CHAPTER

40 COAGULATION AND FIBRINOLYSIS

Ravi Sarode, Craig M. Kessler

OVERVIEW OF HEMOSTASIS, 828 Secondary Hemostasis, 833 ACQUIRED COAGULATION

Laboratory Evaluation of ­DISORDERS, 843
PHYSIOLOGIC HEMOSTASIS, 828
Hemostasis, 833 Disseminated Intravascular
Endothelium and Platelets, 828
Approach To Patients With Coagulation (DIC), 843
Coagulation Protein System, 829
Coagulation Disorders, 836 Liver Disease, 844
The Formation of Fibrin Clot and
HEREDITARY COAGULATION Vitamin K Deficiency, 844
the Fibrinolytic System, 830
­PROTEIN DEFECTS, 836 Massive Transfusion, 845
The Anticoagulation Protein
Systems, 831 Deficiency of Factor VIII (Hemophilia A) Acquired Coagulation Factor
Or Factor IX (Hemophilia B), 837 Deficiencies, 845
Current Hypothesis For Initiation of
The Hemostatic System, 832 Hereditary Deficiencies of Other SELECTED REFERENCES, 846
Coagulation Factors, 840
CLINICAL HEMOSTASIS, 833
Primary Hemostasis, 833

KEY POINTS PHYSIOLOGIC HEMOSTASIS

•  hysiologic hemostasis consists of endothelium, platelets, plasma co-
P
agulation proteins, natural anticoagulants, and the fibrinolytic system.
•  rimary hemostasis involves platelets and von Willebrand factor.
P its cellular component, which consists mostly of platelets and endothe-
Secondary hemostasis is initiated by the tissue factor pathway and
then amplified and propagated by the intrinsic pathway to generate
thrombin that converts fibrinogen to fibrin.
•  outine coagulation tests (prothrombin time and activated partial
R rally occurring serine protease inhibitors (anticoagulation) that termi-
thromboplastin time) may help identify a hemostatic defect in a bleed-
ing patient, but mild to moderate abnormalities of these tests do not
predict a bleeding tendency in a nonbleeding patient.
•  cquired coagulopathy often reflects multiple coagulation defects
A ENDOTHELIUM AND PLATELETS
rather than specific protein defects.
OVERVIEW OF HEMOSTASIS
Hemostasis is a physiologic response to a vascular injury to limit blood
loss. Therefore, it is initiated rapidly, is localized, and is well regulated.
Recently, there has been an evolution in our understanding of the physi-
ologic hemostatic system (Fig. 40.1). Initially, the hemostatic system was
referred to as the coagulation cascade based on the waterfall model of Ratnoff
and Davies; MacFarland published simultaneously a sequence of proteo-
lytic reactions beginning with factor XII (FXII; Hageman factor) activation
and leading to thrombin formation that proteolyzes fibrinogen to form
a clot (MacFarland, 1964; Ratnoff & Davies, 1964). However, the physi-
ologic basis of this pathway was questioned because patients with FXII
deficiency did not bleed. Furthermore, in the mid-­1970s, the cofactors for
FXII activation—prekallikrein and high-­ molecular-­weight kininogen—
were identified and their deficiencies also did not cause bleeding (Weup-
pers & Cochrane, 1972; Colman et al., 1975; Saito et al., 1975). Later, the
physiologic role of tissue factor (TF) and FVIIa was recognized along with
its regulator tissue factor pathway inhibitor (TFPI) (Osterud & Rappaport,
1977). Thus, the physiologic hemostatic system is a cell-­based model in
which cellular elements such as endothelium, platelets, neutrophils, mono-
cytes, and macrophages express TF and provide phospholipid (PL) surface
for various coagulation proteins for attachment and sequential activation
of coagulation factors to generate thrombin at the site of injury. Throm-
bin converts fibrinogen to a fibrin clot. Natural anticoagulants regulate
thrombin generation, and the fibrinolytic system dissolves the clot once it
has served its purpose.
The physiologic hemostatic system, a tightly regulated balance between
formation and dissolution of hemostatic plugs modulated by a series of
enzymes and scaffolding proteins, has two major parts. The first part is
lial cells but also includes neutrophils and monocytes. The second part
is a large group of plasma proteins, which participate in clot formation
(coagulation), dissolution of clots (fibrinolysis), and the action of natu-
nate the activity of several enzymes of the coagulation and fibrinolytic
systems.
Normal hemostasis involves interplay between the cellular components
and proteins involved in clot formation and lysis. Intact endothelium
that lines the vascular wall in a physiologic state exerts a hemostatic bal-
ance. When vascular integrity is breached, blood coagulation is initiated.
Intact endothelial cells secrete an ectonucleotidase, CD39, that degrades
adenosine diphosphate (ADP) (Pinsky et al., 2002). Endothelial cells also
secrete prostacyclin and nitric oxide (NO), which prevent platelet activa-
tion and aggregation, thereby reducing the risk of arterial thrombosis
(Hong, 1980; Palmer et al., 1987; Shariat-­Madar et al., 2006). Endo-
thelial cells additionally bind plasminogen, tissue plasminogen activa-
tor, and single-­chain urokinase, all of which contribute to fibrinolysis
and maintenance of hemostatic balance (Barnathan et al., 1988; Cesar-
man et al., 1994). The glycosaminoglycans present on endothelium bind
antithrombin (AT), which then neutralizes thrombin and FXa (Marcum
et al., 1984). Thrombomodulin on endothelium binds to thrombin; that
complex then converts protein C to activated protein C (APC) (Esmon
& Owen, 1981).
When a vessel wall is injured, subendothelial collagen is exposed and plate-
lets adhere to the site of injury (Van der Meijden et al., 2009). Von Willebrand
factor helps platelets adhere to the injured vessel wall by binding to exposed
collagen at the A3 domain and to the platelet receptor GP Ib/IX/V complex
(He et al., 2003). This adhesion event activates platelets, initiating a signaling
cascade within them (Zaffran et al., 2000). The stimulated platelets release the
contents of their granules, recruit additional platelets to aggregate to those
already adherent and provide a phospholipid surface for the activation of mul-
tiple coagulation factors. Furthermore, in flowing blood, FXII autoactivates if
exposed to collagen or in the milieu of activated platelets, leading to the cas-
cade of proteolytic events producing thrombin (Van der Meijden et al., 2009).
Once prothrombin has been activated to thrombin in the intravas-
cular compartment, the resulting thrombin stimulates endothelium to

828
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XII VII
Tissue factor
Surface
HK
PK XI
XIIa VIIa
Tissue factor/VIIa
IX
XIa
PL, Ca++
Ca++
IXa X
PL, Ca++
Ca++
VIII VIIIa
Xa
Prothrombin
PL, Ca++
Va V and clot retraction (Heemskerk et al., 2013).
THROMBIN
Fibrinogen XIII
Soluble fibrin monomers Ca++
XIIIa
Polymerized fibrin clot
Covalently crosslinked fibrin clot
Figure 40.1 Schematic diagram of physiologic blood coagulation. Note that labels
for the “contact phase” factors constituting factor XII (FXII), high-­molecular-­weight
kininogen (HK), and prekallikrein (PK) are in gray. Although FXI is activated by FXIIa
on a surface under artificial in vitro conditions, such as in activated partial thrombo-
plastin time (APTT), this pathway is not believed to contribute to normal physiologic
hemostasis. Similarly, whereas tissue factor (TF)/FVIIa can directly activate X to Xa in
the in vitro prothrombin time (PT) test under conditions in which supraphysiologic
concentrations of TF are employed, this reaction is shown as grayed because it does
not contribute significantly to clot formation under normal in vivo physiologic con-
ditions. Normal clotting in vivo is initiated when sufficient TF/FVIIa becomes avail-
able to activate FIX to FIXa. Subsequently, FIXa in the presence of FVIIIa activates FX
to FXa, which, in turn, activates prothrombin to thrombin in the presence of FVa.
Thrombin not only then proceeds to clot fibrinogen and to activate platelets but ad-
ditionally exerts critically important positive feedback by activating both factor VIII
and factor V. Physiologic amplification of thrombin formation is believed to result
from thrombin activating FXI to FXIa, thereby providing an additional pathway for
the activation of FIX. Phospholipid (PL) protein complexes present on the surface
membranes of platelets in vivo. Ca++, Calcium ions.
upregulate TF, and TF forms a complex with FVIIa. The complex of
FVIIa–TF activates FIX to FIXa, which converts zymogen FX to enzy-
matically active FXa (see Fig. 40.1). In the presence of FVa from liver
or injured endothelium, FXa activates prothrombin (FII) to thrombin
(FIIa). Under normal circumstances, the rate-­limiting component of pro-
thrombinase complex formation and the ultimate generation of throm-
bin activity is the concentration of FXa (Rand et al., 1996). Thrombin
then proteolyzes fibrinogen to form fibrin. In static in vitro systems, as
little as 5 to 10 pM TF is sufficient to induce clot formation, leading to
a 1000-­to 2000-­fold amplification of the process, which increases the
concentration of thrombin to 10 to 20 nM, which is sufficient to initiate
clot formation (Mann, 2003). In a static model of blood coagulation, the
addition of 5 pM TF results in an average clot time of approximately 5
minutes—sufficiently fast for physiologic hemostasis. Thrombin also is
a major physiologic activator of platelets together with other agonists,
such as collagen, ADP, platelet-­activating factor, and epinephrine. Inacti-
vated platelets probably circulate without promoting coagulation. When
platelets are activated (see Chapter 41), their membranes become a site
for additional thrombin formation. According to a cell-­based model of
coagulation (Hoffman & Monroe, 2001), activated platelets offer the most
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TABLE 40.1
Proteins of the Plasma Coagulation System
Surface-­Bound Vitamin K– Cofactors/
Zymogens Dependent Zymogens Substrates
Factor XII Factor VII High-­molecular-­
PART 5
weight kininogen
Prekallikrein Factor IX Factor VIII
Factor XI Factor X Factor V
Factor II Fibrinogen
Protein C Protein S
important source of negatively charged phospholipids (mainly, phospha-
tidylserine) and procoagulant membranes (Furie & Furie, 2005) needed
for coagulation factor action in the propagation phase. This pathway for
thrombin formation conjoins with FXII autoactivation on exposed ves-
sel collagen and downstream elaboration of thrombin as well (Van der
Meijden et al., 2009). Critical reactions to form FXa and thrombin are
accelerated 300,000-­fold on the platelet surface. Platelets control three
important phases of coagulation: thrombin generation, fibrin formation,
COAGULATION PROTEIN SYSTEM
Characterization of Coagulation Proteins
The proteins that constitute the coagulation system consist of zymo-
gens and cofactors (Table 40.1). The zymogens (proenzymes) of the
coagulation system can be grouped into the phospholipid-­bound and
the surface-­bound categories. Phospholipid-­bound zymogens make up
the physiologically important hemostatic system. These proteins are vita-
min K dependent. Vitamin K is required for an essential γ-­carboxylation
reaction that takes place on the glutamic acid residue of each of these pro-
teins located in their amino-­terminal regions (see Chapter 43, Fig. 43.1).
This carboxylation reaction allows these proteins to bind to phospholipid
and cell membranes, where they are activated. Without this carboxyl-
ation reaction, these proteins do not function normally in the hemostatic
system. Proenzymes include FX, FIX (Christmas factor), FVII, and FII
(prothrombin). Protein C is a vitamin K–dependent phospholipid-­bound
zymogen; however, when activated (APC), it functions as an antico-
agulant by inactivating FVa and FVIIIa (see Chapter 42). Protein S is
another natural anticoagulant that is also vitamin K dependent and acts
as a cofactor for APC.
The surface-­bound proenzymes are proteins of the plasma kallikrein/
kinin system (see Table 40.1). Surface-­bound proenzymes include FXII
(Hageman factor), prekallikrein (Fletcher factor), and FXI. These protein
zymogens are also known as the contact system because FXII autoactivates
when associated with a negatively charged surface, such as a glass tube and
several physiologic substances (Wiggins & Cochrane, 1979; Miller et al.,
1980). The autoactivation phenomenon of FXII allows for a common labo-
ratory test, activated partial thromboplastin time (APTT), which is used to
assess the integrity of the coagulation system. Although absolutely essen-
tial for a normal APTT, the proteins of the plasma kallikrein/kinin system
do not have a role in hemostasis. These proteins (FXII, prekallikrein, and
high-­molecular-­weight kininogen) may participate in blood pressure regu-
lation, fibrinolysis, inflammation, angiogenesis, and thrombosis (Schmaier,
2002, 2003, 2004; Shariat-­Madar et al., 2006; Maas et al., 2008; LaRusch
et al., 2010).
Although deficiencies of FXII, prekallikrein, and high-­ molecular-­
weight kininogen are not associated with bleeding, investigations indicate
that they may have a role in arterial and venous thrombosis. Recently, acti-
vation of the contact factor coagulation pathway has been shown to occur
in vivo from negatively charged surfaces such as polyphosphate, extracellu-
lar RNA, and collagen. In detail, polyphosphate (PolyP) in humans consists
of 60 to 100 phosphate units, and it is released from platelet dense granules
upon platelet activation. PolyP has prohemostatic, prothrombotic, and
proinflammatory properties. It has been identified as an activator of the
contact phase (Wang et al., 2019). It is a potent activator of thrombin and
coagulation FXI, and it accelerates FV activation and abolishes the inhibi-
tory effect of TFPI.
TF (47-­kDa protein) is an essential cofactor for activated FVIIa. It is
found in most tissues and cells. Upregulation of TF results in the forma-
tion of complexes with FVII that produce the initiation of hemostatic reac-
tions. FVIII (antihemophilic factor) is a 330-­kDa protein. When activated
829

CHAPTER 40 COAGULATION AND FIBRINOLYSIS
A Thrombin
B Beta knob
C D
Cross-link
D D
E E
D D
D
Figure 40.2 Formation of a fibrin clot. A, Sche-
matic of fibrinogen. B, Thrombin proteolyzes fi-
brinopeptides A and B from fibrinogen to leave
soluble fibrin monomer. Soluble fibrin monomer
then associates side to side (C) and end to end
(not shown, for clarity) to form fibrin polymers.
D, Thrombin-­activated factor XIII (factor XIIIa)
covalently cross-­links the fibrin polymers into an
increasingly complex structure and an ultimate-
ly insoluble clot (E). Note that “E” corresponds
to the central domain of the original fibrinogen
molecule and “D” to the peripheral domains.
(Modified with permission from Doolittle RF: Fi-
brinogen and fibrin, Sci Am 245:126–135, 1981.) E
to FVIIIa, it is a cofactor for FIXa in the activation of FX. Its absence
is associated with the most severe clinically recognized bleeding disor-
der, hemophilia A. FV is also a 330-­kDa protein with homology to FVIII.
When activated to FVa, it serves as a cofactor for FXa in the activation of
FII (prothrombin) to thrombin. Fibrinogen is a 330-­kDa protein that not
only is the main substrate of thrombin (FIIa) but is also the principal adhe-
sive molecule for platelet aggregation. When fibrinogen is proteolyzed by
thrombin, a fibrin monomer is formed. These monomers associate end to
end and side to side to form a polymerized fibrin clot. The clot is stabilized
by activated FXIII, a tissue transglutaminase that cross-­links the strands of
associating fibrin (Fig. 40.2).
Physiologic Protein Assemblies
Certain critical protein assemblies in hemostatic reactions accelerate
these proteolytic events. The proteins of the coagulation system that
are essential for hemostasis or control of bleeding were originally iden-
tified by observation of affected patients and, more recently, through
mouse knockout studies. Deficiencies in coagulation factors VIII and
IX are the most prominent bleeding disorders that occur in patients
who survive gestation and birth. Rare patients who have congenital
deficiencies of coagulation factors VII, X, V, and II usually do not have
severe bleeding states. In contrast, murine models have demonstrated
that mouse embryos containing complete genetic knockouts of factor
VII, X, V, or II die of massive hemorrhage during gestation or at birth
(Cui et al., 1996; Rosen et al., 1997; Sun et al., 1998; Dewerchin et al.,
2000). This information suggests that the human patient who has a
deficiency of factor VII, X, V, or II may have some factor, albeit less
than 1%, to allow for a milder clinical phenotype than that seen in
mouse models. Alternatively, other compensatory mechanisms may be
operative in the human patient. All of these proteins participate in two
critically important assemblies that are essential for normal hemostasis:
tenase and prothrombinase complexes.
Extrinsic tenase involves TF and FVIIa complex on the PL surface,
converting FX to FXa. The intrinsic tenase complex comprises the assem-
bly of FIXa and FVIIIa on phospholipid surfaces to convert FX to FXa.
When all of these components are present, the rate of FX activation by
FIXa is increased 1.4 × 108-­fold over the rate of FX activation by FIXa
alone. The prothrombinase complex comprises the assembly of FXa and
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E
D D
Fibrinopeptide A
E Alpha knob
D D
Fibrinopeptide B
D E D
E
D
E D D E D D E
E
D D D D
FVa on phospholipid membranes or cell membranes in an ordered struc-
ture with FII (prothrombin) to accelerate its activation to FIIa (thrombin).
When all of these components are present, the rate of FII activation by
FXa is increased 1.7 × 108-­fold over the rate of FII activation by FXa alone.
Because these coagulation reactions occupy critical regulatory points
within the physiologic hemostatic system, they are also the target of a num-
ber of anticoagulant agents for venous thrombosis that are currently in use,
that is, direct FXa and thrombin inhibitors.
THE FORMATION OF FIBRIN CLOT AND THE
FIBRINOLYTIC SYSTEM
The six peptide chains of the fibrinogen molecule are organized into
a structure described as having a central E domain and two terminal
D domains. When thrombin is formed, it cleaves fibrinopeptide A
from the Aα chain and fibrinopeptide B from the Bβ chain of fibrino-
gen in the E domain region (Doolittle, 1981). The remainder of this
thrombin-­proteolyzed fibrinogen is called a soluble fibrin monomer. Sol-
uble fibrin monomers then assemble with an end-­to-­end and side-­to-­
side association to form a noncovalent fibrin polymer (see Fig. 40.2).
Activated FXIII, a transglutaminase, cross-­ links fibrin monomeric
subunits into an insoluble, cross-­linked fibrin clot. When insoluble,
cross-­linked fibrin is made, a new linkage between the D domains of
two adjacent fibrin monomers occurs. In the process, a neo-­epitope of
interaction is formed (see Fig. 40.2).
The fibrinolytic system regulates fibrin clot degradation and clot dis-
solution. In fibrinolysis, the circulating zymogen plasminogen is converted
to its active form plasmin mainly by endogenous tissue plasminogen acti-
vator (tPA) (Draxler & Medcalf, 2015). Two other plasminogen activators
are single-­chain urokinase plasminogen activator (ScuPA) and two-­chain
urokinase plasminogen activator (TcuPA). These activators are found in
the endothelium as well as in neutrophils and monocytes. The natural
plasminogen activators tPA, ScuPA, and TcuPA convert zymogen plas-
minogen to the active enzyme plasmin (Fig. 40.3). tPA is produced con-
stitutively, and ScuPA is increased in inflammatory states. Plasminogen
activator inhibitor-­1 (PAI-­1) is the major inhibitor of tPA and TcuPA. α2-­
Antiplasmin, a serine protease inhibitor (serpin), is the most potent and
highly selective inhibitor of plasmin. However, the plasma concentration
of α2-­antiplasmin, even if high (approximately 1 mM), is only about half
the plasma concentration of plasminogen. The fibrinolytic system is also
regulated by thrombin-­activatable fibrinolysis inhibitor (TAFI) (Vercau-
teren et al., 2013). TAFI is a carboxypeptidase able to remove lysine resi-
dues from fibrin, such that it indirectly controls plasmin by interfering with
molecular mass by eliminating portions of the α-­chain (Marder & Budzyn-
ski, 1974). Plasmin then cleaves the X fragment asymmetrically between
the D and E domains to produce fragment Y. Plasmin further degrades
the Y fragment to produce soluble D and E domains that are called soluble
fibrinogen degradation products or, in the case of fibrin digestion by plasmin,
fibrin degradation products. Their presence indicates that plasmin has been

