Hemostatsis (Fibrinolysis - Plasminogen)

Blood Physiology

1. Fibrinolysis ,
Activation of
plasminogen and
Inhibitors of Plasmin
Pradeep Singh, Shalu
Singh
M.Sc. Medical
Biochemistry
HIMSR, Jamia Hamdard

2. Hemostasis
 Hemostasis is the cessation of bleeding from a cut.
 When a blood vessel is injured, the injury initiates a series of
reactions resulting in hemostasis.
 It occurs in following stages :-
1.Vasoconstriction
2.Platelet Plug Formation
3.Coagulation of Blood
4.Fibrinolysis

4. Von Willebrand factor

6. Fibrinolysis
•Process of removing fibrin from the vasculature.
•After a clot or thrombus is formed, it is normally dissolved
by the fibrinolytic system.
•In 1937 MaCfarlane reported that damage tissues release
a substance (activator that activates the inert precursor
called Plasminogen).

7. Plasmin
•The central enzyme in fibrin lysis is plasmin (Pn), a serine
protease derived from its inactive precursor, plasminogen
(Plg), by the action of activators.
•Plasmin is a non-specific Proteolytic enzyme capable of
degrading fibrin as well as fibrinogen and factors V & VIII.
•Any small amount of plasmin that is formed in fluid phase
under physiologic conditions are rapidly inactivated by the
fast acting plasmin inhibitor, α2 - antiplasmin

8. •Pn also cleaves a variety of other substrates including extracellular
matrix proteins, and activates other proteases and growth factors.

9. Plasmin's RolesPlasmin's Roles CommentsComments
Activates FibrinolysisActivates Fibrinolysis Cleaves Fibrin and Fibrinogen to Cleaves Fibrin and Fibrinogen to
fibrin (ogen) degradation products fibrin (ogen) degradation products
X, Y and D-E X, Y and D-E
Activates Intrinsic Coagulation Activates Intrinsic Coagulation
PathwayPathway
Factors XII----XIIa is amplified Factors XII----XIIa is amplified
indirectly by plasmin indirectly by plasmin
Interferes with intrinsic and Interferes with intrinsic and
common pathways common pathways
Destroys factors Destroys factors VIII and VVIII and V
Block Thrombin conversion of Block Thrombin conversion of
fibrinogen to fibrinfibrinogen to fibrin
FDP interfere with thrombin FDP interfere with thrombin
influence on fibrinogeninfluence on fibrinogen

10. Activation of Plasminogen
•Activators of plasminogen cleave at Arg – Val bond in
plasminogen to produce two chain serine protease ,
plasmin .
• The specificity of plasmin for fibrin is another mechanism
to regulate fibrinolysis.

11. •Via one of its kringle domains , Plasmin (ogen) specifically
binds to lysine residues on fibrin and so is increasingly
incorporated into the fibrin mesh as it cleaves it.

12. Sequence of events involved in
the activation of Plasminogen
•During intravascular clotting , the endothelium of the
blood vessels secrete a thrombin binding protein ,
thrombomodulin .It is secreted by endothelium of all blood
vessels except minute vessels of the brain .
•Thrombomodulin combines with thrombin and forms a
thrombomodulin – thrombin complex.
•Thrombomodulin – thrombin complex activates Protein C

13. •Activated Protein C inactivates factor V and factor VIII in
the presence of a cofactor protein S.
•Protein C also inactivates the t-PA inhibitor.
•Now the t-PA becomes active.
•Activated t-PA and lysosomal enzymes activate
plasminogen to plasmin. Plasminogen is also activated by
thrombin and u – PA .

14. •When Plasminogen is activated it unfolds to expose its potent
enzymatic domains.

15. Flow chart:

16. •Activation of Plasminogen can occur due to
- Intrinsic Plasminogen Activation
- Extrinsic Plasminogen Activation
- Exogenous Plasminogen Activation
- Plasminogen Activation in Secretory Ducts.

18. FIBRIN(OGEN) DEGRADATION by
PLASMIN
•In the process of fibrinogen or fibrin degradation by
plasmin within a clot, specific molecular fragments are
produced called Fibrin (ogen) Degradation Products (FDP)
or Fibrin (ogen) Split Products (FSP)
•These degradation products are removed by the
reticuloendothelial system and other organs.
•The sequence of reaction in the degradation of
Fibrin(ogen) by plasmin are X, Y, D (D-D dimer) and E.

