2.
Plasmin
Fibrinolysis
Besides having a system for clot formation, the body also has a means by
which the fibrin clots may be removed and the flow of blood reestablished.
As soon as the clotting process has begun, fibrinolysis is initiated to break
down the fibrin clot that is formed. Normally, the fibrinolytic system functions
to keep the vascular system free of fibrin clots or deposited fibrin. Evidence
indicates that the fibrinolytic system and the coagulation system are in
equilibrium in normal persons. As a general rule, fibrinolysis is increased
whenever coagulation is increased.
The active enzyme that is responsible for digesting fibrin or fibrinogen is
plasmin. Plasmin is not normally found in the circulating blood but is present
in an inactive form, plasminogen. Plasminogen is converted to plasmin by
certain proteolytic enzymes. These are the plasminogen activators which
are found in small amounts in most body tissues, in very low amounts in
most body fluids, and in urine. The decomposition products of fibrin and
fibrinogen, called fibrin degradation products(FDPs) or fibrin split
products FSPs), are formed during fibrinolysis and are removed from the
blood by the mononuclear phagocytic system. As breakdown products of
fibrin, D-dimers result only when fibrin that has been stabilized by FXI
crosslinking has been digested.
4 Components Fibrinolytic System
Synthetic recombinant TPAs, which mimic natural TPA, are a family of
1. Plasminogen
“clot-busting” drugs used to dissolve pathologic clots that form in
venous and arterial thrombotic disease.
● 92,000 Daltons; from the liver; a single chain protein with 5
glycosylated loops called kringles.
● Kringles enable plasminogen, along with activators TPA and UPA,
to bind the lysine moieties on the fibrin molecule during the
polymerization process. This fibrin-binding step is essential to
fibrinolysis.
● Fibrin-bound plasminogen is converted into a two-chain active
plasmin molecule when cleaved between arginine at position 561
and valine at position 562 by neighboring fibrin-bound TPA or UPA.
● is a serine protease that systematically digests fibrin polymer by the
hydrolysis of arginine-related and lysine –related peptide bonds. As
fibrin becomes digested, the exposed carboxy-terminal lysine
residues bind additional plasminogen and TPA, which further
accelerates clot digestion. Also, FXa, FXIIa, and kallikrein produce
an alternative mechanism of plasminogen activation.
● Bound plasmin digests clot and restores blood vessel patency
(openness). Its localization to fibrin through lysine binding prevents
systemic activity. However, free plasmin can be found in the
circulation and is capable of digesting Factors 1, V, VIII, and
fibronectin. Plasma alpha2- antiplasmin rapidly binds and inactivates
any free plasmin.
3. Plasminogen Activators:
A. Endogenous
a. Tissue Plasminogen Activator (TPA)- secreted by the
endothelial cells , hydrolyze fibrin- bound plasminogen,
converting it to plasmin. . It has 2 glycosylated kringle
regions, forms covalent lysine bonds with fibrin during
polymerization and localizes at the surface of the thrombus
with plasminogen to convert the latter to plasmin. Circulating
TPA is bound to inhibitors such as PAI-1. These complexes
are cleared from the circulation.
b. Urokinase Plasminogen Activator (UPA) - secreted by
urinary tract epithelial cells, monocytes, and macrophages.
Circulates in the plasma at a concentration of 2-4ng/mL and
becomes incorporated into the mix of fibrin-bound
plasminogen and TPA at the time of thrombus formation.
UPA converts plasminogen to plasmin. Because it has only
one ( 1) kringle region, UPA does not bind firmly to fibrin and
has a relatively minor physiologic effect. Like TPA, purified
UPA preparations called urokinase are used therapeutically
to dissolve thrombi in patients with myocardial infarction (MI),
stroke and deep vein thrombosis (DVT).
B. Exogenous
 a. Streptokinase
b. acyl- plasminogen streptokinase activator complex
(APSAC) - inactive complex of human plasminogen and
streptokinase. When this is injected, a controlled
deacylation( removal of acyl group) of the catalytic center