the binding of plasminogen and tPA to fibrin.
The binding of both plasminogen and tPA to fibrin can increase plas-
min generation by two orders of magnitude. The active plasmin molecule
is a potent protease, and it recognizes multiple substrates. Plasmin will
degrade soluble fibrinogen to produce fibrinogen degradation products
(Fig. 40.4A). Plasmin cleaves fibrinogen into an X fragment of similar
PART 5
formed. Plasmin will also degrade insoluble, crossed-­linked fibrin (Fig.
40.4B). When it does, it liberates a D-­D-­dimer domain formed as a result
of the neo-­epitope between these two domains (see Fig. 40.4B) (Marder
et al., 1976). The presence of soluble D-­dimer indicates that, first, throm-
bin has been formed, followed by clotting, then the clot has been cross-­
linked by FXIIIa, and, finally, plasmin has been formed and has cleaved the
insoluble, cross-­linked fibrin clot.

THE ANTICOAGULATION PROTEIN SYSTEMS

Plasminogen Coagulation is tightly regulated by natural anticoagulants and the fibri-
nolytic system to avoid thrombus formation and extension. Three major
tPA Fibrin(ogen) anticoagulant systems regulate the enzymes of the coagulation protein
ScuPA TcuPA Degradation Products (FDP) system to help to inhibit clot formation. These systems are the protein C/
protein S system, the plasma serine protease inhibitor system, and TFPI,
a Kunitz-­type serine protease inhibitor. AT is the main serine protease
Plasmin inhibitor of coagulation enzymes of the plasma serine protease inhibitor
Fibrin, fibrinogen system. Each of these systems plays a critical role in the proper regulation
of the coagulation system. Moreover, in murine deletion models, com-
Figure 40.3 Fibrinolysis. Zymogen plasminogen is converted to plasmin by tissue
plete deficiencies of protein C, protein S, AT, and TFPI are incompat-
plasminogen activator (tPA), single-­chain urokinase plasminogen activator (ScuPA), ible with the survival of mammalian gestation, delivery, or life ex utero
and two-­chain urokinase plasminogen activator (TcuPA). Formed plasmin degrades (Huang et al., 1997; Jalbert et al., 1998; Hayashi et al., 2006; Burstyn-­
fibrinogen or fibrin to form fibrinogen or fibrin degradation products, respectively. Cohen et al., 2009).

αC αC
Plasmin
E
D D Fibrinogen

Plasmin
E
D D Fragment X

Plasmin
E
D D Fragment Y

E
D D Fragments
A
D and E

D D D D
E E E
E
D D D Figure 40.4 A, Plasmin-­ cleaved soluble fibrinogen or fibrin.
D When plasmin cleaves fibrinogen, initially small portions from
the α-­chain (αC) are removed to make Fragment X. Fragment X
is then asymmetrically cleaved into Fragment D and Fragment Y.
Fragment Y is further cleaved by plasmin into Fragments D and E.
(Modified with permission from Greenberg CS, Lai T-­S: Fibrin for-
mation and stabilization. In Loscalzo J, Schafer AI, editors: Throm-
bosis and hemorrhage, ed 3, Philadelphia, 2003, Lippincott Wil-
liams & Wilkins, p 83, Fig. 5.3B.) B, Plasmin-­cleaved insoluble,
D D cross-­linked fibrin. When insoluble, cross-­linked fibrin is prote-
olyzed by plasmin, the neo-­epitope between the D-­domains is
E preserved, and the liberated fragment consists of the D-­dimer to-
gether with an E domain. (Modified with permission from Doolit-
B tle RF: Fibrinogen and fibrin, Sci Am 245:126–135, 1981.)

831
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 Protein C/Protein S System
CHAPTER 40 COAGULATION AND FIBRINOLYSIS
When activated, protein C, a 62-­kDa vitamin K–dependent protein, is
an enzyme that functions as an inhibitor. When free thrombin binds to VII
thrombomodulin on endothelium, this complex then converts protein C
to APC. Its activation is increased when protein C binds to the endothe- XI XIa Tissue factor
lial protein C receptor (EPCR). The complex formed by APC+EPCR
needs to dissociate in order to allow binding of APC with protein S to VIIa
inactivate FVa and FVIIIa (Esmon, 2006). Protein S, a 69-­kDa vitamin IX
K–dependent protein, is a cofactor, or receptor, for APC on cell mem- VIIIi Tissue factor/VIIa
branes. It allows APC to bind to cell surfaces in such a manner as to PL, Ca++
Ca++ Tissue
orient itself to inactivate FVa and FVIIIa. The enzyme uses this cofactor
IXa X factor
as a receptor to localize its activity to perform its inhibitory function.
pathway
Plasma protein S is in equilibrium between the free form (40%) and PL, Ca++ inhibitor
a bound form (60%) that is complexed to C4b-­binding protein; only (TFPI)
the free form functions as a cofactor for APC. APC also binds to three VIII VIIIa
Xa
receptors on endothelial cells: (1) endothelial cell protein C receptor Vi
to activate (2) protease-­activated receptor (PAR) 1 and (3) apolipo- Prothrombin
protein E receptor 2 (ApoER2) (Esmon, 1999; Coughlin, 2000; Yang Antithrombin PL, Ca++
et al., 2009). Activation of PAR1 on endothelial cells may contribute
to the anticoagulant function of activated protein C by liberating tPA. Va V
APC on endothelial cells also has an inflammatory effect via activation
of sphingosine-­1-­phosphate receptor transactivation and activation of Thrombin
ApoER2 (Finigan et al., 2005; Yang et al., 2009). Thus, APC reduces
thrombin formation, stimulates fibrinolysis, and initiates inflammation
to reduce thrombosis risk. Thrombin Thrombomodulin Cell surface

Antithrombin
This serpin is a 58-­kDa protein that inhibits each of the following hemo-
static enzymes: IIa, Xa, VIIa, IXa, XIa, kallikrein, and XIIa. It exerts its
anticoagulant effect primarily by inhibiting FIIa and FXa (Fig. 40.5). Anti-
thrombin has two functional sites: the heparin-­binding site (at the amino
terminus of the protein) and the reactive site (Arg329-­Ser394). The ability
of antithrombin to function as an inhibitor of coagulation protein enzymes
is potentiated by endogenous and exogenous heparins. In fact, it is the
presence of antithrombin that gives heparin its anticoagulant properties.
In the presence of heparins, antithrombin undergoes a conformational
change that increases from 1000-­fold to 4000-­fold its inhibitor effect on
FIIa (Perry, 1994). Trace amounts of circulating TF or TF transiently
exposed by damaged vessels contribute to FVIIa inactivation by antithrom-
bin (Vatsyayan et al., 2014).
In addition to antithrombin, other serpins regulate the enzymes
of the hemostatic and inflammatory systems. Heparin cofactor II is a
serpin that specifically inhibits thrombin in the presence of dermatan
sulfate. Protein Z inhibitor is a serpin that specifically inhibits factor
Xa in the presence of its cofactor protein Z—a vitamin K–dependent
protein. C1 inhibitor (C1 esterase inhibitor) is the most potent inhibitor
of FXIIa, kallikrein, and FXIa in plasma. Its main function is to regu-
late the amount of free bradykinin in the intravascular compartment and
reduce inflammatory events (Han et al., 2002; Schmaier, 2002; Maas
et al., 2008). The absence of C1 inhibitor is the causative factor for types
I and II hereditary angioedema, a disorder associated with excessive bra-
dykinin delivered to tissues.
Protein C
Protein C
Activated
protein C
Protein S
Protein S
C4b-binding protein
Figure 40.5 Natural inhibitors of coagulation: antithrombin (AT); components of
the protein C pathway (thrombomodulin, protein C, protein S); and tissue factor
pathway inhibitor (TFPI). Three major anticoagulant systems are recognized. An-
tithrombin inhibits factor Xa and thrombin to prevent thrombosis. It is important to
note that antithrombin inhibits every enzymatic form of blood coagulation serine
proteases (factor XIIa, plasma kallikrein, factor XIa, factor IXa, and factor VIIa), even
though this is not indicated in the figure for readability purposes. The protein C
system requires that this zymogen is activated to activated protein C by thrombin
when bound to the endothelial cell membrane protein thrombomodulin. Protein S
is a cofactor for activated protein C to inactivate factors Va and VIIIa. C4b-­binding
protein regulates protein S activity. TFPI makes a quaternary complex with tissue
factor/factor VIIa and factor Xa to inhibit both enzymes.

Tissue Factor Pathway Inhibitor
In addition to the serpins, there is the Kunitz-­type serine protease inhibi-
tor, TFPI (Crawley & Lane, 2008). TFPI is the most potent inhibitor of the
FVIIa–tissue factor complex. Under physiologic conditions, TFPI exerts
its inhibitory effects by forming a quaternary complex with FVIIa, TF,
and FXa (see Fig. 40.5). It is noteworthy that the murine TFPI knockout
is embryonically lethal (Jalbert et al., 1998). TFPI is produced by micro-
vascular endothelium, and it seems mainly associated with glycosaminogly-
cans on the endothelial wall. Only approximately 20% of TFPI circulates
in plasma associated with lipoproteins (Caplice et al., 2001). Another fam-
ily of Kunitz-­type serine protease inhibitors, the amyloid β-­protein pre-
cursor (AβPP) and related members, is present in platelets and brain and
regulates factors XIa, IXa, Xa, VIIa/TF, and plasmin (Van Nostrand et al.,
1990; Schmaier et al., 1993, 1995; Mahdi et al., 1995, 2000). This inhibitor
does not inhibit thrombin. The exact function of this inhibitor is not com-
pletely known, but it is believed to be a cerebral anticoagulant (Del Zoppo,
2009). AβPP and amyloid precursor-­like/protein 2 gene–deleted mice sur-
vive gestation but have an accelerated rate of thrombosis in a carotid artery
thrombosis model (Xu et al., 2009).
Prostacyclins, Nitric Oxide, and Thrombospondin-­5
In physiologic conditions, endothelial cells produce vasoactive hormones
able to control the primary phase of hemostasis. Endothelial cells express
the cyclooxygenase-­2 enzyme that produces prostacyclins (PGI2) from
arachidonic acid (Mitchell et al., 2008). PGI2 is a vasodilator and inhib-
its platelet aggregation. The actions of prostacyclins are mediated by
cell membrane prostacyclin (IP) receptors and intracellular peroxisome
proliferator-­activated receptors (PPARβ and PPARγ). Endothelial cells
also express the NO synthase enzyme (eNOS, NOS3), and they release
NO derived from l-­arginine. NO is a potent vasodilator that also inhib-
its platelet adhesion and aggregation. NO activates guanylate cyclase and
cGMP. Activated platelets adhering to collagen seem also able to produce
NO under shear flow (Cozzi et al., 2015), thus, controlling platelet adhe-
sion and vasoconstriction directly at the sites of vessel damage.
Thrombospondin-­ 5, also known as cartilage oligomeric matrix protein
(COMP), is an extracellular protein able to control vascular tone. COMP is
released by a variety of cartilage and muscle tissues and activated platelets.
COMP inhibits thrombin and acts as a natural anticoagulant in mice (Liang
et al., 2015). It determines a dose-­dependent prolongation of thrombin time.
CURRENT HYPOTHESIS FOR INITIATION OF THE
HEMOSTATIC SYSTEM
More proteins participate in coagulation reactions in vitro than are critical
for hemostatic reactions in vivo. The original coagulation cascade waterfall

