ANTICOAGULANTS & ANTIPLATELETS

DR. VAL HILTON

HAEMOSTASIS
 Vital cascade system
 Balance between haemostasis and fibrinolysis
 Classical haemostasis model
 Cell based model of coagulation – in the last 10 – 15 years.

CLASSICAL MODEL
HEMOSTASIS – can be described as the process by which the body ceases bleeding or haemorrhage. It involves a
complicated interplay with the system of vascular, platelets as well as various proteins in plasma that together create blood clots.
The process of blood clot formation, is coordinated by series of responses to vessel injury. It requires complex interaction
between platelets, the clotting cascade blood flow and shear, endothelia cells and fibrinolysis.

1. Vascular Phase [Vasoconstriction – immediately after a blood vessel has been damaged, it expands to limit the
flow of blood and reduce the loss of blood. It is a temporary and quick reaction.

2. Platelet Phase [Platelet Plug Formation] – Platelet adhere to collagen exposed and other elements from the affected
vessel. After activation, they release substances that draw more platelets to the area. The platelets accumulate, creating
the temporary ‘’Platelet plug’’ over the wound.
 3. The coagulation Phase [Blood Clot formation] – it involves a series of enzymatic processes that will result in
transformation of fibrinogen from plasma into fibrin threads which pass through the plug of platelets, forming and
stabilizing the clot into an even more robust blood clot.

When the injury that caused it is repaired, another process known as fibrinolysis breaks down the clot, making sure that blood
flow returns to normal levels.
Hemostasis assures that the body is able to prevent excessive loss of blood due to injuries and also ensures that blood is clear and
unhindered when there are no vessel injuries.
PLATELET PLUG FORMATION

After platelets are activated, they undergo significant morphologic changes, producing elongated pseudopods. They also become
extremely adhesive. The functional response of activated platelets involves four distinct processes:
 Adhesion [deposition of platelets on subendothelial matrix]
 Aggregation [cohesion of platelets]
 Secretion [release of platelets granule proteins] and
 Procoagulant activity [enhancement of thrombin generation]

Platelets are activated at the site of vascular injury to form a plug to stop bleeding. Physiological platelet stimuli include:
 Adenosine Diphosphate (ADP).
 Epinephrine
 Thrombin and
 Collagen.
Weak Platelet Stimuli Strong Agonists
ADP Thrombin
Epinephrine Collagen
Thrombin activation is mediated by G-protein-couple proteases-activated receptors (PAR-1) – specifically PAR-1 and PAR-4.

1. Thrombin cleaves the external domain of the PAR to initiate transmembrane signalling (Figure 1).

Figure 1: Thrombin activation is mediated by G protein-coupled proteases-activated receptor (PAR). Thrombin cleaves the NH 2-
terminal exodomain of the PAR, exposing a new NH 2 terminus, which then serves as a tethered ligands to bind intramolecularly
to the body of the receptor to initiate transmembrane signalling.

2. Platelet responses to ADP require the coordinated activation of two G-protein-coupled receptors, P2Y1 and P2Y12,
which lead to activation of phospholipase C and suppression of cyclic adenosine monophosphate (cAMP) formation,
respectively. Antiplatelet drugs such as ticlopidine and clopidogrel block activation of P2Y12.

3. There are also specific receptors for:

 Epinephrine
 Thromboxane A2 and
 Collagen.
Platelet activation involves 4 distinct processes:
 Adhesion (deposition of platelets on subendothelial matrix)
 Aggregation (cohesion of platelets)
  Secretion (release of platelet granule protein) and
 Procoagulant activity (enhancement of thrombin generation)- Figure 2.

Figure 2: After platelets are activated, they undergo significant morphologic changes, producing elongated pseudopods. They
also become extremely adhesive. The functional response of activated platelets involves four distinct processes:
 Adhesion [deposition of platelets on subendothelial matrix]
 Aggregation [cohesion of platelets]
 Secretion [release of platelets granule proteins] and
 Procoagulant activity [enhancement of thrombin generation]

ADHESION
Platelet adhesion is primarily mediated by the binding of platelet surface receptors glycoprotein (GP) 1b-IX-V complex to
adhesive protein von Willebrand factor (vWF) in the subendothelial matrix.

4. Deficiency of GP1b-IX-V complex or vWF leads to two congenital bleeding disorders.

 Bernard-Soulier disease and
 Von Willebrand disease
Other adhesive interactions – eg binding of platelet collagen receptor GP1a-II to collagen fibrils in the matrix also contribute to
platelet adhesion.

5. AGGREGATION
Platelet aggregation involves binding of fibrinogen to the platelet fibrinogen receptor (i.e., the GPIIb-IIIa complex). GPIIb-
IIIa (also termed aIIbb3) is a member of a superfamily of adhesive protein receptors, called integrins, which are found in
many different cell types. It is the most abundant receptor on the platelet surface. GPIIb-IIIa does not bind fibrinogen on
nonstim-ulated platelets. After platelet stimulation, GPIIb-IIIa undergoes a conformational change and is converted from a
low-affinity fibrinogen receptor to a high-affinity receptor in a process termed inside-out signaling. Fibrinogen, a divalent
molecule, serves to bridge the activated platelets [see Figure 3].
Figure 3: Platelet aggregation involves binding of the divalent molecule fibrinogen to the platelet fibrinogen receptor(the GPIIb-
IIIa complex). After platelet stimulation, GPIIb-III a is converted from a low-affinity fibrinogen receptor to a high affinity
receptor (inside-out signalling). The cytosolic portion of the activated GPIIb-IIIa complex can mediate platelet spreading and clot
retraction (outside-in signalling).

The cyto-solic portion of the activated GPIIb-IIIa complex binds to the platelet cytoskeleton and can mediate platelet spreading
and clot retraction (in a process termed outside-in signaling).6 Congenital deficiency of GPIIb-IIIa or fibrinogen leads to
Glanzmann thrombasthenia and afibrinogenemia. The GPIIb-IIIa-fibrinogen pathway is the final common course for platelet
aggregation. Blockade of this pathway is the basis of an important class of antiplatelet drugs.