occurs, activating the complex so that thrombolysis may
begin.
TPA,purified UPA preparations called urokinases are used therapeutically to
dissolve thrombi in patients with myocardial infarction, stroke, and deep vein
thrombosis.
4. Inhibitors of Fibrinolysis
● regulates fibrinolysis so that overreaction or under reaction is
avoided
a. Plasminogen Activator Inhibitor-1 – the principal inhibitor of
plasminogen activation, inactivating both TPA and UPA, thus
preventing them from converting plasminogen to plasmin.Produced
by endothelial cells, megakaryocytes, smooth muscle cells,
fibroblasts, monocytes, adipocytes, hepatocytes, and other cell
types. Platelets store more than 50% of the total PAI-1. Present in
excess of the TPA concentration in plasma, and circulating TPA
normally becomes bound to PAI-1. Only at times of EC activation,
such as after trauma, does the level of TPA secretion exceed that of
PAI-1 to initiate fibrinolysis. Binding of TPA to fibrin protects TPA
from PAI-1 inhibition. Its deficiency is associated with chronic
bleeding caused by increased fibrinolysis. It is an acute phase
reactant and is increased in many conditions, including metabolic
syndrome, obesity, atherosclerosis, sepsis, and stroke. Increased
levels correlate with reduced fibrinolysis and increased risk of
thrombosis.
b. Alpha-2 antiplasmin - synthesized in the liver and is the primary
inhibitor of free plasmin. It is a SERPIN. Free plasmin produced by
activation of plasminogen can bind either to fibrin, where it is
protected by AP( alpha-2 antiplasmin). Therapeutic lysine analogs,
tranexamic acid and e-aminocaprioc acid, are similarly antifibrinolytic
through their affinity for kringles in plasminogen and TPA. Both inhibit
the proteolytic activity of plasmin, thereby reducing clinical bleeding
caused by excess fibrinolysis.
c. Thrombin Activatable Fibrinolysis Inhibitor (TAFI) - a plasma
procarboxypeptidase produced by the liver that becomes activated
by the thrombin-thrombomodulin complex. This is the same complex
that activates the Protein C pathway; however, the functions are
independent. Activated TAFI functions as an antifibrinolytic enzyme.
i. It inhibits fibrinolysis by cleaving carboxy-terminal lysine
residues from partially degraded fibrin, thereby preventing
the binding of TPA and plasminogen to fibrin and blocking
the formation of plasmin. In coagulation factor –deficient
states, such as hemophilia, decreased thrombin production
may reduce the activation of TAFI. The resulting decreased
fibrinolysis may contribute further to thrombosis. It may also
play a role in regulating inflammation and wound healing.
d. Alpha2 macroglobulin - inactivates plasmin not inhibited by alpha2
antiplasmin
e. Thrombomodulin - released by platelets, inhibits activation of
fibrin-bound plasminogen
FDPs/ FSPs
● Plasmin cleaves fibrin (and fibrinogen) producing a series of
identifiable fibrin fragments: X, Y,D,E and D-D. Several of these
fragments inhibit hemostasis and contribute to hemorrhage by
preventing platelet activation and by hindering fibrin polymerization.
FDPs are antithrombin so that excessive amounts can cause
bleeding.
● Fragment X is described as the central E domain with the 2 D
fragments. (D-E-D), minus some peptides cleaved by plasmin.
Fragment Y is the E domain after cleavage of one D domain (D-E).
Eventually these fragments are further digested to individual D and
E domains.
● The D-D fragment, called D-dimer, is composed of two D domains
from separate fibrin molecules ( not fibrinogen) cross-linked by the
action of FXIIIa. Fragments X,Y,D, and E are produced by digestion
of either fibrin or fibrinogen by plasmin, but D-dimer is a specific
product of digestion of cross-linked fibrin only and is therefore a
marker of thrombosis and fibrinolysis.
D- dimer
D- dimer is present only when the following 3 processes had occurred:
1. Thrombin generation
2. Polymerization of fibrin clot by FXIIIa
3. Fibrinolysis of the clot by plasmin
Assessing D-dimer levels is an important diagnostic tool to identify DIC and
to rule out venous thromboembolism and pulmonary embolism.
 DEGRADATION OF CROSS-LINKED FIBRIN BY PLASMIN
Another way of classifying Plasminogen activators