832
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hypothesis initiated by FXII has been replaced by one whereby TF and
FVIIa are initiators (Gailani & Broze, 1993). TF is ubiquitous throughout
the body, although it is rich in the brain, lung, and placenta. Its expression
is upregulated after injury. Regulation of the expression of TF is a major
control mechanism for the initiation of hemostasis. TF is not fully active
on intact cells; it becomes able to activate coagulation when cell membrane
properties are modified. This process is called decryption of encrypted TF and
is still not completely understood. Different mechanisms of TF decryption
have been proposed: an exposition of phosphatidylserine, dimerization of
TF, and oxidation of a specific disulfide bond in TF (Smith et al., 2015).
In conditions in which a vessel wall injury is limited to endothelial activa-
tion, blood-­borne TF has been characterized on microparticles released
by leukocytes and involved in the initiation of coagulation (Furie & Furie,
2005). Activated macrophages have been shown to express TF on their
cell membrane and microparticles involved in physiologic hemostasis and
thrombosis (Rak, 2014). There is a developing notion that endothelial cell
TF is the initial nidus that leads to hemostasis and thrombosis (Furie, 2009;
Ivanciu et al., 2014).
The FVIIa–TF complex activates FIX, leading, in turn, to FX acti-
vation. It must be noted that at the concentrations of factors ordinar-
ily present in the body, FVIIa/TF does not directly activate appreciable
amounts of FX because of the presence of TFPI—a point emphasized
diagrammatically in Figure 40.1 by the graying of this reaction. Once FX
has been activated to FXa, this FXa, in turn, contributes to the activation
of thrombin from prothrombin. Thrombin then proteolyzes fibrinogen
to form fibrin. Although TF-­dependent coagulation reactions are rapidly
inhibited by TFPI, if the stimulus for thrombin formation is sufficiently
strong, coagulation is maintained through the activation of FXI by throm-
bin (Meijers et al., 2000). Activation of FXI to FXIa results in increased
activation of FIX and eventually increases the formation of thrombin.
Further modulation of hemostatic balance in the direction of clot forma-
tion is exerted by TAFI (also known as carboxypeptidase U). Among its
actions, TAFI cleaves C-­terminal lysine residues from fibrin, thereby
decreasing the binding of plasminogen to the clot and diminishing clot
lysis (Leurs et al., 2005).
CLINICAL HEMOSTASIS
PRIMARY HEMOSTASIS
At the site of endothelial cell injury, subendothelial collagen and von Wil-
lebrand factor (vWF) are exposed. The disrupted endothelium alters lami-
nar blood flow at the site of injury; thereby, platelets undergo shape change
from discoid to spherical and produce pseudopodia that will breach the
site of endothelial damage by binding to vWF via GPIb/IX/V receptors.
This step is called platelet adhesion (attachment of platelets to a nonplatelet
surface), which is followed by secretion. During secretion, contents of alpha
and dense granules from platelets are released along with the generation
of thromboxane A2 (TXA2) via the cyclooxygenase-­1 (COX-­1) pathway.
TXA2 causes vasoconstriction and activates platelets via the TXA receptor.
During the next step of platelet activation, the signal transduction within
platelets leads to a conformational change in GPIIb/IIIa, allowing fibrino-
gen (acts as a ligand) to bring adjacent platelets together, causing aggrega-
tion (attachment of one platelet to another). Activation of platelets leads
to exposure of phosphatidylserine (PS) similar to altered endothelium that
also exposes PS for secondary hemostasis that begins simultaneously to
generate thrombin.
SECONDARY HEMOSTASIS
Secondary hemostasis can be divided into two different phases: (1) the
TF pathway (previously called the extrinsic pathway), which is the ini-
tiator of coagulation; and (2) the intrinsic pathway, which amplifies and
propagates thrombin generation. The TF pathway begins with endo-
thelial injury and expression of TF on the cell surface, which binds the
small amount of FVIIa present in circulation to form a TF+FVIIa com-
plex. This complex is known as extrinsic tenase because this will activate
FX to FXa on the cell surface in the presence of Ca++. This FXa along
with FVa on the cell surface (PL) and Ca++ forms a prothrombinase com-
plex, converting prothrombin to thrombin. As soon as a small amount of
thrombin is generated by the TF pathway, this thrombin activates the
TFPI, which shuts down the TF pathway by inhibiting the TF+FVIIa
and FXa complex. The same thrombin also initiates the intrinsic path-
way by activating FXI to FXIa and FIX to FIXa on the cell surface (PL)
with Ca++. FIXa along with FVIIIa on the cell surface (PL) and Ca++
forms the intrinsic tenase and converts FX to FXa, which, along with FVa,
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forms the prothrombinase complex converting prothrombin to throm-
bin. This intrinsic pathway is the primary pathway of thrombin forma-
tion. Thus, the TF pathway is the initiator and the intrinsic pathway is
the amplifier and propagator of thrombin, and these two pathways are
interrelated. The previous concept of independent extrinsic and intrin-
sic pathways is not physiologic because if these two were independent
PART 5
pathways producing thrombin, then patients with hemophilia A and B
would not have bleeding tendencies. The independence of extrinsic and
intrinsic pathways to produce thrombin is “true only in the test tubes”
where prothrombin time (PT) and activated partial thromboplastin time
(APTT), respectively, assess them. Thrombin is the main enzyme in the
coagulation system because it activates FV and FVIII to their activated
forms to amplify further thrombin generation and converts fibrinogen
to fibrin monomers. It also activates FXIII to FXIIIa to cross-­link these
fibrin monomers to stabilize the clot.
LABORATORY EVALUATION OF HEMOSTASIS
Laboratory evaluation of primary hemostatic defects presenting as
mucocutaneous type bleeding due to platelets and vWF is described in
Chapter 41. Patients with bleeding tendency due to secondary hemosta-
sis defects often present with ecchymoses, soft tissue, and joint bleeds.
Bleeding tendencies could be congenital or acquired. Therefore, an
elaborate personal and family history of bleeding is the prerequisite for
appropriate laboratory testing. Most congenital bleeding disorders often
are diagnosed early in childhood; however, mild bleeding disorders may
not be revealed until later in life when there is a robust hemostatic
challenge.
Routine coagulation screening tests are useful to identify a hemo-
static defect in a bleeding phenotype. However, performing these tests
to assess hemostasis in a nonbleeding patient is not clinically useful—it
often may lead to unnecessary expensive further testing and could delay
surgeries.
Prothrombin time (PT), APTT, fibrinogen, thrombin time and
D-­dimers are considered screening coagulation tests along with platelet
count for initial evaluation of a bleeding patient. Whole blood is col-
lected in 3.2% sodium citrate (blue top) in a 9:1 ratio (blood:citrate) by
a single needlestick without trauma or prolonged stasis and mixed well
by gently inverting 3 to 5 times (avoid vigorous mixing that may activate
platelets) (Adcock, 1998). The specimen should be transported to the
laboratory at room temperature, and the testing should be completed
within 4 hours of collection. However, if the testing cannot be per-
formed within 4 hours of collection, then ideally the specimen should
be centrifuged immediately to separate plasma from cellular compo-
nent and frozen at −20°C and then transported on dry ice for testing
to the laboratory. The separated plasma ideally should be platelet free
(<10 K) before freezing because frozen plasma that is rich in platelets
upon thawing will cause disruption of platelets releasing platelet factor
4, which binds to heparin and may give false low anti-­FXa activity, or
phospholipids released from platelet membranes bind to lupus antico-
agulant and may give false-­negative results. The specimen should not
be held at room temperature for more than 4 hours to avoid loss of
FVIII (and, to some extent FV, both being heat-­labile factors) to avoid
getting spuriously long APTT and low FVIII (and vWF) that might
result in a further unnecessary investigation. Improper sample handling
is often encountered in office clinics that send out their specimens to
remote reference laboratories for coagulation testing. Thus, the pre-
analytic part of coagulation testing is crucial for accurate diagnosis and
management.
Prothrombin Time
PT is a simple test in which the patient plasma is mixed with a PT reagent
that contains tissue thromboplastin and CaCl2. Tissue thromboplastin
consists of tissue factor and phospholipid. The source of tissue throm-
boplastin is important because nonhuman sources (e.g., rabbit brains)
may have lower sensitivity to various vitamin K–dependent coagulation
factors as compared with human sources (e.g., placenta) or recombinant
tissue thromboplastin. The international normalized ratio (INR) was
developed to harmonize PT results for monitoring vitamin K antagonist
(VKA) therapy. The INR is a calculated value obtained by first calcu-
lating the prothrombin time ratio (PTR) by dividing the patient PT by
the control PT, and then raising this PTR by an international sensitiv-
ity index (ISI) [INR = (PTR) ISI]. Ideally, the INR should be used only
for monitoring VKA therapy. However, due to the inclusion of the INR
in the model of end-­stage liver disease (MELD) score calculation, it is
often also used to assess hemostasis in patients not on VKA for no good
833
 reason other than the convenience. When the INR was used for MELD
CHAPTER 40 COAGULATION AND FIBRINOLYSIS
score calculation, the PT reagents most likely had a higher ISI with less
sensitivity for vitamin K–dependent factor deficiencies than most current
PT reagents, especially recombinant tissue thromboplastin. With differ-
ent ISI, the INR can be different; hence, the MELD score can also be
significantly different.
Isolated prolonged PT is usually due to an FVII deficiency and may also
be prolonged due to mild deficiencies of common pathway FII, FV, and FX
depending on the reagent’s sensitivity. Recombinant tissue thromboplastin
is very sensitive to even mild reduction of FVII (40%–45% activity), which
is not clinically relevant since most patients with mild to moderate FVII
deficiency are clinically asymptomatic and are often diagnosed when there
is an incidental prolonged PT. Hemostatic levels of FVII for major sur-
gery are 10% to 20%; however, even mild prolongation of PT/INR often
results in unnecessary plasma transfusion to correct a laboratory value that
is not clinically relevant. Because of rebalanced hemostasis in patients with
cirrhosis (discussed later), several societies have recommended ignoring
PT/INR values obtained preprocedurally in cirrhotics.
Activated Partial Thromboplastin Time
APTT is another routinely performed test to assess clinical hemosta-
sis with poor sensitivity and specificity in a nonbleeding patient. APTT
generally assesses coagulation factors in the intrinsic pathway as well as
the common pathway. In APTT, a contact activator (e.g., ellagic acid,
silica, kaolin) is added to the patient’s plasma to initiate the activation
of contact factors (prekallikrein, HMWK, and FXII) in the presence of
PLs. After incubation for 3 to 5 minutes, the plasma is recalcified and the
clotting time is recorded (usual range, 23–35 sec). Unfortunately, APTT
reagents are not well standardized; hence, there is significant variation
from one reagent to another and when we include coagulation instru-
ments from different companies, the variability in APTT is even more
significant. The APTT reagents come with three different quantities of
PL with different sensitivities for various needs. APTT-­factor sensitive
(APTT-­FS) has the highest amount of PL, making this reagent very sen-
sitive to detect even slightly lower levels of coagulation factors in the
intrinsic pathway. APTT-­LA (lupus anticoagulant sensitive) reagent has
the smallest amount of PL, making it very sensitive to detect even a weak
LA. APTT-­FSL (factor and lupus sensitive) has an intermediate amount
of PL; hence, it should be used in daily practice because this will detect
clinically relevant decreased amounts of coagulation factors and lupus
anticoagulant.
Isolated prolonged APTT is a common clinical finding. Further inves-
tigation should be dictated by a personal and family history of bleeding
if that is the presenting symptom, whereas in a nonbleeding patient it is
essential to check previous APTTs. If the previous APTTs were normal,
then the current prolongation is due to an acquired defect, most likely
lupus anticoagulant. However, if the previous APTTs are also prolonged,
then the contact factor deficiency should be considered and investigated
because FXII-­, HMWK-­, and PK-­deficient patients do not bleed.
In a bleeding patient, if APTT is prolonged, unfractionated heparin
should be excluded. If the previous APTTs were also prolonged, then a
congenital bleeding disorder of intrinsic pathway should be considered
and investigated by measuring FVIII first (common thing first!) followed
by FIX or, rarely, FXI. If the previous APTTs were normal and current
presentation includes soft tissue hematomas, easy bruising, and other con-
ditions, with a prolonged APTT, then acquired hemophilia should be con-
sidered due to autoantibody against FVIII.
Thrombin Time (TT)
Thrombin time (TT) is usually not a commonly ordered test because of its
limited utility in laboratory assessment of most bleeding disorders. In the
TT test, bovine thrombin (3–5 U/mL) is added to the patient’s plasma and
the clotting time recorded. The upper limit of the normal range should be
adjusted close to 25 sec to make TT more sensitive to detect dysfibrino-
genemia. The most frequent cause of prolonged TT is heparin followed by
hypofibrinogenemia and then dysfibrinogenemia. Direct thrombin inhibi-
tors (oral and parenteral) significantly prolong TT.
Fibrinogen Assay
Fibrinogen assay should be measured routinely in a bleeding patient
and patients with cirrhosis. The fibrinogen assay is based on TT (Clauss
method) in which a standard curve is generated by using known calibrators
and bovine thrombin at a very high concentration of 50 U/mL (for TT 3–5
U/mL) so that the clotting time is independent of thrombin concentration.
Bovine thrombin is added to the patient’s plasma and the clotting time
obtained is used to extrapolate fibrinogen value from the standard curve.
834
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A very high amount of the direct thrombin inhibitor will give false low
fibrinogen levels.
If there is clinical suspicion for hypofibrinogenemia, the direct fibrino-
gen assay should be performed rather than relying on PT and APTT to
be prolonged because, in the majority of patients with mild to moderate
hypofibrinogenemia, PT and APTT are likely to be normal. When PT and
APTT were performed manually in the past, the endpoint was a solid, firm
clot formation on the side of the test tube. However, with automation, the
endpoint of PT/APTT is the change in optical density or turbidity due to
formation of fibrin strands. Therefore, the endpoint is often sooner than
the manual method; hence, automated PT and APTT are usually normal,
with mild to moderate hypofibrinogenemia.
D-­Dimers
D-­Dimers are generated by plasmin that cleaves the cross-­linked fibrin
clot. Increased D-­dimers suggest that a clot is undergoing lysis in the cir-
culation. In the D-­dimer assay, a monoclonal antibody against the D-­dimer
molecule is attached to latex particles that agglutinate in the presence of
D-­dimers, causing a change in optical density. The optical density is then
read off a standard curve to obtain a D-­dimer value. In the appropriate
clinical setting, it is useful for a diagnosis of disseminated intravascular
coagulation (DIC), where it is elevated, and to rule out deep vein throm-
bosis (DVT) and pulmonary embolism (PE) in patients with low clinical
probability, where the D-­dimer is not elevated. A large hematoma will also
increase D-­dimers while undergoing resolution.
APTT Mixing Study
In the past, when there were no specific tests to detect lupus anticoagulant
(such as dilute Russell viper venom time, PTT-­LA with hexagonal PL neu-
tralization, and silica clotting time), a mixing study involving equal parts of
patient plasma and normal pooled plasma (NPP) has been often helpful. A
circulating inhibitor was suspected when there was incomplete correction,
and a factor deficiency was suspected when there was complete correction of
the prolonged APTT. However, it must be emphasized that the routine use
of such a mixing study for mildly prolonged APTT can lead to a missed or
wrong diagnosis (Yates et al., 2019). This is due to several reasons, includ-
ing poorly standardized mixing study protocols. Often, these studies are
performed without proper controls and there is often a lack of understand-
ing of how many normal plasmas should be used to prepare NPP. Also,
performing a mixing study only on immediate mixing and without 2-­hour
incubation at 37°C leads to missing an autoantibody against FVIII, the
cause of acquired hemophilia. Investigation of isolated prolonged APTT
should be based on a thorough clinical history of bleeding or no bleeding/
clotting. Mildly prolonged APTT due to lupus anticoagulant can be cor-
rected with NPP, resulting in misinterpretation of a factor deficiency and,
hence, unnecessary measuring intrinsic pathway factors. The presence of a
lupus anticoagulant may influence clot-­based assays for intrinsic pathway
factor levels, especially FVIII. However, if one looks at factor assay curves,
especially FVIII assay, there is an inhibitory pattern observed indicating
that there is interference by the lupus anticoagulant. The lupus anticoagu-
lant effect is revealed by the observation that with each subsequent dilution
of the patient’s plasma during FVIII assay, the FVIII activity increases by
>20%. Such patients generally do not have a bleeding tendency and have
had normal APTTs in the past.
Acquired hemophilia is not an uncommon clinical condition and gener-
ally presents with a prolonged APTT with significant clinical bleeding in
the soft tissues. Such a patient with normal APTTs in the past and in the
right clinical setting should be tested first for FVIII level, which is often
diagnostic of acquired hemophilia. Later, a proper two-­step mixing study
can be done to identify a time and temperature-­dependent inhibitor. In a
two-­step mixing study, the patient’s plasma is first mixed with the NPP
and immediately tested for APTT (which often corrects, unless the patient
has concomitant LA). The second tube with the patient’s plasma and NPP
is incubated at 37°C for 2 hours and then tested for APTT, which shows
further prolongation as compared with the immediate mix.
A routine mixing study for PT is not warranted because isolated pro-
longed PT due to autoantibody against FVII is extremely rare. Most of
these patients with isolated prolonged PT/INR generally have vitamin K
deficiency. Therefore, these patients should be given intravenous vitamin
K (10 mg), which is both diagnostic and therapeutic, confirming vitamin
K deficiency, especially in sick patients due to poor diet and antibiotic
use.
Heparin Monitoring
Therapeutic heparin monitoring is required to avoid over-­and under-­
anticoagulation because of unpredictable pharmacokinetics (PK) and
pharmacodynamics (PD) of unfractionated heparin (UFH). The unpre-
dictable PK and PD are due to the presence of many larger polysaccharide
molecules that get attached to the endothelium, platelets, and acute-­phase
reactant proteins, such as vWF and fibrinogen, causing reduced availability
for their action on antithrombin to act as an anticoagulant in the first 24 to
72 hours of therapy in patients with DVT/PE. Therefore, UFH is given
as a bolus to saturate these cells and proteins. The continuous intravenous
infusion is then monitored every 6 hours until the APTT is within the thera-
peutic range. The APTT therapeutic range has to be established for each
new lot number of APTT reagent in each laboratory, that is, every 6 to 12
months. This therapeutic range is established by measuring APTT and anti-
­Xa activity simultaneously on 50 patients on stable UFH (but not on VKA)
and extrapolating corresponding APTT for anti-­Xa activity of 0.3 to 0.7 U/
mL from a linear regression curve. However, this therapeutic range is not
adjusted for different lots of UFH obtained by the hospital pharmacy!
Heparin resistance is often encountered when APTT is used to monitor
UFH therapy in patients with very high levels of FVIII due to an acute-­phase
effect or those with low AT levels (often acquired and rarely due to con-
genital deficiency). The very high levels of FVIII can be suspected when the
baseline APTT is either at the lower end of the normal range or even shorter
than the lower limit of normal. Most clinicians often do not pay attention
to this phenomenon, which results in patients getting higher amounts of
UFH. This increases anti-­Xa activity, which may lead to severe bleeding
despite APTT showing resistance. In the case of heparin resistance due to
high FVIII, an anti-­Xa assay should be used to monitor UFH. However, if
anti-­Xa activity is also low despite higher UFH, then AT deficiency should
be suspected. If the AT activity is <50%, then AT concentrate may be given;
that will prolong APTT appropriately as well as improve ant-­Xa activity.
Anti-­Xa Activity Assay for Heparin Monitoring
This assay provides an accurate measurement of both UFH and low-­
molecular-­weight heparin (LMWH) levels for therapeutic monitoring.
There are two types of anti-­Xa assays, one with and the other without
exogenously added AT to the reagent. The assay without added AT
that relies on AT in the patient’s plasma is a better assay because it will
detect heparin resistance due to AT deficiency by showing lower anti-
­Xa activity despite adequate UFH dose. UFH/LMWH present in the
patient’s plasma binds to the patient’s own AT and neutralizes the known
amount of FXa present in the reagent. The remaining free FXa acts on
a chromogenic substrate to generate a color. The higher the amount of
heparin in plasma the lower the amount of free FXa and, hence, lighter
the color. This intensity of the color is read as an optical density, and the
amount of anti-­Xa activity is read off of a standard curve generated with
commercial calibrators. This assay is quite robust and, currently, liquid
reagents with 7 days’ stability on board the coagulation instrument have
resulted in replacing APTT in monitoring UFH routinely. The heparin
assay is known to reduce the number of times that heparin dose has to be
adjusted as compared with APTT, thus saving both nursing and pharmacy
time. Therefore, it is very cost-­effective for the hospitals.
Coagulation Factor Assays
One-­stage clot-­based assays are the most commonly used methods for
coagulation factors. The basic principle is similar for all coagulation
factor assays. A standard curve is prepared by making serial dilutions of
NPP with the factor-­deficient plasma that is to be measured (e.g., FVIII-­
deficient plasma for FVIII assay) to obtain 100%, 50%, 25%, and 12.5%
factor activity. Then, either PT (for FVII, II, V, and X assays) or APTT
(for FXII, XI, IX, and VIII assays) are performed and plotted against
appropriate dilutions (Figs. 40.6 and 40.7). The patient’s plasma is then
similarly diluted with that particular factor-­deficient plasma, and corre-
sponding PTs or APTTs are measured. The value of factor activity in the
patient’s plasma is then extrapolated from the standard curve using PT or
APTT. The rationale for making serial dilution of patient plasma is mostly
to dilute out the effect of lupus anticoagulant interference in the APTT-­
based assay; with each subsequent dilution of patient plasma, the effect of
LA is diluted whereas the factor activity increases. This curve obtained in
the presence of LA is known as a nonparallel curve. This pattern alerts the
laboratory technician to make a further dilution of the patient plasma until
the LA effect has disappeared and the correct factor level is obtained. If this
step is not performed, then patients with isolated prolonged APTT may
have spuriously low FVIII activity, which may lead to misdiagnosis and
mismanagement of that patient.
Chromogenic FVIII Assay
There are caveats with the one-­stage FVIII assay at present, with many
extended half-­life (EHL) FVIII concentrates on the market that often show
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Surface Tissue
Activation Thromboplastin
Intrinsic Extrinsic
XII, PK
PART 5
HK
XI VII
IX
VIII
Common
X
Partial Prothrombin
V
Thromboplastin Time
Time II
Fibrinogen
Thrombin Time
Fibrin
Figure 40.6 Organization of the coagulation system based on current assays. The
intrinsic coagulation system consists of the protein factors XII, XI, IX, and VIII and
prekallikrein (PK) and high-­molecular-­weight kininogen (HK). The extrinsic coagu-
lation system consists of tissue factor (tissue thromboplastin) and factor VII. The
common pathway of the coagulation system consists of factors X, V, and II, and
fibrinogen (I).
lower than expected recovery in patient plasma. This lower recovery of
EHL-­FVIII is usually attributed to variable amounts of phospholipid pres-
ent in various APTT reagents and variable sensitivity of modified FVIII
to phospholipids in APTT reagents. Similarly, hemophilia A patients on
emicizumab have normal APTT; hence, FVIII levels or FVIII inhibitor
titer based on the one-­stage APTT cannot be measured. Therefore, the
chromogenic FVIII assay is used in such situations. In the chromogenic
assay, activated FIX is added to the patient plasma, which then binds to
FVIIIa in patient plasma (the bovine thrombin in the reagent converts
FVIII to FVIIIa). The FVIIIa and FIXa complex then acts on FX to give
FXa, which, in turn, cleaves a chromogenic substrate to generate a color.
The amount of color is proportional to FVIII levels and reads off a stan-
dard curve. The other advantage of chromogenic FVIII assay is that it is
unaffected by the presence of LA.
Viscoelastic Measurement of Real-­Time Clot
PT and APTT are often useful screening tests to assess hemostasis affect-
ing coagulation factors in a bleeding patient. However, they have many
limitations in assessing global hemostasis in real time in a bleeding patient
who needs blood component therapy. Also, the turnaround time for PT
and APTT is not fast enough for surgeons/anesthesiologists to make a
timely decision in the operating room (OR).
Thromboelastography (TEG) and rotational thromboelastometry
(ROTEM) are two global hemostasis assays with similar principles for
viscoelastic measurement of a clot in real time (Agren, 2013). Both use
whole blood and should be used to differentiate between coagulopathic
and surgical bleeding. They cannot predict a bleeding tendency in a non-
bleeding patient. There is a pin in the cup that records the torsion during
the rotational movement while a clot is formed and various parameters
are reflected on a tracing. In TEG, the pin is fixed and the cup rotates; in
ROTEM, the cup is fixed and the pin rotates. The tracing is displayed on
a monitor in the OR thus being a point-­of-­display rather than a point-­of-­
care test because current machines are kept outside the ORs in the United
States. The clotting time (CT) in ROTEM or reaction time (R) in TEG
reflects the amount of thrombin generated. If CT or R is prolonged, this
suggests clotting factor(s) deficiency and plasma transfusion is indicated
in a bleeding patient. The maximum clot firmness (MCF) in ROTEM or
maximum amplitude (MA) in TEG reflects the strength of the clot pro-
vided by both fibrinogen and platelets. The deficiency of either will reduce
835
CHAPTER 40 COAGULATION AND FIBRINOLYSIS
Add surface XII Add tissue VII
activator, Surface Tissue factor
thromboplastin
exogenous HK
(tissue factor
phospholipid PK XI and exogenous
VIIa
and Ca++ XIIa phospholipid)
and Ca++ Tissue factor/VIIa
IX
XIa
PL, Ca++