19. •Fragment X and Y are referred to as early degradation
products
•Fragment D and E are late degradation products
•Fragment X is the first and the largest fragment formed
(Mwt 250,000 D)
•Fragment X is the results of Plasmin (P) cleavage of the
terminal portion of the alpha (α) chains from a fibrin
polymer
•Fragment X is cleaved by Plasmin (P) to form two fragments
called Y (YY) and an intermediate complex (DXD)

20. •This complex is further cleaved into intermediate
complexes DED and DY/DY until finally, fragment E
and D (D-D dimer) are formed.
•A single fragment D has Mwt 90,000 D and that the D-
D dimer is 180,000 D
•Presence of D-D dimer is a specific indication of in
vivo fibrinolysis, namely, intravascular thrombin
formation leading to fibrin formation and its
subsequent degradation

22. Pathologic Effect of FDPs
The FDPs are significant because of their haemostatic effects, which
include;
•Anti-thrombin activity
•Interference with polymerization of fibrin monomer
•Interference with platelet activity

23. •The early and large fragments (X and Y) along with the
intermediate FDPs, are important in exerting anticoagulant
Effect
• Fragment Y and D inhibit fibrin polymerization
• Fragment E is a powerful inhibitor of thrombin
•All four fragments, but particularly low molecular weight FDP,
have an affinity coating platelet membrane and therefore,
cause a clinically significant platelet dysfunction by inhibiting
aggregation.

24. PLASMINOGEN
ACTIVATORS

25. •The activators of plasminogen (Plg) are the serine
proteases t-PA or u-PA and baсterial proteins that
acquire proteolytic activity after the interaction with
human Plg or Pn, streptokinase (SK), and
staphylokinase (SAK).
•t-PA and SAK are fibrin-selective, remaining bound to
fibrin and protected from rapid inhibition, while SK and
two-chain u-PA are non-fibrin-selective enzymes,
activating both Plg in the circulating blood and fibrin-
bound Plg.

26. Tissue-type Plg activator
•Tissue-type Plg activator (t-PA) is a 70-kDa glycoprotein
with a circulating level in plasma of about 5 ng/mL .
• It consists of finger, epidermal growth factor-like, two
kringle, and C-terminal trypsin-like catalytic domains.
• The single-chain t-PA (sct-PA) can be converted to the
two-chain form (tct-PA) by Pn-catalyzed cleavage, but
the single-chain form itself exhibits considerable catalytic
activity.

27. •Both sct-PA and tct-PA act by forming a ternary complex with fibrin
and Plg, undergoing conformational changes, and catalyze the
conversion of Plg to active Pn by cleaving the Arg561-Val562 bond.

28. Urokinase-type Plg activator
•Urokinase-type Plg activator (u-PA) is a 53-kDa glycoprotein, with
concentrations in plasma of 2–4 ng/mL , and it is also found in urine.
• The single-chain inactive form (pro-u-PA or scu-PA) is converted to a
two-chain active enzyme (tcu-PA) by Pn and other proteases.

29. • tcu-PA activates both circulating and fibrin-bound Plg by cleaving the
Arg561-Val562 bond with a similar rate .
•u-PA consists of an amino-terminal growth factor domain, a kringle
domain, and a C-terminal serine protease domain

30. Streptokinase
•Streptokinase (SK) is a 47-kDa protein produced by
various strains of β-hemolytic Streptococci.
•Unlike u-PA and t-PA, which possess proteolytic activity
themselves, SK is not an enzyme but acquires the ability
to activate human Plg indirectly by forming a 1 : 1
complex with Plg or Pn, in which the zymogen catalytic
site is activated non-proteolytically by an intramolecular
cleavage of the Arg560-Val561 bond.

31. INHIBITORS

32. 1.Alpha-2- Anti-plasmin (α2 anti-plasmin)
•An (α2) glyco-protein
• Most important naturally occurring inhibitor
•The principle inhibitors of fibrinolysis by binding with
plasmin that is free in the plasma (neutralizing plasmin)
•Inhibits the clot-promoting activities of plasma kallikrein
•Inhibits the serine proteases Xlla, XIa, IIa and Xa
•Hereditary deficiencies have been associated with,
Excessive clotting (DIC)
Excessive fibrinolysis

33. 2. Alpha-2 Macroglobulin
•Large naturally occurring plasma GP
•Inhibits component in both the fibrinolysis and coagulation
systems
•Inhibits plasmin after alpha 2 anti-plasmin depletion
3. Alpha-1 Antitrypsin
•The third most important naturally occurring inhibitor of
fibrinolytic system. Inactivates plasmin slowly and does not bind
plasmin until both alpha2 anti-plasmin and alpha 2 macroglobulin
are saturated
•Inhibits coagulation by its potent inhibitory effects on factor XIa

34. Other Inhibitors:
•Anti-thrombin III, inhibits fibrinolysis by inhibiting plasmin and
kallikrein
•The C1 inactivator also inhibits plasmin.

36. THANK YOU