PROTEIN SECRETION
After stimulation, platelet granules release ADP and serotonin, which stimulate and recruit additional platelets; adhesive
proteins such as fibronectin and thrombospondin, which reinforce and stabilize platelet aggregates; factor V, a component of the
clotting cascade; thromboxane, which stimulates vasoconstriction; and growth factors such as platelet-derived growth factor
(PDGF), which stimulate proliferation of smooth muscle cells and mediate tissue repair. PDGF may also contribute to the
development of atherosclerosis and reocclusion after coronary angioplasty.

PROCOAGULATION
Platelet procoagulation involves the assembly of the enzyme complexes of the clotting cascade on the platelet surface. It is an
important example of the close interrelationship between platelet activation and the activation of the clotting cascade.

CLOTTING CASCADE
The central feature of the clotting cascade is the sequential activation of a series of proenzymes (zymogens) to enzymes,
ultimately generating fibrin and reinforcing the platelet plug. Another key feature, amplification, ensures rapid response for
effective hemostasis but demands tight regulation to prevent untoward thrombosis.
The clotting cascade is usually depicted as comprising intrinsic and extrinsic pathways [see Figure 4]. The intrinsic pathway is
initiated by the exposure of blood to a negatively charged surface (e.g., glass), whereas the extrinsic pathway is activated by
tissue factor or thromboplastin. Both pathways converge on the activation of factor X, which then activates prothrombin (factor
II) to thrombin, the final enzyme of the clotting cascade.
Although this classic view of the clotting cascade has been useful in the interpretation of clotting times, it is not completely
accurate. Patients who are severely deficient in factor XII—as well as many patients deficient in factor XII—do not bleed
clinically, which indicates that the initiation part of the intrinsic path way (the contact phase) is not important in vivo. It is now
established that generation or exposure of tissue factor at the wound site is the primary physiologic event that initiates clotting
[see Figure 4].7 Tissue factor functions as a cofactor that is absolutely required by factor VII/factor VIIa to initiate clotting.
Factor VIIa activates factor X directly and indirectly via the activation of factor IX. This dual pathway of factor X activation is
necessary apparently because of the limited amount of tissue factor generated in vivo and the presence of the tissue factor
pathway inhibitor (see below), which, when complexed with factor Xa, inhibits the tissue factor/factor VIIa complex.
All of the procoagulants are synthesized in the liver except vWF, which is synthesized in megakaryocytes and endothelial cells.
The vitamin K-dependent procoagulants are prothrombin, factor VII, factor IX, and factor X; the vitamin K-dependent
anticoagulants are protein C and protein S. For these factors, the formation of a-carboxyglutamic acid residues by vitamin K-
dependent carboxylation of glutamic acid residues endows them with calcium-binding properties and the ability to interact with
phospholipid membrane surfaces, which are required for biologic activity.

INTERACTION BETWEEN ACTIVATED PLATELETS AND THE CLOTTING CASCADE

There is an extremely close interaction between the clotting cascade and activated platelet surface in vivo. When platelets are
activated, anionic lipids become exposed on the platelet surface, and factor V (stored in platelet granules) is released and bound
on the anionic lipids. The factor V on the platelet surface is activated to factor Va and acts as an assembly site for the binding of
factor Xa (enzyme) and prothrombin (substrate) known as the prothrombinase complex. At the assembly site, thrombin
generation by the prothrombinase complex is approximately 300,000 times more efficient than thrombin generation by fluid-
phase factor Xa and prothrombin alone, and the platelet plug keeps the thrombin localized. Factor Xa bound on factor Va is also
relatively protected from inhibition by circulating inhibitors such as an-tithrombin III (AT-III) (see below). Similar enzyme
complex assembly applies to the activation of factor X by factor VIIIa (cofac-tor) and factor IXa (the intrinsic tenase). The result
of these processes is efficient amplification and localization of clotting.

CONTROL MECHANISMS
Coagulation is modulated by a number of mechanisms: dilution of procoagulants in flowing blood; removal of activated factors
through the reticuloendothelial system, especially in the liver; and control by natural antithrombotic pathways. At least seven
separate and distinct control systems modulate each phase of hemostasis and protect against thrombosis, vascular inflammation,
and tissue damage [see Table 1]. Antithrombin III, protein C, protein S, and tissue factor pathway inhibitor (TFPI) collectively
regulate the clotting cascade; prostacyclin and nitric oxide modulate vascular and platelet reactivity; ecto-ADPase inhibits platelet
recruitment; and fibrinolysis removes the fibrin clot.

TABLE 1 – NATURAL ANTITHROMBOTIC MECHANISMS OF ENDOTHELIAL CELLS

Regulation of clotting cascade Tissue factor pathway inhibitor Antithrombin III
Protein C/Protein S
Modulation of vessel and platelet reactivity Prostacyclin Nitric oxide
Inhibition of platelet recruitment Ecto-ADPase (CD39)
Removal of fibrin clot Fibrinolysis
ANTITHROMBIN III HEPARIN SULFATE SYSTEM
Antithrombin III is a circulating plasma protease inhibitor. It inhibits thrombin and factor Xa, the two key enzymes in the
clotting cascade. AT-III also inhibits activated factor XII and factor XI. In the absence of the glycosaminoglycan heparin, AT-III
inhibits thrombin and factor Xa relatively slowly (complete inhibition requires a few minutes). When present, heparin binds to a
discrete binding site on AT-III that causes a conformational change in AT-III, which then inhibits thrombin instantaneously and
irreversibly. This augmentation of the inhibition of throm-bin and factor Xa is the basis for the therapeutic use of heparin as an
anticoagulant. Heparan sulfate proteoglycans on the luminal surface of endothelial cells appear to activate AT-III in a manner
similar to that of heparin [see Figure 5].9

Figure 5 In the absence of heparan sulfate (HS), antithrombin III (AT-III) inhibits thrombin slowly. When HS is present, it binds
to a specific site on AT-III that causes a conformational change in AT-III, allowing it to reach the active site of thrombin and
inhibit the enzyme instantaneously. HS also binds to a specific site on thrombin, positioning it for optimal inhibition by AT-III.