Partial Prothrombin Time X

Thromboplastin IXa X (PT) Ca++
Time (PTT) PL, Ca++ Ca++
VIII VIIIa Xa
Xa Prothrombin
Prothrombin PL, Ca++
PL, Ca++
Va V
Va V
Thrombin
Thrombin Fibrinogen
Fibrinogen

Soluble fibrin monomers Soluble fibrin monomers

A Polymerized fibrin clot Polymerized fibrin clot B

Add Thrombin
Fibrinogen
exogenous
thrombin
and Ca++
Soluble fibrin monomers

Thrombin Time
Polymerized fibrin clot
C
Figure 40.7 Coagulation screening tests. A, Activated partial thromboplastin time (APTT, “PTT” on the figure) requires the presence of every protein except tissue factor and
factor VII. B, Prothrombin time (PT) requires tissue factor and factors VII, X, V, and II and fibrinogen. C, In thrombin time (or thrombin clotting time), exogenous thrombin
is added to plasma to proteolyze (clot) fibrinogen. The endpoint in each of these tests is the number of seconds until detection of a clot, following addition of the indicated
reagents to citrated platelet-­free patient plasma. Because virtually all measuring systems employed detect the formation of a polymerized fibrin clot, whether or not any
cross-­linking of fibrin occurs, none of these tests will provide information with respect to factor XIIIa activity.

these values. In ROTEM, FIBTEM is another test run simultaneously
that contains cytochalasin B to block platelet function. MCF in FIBTEM
reflects the contribution of only fibrinogen to the clot firmness; if MCF is
low, that indicates the need for cryoprecipitate transfusion.
On the other hand, if the MCF is normal in FIBTEM but low in
EXTEM, it suggests a platelet defect (either number or function), which
indicates the need for platelet transfusion in a bleeding patient. In TEG,
if MA is low, then a platelet mapping test can be performed to differenti-
ate between fibrinogen and platelet defect. In ROTEM, if INTEM CT
is prolonged (EXTEM CT being normal), it suggests the presence of
heparin, which is then confirmed by HEPTEM that contains hepzyme to
neutralize heparin and correct prolonged INTEM CT. When EXTEM
shows increased clot lysis (>15%), APTEM is performed. APTEM con-
tains aprotinin, which inhibits plasmin and shows correction of lysis
parameters. This finding indicates the use of a fibrinolytic agent such as
tranexamic acid or Amicar. FXIII deficiency can also affect clot stability
and show increased clot lysis.
APPROACH TO PATIENTS WITH COAGULATION
DISORDERS
A thorough clinical personal and family history is absolutely essential
for appropriate testing and management of patients with a bleeding
disorder. A systematic step-­by-­step approach is often rewarding and
cost-­effective (Table 40.2). As mentioned before, preanalytic variables
should be kept in mind when interpreting unexpected test results and
repeating tests to show that reproducibility is a better practice to avoid
patient harm. Routine coagulation tests often guide subsequent spe-
cific testing in a bleeding patient or patients with a positive history
of bleeding. Investigation of incidentally encountered abnormal coagu-
lation tests should be taken up judiciously to both avoid unnecessary
testing and health care cost, and patient harm by using blood compo-
nent (often plasma) to correct an abnormal test result. Access to prior
coagulation test results is often useful to differentiate congenital from
acquired bleeding disorders because prior tests are likely to be normal
in acquired disorder versus mostly abnormal in congenital disorders
(Table 40.3).
HEREDITARY COAGULATION PROTEIN
DEFECTS
Protein or factor deficiencies can be quantitative or qualitative. In quan-
titative disorders, the factor level determined by routine clot-­based meth-
ods (functional activity assays) is similar to that obtained by immunologic
(antigen) assays. In qualitative disorders, the functional assay result is
decreased, but the antigen level is significantly higher or normal, indicat-
ing the presence of a dysfunctional protein or an inhibitor to the function
of that protein.