Intrinsic activators Extrinsic activators Exogenous

activators present in activators found in activators found

the blood (Plasma) body tissues outside the physiologic
milieu

1. Factor XIIa 1. Tissue 1. Urokinase –

2. Kallikrein plasminogen Enzyme
3. HMWK activators present in
2. Urokinase urine, blood,
plasminogen extracellular
activators matrix
2. Streptokinase
– enzyme
produced by
certain
streptococcal
bacteria and
converts
plasminogen
to plasmin

DEGRADATION OF FIBRINOGEN AND NON-CROSS- LINKED FIBRIN

BY PLASMIN

Lab. Tests for Fibrinolysis

1. D-dimer immunoassay

● The presence of FDPs, which signifies fibrinolysis , means there was

thrombosis. Normally, FDPs, including D- dimer, circulate at a
concentration of less than 2 ng/mL. Fibrinolysis generates FDP and
D-dimer at concentrations greater than 200 ng/mL. More than this
level indicates acute and chronic DIC, systemic fibrinolysis, deep
vein thrombosis, and pulmonary embolism, and may predict stroke.
FDPs , including D-dimer, are also detected in plasma after
thrombolytic therapy.

2. Quantitative D-dimer assay- results are within 30 minutes
Method:
● Microlatex particles in buffered saline are coated with monoclonal
anti- D- dimer antibodies + Patient’s plasma (D-dimer) ---
turbidimetry (using turbidimetry or nephelometric technology)
● Most methods detect concentrations as low as 10 ng/mL
● Results may be reported in D-dimer units (DDU) or fibrinogen
equivalent units (FEU)
3. Fibrin Degradation Product Immunoassay
● Had been replaced by automated D- dimer assay.
Method:
a. Thrombo-Wellcotest (Thermo-Fisher)- slide agglutination test.
● 1 gtt patient’s serum + 1 gtt latex suspension (polystyrene
latex particles in saline are coated with polyclonal antibodies
specific for D and E fragments calibrated to detect FDPs at
a concentration of 2 ug/mL or greater by the appearance of
visible agglutination.
● Performed in urine/ serum collected in special tubes that
promote clotting and prevent in vitro fibrinolysis.
● Owing to inadequate sensitivity at low concentrations,
qualitative FDP assays are confined to diagnosis and
monitoring of DIC.
4. Plasminogen assay
● When bound to fibrin, plasminogen is converted to plasmin by the
action of nearby TPA. Bound plasmin degrades fibrin, whereas
circulating
● Inhibitor, alpha2- antiplasmin, rapidly inactivates free plasmin.
● Excessive fibrinolysis occurs in trauma and inflammation, reflected
in a radical increase in circulating plasmin that may be associated
with hemorrhage. Bone trauma, fractures, and surgical dissection of
bone, as in cardiac surgery, also increase fibrinolysis.
● Hyperfibrinolysis may be documented using a global assay such as
ROTEM or TEG (Thromboelastograph)
● Hypofibrinolysis occurs when TPA or plasminogen levels become
depleted or when excess secretion of plasminogen activator
inhibitor-1 (PAI-1) suppresses TPA activity.
● Congenital plasminogen deficiencies B are associated with
thrombosis in some families.
● Acquired plasminogen deficiencies are seen in DIC and acute
promyelocytic leukemia.
● Thrombolytic therapy is ineffective when plasminogen activity is low.
5. Plasminogen Chromogenic Substrate Assay
● Principle: Mimics the natural process, where plasminogen is
converted to plasmin which cleaves its substrate fibrinogen..
Streptokinase is the plasminogen activator. To detect plasmin,its
natural substrate fibrinogen is mirrored in the synthetic chromogenic
substrate S- 2251, composed of H-D-Val- Leu- Lys- pNA.
Streptokinase
● It is added to patients' plasma, where it binds and activates to form
plasmin. The resulting streptokinase-plasmin complex hydrolyzes a
bond in the S-2251 valine-leucine-lysine ( Val-Leu-Lys) sequence.
pNA which is covalently bound to the carboxyl terminus of the
oligopeptide substrate is released on digestion producing a color.
Color intensity is proportional to the plasminogen concentration.
Results of patient specimens and controls read from a calibration
curve are recorded.
● Reference Interval of plasminogen: 5-13.5mg/L.
● Decreased in : thrombolytic therapy, DIC, hepatitis, and cancer, or
may be hereditary. Decreased levels is associated with thrombosis
● Increased: systemic fibrinolysis, acute inflammation, and during
pregnancy, and hemorrhage.
Tissue Plasminogen Activator Assays
● The 2 physiologic human plasminogen activators are: TPA and
urokinase.
● TPA: synthesized in vascular endothelial cells and released into the
circulation
● Half –life – 3 minutes
● Plasma concentration on the average - 5ng/mL
● Urokinase: produced in the kidney and vascular endothelial cells
● Half-life- 7 minutes
● Plasma concentration - 2-4ng/mL
● Both activators are serine proteases that form ternary complexes(
composed of 3 proteins bound together) with bound plasminogen at
the surface of fibrin, activating plasminogen to form plasmin and
initiating thrombus degradation.
● The endothelial- secreted PAI-1 covalently inactivates both.