Thus, the endothelial surface is normally coated with a layer of AT-III that is already activated by the endogenous heparan sul-
fate. Because 1 ml of blood can be exposed to as much as 5,000 cm2 of endothelial surface, the AT-III-heparan sulfate system is
poised to rapidly inactivate any thrombin in the general circulation.
PROTEIN C AND PROTEIN S-THROMBOMODULIN SYSTEM
Thrombomodulin is an integral membrane protein found on the luminal surface of the vascular endothelium in the microcir-
culation. The binding of thrombin to thrombomodulin results in a remarkable switch in thrombin’s substrate specificities: it no
longer clots fibrinogen or activates platelets [see Figure 6]. On the other hand, it acquires the ability to activate protein C in
plasma.10 A distinct endothelial receptor for protein C has been found that enhances the activation of protein C by the thrombin-
thrombomodulin complex.11 Activated protein C degrades factor Va and factor Villa, the two cofactors responsible for the
assembly of the prothrombinase and intrinsic tenase complex in the clotting cascade. Protein S serves as a cofactor for activated
protein C. Deficiencies of AT-III, protein C, and protein S are important causes of a hypercoagulable state.

Protein C and protein S both show some structural similarity to the vitamin K-dependent clotting factors (prothrombin, factor
VII, factor IX, and factor X). Protein S circulates in two forms: a free form, in which it is active as an anticoagulant, and a bound,
inactive form, in which it is complexed to C4b-binding protein of the complement system. C4b-binding protein acts as an acute-
phase reactant. The resultant increase in inflammatory states reduces the activity of free protein S, enhancing the likelihood of
thrombosis.

TISSUE FACTOR PATHWAY INHIBITOR

Tissue factor pathway inhibitor is a circulating plasma protease inhibitor that is synthesized by the microvascular en-
dothelium. Unlike AT-III, TFPI has a very low plasma concentration. TFPI inhibits factor Xa. The TFPI/factor Xa complex
becomes an effective inhibitor of tissue factor/factor VIIa, thus mediating feedback inhibition of tissue factor/factor VIIa [see
Figure 7]. Animal studies have shown that depletion of the endogenous TFPI sensitizes the animals to the development of
disseminated intravascular coagulation induced by tissue factor or endotoxin.12
TFPI is primarily synthesized by the microvascular endotheli-um. Approximately 20% of TFPI circulates in plasma associated
with lipoproteins; the majority remains associated with the en-dothelial surface, apparently bound to the cell-surface
glycosaminoglycans. The plasma level of TFPI is greatly increased after intravenous administration of heparin. This release of
en-dothelial TFPI may contribute to the antithrombotic efficacy of heparin and low-molecular-weight heparin. Recombinant
TFPI is now in early clinical trials.

PROSTACYLIN
Upon cell perturbation, the fatty acid arachidonic acid is released from cell membrane phospholipids by phospholipase A2. The
enzyme prostaglandin endoperoxide H synthase-1 (PGHS-1) converts arachidonic acid into prostaglandin endoperoxides and
finally to thromboxane A2 (TXA2) in platelets and prostacy-clin (PGI2) in endothelial cells.

TXA2 and PGI2 have opposite functions. TXA2 is a potent stimulator of platelet aggregation and causes vasoconstriction,
whereas PGI2 inhibits platelet aggregation and induces vasodilatation. PGI2 functions by activating adenylate cyclase, which
leads to an increase in intracellular cAMP [see Figure 8].
Figure 8 Significant synergism exists between nitric oxide (NO) and prostacyclin (PGI2), leading to platelet inactivation and
vasodilatation. The enzyme prostaglandin endoperoxide H synthase-1 (PGHS-1) converts arachidonic acid into PGI2 in
endothelial cells. PGI2 activates adenylate cyclase, which leads to an increase in intracellular cyclic adenosine monophosphate
(cAMP), inhibiting platelet aggregation and inducing vasodilatation. NO, formed from L-arginine, stimulates production of cyclic
guanosine monophosphate (cGMP). Cyclooxygenase-2 (COX-2) is the induced isoform of PGHS; its formation presumably
results from hemodynamic shear in the circulation. NO formation is catalyzed by NO synthases (NOS).

Cyclooxygenase-1 and Cyclooxygenase-2

Cyclooxygenase-1 (COX-1) is the constitutive isoform of PGHS. Cyclooxygenase-2 (COX-2) is an inducible isoform of PGHS.
COX-2 is undetectable in most tissues. However, it can be rapidly induced in response to growth factors, endotoxins, and cy-
tokines in endothelial cells and monocytes (although not in platelets).14 Recent evidence indicates that endothelial COX-2 is a
major source of PGI2 under physiologic conditions in humans, perhaps because of continual COX-2 induction by hemodynam-ic
shear in the circulation.15 Aspirin acetylates and irreversibly inhibits both COX-1 and COX-2. Other nonsteroidal anti-
inflammatory drugs (NSAIDs) also inhibit COX-1 and COX-2, although not permanently. Selective COX-2 inhibitors are now
available as a new generation of NSAIDs.16
Because aspirin irreversibly inhibits COX-1 and because platelets cannot make new COX-1, brief exposure to aspirin will
permanently inhibit TXA2 production for the life span of affected platelets.