836
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 TABLE 40.2
Patterns of Clinical Bleeding in Disorders of Hemostasis
Characteristics Primary Hemostasis (Platelet/Vascular Problem)
Onset Spontaneous, immediate after trauma
Sites Skin, mucous membranes
Form Petechiae, ecchymosis
Mucous membrane Common (nasal, oral, gastrointestinal, genitourinary)
Other sites Rare
Clinical examples Thrombocytopenia, platelet defects, von Willebrand disease, scurvy
TABLE 40.3
Differential Diagnosis of Abnormal Coagulation Screening Tests
Abnormal Activated Partial Thromboplastin Time (APTT) Alone
Associated with bleeding: VIII, IX, and XI defects
Not associated with bleeding: XII, prekallikrein (PK), high-­molecular-­
weight kininogen, lupus anticoagulants
Abnormal Prothrombin Time (PT) Alone
Factor VII defects
Combined Abnormal APTT and PT
Medical conditions: Anticoagulants, disseminated intravascular coagula-
tion (DIC), liver disease, vitamin K deficiency, massive transfusion
Rarely, dysfibrinogenemia; factor X, V, and II defects
DEFICIENCY OF FACTOR VIII (HEMOPHILIA A) OR
FACTOR IX (HEMOPHILIA B)
Hemophilia A (FVIII deficiency) is the most common severe congenital
bleeding disorder, affecting 1 in 5000 to 10,000 males without ethnic
predilection. Hemophilia B (FIX deficiency, Christmas disease) is also
a severe congenital bleeding disorder, affecting 1 in 25,000 to 30,000
males. The FVIII and FIX genes are both located on the X chromosome,
with the result that hemophilia A and B are X-­linked recessive disorders
primarily affecting males. Females carrying a hemophilia mutation on
one of their two X chromosomes are carriers. Hemophilia A can affect
females when carriers have imbalanced lyonization of the normal X chro-
mosome or Turner syndrome (XO), or when they are daughters of an
affected male and a carrier female.
Hemophilia is defined as mild, moderate, or severe depending on the
baseline factor activity level (Blanchette et al., 2014) (Table 40.4). Factor
levels correlate with the degree of bleeding symptoms (Pavlova & Olden-
burg, 2013). However, among patients, there is considerable variability in
bleeding pattern.
Severe hemophilia A and B usually manifest very early in life, often as
cephalohematomas at birth (whether a vaginal or cesarean section delivery),
postintramuscular vitamin K hematomas, or prolonged bleeding with new-
born heel sticks. The classic presentation of the hemophilias was provided
in the Talmud, which emphasized the risk of bleeding associated with cir-
cumcision. Surprisingly, it is uncommon to observe bleeding at the umbili-
cal stump in those affected by hemophilia A or B. As the child progresses
into toddler age, bleeding predominantly manifests as acute and recurrent
hemarthroses, muscle and soft-­tissue hematomas, and then gum and den-
tal bleeding associated with loss of deciduous teeth. In childhood through
adulthood, the bleeding frequency increases in proportion to activity
intensity and trauma. However, even the frequency of spontaneous bleed-
ing increases—for example, hemorrhage unassociated with known trauma.
This suggests that even the stress of weight-­bearing, normal torque on the
joints and supporting structures can precipitate bleeding events. The hinge
joints remain the primary sites of hemophilia-­related bleeding complica-
tions throughout life, such as ankles, knees, and elbows, although even the
ball-­and-­socket joints can bleed occasionally. Repeat hemorrhage into the
same joint often leads to restricted range of motion, support muscle atro-
phy with disability, acute and chronic pain, and structural deterioration.
Iron uptake into the joint synovium triggers the local release of proinflam-
matory and proteolytic cytokines, particularly IL-­1 beta and IL-­6, which,
in turn, induce chondrocyte apoptosis and cartilage damage (Van Vulpen
et al., 2015) via impaired scavenging, degradation, and transport of iron
from the joint synovium. Life-­threatening hemorrhage usually occurs in
the brain and paratracheal regions and is typically related to trauma (see
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Secondary Hemostasis (Coagulation Factor Problem)
Delayed after trauma
PART 5
Deep tissues
Hematomas
Less common
Joint, muscle, central nervous system, retroperitoneal space
Factor deficiency, liver disease, acquired inhibitors
TABLE 40.4
Classification of Hemophilia A and B
CLASSIFICATION
Severe Moderate Mild
Percentage of 50–70 10 30–40
patients
Factor VIII or factor <1 1–5 6–30
IX activity, %
Pattern of bleeding ∼2–4 per ∼4–6 per year Uncommon
episodes month
Cause(s) of bleeding Spontaneous Minor trauma Major trauma
Surgery
Table 40.2). The treatment of severe hemophilia (FVIII or FIX activ-
ity <1% of normal) has traditionally required frequent administration of
plasma-­derived or genetically engineered products containing the specific
missing clotting factor, using strategies that reverse active bleeding or pre-
vent spontaneous and minor traumatic hemorrhage (primary or secondary
prophylaxis). Newer approaches utilize “disruptive” therapies, which pro-
mote thrombin generation and hemostasis by circumventing the need for
the deficient clotting factor (Weyland and Pipe, 2019).
Mild hemophilia (FVIII or FIX activity >5% of normal) is rarely symp-
tomatic for spontaneous bleeding. However, it manifests with prolonged
bleeding after trauma or major surgery, and patients rarely need factor
replacement. Moderate severity hemophilia (FVIII or FIX activity 1% to
5% of normal) is characterized by an intermediate bleeding phenotype.
Hemophilia is suspected based on bleeding symptoms or a family his-
tory of hemophilia. About one-­third of hemophilia A cases arise from
spontaneous mutations. Laboratory evaluation of patients with such a
bleeding history should include APTT, PT, and platelet count. Diagnosis
is confirmed by an FVIII or FIX assay. Hemophilia can be diagnosed as
early as the birth of a male neonate with a positive family history because
FVIII and FIX levels can be directly measured in cord blood.
It is important to appreciate that in the coagulation laboratory, mea-
surement of at least two dilutions of patient-­citrated plasma should be
assayed in duplicate against standard curves for FVIII or FIX coagulant
activity in order to fully characterize the severity of disease in those with
severe FVIII or FIX deficiency because the dilution curves are not linear
at lower values. FVIII levels above 10% are easier to evaluate because the
standard curve spans between 10% and 150% FVIII/FIX activity. Varia-
tions of the one-­stage assay method are the most commonly employed
techniques to measure FVIII or FIX activities in plasma.
The type of clotting factor concentrate administered can affect assay of
the factor—for example, assay discrepancies of up to 40% are possible dur-
ing clinical monitoring of recombinant FIX and plasma-­derived FIX con-
centrates (EMA, 2014) and between modified recombinant FVIII (rFVIII)
extended half-­life products, such as pegylated FVIII and Fc fusion, versus
standard (or B-­domain deleted) half-­life recombinant FVIII concentrates
(Nagoya et al., 2019).
Several additional comments regarding hemophilia care are pertinent:
1. Only 25% of the world’s hemophilia population receives adequate care,
and opportunities are still available to provide enough clotting factor
concentrate to prevent joint damage, long-­term morbidity from bleed-
ing complications, and death from bleeding.
2. The standard of care for severe hemophilia A and B in the resource-­
rich world now includes the initiation of primary prophylaxis with the
administration of clotting factor replacement therapy either before or
shortly after the first bleeding event in childhood. Prophylaxis regi-
837
 mens should be maintained indefinitely, although some young adults
CHAPTER 40 COAGULATION AND FIBRINOLYSIS
can do well off prophylaxis. The target FVIII or FIX trough activity
level to maintain should be individualized according to the patient’s
lifestyle.
3. Primary prophylaxis is not perfect in its ability to prevent joint deterio-
ration, even when the individual has not experienced any clinical hem-
orrhage. In addition, there are those rare individuals who manifest no
joint deterioration despite recurrent bleeding events (Manco-­Johnson
et al., 2007).
4. All of the currently available clotting factor concentrates for FVIII or
FIX replacement are viral safe, without any reported transmission of
human immunodeficiency virus, hepatitis C virus, hepatitis B virus, and
so on. The viral attenuation processes in the pooled human plasma-­
derived factor concentrates have rendered these products free from
transmitting such lipid-­ enveloped infectious agents as severe acute
respiratory syndrome, West Nile virus, and so forth. However, these
products still may potentially transmit non-­lipid-­coated viruses, such as
parvovirus B19, despite ultrafiltration.
5. The most common cause of severe hemophilia A is a partial inversion
of the FVIII gene up to and including intron 22, which accounts for
up to 40% to 45% of such patients (Naylor et al., 1992; Lakich et al.,
1993). These inversions are due to homologous recombination between
the region that includes the F8A gene in intron 22 and one of the two
other homologous regions located more than 400 kb 5′ (telomeric) to
the FVIII gene (Lakich et al., 1993; Kaufman et al., 2001). Several types
of these inversions have been described (Kaufman et al., 2001). If the
intron 22 inversion is not detected, the definition of the responsible
mutation is difficult because of the large size of the FVIII gene (ap-
proximately 186,000 bp long). In contrast, the FIX gene is one-­fifth the
size of the FVIII gene, and the mutations responsible for hemophilia B
are easier to define by genetic analysis.
More diagnostic testing may be required for patients with mild to mod-
erate deficiency of FVIII for whom a diagnosis of hemophilia A may be
less apparent (e.g., a female patient with low FVIII and apparent auto-
somal inheritance). In patients with von Willebrand disease, a secondary
deficiency of FVIII may occur (see Chapter 41) because FVIII is normally
bound to vWF in the plasma (Weiss et al., 1977). Further, a unique vWF
abnormality has been described in which a missense mutation in vWF
impairs its capacity to bind to and promote FVIII secretion into plasma.
This abnormality, first described in a French patient from Normandy, has
been named von Willebrand disease “Normandy,” or type 2N. It should
be suspected in a patient with normal vWF antigen and functional studies
(see Chapter 41) but reduced FVIII levels, whose inheritance is autosomal
recessive rather than sex linked (Schneppenheim et al., 1996).
FIX activity levels during childhood remain at about 75% of adult lev-
els. A 25% increment in FIX expression begins at puberty in both sexes
(Andrew et al., 1992). It has been assumed that this is a result of steroid
hormone action. It is interesting to note that a rare form of FIX deficiency,
hemophilia B Leyden, undergoes postpubertal phenotypic resolution.
Patients with this condition present with hemophilia B in early childhood,
with FIX activity ranging from less than 1% to 13% of normal. Plasma
levels rise to as high as 70% of normal after the onset of puberty with reso-
lution of bleeding complications (Reitsma, 2001). Mutations in hemophilia
B Leyden have been identified in the promoter region of the FIX gene,
within which a consensus sequence for steroid receptor binding is located
(Crossley et al., 1992).
Evaluation of Carriers
Detection of carriers is useful for the prediction of symptoms and prena-
tal counseling. Carriers are expected to have approximately 50% of nor-
mal factor activity, a value generally able to prevent bleeding. However,
some hemophilia carriers may experience menorrhagia and other bleeding
symptoms, phenotypically like males with mild hemophilia (Arun & Kes-
sler, 2001; Plug et al., 2006). In hemophilia A, if the mutation is known, the
potential carrier may be tested for that mutation. Otherwise, the woman
should be tested for the common intron 22 inversion or get whole exo-
some sequencing. A detailed genetic analysis of the woman, the affected
male, and intervening family members may define the mutation. These
techniques provide an extremely high likelihood of detection of carriers
when all family members are available for study. A less optimal, but more
readily available and cost-­effective approach is the calculation of the ratio
of FVIII activity to the level of vWF antigen to predict carrier status. The
ratio of FVIII/vWF had a median value of 0.71 (range, 0.18–2.2) in a group
of 147 carriers versus a median value of 1.39 (range, 0.58–2.48) in a group
of 25 noncarriers. A value of FVIII/vWF:Ag ratio ≤0. 9 was found in only
one noncarrier (4%) versus in 113 (76.9%) carriers (Labarque et al., 2017).
838
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Prenatal diagnosis of the hemophilias can be performed at a number of
high-­risk obstetric centers by chorionic villous sampling at 9 to 14 weeks’
gestation or by amniocentesis after the 16th week. If the genetic basis of
the disease is known within a family, then mutational analysis or exami-
nation for the intron 22 inversion can be performed on the cell samples
(Ljung, 1999; Arun & Kessler, 2001). Fetoscopic blood sampling can be
performed at 20 weeks’ gestation for coagulation factor analysis, although
it carries a higher risk and is less precise. However, because fetal deoxy-
ribonucleic acid is detectable in maternal circulation within 10 days of
conception, collection of maternal plasma should be sufficient to make a
prenatal diagnosis of hemophilia presently, obviating the use of the inva-
sive diagnostic techniques listed earlier. In hemophilia B, 30% to 40% of
carriers will be missed by measurement of FIX activity, and mutation anal-
ysis is important for defining carrier status (Graham, 1979; Ljung, 1999).
Similar to hemophilia A, hemophilia B arises spontaneously in one-­third
of affected individuals.
Treatment of Hemophilia
The primary principle of hemophilia treatment is to prevent or reverse
bleeding episodes, spontaneously occurring, traumatic, or surgically
induced. The secondary goals of hemophilia treatment are to avoid the for-
mation of alloantibody inhibitors, which neutralize the coagulation func-
tion of exogenously administered replacement products and to improve the
convenience of treatment in order to improve adherence to any long-­term
treatment regimens. Well-­conducted prospective clinical trials have indi-
cated that the development of FVIII inhibitors is the most serious compli-
cation of FVIII replacement therapy and affects 29% to 45% of previously
untreated patients (PUPs) with severe hemophilia A. In contrast, there is a
1.5% to 3% incidence of neutralizing alloantibody inhibitor development
in PUPs with severe hemophilia B. They predominate in those individuals
with large FIX gene deletions.
The landscape of hemophilia treatment has expanded rapidly over the
past few years and bioengineering innovations have yielded modifications
of standard recombinant replacement products that extend their circu-
lating half-­lives. New technologies have also been applied to develop
“disruptive” treatment strategies, which circumvent the need to replace
the specific deficient clotting protein by promoting thrombin generation
using “mimetic” proteins or by “rebalancing” normal coagulation through
interfering with the natural modulators of the coagulation pathways, that
is, tissue factor pathway inhibitor, antithrombin, and so on. Lastly, gene
therapy is emerging as a potential “cure” for the hemophilias.
All modern replacement products for hemophilia A and B are viral safe
and equivalently efficacious in treating and preventing bleeding events. No
transmission of blood-­borne pathogens (including prion-­related disease)
has been documented for any of the currently available products. Cryopre-
cipitate for FVIII replacement or fresh frozen plasma for FIX replacement
is no longer considered the standard of care in most of the world.
FVIII replacement concentrates are characterized as plasma-­derived
(pd) from pooled normal plasma from tens of thousands of donors or as
recombinant genetically engineered products. The plasma-­derived (pd)
products are available as high specific activity, immunoaffinity processed,
highly purified FVIII without vWF protein or as low specific activity pd
FVIII concentrates containing substantial amounts of vWF protein. Stan-
dard recombinant FVIII concentrates are all ultra-­purified and very high
specific activity products, further defined by the presence or absence of
extraneous human or animal proteins in the media bathing the murine-­
derived cell cultures synthesizing the FVIII or at the end commercial
product. They can be full-­length native FVIII protein or B-­domain deleted
truncated FVIII coagulant protein.
The category of EHL rFVIII concentrates is further characterized
according to how they are bioengineered to convey longer circulating activ-
ity levels. These techniques include pegylation of the B-­domain deleted
FVIII construct; covalent bonding of the heavy and light chains of FVIII into
a single-­chain structure; or B-­domain deleted rFVIII fused to the IgG-­1 Fc-­
domain. EHL rFVIII products are also distinguished according to whether
they are synthesized by murine-­derived Chinese hamster ovary or baby ham-
ster kidney cell lines or synthesized in human embryonic kidney cell lines.
Replacement FIX concentrates are available as pd, high specific activity, or
immunoaffinity or dual affinity chromatographically purified products. They
are all nano-­filtered and solvent detergent treated to enhance viral safety.
Standard recombinant FIX concentrates are synthesized by Chinese
hamster ovary (CHO) cell lines. EHL rFIX concentrates are synthesized
in CHO or human embryonic kidney cell lines and are further character-
ized as pegylated, albumin-­fusion, or IgG-­1 Fc domain fusion proteins.
Although plasma-­derived FVIII and FIX replacement products remain
commercially available and are very effective in promoting hemostasis in
individuals with hemophilia A and B, they are believed capable of transmit-
ting nonenveloped viruses such as parvovirus B19 or prions, the agents
causing Creutzfeldt-­Jakob disease (CJD) and variant CJD (vCJD). These
pathogens are not eliminated by current viral inactivation and product
purification technologies and are emblematic of the hypothetical poten-
tial of these products to transmit yet undiscovered or unrecognized blood-­
borne infectious agents in the future. Thus, in 2014, the Medical and
Scientific Advisory Council of the National Hemophilia Foundation advo-
cated recombinant FVIII and FIX concentrates as the treatment of choice
for individuals with hemophilia A and B.
The important Survey of Inhibitors in Plasma-­Product Exposed Tod-
dlers (SIPPET) study (Peyvandi et al., 2016) reported a cumulative allo-
antibody FVIII neutralizing inhibitor incidence of 44.5% (28.4% high
titer) in PUPs treated with recombinant FVIII concentrates produced in
hamster cell lines and 26.8% (18.6% high titer) with plasma-­derived FVIII
(pdFVIII) products containing vWF. As a result of these data, many clini-
cians believe strongly that PUPs with severe hemophilia A should receive
plasma-­ derived products containing vWF to reduce the likelihood of
inhibitor development. The caveat here is that the individual PUP’s FVIII
gene mutation may be critical to determining susceptibility to inhibitor
formation (in the SIPPET study, no inhibitors occurred in PUPs with a
low genetic risk [non-­null mutations] who received pdFVIII versus 31%
inhibitor incidence in PUPs with high genetic risk [null mutations] treated
with pdFVIII). In contrast, the risk of inhibitor formation in PUPs with
either low-­or high-­risk FVIII gene mutations did not differ when treated
with rFVIII (43% and 47%, respectively). Furthermore, the fact that the
use of pdFVIII did not eliminate inhibitor formation in PUPs implies that
the replacement factor choice is only one of the multiple variables involved
in inhibitor formation. A second caveat is that the results from the SIPPET
study do not apply to inhibitor formation in previously treated hemophilia
A patients in whom rFVIII products have not been associated with increased
inhibitor incidence. Lastly, SIPPET concerns cannot be extrapolated at
this time to the newer generations of EHL FVIII concentrates as these
products have not been examined in similar prospective randomized trials.
Replacement clotting factor concentrates are dosed on the basis of
weight and desired plasma activity. Plasma with 100% clotting factor activ-
ity has 1 U/mL of that clotting factor. By convention, one unit of a coagu-
lation factor is the amount of that factor present in 1 mL of normal pooled
human plasma. Each unit of intravenously administered pd or recombinant
FVIII/kg ideally raises the plasma FVIII activity by about 2%, and each
unit of recombinant FIX/kg raises the plasma FIX activity by about 1%.
To raise the FVIII plasma activity of a 70-­kg man by 100%, the calculated
dose will be as follows:
The mean plasma half-­life of standard exogenously administered FVIII
to adults with severe hemophilia A is about 8 to 12 hours. EHL recombi-
nant FVIII concentrates have mean circulating half-­lives of about 18 to 24
hours (about 1.5 times normal).
To raise the FIX plasma activity of a 70-­kg man by 100%, the calcu-
lated dose will be as follows (Shapiro et al., 2005):
About one-­third of FIX-­deficient patients have a lower recovery after
recombinant FIX infusion, requiring 20% higher dosing. The mean plasma
half-­life of exogenously administered pd or standard recombinant FIX to
adults with severe hemophilia B is about 18 to 24 hours. EHL recombinant
FIX concentrates in adults have mean circulating half-­lives ranging between
82 to 110 hours (about 5 times normal). The mean circulating half-­lives of
FVIII or FIX concentrates in young children up to 6 years old are about 15%
to 20% lower than in adults due to accelerated plasma clearances. Dosing
strategies in young children should take these pharmacokinetic differences
into consideration. The very active person with hemophilia may wish to
maintain a trough clotting factor activity level of 15% to minimize bleeding
events during rigorous athletics or work-­related requirements. In contrast,
for the sedentary individual, a trough level of 1% may be sufficient.
Complications of Treatment
An important complication of replacement therapy, occurring mainly
in patients with severe hemophilia, is the development of alloantibodies
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(inhibitors) that interfere with the coagulation factor function. Inhibi-
tors occur in 20% to 30% of patients with severe hemophilia A and 3%
to 5% with severe hemophilia B. They are quite uncommon in patients
with moderate or mild hemophilia, probably because the infused factor is
not recognized as a foreign antigen in these individuals (Eckhardt et al.,
2013). Inhibitors reduce or abolish/neutralize, depending on their titer or
PART 5
the response to replacement therapy. Periodic inhibitor screening is very
important for effective and prompt management of this complication (Sou-
cie et al., 2014).
Characterization of a specific inhibitor requires careful quantitation. In
general, lower-­titer FVIII inhibitors (<5 Bethesda units [BU]) can still be
treated with replacement factor. In contrast, higher-­titer inhibitors (>10
BU) typically need the administration of the so-­called bypassing agents:
recombinant FVIIa, FEIBA (FVIII inhibitor bypassing agent that is an
activated prothrombin complex concentrate [PCC]), and recombinant por-
cine FVIII (Hay et al., 2006). A simple mixing test as described previously
for the APTT can underestimate the presence of an inhibitor. Further, as
indicated earlier, there is no unanimity on how such mixing assays should
be performed. In order to diagnose the presence of a FVIII or FIX inhibi-
tor, a formal FVIII or FIX “Bethesda” inhibitor assay should be performed
(Kasper et al., 1975). In the classic Bethesda assay, the patient’s plasma is
serially diluted with imidazole buffered saline (IBS), then one part patient
plasma is mixed with one part normal pooled plasma and incubated for
2 hours at 37°C. The control is a 1 : 1 mix of normal pooled plasma with
IBS. In the Nijmegen modification, the IBS is replaced with FVIII defi-
cient plasma for serial dilution, which makes this method more sensitive
to detect low-­titer inhibitors (Verbruggen et al., 1995). After incubation,
FVIII is measured in patient plasmas and the control plasma, and the per-
centage of residual FVIII activity is determined. One BU is defined as the
amount of inhibitor antibody in patient plasma that inhibits half the FVIII
activity in the control plasma (Fig. 40.8). Therefore, patient plasma pro-
ducing a residual FVIII activity of 50% is considered to contain 1 BU of
inhibitor per milliliter. If this same plasma had been diluted tenfold before
assaying for the inhibitor, then this result would indicate an inhibitor value
of 10 BU/mL.
This assay is sensitive, but at times it is difficult to reproduce. A com-
mon observation is that varying dilutions of plasma may yield different
estimates of the inhibitor titer. For assigning the level of BUs, the dilution
that comes closest to the 50% residual factor is chosen for the calcula-
tion of the FVIII inhibitor titer. Values on the standard curve should be
read-­only at between 25% and 75% residual FVIII activity. If below 25%,
higher dilutions of the sample are needed. If residual activity using the 1:1
diluted patient specimen is above 75%, a demonstrable inhibitor may not
be present. It is, in fact, important for the laboratory to determine a lower
limit of sensitivity of this assay, typically about 0.5 BU. It is additionally of
note that a plot made of residual FVIII activity versus increasing volume of
original patient plasma in the Bethesda assay may distinguish between an
inhibitor capable of reducing FVIII activity into the unmeasurable range
(type I inhibitor) and an inhibitor that preserves some degree of residual
FVIII activity, even at high inhibitor titer (type II inhibitor), presumably
reflecting a target epitope at a less critical region of the FVIII molecule
(Gawryl & Hoyer, 1982; Luna-­Zaizar et al., 2009).
Treatment of Acute Bleeds Associated With Neutraliz-
ing Alloantibody Inhibitors in Hemophilia A and B
The approach to the reversal/treatment of acute hemorrhagic episodes is
predicated on circumventing the neutralizing effects of antibodies on exog-
enous replacement therapies. In the context of low responding inhibitors
(≤5 BU and no anamnestic response to reexposure to exogenous FVIII or
FIX), high doses of any construct human FVIII or FIX concentrate (200
IU/kg) can be administered to saturate and “overwhelm” the respective
inhibitor. The caveat to this approach is that the incremental increase
in clotting factor activity (recovery) and the circulating half-­life will be
blunted by the effects of the inhibitor. Second, for hemophilia B patients,
particularly those with large FIX gene deletions, there is a small but sig-
nificant incidence of developing potentially serious and life-­threatening
allergic or anaphylactic reactions to FIX and some also develop nephrotic
syndrome. In these patients, as well as those with high responding allo-
antibody inhibitors to FVIII or FIX (>5 BU and anamnestic responses to
reexposure to the clotting factor), the treatment of choice for acute bleeds
is a “bypassing agent,” such as FVIII inhibitor bypass activity (FEIBA) or
recombinant FVIIa.
On the other hand, recombinant porcine B-­domain deleted FVIII con-
centrate, synthesized in CHO cells to treat acute bleeding in acquired auto-
antibody FVIII inhibitor patients, has also been used in phase II clinical
839
 100
CHAPTER 40 COAGULATION AND FIBRINOLYSIS
75
50
Residual factor VIII (%)
25
10
0 1.0
Bethesda units per mL
Figure 40.8 Expression of factor VIII inhibitor titer in Bethesda units. In this assay
for an inhibitor to factor VIII, the percent of residual factor VIII activity in normal
plasma is determined after incubation with patient plasma. By convention, 1 in-
ternational Bethesda unit of inhibitor is the amount of antibody that destroys 0.5
U factor VIII activity after 2 hours of incubation at 37°C. As shown in the figure, the
percent of residual factor VIII activity on a log scale is plotted against Bethesda units/
mL in a linear scale. For a sample to be evaluable in this assay, the level of factor VIII
activity should fall between 25% and 75%. Patient samples that produce residual
factor VIII activity below 25% need to be diluted further so that the 50% residual
factor VIII activity point can be found. (Redrawn with permission from Bockenstedt
PL: Laboratory methods in hemostasis. In Loscalzo J, Schafer AI, editors: Thrombosis
and hemorrhage, ed 3, Philadelphia, 2003, Lippincott Williams & Wilkins, p 370,
Fig. 21.7.)
trials to treat bleeding in allo-­FVIII antibody inhibitor patients with con-
genital hemophilia A. The product has demonstrated good efficacy and
safety (Mannucci & Franchini, 2017) but has not been licensed by the FDA
for use in congenital hemophilia A with inhibitors. Recombinant porcine
FVIII was developed on the premise that there is a lower cross-­reactivity of
porcine FVIII with antihuman FVIII antibodies.
Disruptive Therapies for the Hemophilias
The concept of “rebalancing” the coagulation cascade to generate throm-
bin and to establish hemostasis without specific replacement of the defi-
cient coagulation factor protein has evolved rapidly and has yielded novel
therapeutic strategies for hemophilia A and B with and without alloanti-
body inhibitors.
Emicizumab-­kxwh (Hemlibra, Genentech/Roche) is the first commer-
cially available agent in this category. It is indicated for the prevention of
bleeding in any individual (infants and adults, but there are no clinical efficacy
or safety data in patients less than 1 year of age) with severe hemophilia A
with and without inhibitors (Barg et al., 2019). It has the advantage of being
subcutaneously administered in weekly, every other week, or every 4 weeks
dosing regimens. Emicizumab is a genetically engineered bispecific IgG anti-
body that functions as an “FVIII mimetic” by substituting for the severely
deficient FVIII and serving as a cofactor to produce the tenase complex in
the coagulation cascade. As a substitute for FVIIIa, it bridges FIXa and FX to
facilitate hemostasis. This is critical to the ultimate thrombin generation and
multiple well-­conducted clinical trials have demonstrated impressive reduc-
tions in annualized bleeding rates in severe hemophilia A with or without
inhibitors (Mahlangu et al., 2018, Oldenburg et al., 2017). The most con-
cerning adverse events within these clinical trials, only in inhibitor patients
who received repeated and large amounts of FEIBA for breakthrough bleeds,
was the development of thrombotic complications in 5 individuals. A unique
feature of these hypercoagulable events is the presence of thrombotic micro-
angiopathic anemia and the unusual location of some of the thrombi.
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Laboratory monitoring of emicizumab is not required while on routine
prophylactic dosing. In fact, the APTT will be artefactually decreased and
the one-­stage FVIII activity assay will be misleadingly elevated and will
not accurately correlate with the clinical hemostatic status of the patient.
Chromogenic FVIII activity assays that use bovine reagents do measure
FVIII activity in emicizumab patients who have received an exogenous
FVIII replacement. Similar limitations occur with the clot-­based Bethesda
assay; a bovine-­reagent chromogenic-­based Bethesda inhibitor assay is
available. Emicizumab can be measured in plasma, but this does not pro-
vide a reliable “FVIII-­equivalence” assay to predict in vivo hemostasis.
Another emerging disruptive therapy for hemophilia A or B with or
without inhibitors is fitusiran (Sanofi-­Genzyme), which interferes with
antithrombin synthesis in the liver and, thus, leads to increased thrombin
generation in vivo. Fitusiran binds to and degrades messenger ribonucleic
acid for antithrombin and thereby interferes with AT translation by the
hepatocyte. This synthetic, double-­stranded small interfering ribonucleic
acid oligonucleotide is also administered subcutaneously as a weekly or
monthly therapeutic; clinical trials have demonstrated reduced annualized
bleeding rates in patients with hemophilia A and B, with or without inhibi-
tors (Pasi et al., 2017). In these studies, two severe hemophilia A patients
have developed a fatal thrombosis after concurrent repeated administra-
tion of high-­dose factor VIII concentrate. Theoretical benefits have been
speculated for fitusiran to prevent bleeding complications in other rare
bleeding disorders, such as severe deficiency of factors V, VII, X, and XI.
No clinical trials in these latter coagulopathies have been conducted as yet.
Concizumab (Novo Nordisk) is the third disruptive therapy for hemo-
philia A and B with and without inhibitors. It is a daily subcutaneously
administered high-­affinity, anti-­TFPI monoclonal antibody, which results
in an increased generation of FXa and, ultimately, increased thrombin gen-
2.0 eration. Clinical trials have demonstrated decreased annualized bleeding
rates with few side effects (Shapiro et al., 2019). That being said, the clini-
cal trials with a different anti-­TFPI construct (Bayer) were terminated due
to thrombotic complications.
Gene Therapy for the Hemophilias
Congenital hemophilia A and B appear to be ideal disorders for gene ther-
apy for the following reasons:(1) FVIII and FIX genes were cloned in the
early 1980s, (2) the proteins expressed by the genes (FVIII/FIX) are easily
measured in small amounts of plasma, (3) incremental increases in pro-
tein expression due to gene therapy could change the phenotypic bleeding
profile in a significant manner, (4) hemophilic animal models have been
developed to provide proof of principle that gene therapy would be feasible
and useful, and (5) individuals with congenital severe hemophilia A and
B have been enthusiastic and willing to participate in gene therapy clini-
cal trials with the potential that their bleeding rates and the consequences
therefrom could be eliminated or at least ameliorated.
The most successful clinical gene therapy studies currently employ
nonintegrating recombinant adenoassociated viral (AAV) vectors to carry
the genetic “payload” to the targeted hepatocytes for cellular transduction.
Although no long-­term immunologic or oncologic toxicities have been
observed with either FVIII or FIX gene therapies to date, longitudinal
vigilance is indicated. In addition, gene therapy trials have been hampered
somewhat by the presence of preexisting neutralizing antibodies to the
AAV capsid in potential participants. Also, all of the gene therapy trials
have observed some degree of lost transgene expression over time, attrib-
uted to either episomal latency within the hepatocyte or by yet to be fully
elucidated immunologic mechanisms.
The most successful FIX gene therapy results have been accomplished
in trials that incorporate a FIX molecule with a gain-­of-­function FIX
mutation (FIX-­Padua) with AAV capsids (Batty & Lillicrap, 2019). Some
patients have experienced sustained FIX activity levels in the 30% to 40%
range (compared to baseline levels of ≤2%). Gene therapy trials in severe
hemophilia A have also achieved clinically significant incremental increases
in FVIII levels, frequently in the normal range (≥50% activity). The dura-
bility of the response has emerged as an issue to watch over time in these
patients (Pasi et al., 2020).
HEREDITARY DEFICIENCIES OF OTHER COAGULA-
TION FACTORS
The other hereditary deficiencies of coagulation factors have an autosomal
inheritance (Table 40.5). With the exception of FXI deficiency, these dis-
orders are very rare. However, they have a higher prevalence in areas where
consanguineous marriage is practiced (Mannucci et al., 2004). Deficiencies
of factors XIII, X, VII, V, and II are defined rare inherited coagulation
 TABLE 40.5
Characterization of Coagulation Factors and Their Deficiencies
Molecular Normal Circulating
Factor Weight, kDa Gene Location Half-­Life Incidence Inheritance Bleeding Severity