Specimen Collection for the TPA Assay
● TPA activity exhibits diurnal variation (peak in the morning and
lessens during night time) and rises upon exercise.
● It is unstable in vitro because it rapidly binds PAI-1 after collection.
Precautions in blood collection:
1. Patients should be at rest
2. tourniquet application should be minimal
3. The phlebotomist should record the collection time, and immediate
acidification of the specimen in acetate buffer is necessary( using
Biopool Stability acidified citrate tube. Supernatant plasma may be
frozen at -70 C until the assay is performed.
TPA ASSAY
Principle: EIA
● To measure TPA activity, a specified concentration of reagent
plasminogen is added to the patient plasma.Plasma TPA activates
the plasminogen, and the resultant plasmin activity is measured
using a chromogenic substrate. The resulting color intensity is
proportional to TPA activity. The system may incorporate soluble
fibrin to increase TPA activity.
Clinical significance of TPA
● TPA is the primary mediator of fibrinolysis and is the model for
therapeutic, synthetic TPA ( Activae, alteplase).
● Decreased TPA levels may correlate with increased risk of
myocardial infarction (MI), stroke, or deep vein thrombosis (DVT).
● Impaired fibrinolysis in the form of TPA deficiency or PAI-1 excess is
also associated with DVT and MI.
Reference interval:
● upper limit for TPA activity: 1.1 units/ mL
● upper limit for TPA antigen concentration- 14ng/mL
PAI-1 Assay
● PAI-1- produced by endothelial cells and hepatocytes and circulates
in plasma bound to vitronectin at an average concentration of
10ng/mL with diurnal variations.
● An inactive form of PAI-1 is stored in high concentrations in
platelets.
● PAI-1 inactivates TPA by covalent binding.
● Elevated PAI-1: venous thrombosis, cardiovascular risk factor,
marker of senescence
● PAI-1 deficiency:
● Hemorrhage which occurs only when homozygous or compound
heterozygous SERPINE 1 mutation is detected.
● Principle: Blood is collected from patients at rest into an acidified
citrate tube( Stabilyte) and centrifuged immediately to make platelet
poor plasma , to avoid in vitro release of platelet PAI_1
● Immunologic substrate- estimation of PAI-1 antigen
● Chromogenic substrate-PAI-1 activity
● One enzyme immunoassay for functional PAI-1 uses urokinase to
bind PAI-1. The urokinase-PAI-1 complex is immobilized with
solid-phase monoclonal anti urokinase immunoglobulin as detecting
antibody.
Traditional Methods for Fibrinolysis
1. Whole Blood Clot Lysis Time
2. Euglobulin Clot Lysis Time
○ Euglobulin fraction of the plasma contains fibrinogen,
plasminogen, and all plasminogen activators but only traces
of antiplasmins.
3. Diluted Blood Clot Lysis Time
4. Diluted Plasma Clot Lysis Time
5. Quantitative Assay of Fibrin- Fibrinogen Degradation Products
6. Protamine Sulfate Turbidity Test
7. Latex Bead Agglutination
8. D-dimer test
○ Principle: A monoclonal antibody reagent directed against a
unique neoantigen of covalently crosslinked D fragments
resulting from fibrinolysis
○ This test is superior in sensitivity & specificity that that of
conventional FDP assay ( sensitive to as little as 20 ng/mL,
as compared with 10u/mL for conventional latex FDP).
D-dimer is positive in early DIC. This is specific for
cross-linked D-dimer fragments of thrombin.
9. Prothrombin Fragment 1.2 (F1.2)- sensitive biologic marker of
thrombin generation and FXa activity because generation of F1.2
precedes thrombus formation.
○ Elevated levels are seen in: Predisposition to thrombotic
risk( cancer, heart disease, orthopedic surgery)
10. Tanned red cell hemagglutination inhibition immunoassay -
positive is w/out agglutination. Principle: FDP present in pt’s serum
 neutralizes anti fibrinogen antiserum, thereby preventing the
antiserum from agglutinating fibrinogen-coated erythrocytes.
Global Coagulation Assay
2. Red cell morphology (in stained blood smear) - DIC- with normal
morphology, in primary fibrinolysis- fragmentocytes, schistocytes are
common because the fibrin clots in the blood vessels destroy S
RBCs as they pass through them.