Nitric oxide
Nitric oxide (NO) is formed from L-arginine in endothelial cells. NO stimulates guanylate cyclase, leading to an increase in
cyclic guanosine monophosphate (cGMP) in target cells; causes vasodilatation; and inhibits platelet adhesion and aggregation
[see Figure 8].17 NO is rapidly destroyed by hemoglobin and thus functions as a local (i.e., paracrine) hormone. Intravenous
infusion of an arginine analogue that blocks NO production leads to an immediate and substantial rise in blood pressure. This
phenomenon suggests that NO is released continually and basally to regulate vascular tone (in contrast to the production of PGI2,
which is more stimulus-responsive). There is significant syner-gism between NO and PGI2. Formation of NO is catalyzed by NO
synthases, which exist in different isoforms in various tissues. In addition to regulating vascular events, NO has a wide range of
biologic effects (e.g., neurotransmittal function in the central nervous system).

ECTO-ADPase (CD39)
CD39 is an integral membrane protein found on the endothe-lial cell surface. It is an
active enzyme that rapidly hydrolyzes ADP to AMP, thus functioning as a cell-bound
ecto-ADPase. It limits the recruitment of additional platelets into the growing platelet
plug by removing ADP released from the dense granules of activated platelets and from
damaged erythrocytes and endothelial cells.

Fibrinolysis
Tissue plasminogen activator (t-PA) is released from perturbed endothelial cells near the site of vascular injury. t-PA converts
plasminogen to plasmin. Like the AT-III interaction with thrombin, which is accelerated in the presence of endothelial cell
surface heparan sulfate, generation of plasmin takes place optimally on a surface (in this case, the fibrin clot). Both t-PA and
plasminogen bind to fibrin (via recognition of lysine residues), which facilitates plasmin generation and localized fibrinolysis
[see Figure 9].
Plasmin cleaves the polymerized fibrin strand at multiple sites, releasing fibrin degradation products. One of the major fibrin
degradation products is D-dimer, which consists of two D domains from adjacent fibrin monomers that have been cross-linked by
activated factor XIII [see Figure 10]. Plasmin has a broad substrate specificity and, in addition to fibrin, cleaves fibrinogen and a
variety of plasma proteins and clotting factors. Plasmin bound on the fibrin clot is protected from inactivation, whereas plasmin
released into the circulation is rapidly inactivated by plasma a2-antiplasmin. Thus, localized fibrinolysis is achieved, but
nonspecific plasmin degradation of plasma proteins is prevented. In rare cases, patients have bleeding problems caused by a
congenital deficiency in a2-antiplasmin.
Urokinase is the second physiologic plasminogen activator. It is present in high concentration in the urine. Although t-PA is
largely responsible for initiating intravascular fibrinolysis, uroki-nase is the major activator of fibrinolysis in the extravascular
compartment. Urokinase is secreted by many cell types in the form of prourokinase, also termed single-chain urokinase-type
plasminogen activator (scu-PA). Prourokinase is converted to urokinase by plasmin. Urokinase lacks fibrin specificity in
converting plasminogen to plasmin, whereas prourokinase displays such specificity.
The major physiologic inhibitor of t-PA and urokinase plas-minogen activator (u-PA) is plasminogen activator inhibitor-1
(PAI-1).19 Substantial amounts of PAI-1 are found in platelets. PAI-1 is also released from endothelial cells. PAI-1 deficiency is
associated with bleeding diathesis, usually related to trauma or surgery.20 A second inhibitor, PAI-2, is normally secreted by
monocytes. During pregnancy, PAI-2 levels are greatly increased because of synthesis by the placenta. The biologic importance
of PAI-2 remains to be established.

Thrombin-Activatable Fibrinolysis Inhibitor

Plasma carboxypeptidase is a newly recognized thrombin-ac-tivatable fibrinolysis inhibitor (TAFI) [see Figure 11].21,22 TAFI
is the second known physiologic substrate for the thrombin-thrombomodulin complex. One may envisage that after the initial
fibrin clot is formed by thrombin at the site of a vascular wound, thrombin binds to thrombomodulin on the nearby intact
endothelial surface. The thrombomodulin-bound thrombin leads to the generation of activated protein C, which dampens the
clotting cascade and prevents excessive thrombin generation. At the same time, the thrombomodulin-bound thrombin activates
TAFI, thus slowing down the lysis of the existing clot. In hemophilia, the decreased generation of thrombin may lead to
suboptimal activation of TAFI and result in premature clot lysis, which contributes to the delayed bleeding observed in these pa-
tients.22 Whether excessive TAFI activity leads to thrombosis is unknown at present.

Overview of Blood Coagulation

The clotting cascade is initiated by the exposure of tissue factor at a vascular wound,
which leads to the generation of thrombin and the deposition of a fibrin clot [see Figure
12]. Simultaneously, the damaged endothelium releases t-PA, which converts
plasminogen to plasmin, which then lyses the clot.

Figure 9 Tissue-type plasminogen activator (t-PA), released from perturbed

endothelial cells near an injured blood vessel, converts plasminogen to plasmin.
Free plasmin is rapidly inactivated by plasma a2-antiplasmin; plasmin bound to
the fibrin clot is protected from inactivation.
Figure 10 The transformation of fibrinogen to fibrin is initiated by thrombin cleavage of fibrinopeptides A and B from the E
domains of fibrinogen to form fibrin monomer. The cleavage apparently changes the overall negative charge of the E domain to a
positive charge. This change in charge permits the spontaneous polymerization of fibrin monomers, because the positively
charged E domain assembles with the negatively charged D domains of other monomers. The polymer is initially joined by
hydrogen bonds. Thrombin activates factor XIII, which catalyzes the formation of covalent bonds between adjacent D domains in
the fibrin polymer. Plasmin cleaves the polymerized fibrin strand at multiple sites and releases fibrin degradation products,
including D-dimer.

Both pathways are regulated: TF/factor VIIa is regulated by the TFPI/factor Xa

complex, and thrombin is regulated by protein C and protein S. Similarly, the activity of
t-PA is regulated by PAI-1. Thrombin and plasmin are under the control of their
respective inhibitors, AT-III and a2-antiplasmin. When these two pathways work in
coordinated symmetry, a clot is laid down to stop bleeding, and clot lysis and tissue
remodeling follow. Diminished thrombin generation (as in factor VIII deficiency) or
enhanced plasmin production (as in a2-antiplasmin deficiency) causes hemorrhage [see
5:XIII Hemorrhagic Disorders]. Conversely, excessive production of thrombin (as in AT-
III or protein C deficiency) leads to thrombosis [see 5:XIV Thrombotic Disorders].