Fibrinogen 330 4q31.3-­q32.1 2–4 days 1 : 1 million Recessive Mild–severe*

PART 5
II 72 11p11.2 3–4 days Very rare Recessive Mild–moderate
V 330 1q24.2 36 hours 1 : 1 million Recessive Moderate
V and VIII com- — LMAN1:18q21.32 36 hours for FV; 10–14 1 : 2 million Recessive Mild–moderate
bined MCFD2:2p21 hours for FVIII
VII 50 13q34 3–6 hours 1 : 500,000 Recessive Mild–severe
VIII 330 Xq28 10–14 hours 1 : 10,000 Sex-­linked Mild–severe
IX 56 Xq27 18–24 hours 1 : 30,000 Sex-­linked Mild–severe
X 58 13q34 40–60 hours 1 : 500,000 Recessive Mild–severe
XI 160 4q35.2 40–70 hours Rare† Recessive Mild–moderate
XII 80 5q33-­qter 50–70 days Rare Recessive No bleeding
PK 88 4q33-­q35 Not known Very rare Recessive No bleeding
HK 120 3q27 9–10 hours Extremely rare Recessive No bleeding
XIII 320 A:6p25.1 11–14 days <1 : 1 million Recessive Moderate–severe
B:1q31.3

HK, High-­molecular-­weight kininogen; PK, prekallikrein.

*May be associated with thrombosis.
†Rare except in those of Ashkenazi Jewish descent

disorders (RICDs) that represent 2% to 5% of inherited coagulation fac-
tor defects in the general population. The most common RICDs include
deficiencies of FXI and FVII, followed by FV, FX, fibrinogen, and FXIII
(Ruiz-­Saez, 2013).
In general, most patients with RICDs have partial defects because they
have relatively mild bleeding disorders. Most coagulation-­based assays
have difficulty detecting these factors once their levels are below 5% to
10%. In vivo, however, the presence of even this small level of coagulation
factor can greatly influence bleeding risk. In contrast, findings in knock-
out mice indicate that a true null of each of these proteins is associated
with virtually 100% mortality from hemorrhage before or at the time of
birth. With hereditary factor deficiencies, heterozygous-­ deficient indi-
viduals have approximately 50% (most commonly, 30%–60%) of the nor-
mal level of the affected factor. The symptoms of these disorders are quite
variable. Coagulation factor deficiencies may be suspected on the basis of
symptoms, family history, or abnormal screening tests, and the laboratory
diagnosis is confirmed by specific factor assays. Once a specific protein
deficiency is recognized, clinical history usually distinguishes a congenital
from an acquired defect.
Disorders with Prolonged APTT and Normal PT
Factor XI
FXI deficiency is common in Ashkenazi Jews, in whom heterozygote
frequency is 8% (Asakai et al., 1991; Emsley et al., 2010). This ethnic
population constitutes about 50% of the FXI-­deficient patients seen in
the United States. Most patients with FXI deficiency rarely have a spon-
taneous hemorrhage. This notion is further supported by the lack of any
spontaneous bleeding reported in a recent trial in which antisense treat-
ment was administered to decrease the activity of FXI to 25% normal in
knee replacement surgery with the aim to reduce the incidence of venous
thrombosis (Buller et al., 2015). Bleeding typically occurs after trauma
or surgery, particularly involving areas of the body with high fibrino-
lytic activity (mouth, nose, genitourinary tract). Women can experience
menorrhagia and postpartum hemorrhage. Bleeding can occur in het-
erozygotes and does not necessarily correlate with the residual FXI level
(Leiba et al., 1965; Bolton-­Maggs et al., 1988). However, the bleeding
tendency may be modified by additional defects such as hemophilia, von
Willebrand disease, and platelet function defects (Brenner et al., 1997).
Diagnosis depends on the determination of the FXI activity below the
reference range. The most widely used APTT reagents will have pro-
longed APTT results for patient samples with FXI activity below 20%
to 25% and mixed results when levels are 25% to 60%. The lower limit
of the normal range is probably between 60% and 70% (Bolton-­Maggs
et al., 1995). Therefore, a normal APTT does not rule out a mild FXI
deficiency. In cases in which the marked elevation of FVIII may be pres-
ent, APTT can be normalized even when other factors are reduced
(Lawrie et al., 1998). Thrombin generation assay performed in specific
sample conditions appears to be able to identify the bleeding phenotype
of patients with FXI deficiency (Pike et al., 2015). In all patients with
FXI deficiency, preoperative management is critical to prevent bleeding
complications. Patients with a severe deficiency (FXI <10%–20%) should
receive treatment with fresh frozen plasma to raise their FXI levels. Usu-
ally, replacement therapy of 20 mL/kg loading dose with a maintenance
dose of 5 to 10 mL/kg every 24 hours is sufficient to cover patients with
severe FXI deficiency through elective surgery (Kessler et al., 1996).
Patients with levels from 20% to 70% may or may not bleed.
The severity of the surgery, the patient’s past history of bleeding
following prior surgeries, and perhaps the bleeding histories of fam-
ily members with the same level of FXI activity should be considered
when the strategy for bleeding prevention is being formulated. Antifi-
brinolytic therapy with tranexamic acid or epsilon aminocaproic acid
has been a very effective adjunctive therapy when used alone or in com-
bination with replacement therapies. This is particularly useful in the
United States, where only fresh frozen plasma or solvent/detergent (S/D)
treated, pooled human plasma (Octaplas, Octapharma) is available for
FXI replacement therapy.
Pooled human plasma-­derived FXI concentrates are available in France
and the United Kingdom (LFB Hemoleven and BPL FXI), but neither
has been approved by the United States FDA. Rare arterial and venous
thrombotic episodes have been reported with both (or that can have a
prothrombotic effect) (Bauduer et al., 2015; Ling et al., 2016). Off-­label
use of lower-­ dose (15–30 μg/kg) recombinant factor VIIa concentrate
(NovoSeven) has also provided effective and safe hemostasis for surgery in
severe FXI deficiency. No large clinical trials have utilized this approach
(O’Connell et al., 2008, Riddell et al. 2011).
The development of neutralizing antibodies that function as inhibitors
to exogenously administered sources of FXI in patients with severe FXI
deficiency have been described, although these are rare (Salomon et al.,
2003). These patients should be treated with rFVIIa concentrate (Kenet
et al., 2009).
FXII, Prekallikrein, and High-­Molecular-­Weight Kininogen
FXII, prekallikrein, and HMWK deficiencies are associated with pro-
longed APTT but are not associated with any bleeding risk. FXII defi-
ciency is most common, occurring in all racial and ethnic backgrounds. It
is associated with a very long PTT. Prekallikrein deficiency is less common
(Girolami et al., 2014) but is seen in the United States in all ethnic groups.
It is associated with a slightly prolonged APTT that corrects to normal
when sitting on a bench for 1 hour at 37°C. HMWK deficiency is a rare
disorder that also is associated with a very long APTT. These protein defi-
ciencies are not associated with bleeding—a point emphasized diagram-
matically in Figure 40.1 by the graying of this portion of the coagulation
“cascade.” Because no replacement therapy for hemostasis is necessary for
FXII, PK, or HMWK deficiency, it is important to recognize these defects
to prevent unnecessary treatment. Deficiencies of FXII, PK, and HMWK