Review Questions
1. Thromboelastography (TEG) and it's more recent modification,
rotational thromboelastometry (ROTEM) have been original assays
to assess the hemostatic status of patients. They are global
Chapter 35
whole-blood analyzers that measure clotting time and the dynamics
of clot formation and dissolution as affected by the kinetics of
thrombin generation, clot strength, clot stability, and inhibitory effects 1. What intimal cell synthesizes and stores von Willebrand factor
on any aspect. These manual coagulometers are used mainly in (VWF)?
liver surgery, cardiac, and trauma. a. Smooth muscle cell
2. Thrombin Generation Assays - Calibrated Automated b. Endothelial cell
Thrombogram (CAT) and the TechnothrombinTGA- measure the c. Fibroblast
phased kinetics of thrombin generation from initiation and peak d. Platelet
generation through down regulation and inactivation. 2. What subendothelial structural protein triggers coagulation
a. Thrombin generation Assays help to better categorize the through activation of factor VII?
physiologic mechanisms of hemostasis and to better assess a. Thrombomodulin
bleeding risk, thrombotic risk, and their modification by b. Nitric oxide
hemostatic or antithrombotic agents. c. Tissue factor
d. Thrombin
3. What coagulation plasma protein should be assayed when
Genetic Testing for Hemostatic Disorders
platelets fail to aggregate properly?
a. Factor VIII
1. High- throughput genetic sequencing (HTS) - the reference b. Fibrinogen
method to identify the genetic variants that underlie platelet-related c. Thrombin
bleeding disorders. d. Factor X
a. whole –genome sequencing 4. What is the primary role of vitamin K for the prothrombin group
b. whole-exome sequencing factors?
c. gene-targeted methods a. Provides a surface on which the proteolytic reactions of
the factors occur
b. Protects them from inappropriate activation by compounds
Important Notes
such as thrombin
c. Accelerates the binding of the serine proteases and their
DIC and primary fibrinolysis have similar clinical manifestations and cofactors
laboratory profiles. Both have: d. Carboxylates the factors to allow calcium binding
1. Prolonged PT, APTT, and PT results 5. What is the source of prothrombin fragment F1.2?
2. Lower than normal fibrinogen levels a. Plasmin proteolysis of fibrin polymer
3. High levels of FSPs/FDPs b. Thrombin proteolysis of fibrinogen
c. Proteolysis of prothrombin by factor Xa
2 tests which can differentiate them: d. Plasmin proteolysis of cross-linked fibrin
1. Platelet count - low in DIC, within reference interval in primary 6. What serine protease forms a complex with factor VIIIa, and
fibrinolysis what is the substrate of this complex?
a. Factor VIIa, factor X
 b. Factor Va, prothrombin
c. Factor Xa, prothrombin 1. What is the prevalence of venous thrombosis in the United
d. Factor IXa, factor X States?
7. What protein secreted by endothelial cells activates a. 0.01
fibrinolysis? b. 1 in 1000
a. Plasminogen c. 10% to 15%
b. TPA d. 500,000 cases per year
c. PAI-1 2. What is thrombophilia?
d. TAFI a. Predisposition to thrombosis secondary to a
8. What two regulatory proteins form a complex that digests congenital or acquired disorder
activated factors V and VIII? b. Inappropriate triggering of the plasma coagulation system
a. TFPI and Xa c. A condition in which clots form uncontrollably
b. Antithrombin and protein C d. Inadequate fibrinolysis