Heterogeneity of Endothelial Cells and Vascular Bed-Specific Hemostasis

Although the endothelium is generally considered to be a distinct, homogeneous organ system, there are significant differences
between arterial, venous, and capillary endothelial cells in terms of morphology and disease susceptibility. Recent studies have
shown distinct sets of proteins that mark the arterial and venous endothelial cells from the earliest stages of angiogenesis. Ephrin-
B2, an Eph family transmembrane ligand, marks arterial but not venous endothelial cells. Conversely, Eph-B4, a receptor
tyrosine kinase for ephrin-B2, marks veins but not arteries.23
It is also likely that endothelia from different vascular beds are not identical.24 For example, the high endothelium in the post-
capillary venules of lymph nodes and Peyer patches regulates the circulation of lymphocytes from blood to lymphatics and
peripheral tissues. Specific adhesive protein receptors and matrix proteins are highly expressed in these high endothelial venules.
The specialized endothelium representing the blood-brain barrier is another example.
These differences between arterial and venous endothelial cells and the vascular bed-specific endothelium may partly account for
their different susceptibilities to thrombosis. For example, whereas AT-III and protein C deficiencies are usually associated with
deep vein thrombosis of the lower extremities, thrombosis of portal and hepatic veins is frequently associated with
myeloproliferative diseases.25 In both conditions, the underlying defect is a systemic hypercoagulable state, and yet there is a
clear predisposition of thrombosis to specific vascular beds. Thus, clinical thrombosis is attributable to an imbalance between
systemic prothrombotic stimuli and local antithrombotic mechanisms [see 5:XIV Thrombotic Disorders].

Platelet Production and Thrombopoietin

Platelets are derived from megakaryocytes, which arise from pluripotent myeloid
stem cells. Platelet production is controlled by a thrombopoietin that is involved in the
final maturation of the megakaryocyte. Thrombopoietin has multiple actions in
megakaryocyte development.26 It shares some structural homol-ogy with erythropoietin
and is produced principally by the liver. It increases the size and number of
megakaryocytes, stimulates the expression of platelet-specific markers, and is a potent
megakaryocyte colony-stimulating factor. Although throm-bopoietin is clearly a key
factor, stem cell factor (also called kit ligand), interleukin-3 (IL-3), IL-6, and IL-11 all play
contributory roles in controlling megakaryocytopoiesis.
Figure 11 Plasma carboxypeptidase is a thrombin-activatable fibrinolysis inhibitor
(TAFI). When fibrin is degraded by plasmin, new carboxyl-terminal lysines are
exposed in the partially digested clot. These lysines provide additional sites for
plasminogen incorporation and activation in the clot, setting up a positive
feedback loop in clot lysis. Thrombin activates carboxypeptidase-B in plasma,
which removes the exposed carboxyl-terminal lysines and prevents further
plasminogen incorporation into the clot.
Figure 12 Exposure of tissue factor at a vascular wound initiates the clotting
cascade. Generation of thrombin and deposition of a fibrin clot occur
simultaneously with release of t-PA from the damaged epithelium and conversion
of plasminogen to plasmin. Plasmin then lyses the clot. When these two
pathways work in coordinated symmetry, a clot is laid down to stop bleeding, and
clot lysis and remodeling follow. (a2-AP—a2-antiplasmin; AT-III—antithrombin III;
PAI-1—plasminogen activator inhibitor-1; PC/PS—protein C/protein S; TF—tissue
factor; TFPI—tissue factor pathway inhibitor; t-PA—tissue-type plasminogen
activator)

Megakaryocytes undergo endomitosis, in which nuclear divisions occur without cell

division and are followed by nuclear fusion, to yield a cell with a chromosomal content of
8n, 16n, or 32n. The megakaryocyte cytoplasm then changes into a series of thin,
cylindrical strands that eventually fragment into small pieces of megakaryocytes, called
proplatelets, that are released into the circulation. Megakaryocyte volume correlates
with ploidy and cytoplasmic maturity; the largest megakaryocytes produce the greatest
number of platelets. Large platelets called megathrombocytes are seen in the peripheral
blood in thrombo-cytopenic states, especially in idiopathic thrombocytopenic pur-pura
[see 5:XIIIHemorrhagic Disorders]. These megathrombocytes probably are young
proplatelets and account for the increase in mean platelet volume that occurs during
response to or recovery from acute thrombocytopenia.
Platelets entering the circulation survive about 8.5 to 10 days and have a half-life of
about 4 days. Approximately 30% to 40% of the platelets are present in a splenic pool
that can freely exchange with the circulation. When the need for platelets
arises,production can increase sevenfold to eightfold. Because there is no marrow pool
of platelets waiting to be released, meeting increased requirements for platelets may
require a few days. Platelets have receptors for thrombopoietin and remove it from
plasma, and the platelet mass functions as a major thrombopoi-etin regulator.27 In
states of megakaryocyte hypoplasia and thrombocytopenia, little thrombopoietin is
metabolized and the plasma thrombopoietin level rises, leading to increased production
of megakaryocytes and platelets. In the setting of thrombocytosis, thrombopoietin
metabolism increases, lowering the plasma thrombopoietin level and decreasing platelet
production.

Coagulation Tests and Their Use

Tests of coagulation cascade

Most coagulation tests measure the time required for fibrino-gen from plasma to form
fibrin strands, which can be detected by either optical or electrical devices. Prolongation
may represent a low factor concentration, inactive factor or factors, or the presence of
inhibitors.