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CHAPTER 40 COAGULATION AND FIBRINOLYSIS
have been associated with reduced thrombosis risk. Deletions of these pro-
teins in mice are associated with delayed times to thrombosis in arterial
thrombosis models (Renne et al., 2005; Stavrou et al., 2015; Merkoulov
et al., 2008). When a deficiency in the proteins of the kallikrein/kinin sys-
tem is suspected, a FXII assay should be performed first because it is the
most common.
Disorders with Prolonged APTT and PT
Prolonged PT and APTT most commonly are due to general medical
conditions described later in the Acquired Coagulation Disorders section.
When hereditary disorders are considered, the following, although much
less common, should be considered.
Disorders of Fibrinogen
Hereditary fibrinogen abnormalities include two categories of plasma
fibrinogen defects: type I, afibrinogenemia or hypofibrinogenemia, with
absent or low plasma fibrinogen antigen levels (quantitative fibrinogen
deficiencies); and type II, dysfibrinogenemia or hypodysfibrinogenemia,
which determines normal or reduced antigen levels associated with the
disproportionately low functional activity (qualitative fibrinogen deficien-
cies). In the type I disorder, null mutations occur in the FGA, FGB, and
FGG fibrinogen encoding genes (de Moerloose et al., 2013).
Fibrinogen deficiencies may prolong the PT and APTT if the plasma
concentration of the protein is sufficiently low, usually less than 100 mg/
dL. Afibrinogenemia is a bleeding disorder of variable severity (Fried &
Kaufman, 1980; Lak et al., 1999). Umbilical stump and mucosal bleeding
are the most common symptoms, as is an increased incidence of muscu-
loskeletal and central nervous system bleeding. Patients also exhibit poor
wound healing. Hypofibrinogenemia is a decreased level of normal fibrino-
gen that has a similar but milder pattern of bleeding. Both afibrinogen-
emia and hypofibrinogenemia are associated with recurrent miscarriage,
as well as with antepartum and postpartum hemorrhage (Goodwin, 1989;
Kobayashi et al., 2000). Paradoxically, reports have described thrombotic
events in patients with afibrinogenemia (Chafa et al., 1995; Lak et al., 1999;
Dupuy et al., 2001).
Dysfibrinogenemia is a qualitative fibrinogen deficiency characterized
by the production of dysfunctional fibrinogen (Miesbach et al., 2010).
Most patients with congenital dysfibrinogenemia are heterozygous
because of molecular defects in produced protein, although rare homozy-
gous cases have been reported. Dysfibrinogenemias are most commonly
acquired in association with liver disease. Acquired dysfibrinogenemias
are mainly due to posttranslational modifications of the fibrinogen pro-
tein as a result of synthesis in an abnormal liver. These defects are com-
mon in patients with hepatitis B and C. Patients with dysfibrinogenemia
are usually asymptomatic or have mild bleeding. In some cases, however,
thrombosis has been reported with or without a bleeding history (Hanss
& Biot, 2001). The thrombin time and reptilase time, which measure
clotting time during the conversion of fibrinogen into fibrin, are often
prolonged in dysfibrinogenemia. When thrombin proteolyzes fibrino-
gen in the thrombin time, fibrinopeptides A and B are released from the
Aα and Bβ chains of fibrinogen. Reptilase clots fibrinogen by liberating
only fibrinopeptide A (Funk et al., 1971). Proteolysis of fibrinopeptide A
from fibrinogen is sufficient to induce clot formation. Fibrinopeptide B
liberation increases the rate of association of the fibrin monomers, but
not the actual physiologic clot (Martinelli & Scheraga, 1980; Nawara-
wong et al., 1991). Bleeding risk is associated only with fibrinopeptide
A release defects, not with fibrinopeptide B release defects. Therefore,
a patient can have long PT and APTT from a fibrinopeptide B release
defect and not have any bleeding risk—hence, the reason for using both
the thrombin time and reptilase time to assess dysfibrinogenemias. Assays
that measure clottable fibrinogen will show lower levels than assays that
measure fibrinogen antigen. A normal fibrinogen has a ratio of clottable
fibrinogen activity to fibrinogen antigen greater than 95%. Ratios less
than this raise the possibility of a dysfibrinogenemia. For many years,
cryoprecipitate has served as a good source of fibrinogen when replace-
ment is needed. A number of virally inactivated fibrinogen concentrates
are now available for therapeutic administration, both in Europe and
North America. Human fibrinogen viral attenuated concentrates (Ria-
STAP [CSL Behring] and Fibryga [Octapharma]) are available for the
treatment and prevention of acute bleeding episodes in persons with con-
genital fibrinogen deficiency, including afibrinogenemia and hypofibri-
nogenemia. Primary prophylaxis is easily achieved with these products;
however, thrombotic complications may arise.
These concentrates are not indicated for acquired noncongenital hypo-
fibrinogenemias, including those with dysfibrinogenemia, DIC, massive
postpartum hemorrhage, and others.
842
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FII (Prothrombin) Deficiency
Prothrombin deficiency may be the rarest inherited coagulation factor
deficiency (1 : 2,000,000) (Bolton-­Maggs et al., 2004). A true prothrombin
deficiency in mice is incompatible with life after birth (Sun et al., 1998).
Hypoprothrombinemia (type I deficiency) manifests as a concomitant
reduction in prothrombin activity and antigen levels. Bleeding symptoms
include mucosal bleeding, hematomas, and hemarthrosis. Dysprothrom-
binemia (type II deficiency) presents with reduced activity and normal
antigen levels. The clinical presentation of dysprothrombinemia is less
predictable—patients may be asymptomatic or may have only mild bleed-
ing manifestations (Bolton-­Maggs et al., 2004). Depending on the sensitiv-
ity of the reagents, both PT and APTT may be prolonged in prothrombin
deficiency. However, a specific FII assay is best if there is clinical suspi-
cion or positive family history in the presence of normal screening tests. A
PT reagent-­based prothrombin assay with factor-­deficient plasma is most
convenient. Purified or recombinant factor II is currently not available.
Prothrombin complex concentrates are the treatment of choice, although
fresh frozen plasma can serve as an alternative source of prothrombin. The
half-­life of the protein is long (2–4 days); thus, replacement therapy every
3 to 4 days is typically sufficient.
FV Deficiency
Heterozygous FV deficiency is usually asymptomatic. Homozygous FV defi-
ciency is rare (1 : 1,000,000), presenting in children with a mild to severe
bleeding phenotype (Girolami et al., 1998; Lak et al., 1999). Individuals with
severe factor V deficiency (FV:c <1%) may well have some functional FV
considering that 50% of true null mice die at the time of development of
the cardiovascular system (days 9 to 11), and the other half die at birth from
hemorrhage (Cui et al., 1996). A biological reason for a milder than expected
bleeding phenotype in FV-­deficient subjects may be related to the presence
of residual factor V activity in platelets (Bouchard et al., 2015). The most
frequent symptoms are mucosal tract bleeding and bleeding after invasive
procedures. FV deficiency is associated with prolongation of both the APTT
and PT (but a normal thrombin time), and it is confirmed by a PT reagent-­
based, single-­stage FV assay. Patients with FV deficiency should also have
an FVIII assay performed to evaluate for combined deficiency of FV and
FVIII (Nichols et al., 1998; Zhang & Ginsburg, 2004). The only suitable
replacement product available is fresh frozen plasma (solvent/detergent if
available). Human platelets contain 20% of total plasma FV; they can also
be a source of this protein in acute bleeding (Tracy et al., 1982). The goal
of replacement therapy should be to elevate the FV level to at least 10% to
15% (Peyvandi & Mannucci, 1999). The half-­life of FV is quite short (16–36
hours); thus, to achieve the hemostatic level of 20%, daily infusions may be
needed. During treatment, secondary volume overload needs to be taken
into account and actively treated. Acquired inhibitors to FV are relatively
common from the surgical use of topical bovine thrombin to aid incision
hemostasis. The bovine thrombin is contaminated with bovine FV; when
the patient makes an antibody against bovine FV, that antibody then cross-­
reacts with human FV, causing the deficiency. Most cases are not severe, but
their management can be challenging due to the lack of specific replacement
therapy (Ardillon et al., 2014). Acquired FV deficiency is treated with plate-
let transfusion because FV in platelets is protected from the antibody and
FV is delivered to the site of injury by platelets. Plasma infusion is ineffective
because the antibody will immediately neutralize the transfused FV.
FX Deficiency
Heterozygous FX deficiency is asymptomatic. Homozygous FX deficiency
is a severe bleeding disorder that presents in infancy. The bleeding phe-
notype correlates well with FX activity levels. Symptoms include umbilical
cord bleeding, mucosal bleeding, severe soft-­tissue hematomas, and hem-
arthroses (Peyvandi et al., 1998a). One-­stage PT-­based FX assays are suf-
ficient for diagnosis, although additional assays are commercially available.
Before the diagnosis of an inherited deficiency is made, it is important to
rule out a vitamin K deficiency or other acquired causes for the FX defi-
ciency. For example, FX deficiency is the most common factor deficiency
associated with primary amyloidosis, occurring in less than 10% of patients
and probably due to adsorption of FX onto amyloid fibrils (Uprichard &
Perry, 2002). Factor X concentrates are available in some countries in
Europe. Bleeding can also be treated with prothrombin complex concen-
trates, fresh frozen plasma, and recombinant FVIIa (Boggio & Green,
2001). The half-­life of factor X (40–60 hours) may require daily treatment
or continuous infusion to keep FX levels above 20%.
Combined Deficiency of FV and FVIII
Combined FV and FVIII deficiency is a rare autosomal-­recessive disor-
der that results from a single gene defect rather than from coinheritance
of defects in both FV and FVIII genes. This defect of the endoplasmic
reticulum results in impaired transport of both FV and FVIII from the
intracellular site of synthesis. Patients typically have FV and FVIII levels
between 5% and 30%, their bleeding phenotype is mild to moderate, and
it is weakly related to the factor level activity. In two-­thirds of patients, this
results from the null expression of LMAN1 (previously known as ERGIC-
­53) (Nichols et al., 1998). LMAN1 shuttles between the endoplasmic retic-
ulum and the Golgi and is believed to facilitate protein trafficking through
the secretion pathway (Zhang et al., 2003). Other patients have a mutation
of MCFD2, which directly interacts with LMAN1 (Zhang et al., 2003).
Bleeding manifestations include epistaxis, easy bruising, menorrhagia,
and postpartum hemorrhage, as well as bleeding following surgery, den-
tal extraction, and trauma (Seligsohn et al., 1982; Peyvandi et al., 1998b).
Testing usually demonstrates disproportionate prolongation of the PTT
compared with the PT. Replacement therapy is usually reserved for surgi-
cal, traumatic, or gynecologic bleeding (Mumford et al., 2014). It includes
both FVIII concentrates and fresh frozen plasma (as a source of FV).
Combined Deficiency of Vitamin K–Dependent Clotting Factors
Vitamin K–dependent clotting factor deficiency affecting multiple vita-
min K–dependent coagulation (II, VII, IX, and X) and anticoagulant
(protein S and protein C) factors occurs with vitamin K deficiency and
hepatic dysfunction. It also results from heritable dysfunction of hepatic
enzyme γ-­glutamyl carboxylase (type I) or the vitamin K epoxide reductase
enzyme complex (type II) (Brenner et al., 1998; Zhang & Ginsburg, 2004).
Severely affected individuals may present as neonates with fatal hemor-
rhages, umbilical stump bleeding, or spontaneous intracranial hemorrhage;
in infancy or early childhood with hemarthroses, soft-­tissue hematomas,
or gastrointestinal hemorrhages; or as adults with easy bruising, mucosal
bleeding, and bleeding following surgery. Diagnosis is established by the
prolongation of both APTT and PT and associated reductions in levels of
vitamin K–dependent clotting factors. These patients may respond to vita-
min K (oral or parenteral) supplementation with normalization of APTT,
PT, and factor levels, as well as the resolution of bleeding symptoms. This
makes the establishment of a diagnosis of an inherited abnormality dif-
ficult in that other acquired causes of vitamin K deficiency (hemorrhagic
disease of the newborn, liver disease, and prolonged use of broad-­spectrum
antibiotics) or factitious warfarin administration could manifest similarly.
For patients who do not respond fully to vitamin K administration, fresh
frozen plasma can be used for acute bleeding or surgery. In addition, pro-
thrombin complex concentrates and combination therapy and vitamin K
supplementation may constitute alternative treatment options (Napolitano
et al., 2010).
Disorders with Normal APTT and Prolonged PT
FVII Deficiency
FVII deficiency is the most common rare hereditary coagulation factor
deficiency (Mariani & Bernardi, 2009). The bleeding manifestations are
variable, with epistaxis, mucosal bleeding, and menorrhagia commonly
reported (Peyvandi et al., 1997). Severe FVII deficiency is autosomal
recessive, often presenting shortly after birth, and may have a dramatic
presentation with intracranial hemorrhage in 15% to 60% of cases (Ragni
et al., 1981). The diagnosis is suspected with the finding of isolated pro-
longation of PT. However, FVII, a vitamin K–dependent clotting factor, is
low in the newborn period and will also be low in the presence of vitamin
K deficiency. Therefore, the reevaluation of infants with mild deficien-
cies is required after they reach a few months of age or after vitamin K
replacement in older patients. Functional FVII activity is measured by a
PT-­based, FVII-­deficient plasma coagulant assay. The use of recombi-
nant human thromboplastin will yield results that are more likely to reflect
in vivo FVII levels. Samples for FVII testing should not be stored at 4°C, as
this can lead to cold activation of FVII as a result of C1 inhibitor inactiva-
tion and FXIIa formation in the tube with FVII activation (Kitchen et al.,
1992). Cold activation of FVII results in an overestimation of the actual
plasma FVII level. Therapeutic options include recombinant FVIIa (15–20
μg/kg), fresh frozen plasma, or 4-­factor prothrombin complex concen-
trates. For major surgery, plasma FVII levels of at least 20% are sufficient
(Bolton-­Maggs et al., 2004).
Disorders with Normal APTT and PT
FXIII Deficiency
Factor XIII stabilizes and cross-­links fibrin (Hsieh & Nugent, 2008). Fac-
tor XIII deficiency can determine delayed bleeding, usually 24 to 36 hours
after surgery or trauma; spontaneous bleeding also occurs. Coagulation
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assays such as PT, PTT, and TT are normal. A reduced plasma factor
XIII activity, determined with an immunoassay for factor XIII, leads to the
diagnosis. Clot dissolution in 5M urea or monochloroacetic acid is useful
for the diagnosis (Lorand et al., 1980), but these assays are not standard-
ized and have low sensitivity. More robust quantitative activity assays are
based on cross-­linking of glycine–ethyl ester into a specific peptide (Fick-
PART 5
enscher et al., 1991), incorporation of an amine substrate into fibrinogen
(Kohler et al., 1998), or incorporation of a biotinylated peptide to spermine
attached to microtiter plates (Hitomi et al., 2009).
Testing for FXIII deficiency is suggested for individuals with a posi-
tive bleeding history, particularly with features such as delayed bleeding,
umbilical stump bleeding, or miscarriage, in which the PT and APTT are
normal (Anwar & Miloszewski, 1999). Of severely FXIII-­deficient patients,
30% die in middle age from spontaneous intracerebral hemorrhage unless
they are receiving prophylactic therapy (Lorand et al., 1980). Acquired
deficiency of FXIII has been described in a variety of diseases, including
Henoch-­Schönlein purpura, isoniazid treatment for tuberculosis, various
forms of colitis, erosive gastritis, and some forms of leukemia (Board et al.,
1993), although FXIII level determination and therapeutic management
are controversial. The half-­life of FXIII is very long; thus, replacement
with cryoprecipitate is usually sufficient for most patients. Plasma-­derived
and recombinant FXIII concentrates are currently commercially available
(Dorey, 2014).
Hereditary Hemorrhagic Disorders of Fibrinolysis
Hereditary bleeding disorders resulting from excessive fibrinolysis are
rare. α2-­Antiplasmin deficiency determines enhanced plasmin activity and
fibrinolysis (Fay et al., 1992; Aoki et al., 1980). Patients typically present
with delayed bleeding. Measurement of α2-­antiplasmin with either immu-
nologic or functional assays leads to the diagnosis. Plasma transfusion and
oral fibrinolysis inhibitors can control bleeding episodes and enable surgi-
cal procedures.
Acquired α2-­antiplasmin deficiency has been described in amyloido-
sis, DIC, acute promyelocytic leukemia malignancies, severe liver disease
(impaired synthesis), nephrotic syndrome (increased renal excretion),
abdominal aortic aneurysm, and following the administration of thrombo-
lytic agents (Okajima et al., 1994). Although not recommended for routine
use as a screening test, the whole-­blood clot lysis time can be helpful as
a first-­order test to detect deficiencies of α2-­antiplasmin. In brief, whole
blood is allowed to clot; then, the time to clot lysis is recorded. In normal
individuals, the presence of α2-­antiplasmin effectively prevents lysis from
occurring in this in vitro setting, even 24 hours after initial clot forma-
tion. In contrast, in the presence of a severe deficiency of α2-­antiplasmin
or in some fibrinolytic states, clot lysis can be observed after several hours.
In such instances, follow-­up testing specific for the α2-­antiplasmin activity
should be employed. Also, a euglobulin lysis time (ELT) can evaluate the
global function of the fibrinolytic system. Plasma is admixed with dilute
acid to precipitate a plasma fraction relatively rich in plasminogen activa-
tor, plasminogen (euglobulin fraction), and fibrinogen but relatively poor
in antiplasmins. This euglobulin fraction is then redissolved in the buf-
fer and clotted by recalcification; the time for clot lysis is then measured.
Euglobulin clot lysis is normally complete in 2 to 5 hours, but the time may
be shortened with increased fibrinolysis associated with the increased plas-
minogen activator activity. A shortened ELT is an expression of a hyperfi-
brinolytic state; also, it can be adopted to rule out acquired disorders such
