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
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 toCleaves Fibrin and Fibrinogen to
fibrin (ogen) degradation productsfibrin (ogen) degradation products
X, Y and D-EX, Y and D-E
Activates Intrinsic CoagulationActivates Intrinsic Coagulation
PathwayPathway
Factors XII----XIIa is amplifiedFactors XII----XIIa is amplified
indirectly by plasminindirectly by plasmin
Interferes with intrinsic andInterferes with intrinsic and
common pathwayscommon pathways
Destroys factorsDestroys factors VIII and VVIII and V
Block Thrombin conversion ofBlock Thrombin conversion of
fibrinogen to fibrinfibrinogen to fibrin
FDP interfere with thrombinFDP 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.
16. • Activation of Plasminogen can occur due to
- Intrinsic Plasminogen Activation
- Extrinsic Plasminogen Activation
- Exogenous Plasminogen Activation
- Plasminogen Activation in Secretory Ducts.
17.
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
21.
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.
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.
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.
Fibrinolysis is a basic defence mechanism of the organism designed to control the deposition of fibrin in the vascular system and elsewhere. When thrombin is generated by the coagulation system it acts on fibrinogen and 'precipitates' it out of solution as a polymer-fibrin. This polymer deposition is regulated by the fibrinolytic system
which acts on the insoluble protein fibrin and, by
splitting a limited number of peptide bonds, renders
it soluble. This solubilisation mechanism which
liquifies the fibrin clot is the fibrinolytic system.
i.e. to keep blood within blood vessels
1) Initial vasoconstriction of injured vessels , causing diminished blood flow distal to the injury
Clot is a meshwork of thin fibrils entangling the blood cells. Fibrils consist of fibrin threads. The fibrin is formed from fibrinogen
Lysis is slow because of fibrin clot stabilization by factor XIII.
Lysis of blood clot inside the blood vessel is called fibrinolysis. It helps to remove the clot from the lumen of blood vessel. This process requires a substance called plasmin or fibrinolysin.
Plasminogen normally circulates in the plasma to its active form called, Plasmin.
Activator – t PA , lysosomal enzyme and thrombin
Plasminogen and plasminogen activator bind selectively to fibrin and on the fibrin surface plasmin is formed which immediately interacts with the fibrin. This interaction occupies the active sites of the plasmin and renders them unavailable for interaction and neutralization by the anti-plasmin in the surrounding plasma.
Plasmin formed away from the fibrin surface immediately interacts with the fast anti-plasmin a2-plasmin inhibitor (a2-PI) and is inhibited.
As a result, in addition to fibrinolysis, Plg and Pn are involved in a number of other physiologic and pathophysiologic processes such as cell migration, wound healing, inflammation, embryogenesis, ovulation, angiogenesis, tumor growth and metastasis, and atherosclerosis
Bond cleaved by all plasminogen activators to give the two chain plasmin molecule. Solid triangle indicates the serine residues of the active site. The two chain plasmin is held together by a disulphide bridge
Kringle domains are common protein motifs of about 100 aa residues in length, that have a specific covalent structure defined by a pattern of 3 sulphide bonds
D-dimer is a protein that represents an area of fibrin which contain cross-linked regions used as clot stabilizers.
D-dimer present in circulation is used as an indicator of a blood clot being formed and broken down somewhere in the body