Partial Thromboplastin Time

The partial thromboplastin time (PTT), sometimes termed the activated PTT (aPTT),
tests the intrinsic coagulation system. A negatively charged surface (e.g., kaolin or
silica), followed by cephalin, is added to whole plasma to activate factors XII and XI.
The PTT is most sensitive to abnormalities and deficiencies in the sequence of the
coagulation cascade before factor X activation. The PTT is also quite sensitive to the
action of heparin. It is used to monitor and adjust anticoagulant therapy with regular
hep-arin but not with low-molecular-weight heparins.

Prothrombin Time
The prothrombin time (PT) is a test of the extrinsic system. It detects deficiencies in
fibrinogen, factor II (prothrombin), factor V, factor VII, and factor X. Tissue factor is
added to whole plasma, leading to fibrin formation, normally in 9 to 12 seconds. Results
are usually reported using the international normalized ratio (INR). The INR is
calculated by using the following equation:
INR = (Log patient PT / Log control PT)C

where C represents the international sensitivity index (ISI). In this way, the
thromboplastin used in an individual laboratory, with its specific ISI, is calibrated against
a standard reference thromboplastin, and the PT is reported as an INR.28 The
presence of a lupus anticoagulant may also interfere with the PT.29

Dilute Russell Viper Venom Time

Russell viper venom contains an enzyme that activates factor X; therefore, the dilute
Russell viper venom time (DRVVT) measures the common pathway of the clotting
cascade. It is sensitive to the presence of a lupuslike anticoagulant that inhibits the
phospholipid-dependent prothrombinase complex.

Thrombin Time
The thrombin time (TT) is used to test abnormalities of the conversion of fibrinogen to
fibrin. It can be prolonged because of hypofibrinogenemia, abnormal fibrinogen
(dysfibrinogen), or the presence of inhibitors (e.g., fibrin degradation products) that
interfere with fibrin polymerization. The clinical factors commonly associated with
prolonged TT are severe liver disease, disseminated intravascular coagulation, and
heparin therapy.

Reptilase Time
Reptilase is a thrombinlike enzyme that converts fibrinogen to fibrin. The reptilase time
(RT) is prolonged under conditions similar to those for prolonged TT, with one
significant difference: reptilase is not inhibited by the AT-III-heparin complex. Therefore,
RT is not prolonged by heparin. A long thrombin time and normal RT suggest a heparin
effect.

Fibrinopeptide A
Thrombin activates fibrinogen by splitting off two peptides, fibrinopeptide A (FPA) and
FPB, from the Aa and Bp chain of fibrinogen and converting fibrinogen to fibrin
monomer. Measurement of FPA in the blood can be used as an index of throm-bin
activity in vivo. Because the clotting cascade can be activated during the blood-sample
collection, however, precautions are required in the measurement and interpretation of
FPA levels.

Fibrinogen
The fibrinogen level in plasma can be measured either anti-genically or more commonly
by clotting assays. The results are reported in mg/dl.

D-Dimer and Fibrin-Fibrinogen Degradation Products

Fibrinogen degradation products (FDP) and fibrin-fibrinogen split products (FSP)
result from plasmin degradation of fibrino-gen and fibrin clot [see Figure 9]. D-dimer is
released by the plas-min-mediated degradation of fully polymerized fibrin. Plasmin
cleavage of fibrinogen or soluble fibrin monomer does not yield the D-dimer. Thus,
elevated D-dimer is a specific measure of in-travascular fibrin deposition and plasmin
degradation characteristic of disseminated intravascular coagulation. The D-dimer test
has largely replaced the FSP test.

Factor XIII
Factor XIII is the only clotting factor whose activity is not assessed in PT or PTT
because the end point for both tests is the formation of fibrin polymers, irrespective of
whether these polymers are cross-linked covalently by activated factor XIII. Factor XIII
deficiency may be suspected in an infant who has significant bleeding after circumcision
or, more rarely, in an adult patient who has unexplained bleeding.

Plasminogen and a2-Antiplasmin

The activation of the plasminogen-plasmin system can be inferred from the findings
of a long TT, a low plasma fibrinogen level, and an elevated D-dimer level. Another
crude test used to measure plasminogen-plasmin activation is the euglobulin lysis time.
The sensitivity and specificity of this test is not well defined, however. During extensive
thrombosis and fibrinolysis, both plasminogen and a2-antiplasmin (the physiologic
inhibitor of plasmin) are consumed. The direct measurement of plasma levels of
plasminogen and a2-antiplasmin is sometimes useful to assess the extent of fibrinolysis
and the requirement for replenishment of these plasma proteins using fresh frozen
plasma.

Tests of platelets and of platelet function

Peripheral Blood Smear Evaluation
This examination provides quick, definitive information to confirm or question a
platelet count. Normally, there are eight to 12 platelets per high-power field (1,000 x
magnification), corresponding to a normal platelet count of 150,000 to 300,000/ml. The
smear also shows platelet granularity and whether mega-thrombocytes are present.

Bleeding Time
This test primarily measures platelet function. A spring-loaded device is used to make a
standard skin incision on the forearm. A prolonged bleeding time with platelets greater
than 100,000/ml suggests impaired function. The bleeding time is difficult to
standardize, and a normal bleeding time does not predict the safety of surgical
procedures or accurately predict hem-orrhage.30 It should not be used as a general
screening test in a preoperative setting. Although once used in the screening of patients
for von Willebrand disease or certain platelet function disorders, for these purposes
bleeding time has been largely replaced by the platelet function-100 assay (PFA-100).

Platelet Function Assay-100

PFA-100 is a newly developed automated test for platelet function. Citrated whole blood
is aspirated through a capillary tube under high shear onto a membrane coated with
collagen and ep-inephrine or collagen and ADP in which a central aperture is made.
The time it takes for blood flow through the membrane to stop is denoted as closure
time and is a measure of platelet function. The closure time is prolonged in patients with
von Wille-brand disease or other platelet functional defects.31 PFA-100 should be
considered the first-line test for platelet function disorders.