as DIC. Plasminogen activator, plasminogen, and plasminogen activator
inhibitor may be assayed directly.
ACQUIRED COAGULATION DISORDERS
Acquired bleeding disorders are due to anticoagulation, DIC, liver disease,
vitamin K deficiency, massive transfusion, and specific inhibitors of coagu-
lation proteins. The topic of anticoagulation is addressed extensively in
Chapter 43. In general, prolongations of PT and APTT arising in a patient
should raise the possibility, as discussed in the following sections, of anti-
coagulants, DIC, liver disease, vitamin K deficiency, and massive transfu-
sion. Only after these clinical states have been excluded should attention be
directed to specific protein defects influencing PT and PTT.
DISSEMINATED INTRAVASCULAR COAGULATION
(DIC)
DIC is a clinicopathologic syndrome in which activation of the coagulation
and fibrinolysis systems results in the simultaneous formation of thrombin
and plasmin with the consumption of coagulation factors and inhibitors of
843
CHAPTER 40 COAGULATION AND FIBRINOLYSIS
TABLE 40.6
ISTH Diagnostic Scoring System for DIC
Risk assessment: Does the patient have an underlying disorder known to
be associated with overt DIC?
• If yes, proceed.
• If not, do not use this algorithm.
Order global coagulation tests (PT, platelet count, fibrinogen,
D-­dimer)
Score the test results:
• Platelet count (>100 k/μL = 0, <100 k/μL = 1, <50 k/μL = 2)
• Elevated D-­dimer (<0.4 μg/mL = 0, 0.4–4.0 μg/mL = 2, >4.0 μg/mL = 3)
• Prolonged PT (<3 sec = 0, >3 sec but <6 sec = 1, >6 sec = 2)
• Fibrinogen level (>100 mg/dL = 0, <100 mg/dL = 1)
Calculate score:
• If ≥5, compatible with overt DIC: Repeat score daily.
• If <5, suggestive (not affirmative) for nonovert DIC: Repeat next 1–2
days.
DIC, Disseminated intravascular coagulation; ISTH, International Society on Throm-
bosis and Haemostasis; PT, prothrombin time.
Note that in this example of score implementation, a D-­dimer test having an upper
normal limit of 0.4 μg/mL was employed. More generally, for the particular analyte
employed as an “elevated fibrin marker,” no increase = 0, moderate increase = 2,
and strong increase = 3.
the system. There are a few scoring systems for DIC diagnosis; however,
they often are not used in clinical practice, probably due to poor sensitivity
and specificity as well as reproducibility in daily practice (Iba et al., 2019,
Yamazaki et al., 2019) (Table 40.6).
These changes result in the clinical features of usually widespread
bleeding (especially from puncture sites, wounds, and surgical sites)
and laboratory findings of prolonged PT, APTT, decreased fibrinogen,
elevated D-­dimers, and thrombocytopenia. The peripheral blood smear
examination may show microangiopathic hemolytic anemia (MHA) with
schistocytes and helmet cells in both acute and chronic DIC. However,
the absence of MHA does not exclude DIC. DIC can be an acute, life-­
threatening, or chronic clinical condition depending on the underlying
cause. Identifying DIC and the underlying condition responsible for it are
critical to adequate management.
DIC can occur in patients with sepsis, malignancy, obstetric complica-
tions, or massive tissue injury (Schmaier, 1991). DIC can also arise during
surgery as a result of the release of thromboplastin material. In circula-
tory arrest operations on the arch of the aorta or the main pulmonary
arteries, DIC is a frequent complication related to hypothermia induced
in the patient and tissue destruction. Abruptio placentae, placenta previa,
amniotic fluid embolism, and HELLP syndrome (hemolysis, elevated liver
enzymes, and low platelets) are associated with acute hemorrhagic DIC,
whereas the retained dead fetus is associated with a DIC that is not hemor-
rhagic but rather prothrombotic. DIC with sepsis is most commonly seen
with gram-­negative infection but can occur with gram-­positive infection
and in the immunosuppressed patient with fungemia.
This clinical laboratory phenotype of DIC most probably results from
a hyperfibrinolytic state and is the form of DIC most commonly recog-
nized. A prothrombotic state as the result of DIC is usually associated with
normal PT, normal to shortened APTT due to high FVIII with mildly
reduced platelet count and normal or elevated fibrinogen (acute-­phase
effect, like FVIII). The D-­dimers are elevated.
Management of DIC
The treatment of the underlying cause often results in the resolution of
DIC. The practice of using plasma, platelets, or cryoprecipitate to cor-
rect an abnormal coagulation test parameter in a nonbleeding patient
can result in worsening of the clinical condition due to exacerbation of
microthromboses as a result of the “adding fuel to the fire” effect. There-
fore, in a nonbleeding patient with only laboratory evidence of DIC, a
low-­dose heparin (3–5 IU/kg/hour) may help to neutralize thrombin
and, thus, downregulate the DIC process. This effect is often observed
as improved platelet count and fibrinogen value, and gradual improve-
ment of PT and APTT—of course, treatment of the underlying cause
is imperative. In a patient with acute DIC and bleeding, again, control
of the underlying cause is essential, along with appropriate use of blood
components, including plasma to correct coagulopathy, platelet transfu-
sion to maintain platelet count >20 × 109/L and cryoprecipitate to main-
tain fibrinogen >100 mg/dL.
844
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LIVER DISEASE
Hepatocytes synthesize all coagulation factors except for FVIII and vWF.
Therefore, both acute and chronic liver diseases affect laboratory coagula-
tion parameters. Chronic liver disease (cirrhosis) is increasing worldwide
due to alcohol, hepatitis C, and other causes. Acute fulminant liver disease
is often associated with drugs, viral infection, sepsis, and more.
Cirrhosis
Patients with cirrhosis often have prolonged PT and mild to moderate
thrombocytopenia. Cirrhotic patients have been shown to have rebalanced
hemostasis, in which, despite abnormal coagulation parameters, they have
normal hemostasis with no excessive bleeding tendency (Tripodi & Man-
nucci, 2011). Large epidemiologic studies have demonstrated that cirrhot-
ics have double the incidence of DVT/PE and portal vein thrombosis than
the general population (Ambrosino et al., 2017; Mancusio, 2016). Most
bleeding in cirrhosis is associated with increased portal pressure caus-
ing variceal bleeding—that is, surgical rather than coagulopathic bleed-
ing—that responds to surgical intervention and not to blood component
transfusion to correct laboratory values. Blood components increase portal
pressure further, leading to worsening of variceal bleeding and harm to
the patients.
Rebalanced Primary Hemostasis
Cirrhotics have mild to moderate thrombocytopenia, which is multifac-
torial, including splenomegaly, decreased thrombopoietin (TPO), bone
marrow suppression due to nutritional deficiency, or infection. However,
there are extremely high levels of vWF with increasing severity of cirrhosis
from Child-­Pugh stage A to C with mean vWF being two-­to fourfold
that of normal (300%–600%). This increased vWF improves the adhesive
function of platelets manyfold and, thus, normalizes primary hemostasis.
A randomized clinical trial using eltrombopag (a TPO mimetic) was dis-
continued due to higher portal vein thrombosis in the treatment arm com-
pared with placebo, suggesting that cirrhotics do not need platelet counts
>50 K for surgical hemostasis (Afdhal et al., 2012).
Also, platelet transfusions often fail to increase platelet count due to
sequestration in the enlarged spleen within minutes of the transfusion.
Multiple platelet transfusions are detrimental due to the presence of ∼300
cc of plasma in each dose of platelet that further increases portal pressure;
the resulting bleeding is often attributed to thrombocytopenia instead.
Rebalanced Secondary Hemostasis
The liver makes both procoagulants and natural anticoagulants. However,
routine coagulation tests, PT/INR, and APTT reflect only decreased pro-
coagulants and not the decreased natural anticoagulants (AT, PC, and PS).
The cirrhotic plasma in the presence of thrombomodulin generates the
same amount of thrombin as normal plasma despite an INR >3, reflecting
rebalanced secondary hemostasis (Tripodi et al., 2005). The viscoelastic
measurements with TEG/ROTEM often show normal reaction time/clot-
ting time (R/CT) values despite very long PT/INR (De Pietri et al., 2016).
TEG/ROTEM should not be used routinely other than for R/CT param-
eters because both MA and MCF are usually lower than normal due to
thrombocytopenia. Primary hemostasis is rebalanced in-­vivo and does not
reflect in MA/MCF due to absence of endothelium in the system.
Thus, it is futile to attempt correction of mild to moderately prolonged
PT/INR (1.5–2.5). If the PT/INR is prolonged >2.5, it could be due to
vitamin K deficiency and/or hypo/dysfibrinogenemia, which is observed in
up to 30% to 45% of cirrhotics. Many patients with cirrhosis have fibrino-
gen <100 mg/dL; hence, fibrinogen should be routinely measured if the
patient is scheduled for a high-­risk procedure or has nonvariceal bleeding.
Hypo/dysfibrinogenemia should be treated with cryoprecipitate.
Rebalanced Fibrinolytic System
Plasminogen and α2-­antiplasmin are reduced, but endothelial-­derived tPA
and PAI-­I are increased; therefore, fibrinolysis is also rebalanced. Usually,
patients with end-­stage cirrhosis may exhibit hyperfibrinolysis with large
ecchymoses and severe hypofibrinogenemia. These patients may benefit
from antifibrinolytics along with cryoprecipitate.
VITAMIN K DEFICIENCY
Vitamin K, a lipid-­ soluble vitamin, is provided by dietary intake of
leafy green vegetables and by the synthesis of intestinal flora. In clinical
practice, vitamin K deficiency is seen most often in acutely ill patients
on antibiotics who have subsisted on parenteral nutrition. Not infre-
quently, intravenous fluids are not supplemented with vitamin K. After
a few weeks of parenteral nutrition and antibiotic treatment, the patient
becomes vitamin K deficient, which is reflected in the gradual increase
in PT/INR from the normal at baseline. Vitamin K deficiency can also
be seen in patients who have an anatomic bypass of the small intestine,
malabsorption, biliary tract obstruction, and, rarely, reduced dietary
intake. For example, alcoholics are often vitamin K deficient. Warfarin
also inhibits the enzymatic pathway necessary for vitamin K utilization
(see Fig. 43.1). Vitamin K has a critical role in the γ-­carboxylation reac-
tion of several glutamic acid residues within coagulation factors II, VII,
IX, and X and proteins C, S, and Z. This γ-­carboxylation is critical for the
proteins to bind to cells and phospholipids so that they can participate
in physiologic coagulation reactions. Thus, patients will have reduced
vitamin K–dependent factors II, VII, IX, and X. If antigen levels of these
patients are measured, they will frequently be higher. All such clinical
scenarios should be treated with 10 mg intravenous vitamin K in 25 to 50
mL of normal saline slowly infused over 15 to 30 minutes. This approach
is both diagnostic and therapeutic, avoiding unnecessary investigations
of prolonged PT/INR. The intravenous route is preferred for its rapid
onset of action, within 2 to 4 hours, as opposed to oral and subcutaneous
routes that have unpredictable absorption.
MASSIVE TRANSFUSION
Massive transfusion is defined as the replacement of more than 1.0 blood
volume or at least 10 units of packed red blood cells in 24 hours, usually
following major trauma. Trauma-­induced coagulopathy (TIC) is unique
and still not well understood (Cohen et al., 2017). TIC is not merely
either dilutional coagulopathy due to infusion of liters of colloids/crys-
talloids or red cell transfusions during the transfer from the trauma
site to the emergency department (ED) of the hospital nor just mas-
sive DIC initiated by the tissue factor released after trauma. TIC exists
in addition to these and may involve endothelial perturbation affecting
thrombomodulin and activated protein C pathway. The coagulopathy
is made worse by hypothermia and acidosis. Therefore, recently, the
use of liquid plasma and low titer O whole blood at the trauma site is
undergoing clinical trials to improve hemostasis and prevent the onset of
coagulopathy. Once the coagulopathy sets in, then it is difficult to catch
up with when the patient arrives in the ED. Massive transfusion proto-
cols (MTPs) have been developed to manage such patients by transfus-
ing balanced amounts of various blood components. ROTEM is also
used as an algorithmic approach to provide goal-­directed transfusion
therapy because of the real-­time tracings displayed in the OR. Plasma
transfusion is indicated when EXTEM CT is prolonged, or transfusion
of cryoprecipitate if the FIBTEM shows a low value along with low
EXTEM MCF, or transfusion of platelets if low EXTEM MCF is asso-
ciated with normal FIBTEM. In places where ROTEM is unavailable,
MTPs consisting of a red cell:plasma:platelet ratio of 1:1:1 or 2:1:1 are
used. Although surgical mishap resulting in massive transfusions during
nontrauma surgery may occur, coagulopathy in such a patient is more
controlled than trauma. Often, goal-­directed transfusion therapy with
ROTEM guidance is useful in these circumstances. However, postpar-
tum hemorrhage seems to require a trauma level transfusion strategy,
especially regarding fibrinogen levels.
ACQUIRED COAGULATION FACTOR DEFICIENCIES
Rarely, spontaneous autoantibody against coagulation protein can be
formed and may present with a bleeding tendency (Kruse-­Jarres et al.,
2017). The most common of these rare acquired bleeding disorders
is acquired hemophilia A (AHA) due to an autoantibody formation
against FVIII. The inhibitor is an IgG that binds to FVIII and causes
its clearance or inhibits its function, resulting in a severe bleeding ten-
dency that, if not diagnosed promptly and treated, can be fatal. AHA is
encountered in two peaks—one in young women during the postpartum
period and the second in the elderly (6th–8th decades). Some patients
may have an underlying autoimmune disorder; otherwise, it is seen as a
primary disorder. Both males and females are affected and present with
significant soft-­tissue hemorrhage (usually in areas with much adipose
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tissue), hematuria, and, rarely, joint bleed. These patients have a pro-
longed APTT with normal PT. In the right clinical setting when there
is significant bleeding and the patient has previously normal APTT, a
stat FVIII level, especially by the chromogenic method, would establish
the diagnosis. If there is no access to stat FVIII assay, then a two-­step
mixing may be performed to show time-­and temperature-­dependent
PART 5
inhibitor. FVIII levels are often reduced to single digits or in the teens
despite a high-­titer inhibitor, suggesting that the assay is probably
detecting dissociated FVIII from its IgG inhibitor in vitro. Hemor-
rhage in these patients is treated with a bypassing agent such as FEIBA
or rFVIIa. Recently, emicizumab and recombinant porcine FVIII have
been other options. AHA generally responds well to immunosuppres-
sion therapy with corticosteroids (1 mg/kg), cyclophosphamide, and
rituximab (both low, 100-­mg weekly × 4 fixed-­dose as well as lymphoma
dose, 375 mg/m2 × 4 weeks) (Aggarwal, 2005). The response to therapy
can be monitored with improvement in APTT initially and then FVIII
levels, and inhibitor titer twice a month.
FX deficiency is rarely encountered in patients with amyloidosis, in
whom the amyloid fibrils selectively adsorb FX from the plasma (Chan &
Ogunsile, 2017). In such cases, patients present with bleeding tendencies
that are associated with prolonged PT and APTT with normal thrombin
time. FX is usually moderate to severely deficient (<5% activity). Plasma
infusion, plasma exchange, and nonactivated PCC have been often ineffec-
tive because, within minutes of infusion, the amyloid fibrils rapidly adsorb
out FX. The best option is to use FEIBA to treat bleeding.
Recombinant human thrombin has mostly replaced bovine thrombin
in clinical practice. However, rarely upon exposure to bovine thrombin,
a patient may make an antibody against either bovine thrombin or bovine
FV that is present as a contaminant in bovine thrombin (Gaava et al.,
2016). These antibodies may cross-­react with human thrombin or human
FV, causing prolonged PT and APTT. Thrombin time is also prolonged
with an antibody against bovine thrombin but not with an antibody against
FV. A mixing study will support a diagnosis of inhibitor due to incomplete
to no correction of prolonged PT/APTT/TT. Most of these patients do
not bleed; however, if there is bleeding, they should be treated with corti-
costeroids that often eliminate the inhibitor(s). These inhibitors are often
transient following exposure to some antibiotics. In severe bleeding cases,
FEIBA or rFVIIa can be used.
Lupus Anticoagulant
This circulating inhibitor was initially identified in patients with sys-
temic lupus erythematosus (SLE); because it prolonged APTT similarly
to heparin, it was called lupus anticoagulant (LA). It was initially thought
to interact with phospholipids in the coagulation cascade during APTT
testing in the test tube. It is triply misnomered because most LAs are (1)
found in non-­SLE patients, (2) are prothrombotic, and (3) interact with
PL bound to a protein moiety such as prothrombin or β2-­glycoprotein
I. Between 1960 and 1980, there was no specific diagnostic test for LA.
Hence, a mixing study was used to differentiate between factor deficiency
and LA as a cause of prolonged APTT. Recently, more specific tests—
such as the dilute Russell viper venom test (DRVVT), silica clotting time,
and PTT-­LA—are available to detect LA (see Chapter 42). Hence, they
should be used to identify the cause of prolonged APTT in a nonbleeding
or thrombotic patient because a weak LA with mildly prolonged APTT
will show complete correction on mixing study, leading to unnecessary
investigation of factor deficiency in the intrinsic pathway (Pengo, 2009).
The sensitivity of each APTT reagent for LA depends on its PL con-
tent. Some patients with a strong LA may also have prolonged PT/INR
that should be recognized at the beginning of VKA therapy because such
patients should be monitored with chromogenic FX rather than INR for
proper anticoagulation therapy.
Rarely, patients with a very strong LA may also have an autoantibody
against prothrombin, causing very prolonged PT and APTT. These
patients with “lupus anticoagulant and hypoprothrombinemia syndrome”
generally have severe prothrombin deficiency, and present with significant
clinical bleeding rather than clotting despite the presence of LA. Most
of these patients are children, who respond well to corticosteroids. The
bleeding should be managed with PCC.
845
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