c. APC and protein S 3. What acquired thrombosis risk factor is assessed in the
d. Thrombomodulin and plasmin hemostasis laboratory?
9. What are the primary roles of VWF? a. Smoking
a. Inhibit excess coagulation and activate protein C b. Immobilization
b. Activate plasmin and promote lysis of fibrinogen c. Obesity
c. Mediate platelet adhesion and serve as a carrier d. Lupus anticoagulant
molecule for factor VIII 4. Trousseau syndrome, a low-grade chronic DIC, is often
d. Mediate platelet aggregation via the GP IIb/IIIa receptor associated with what type of disorder?
10. Most coagulation factors are synthesized in: a. Renal disease
a. The liver b. Hepatic disease
b. Monocytes c. Adenocarcinoma
c. Endothelial cells d. Chronic inflammation
d. Megakaryocytes 5. What is the most common heritable thrombosis risk factor in
11. The events involved in secondary hemostasis: Caucasians?
a. Lead to the formation of a stable fibrin clot a. APC resistance (factor V Leiden mutation)
b. Usually occur independently of primary hemostasis b. Prothrombin G20210A mutation
c. Occur in a random fashion c. Antithrombin deficiency
d. Are the first line of defense against blood loss d. Protein S deficiency
12. Which of the following coagulation factors is activated by 6. In most LAC profiles, what test is primary (performed
thrombin and mediates the stabilization of the fibrin clot? first)because it detects LAC with the least interference?
a. Tissue factor a. Low-phospholipid PTT
b. Factor VII b. DRVVT
c. Factor IX c. KCT
d. Factor XIII d. PT
13. Which of the following endogenous plasma inhibitors is (are) 7. A patient with venous thrombosis is tested for protein S
important for the control of excessive thrombin generation? deficiency. The protein S activity, antigen, and free antigen are
a. AT, TFPI all less than 65%, and the C4bBP level is normal. What type of
b. Platelet factor 4 deficiency is likely?
c. TAT, F1.2 a. Type I
d. a and b b. Type II
c. Type III
d. No deficiency is indicated, because the reference range
Chapter 39
includes 65%
8. An elevated level of what fibrinolytic system assay is
associated with arterial thrombotic risk?
a. PAI-1
b. TPA
c. Factor VIIa
d. Factor XII
9. How does lipoprotein (a) cause thrombosis?
a. It causes elevated factor VIII levels
b. It coats the endothelial lining of arteries
c. It substitutes for plasminogen or TPA in the forming
clot
d. It contributes additional phospholipid in vivo for
formation of the tenase complex
10. What test may be used to confirm the presence of LAC?
a. PT
b. Bethesda titer
c. Antinuclear antibody
d. DRVVT using high-phospholipid reagent
11. What molecular test may be used to confirm APC resistance?
a. Prothrombin G20210A
b. MTHFR 1298
c. MTHFR 677
d. Factor V Leiden
12. What therapeutic agent may occasionally cause DIC?
a. Factor VIII
b. Factor VIIa
c. Antithrombin concentrate
d. Activated prothrombin complex concentrate
13. Which is not a fibrinolysis control protein?
a. Thrombin-activatable fibrinolysis inhibitor
b. Plasminogen activator inhibitor-1
c. a2-Antiplasmin
d. D-dimer
14. What is the most important application of the quantitative
D-dimer test?
a. Diagnose primary fibrinolysis
b. Diagnose liver and renal disease
c. Rule out deep venous thrombosis
d. Diagnose acute myocardial infarction
15. What is the first laboratory assay necessary to detect heparin
induced thrombocytopenia?
a. Platelet count
b. H:PF4 immunoassay
c. Quick turnaround test
d. Serotonin release assay