Platelet Aggregometry
Platelet aggregometers are photometric devices for recording the transmission of light
through a suspension of platelets. When platelets aggregate, light passes through the
suspension more readily. To test aggregation, dilute concentrations of platelet agonists
(e.g., ADP, epinephrine, collagen, and ristocetin) are added to citrated platelet-rich
plasma. With the weak agonists, such as ADP and epinephrine, the initial primary wave
of aggregation is followed by a secondary wave. The secondary wave reflects the
induction of the platelet release reaction, in which platelet granule contents are released
to augment further platelet aggregation. A suboptimal secondary wave is seen with
platelet storage pool defects in which either platelet granule content is diminished or its
release activity is impaired. The latter is commonly associated with aspirin intake or
uremia-related thrombo-cytopathy. Patients with von Willebrand disease will have a
sub-optimal platelet aggregation response to ristocetin but a normal response to the
other agonists. Platelet aggregation testing is labor intensive and expensive and should
be performed only in clinical coagulation laboratories that do this test regularly.

Tests of inhibitors of hemostasis

Mixing Studies
A prolonged clotting time (e.g., PTT of 60 seconds [normal, 28 to 30 seconds]) can
be caused by either a clotting factor deficiency or an inhibitor. An inhibitor is generally
an antibody directed against a specific clotting factor or against a phospholipid-pro-tein
complex, the so-called lupus anticoagulant [see 5:XIV Thrombotic Disorders]. In a
mixing study, one volume of a patient’s plasma is mixed with an equal volume of normal
plasma. The resulting mixture will provide at least 50% of a deficient factor and correct
the abnormality. If the problem is caused by an inhibitor, the resulting plasma mixture
still has a prolonged clotting time. A mixing study should always be done when a
prolonged clotting time is noted.

Antithrombin III
Bioassays and immunoassays are available for assessing AT-III activity. A functional
assay is preferable to an antigenic assay.

Protein C and Protein S

Functional and immunologic methods are available. Because protein C and protein
S are vitamin K dependent, their measurement can be problematic in patients taking
warfarin. It is best to measure protein C or protein S when the patient has been off
warfarin for 3 to 4 weeks.
FIGURE 4

Figure 4 In the classic view of the clotting cascade (left), the intrinsic pathway is
initiated by the exposure of blood to a negatively charged surface (e.g., glass)
and the extrinsic pathway is activated by tissue factor or thromboplastin. In the
modified view (right), generation or exposure of tissue factor at the wound site is
the primary physiologic event that initiates clotting.
FIGURE 5

Figure 5 In the absence of heparan sulfate (HS), antithrombin III (AT-III) inhibits
thrombin slowly. When HS is present, it binds to a specific site on AT-III that
causes a conformational change in AT-III, allowing it to reach the active site of
thrombin and inhibit the enzyme instantaneously. HS also binds to a specific site
on thrombin, positioning it for optimal inhibition by AT-III.
FIGURE 6

Figure 6 The protein C/protein S pathway is complementary to the AT-III pathway.

When thrombin binds to thrombomodulin, thrombin undergoes a conformational
change and no longer clots fibrinogen or activates platelets. However, it acquires
the ability to activate protein C in plasma. Protein S serves as a cofactor for
activated protein C. Activated protein C degrades activated factors V and VIII, the
two cofactors in the clotting cascade.
FIGURE 7

Figure 7 Tissue factor pathway inhibitor (TFPI) binds to and inhibits factor Xa.
After binding to factor Xa, TFPI undergoes a conformational change. The
TFPI/factor Xa complex then mediates feedback inhibition of tissue factor/factor
VIIa.

ANTITHROMBIN III HEPARIN SULFATE SYSTEM

Antithrombin III is a circulating plasma protease inhibitor. It inhibits thrombin and factor Xa, the two key enzymes in the
clotting cascade. AT-III also inhibits activated factor XII and factor XI. In the absence of the glycosaminoglycan heparin, AT-III
inhibits thrombin and factor Xa relatively slowly (complete inhibition requires a few minutes). When present, heparin binds to a
discrete binding site on AT-III that causes a conformational change in AT-III, which then inhibits thrombin instantaneously and
irreversibly. This augmentation of the inhibition of throm-bin and factor Xa is the basis for the therapeutic use of heparin as an
anticoagulant. Heparan sulfate proteoglycans on the luminal surface of endothelial cells appear to activate AT-III in a manner
similar to that of heparin [see Figure 5].9
Figure 5 In the absence of heparan sulfate (HS), antithrombin III (AT-III) inhibits thrombin slowly. When HS is present,
it binds to a specific site on AT-III that causes a conformational change in AT-III, allowing it to reach the active site of
thrombin and inhibit the enzyme instantaneously. HS also binds to a specific site on thrombin, positioning it for optimal
inhibition by AT-III.

Thus, the endothelial surface is normally coated with a layer of AT-III that is already activated by the endogenous heparan sul-
fate. Because 1 ml of blood can be exposed to as much as 5,000 cm2 of endothelial surface, the AT-III-heparan sulfate system is
poised to rapidly inactivate any thrombin in the general circulation.

PROTEIN C AND PROTEIN S-THROMBOMODULIN SYSTEM

Thrombomodulin is an integral membrane protein found on the luminal surface of the vascular endothelium in the microcir-
culation. The binding of thrombin to thrombomodulin results in a remarkable switch in thrombin’s substrate specificities: it no
longer clots fibrinogen or activates platelets [see Figure 6]. On the other hand, it acquires the ability to activate protein C in
plasma.10 A distinct endothelial receptor for protein C has been found that enhances the activation of protein C by the thrombin-
thrombomodulin complex.11 Activated protein C degrades factor Va and factor Villa, the two cofactors responsible for the
assembly of the prothrombinase and intrinsic tenase complex in the clotting cascade. Protein S serves as a cofactor for activated
protein C. Deficiencies of AT-III, protein C, and protein S are important causes of a hypercoagulable state.
Protein C and protein S both show some structural similarity to the vitamin K-dependent clotting factors (prothrombin, factor
VII, factor IX, and factor X). Protein S circulates in two forms: a free form, in which it is active as an anticoagulant, and a bound,
inactive form, in which it is complexed to C4b-binding protein of the complement system. C4b-binding protein acts as an acute-
phase reactant. The resultant increase in inflammatory states reduces the activity of free protein S, enhancing the likelihood of
thrombosis.

TISSUE FACTOR PATHWAY INHIBITOR

Tissue factor pathway inhibitor is a circulating plasma protease inhibitor that is synthesized by the microvascular en-
dothelium. Unlike AT-III, TFPI has a very low plasma concentration. TFPI inhibits factor Xa. The TFPI/factor Xa complex
becomes an effective inhibitor of tissue factor/factor VIIa, thus mediating feedback inhibition of tissue factor/factor VIIa [see
Figure 7]. Animal studies have shown that depletion of the endogenous TFPI sensitizes the animals to the development of
disseminated intravascular coagulation induced by tissue factor or endotoxin.12
TFPI is primarily synthesized by the microvascular endotheli-um. Approximately 20% of TFPI circulates in plasma associated
with lipoproteins; the majority remains associated with the en-dothelial surface, apparently bound to the cell-surface
glycosaminoglycans. The plasma level of TFPI is greatly increased after intravenous administration of heparin. This release of
en-dothelial TFPI may contribute to the antithrombotic efficacy of heparin and low-molecular-weight heparin. Recombinant
TFPI is now in early clinical trials.

PROSTACYLIN
Upon cell perturbation, the fatty acid arachidonic acid is released from cell membrane phospholipids by phospholipase A2. The
enzyme prostaglandin endoperoxide H synthase-1 (PGHS-1) converts arachidonic acid into prostaglandin endoperoxides and
finally to thromboxane A2 (TXA2) in platelets and prostacy-clin (PGI2) in endothelial cells.

TXA2 and PGI2 have opposite functions. TXA2 is a potent stimulator of platelet aggregation and causes vasoconstriction,
whereas PGI2 inhibits platelet aggregation and induces vasodilatation. PGI2 functions by activating adenylate cyclase, which
leads to an increase in intracellular cAMP [see Figure 8].
Figure 8 Significant synergism exists between nitric oxide (NO) and prostacyclin (PGI2), leading to platelet inactivation
and vasodilatation. The enzyme prostaglandin endoperoxide H synthase-1 (PGHS-1) converts arachidonic acid into PGI2
in endothelial cells. PGI2 activates adenylate cyclase, which leads to an increase in intracellular cyclic adenosine
monophosphate (cAMP), inhibiting platelet aggregation and inducing vasodilatation. NO, formed from L-arginine,
stimulates production of cyclic guanosine monophosphate (cGMP). Cyclooxygenase-2 (COX-2) is the induced isoform of
PGHS; its formation presumably results from hemodynamic shear in the circulation. NO formation is catalyzed by NO
synthases (NOS).

Cyclooxygenase-1 and Cyclooxygenase-2

Cyclooxygenase-1 (COX-1) is the constitutive isoform of PGHS. Cyclooxygenase-2 (COX-2) is an inducible isoform of PGHS.
COX-2 is undetectable in most tissues. However, it can be rapidly induced in response to growth factors, endotoxins, and cy-
tokines in endothelial cells and monocytes (although not in platelets).14 Recent evidence indicates that endothelial COX-2 is a
major source of PGI2 under physiologic conditions in humans, perhaps because of continual COX-2 induction by hemodynam-ic
shear in the circulation.15 Aspirin acetylates and irreversibly inhibits both COX-1 and COX-2. Other nonsteroidal anti-
inflammatory drugs (NSAIDs) also inhibit COX-1 and COX-2, although not permanently. Selective COX-2 inhibitors are now
available as a new generation of NSAIDs.16
Because aspirin irreversibly inhibits COX-1 and because platelets cannot make new COX-1, brief exposure to aspirin will
permanently inhibit TXA2 production for the life span of affected platelets.

Nitric oxide
Nitric oxide (NO) is formed from L-arginine in endothelial cells. NO stimulates guanylate cyclase, leading to an increase in
cyclic guanosine monophosphate (cGMP) in target cells; causes vasodilatation; and inhibits platelet adhesion and aggregation
[see Figure 8].17 NO is rapidly destroyed by hemoglobin and thus functions as a local (i.e., paracrine) hormone. Intravenous
infusion of an arginine analogue that blocks NO production leads to an immediate and substantial rise in blood pressure. This
phenomenon suggests that NO is released continually and basally to regulate vascular tone (in contrast to the production of PGI2,
which is more stimulus-responsive). There is significant syner-gism between NO and PGI2. Formation of NO is catalyzed by NO
synthases, which exist in different isoforms in various tissues. In addition to regulating vascular events, NO has a wide range of
biologic effects (e.g., neurotransmittal function in the central nervous system).

Figure 4 In the classic view of the clotting cascade (left), the intrinsic pathway is
initiated by the exposure of blood to a negatively charged surface (e.g., glass)
and the extrinsic pathway is activated by tissue factor or thromboplastin. In the
modified view (right), generation or exposure of tissue factor at the wound site is
the primary physiologic event that initiates clotting.
FIGURE 5

Figure 5 In the absence of heparan sulfate (HS), antithrombin III (AT-III) inhibits
thrombin slowly. When HS is present, it binds to a specific site on AT-III that
causes a conformational change in AT-III, allowing it to reach the active site of
thrombin and inhibit the enzyme instantaneously. HS also binds to a specific site
on thrombin, positioning it for optimal inhibition by AT-III.
FIGURE 6

Figure 6 The protein C/protein S pathway is complementary to the AT-III pathway.

When thrombin binds to thrombomodulin, thrombin undergoes a conformational
change and no longer clots fibrinogen or activates platelets. However, it acquires
the ability to activate protein C in plasma. Protein S serves as a cofactor for
activated protein C. Activated protein C degrades activated factors V and VIII, the
two cofactors in the clotting cascade.