Haemostasis — Clotting & Thrombolysis

Haemostasis Pathway

• collective term for the mechanisms that stop blood loss
• balance of pro-coagulant + anticoagulant systems
  • procoagulants system: promotes coagulation → bioamplification system involving activation of clotting cascade
  • anticoagulant system → regulates or inhibits coagulation

3 main components involved in haemostasis (Virchows triad)

• platelets
• endothelium
  • when damaged → rapidly initiates haemostatic response
  • normal function = prevent haemostasis + promote blood flow
    • inhibition of platelet adhesion:
      • NO + prostacyclin (PGI2)
      • production of adenosine diphosphate (degrades ADP)
    • anticoagulant effects:
      • due to 2 endothelial membrane bound proteins:
      • heparin sulphate (activates ATIII → inactivates thrombin + FXa)
      • thrombomodulin: directly binds thrombin + activates protein C (inactivates FVa + VIIIa)
    • Fibrinolytic effects
      • Secretes tissue plasminogen activator (t-PA) → cleaves proenzyme plasminogen → form plasmin → degrades fibrin clots from endothelial cell surface (fibrinolysis)
• coagulation proteins

Steps involved in haemostasis

Initiation of haemostasis.

Damaged vessel → plasma exposed to:

• Von willebrand factor (vWF): binds platelets to sub-endothelial collagen fibres
• Collagen fibres: platelets bind to collagen + become activated
• Tissue factor (TF): activates plasma coagulation proteins through extrinsic pathway → thrombin

Clot formation

3 key steps:

• Vasoconstriction
  • ↓ blood flow → ↓ platelet plug washed away + ↓ blood loss
• Platelet aggregation
  • Adhesion: vessel damage exposes TF, collagen, vWF → platelet GPIb-V-IX binds to subendothelial collagen via vWF
  • Activation: metabolic process; adhesion triggers GPIb/IIIa activation → irreversible binding to matrix ligands; change shape + activation. Activation results in:
    • Exocytosis of granules: contain: 5-HT, TXA2, ADP, PAF, vWF, fibrinogen, thrombin, Ca2+, PDGF
      • Dense granules: release ADP, adrenaline, 5-HT → reinforce platelet activation
      • α granules: release fibrinogen, β thromboglobulin, PAF-4, FV, vWF, PDGF, thrombospondin → mediate + reinforce platelet aggregation + adhesion
    • Activation of phospholipase A2 to form TXA2
    • Deformation from disc to sphere with long projections
    • Promotion of coagulation cascade
    • Platelet contraction (with clot contraction)
  • Aggregation: activated GPIIb/IIIa mediated aggregation via fibrinogen + vWF
  • Haemostatic plug: plug of degranulated platelets, fibrin mesh, leukocytes, entrapped RBCs
• Coagulation
  • coagulation cascade = biological amplification system involving plasma proteins → formation of thrombin
  • Classical model: intrinsic + extrinsic → common pathway
    • reflects in vitro lab tests but not in vivo haemostasis
    • Exrinsic pathway: activated by TF → FVII → FIIa → activates FX → start of common pathway
    • Intrinsic pathway: activated by contact with -vely charged substances e.g. subendothelial collagen
    • Final common pathway: FXa → converts prothrombin (FII) to thrombin (FIIa) → thrombin
  • New model: cell based
    • Better represents in vivo mechanism of coagulation
    • Initiation phase: coagulation triggered by vessel damage → exposes plasma to TF → FV + FVII activated → activate other nearby clotting factors → formation of thrombin
    • Amplification: further activation of clotting factors + platelets
    • Propagation: occurs on surface of activated platelet → catalyses formation of thrombin+++
  • Thrombin:
    • acts on fibrinogen mesh within platelet plug → hydrolyses soluble fibrinogen → produce insoluble fibrin strands
    • Activation of FXIII: forms covalent crossbridges between fibrin strands in platelet plug → stable clot
    • +ve feedback loop: activates FV + FVIII → feed into cascade to produce more thrombin
    • activation of protein C by thrombin-thrombomodulin complex: inhibitor of coagulation → deactivates FVa and VIIIa

Fibrinolysis

• Physiological mechanism in which the fibrin within blood clots is slowly dissolved
• Normal part of wound healing + important mechanism to keep small vessels patent
• Key points:
  • Plasminogen is a b-globulin (proenzyme synthesised by the liver) → becomes interwoven into the fibrin clot as it is formed → converted to plasmin (serum protease)
  • Main physiological activator of plasminogen is t-PA, expressed by endothelial cells – helps to keep the endothelial cell surface free of fibrin deposits
  • Fibrin cleaved by plasmin → produces fibrin degradation products (FDPs)
    • One of the FDPs is the d-dimer – cleavage product of cross-linked fibrin

• Fibrinolysis pathway can be manipulated:
  • Thrombolysis eg streptokinase promotes conversion of plasminogen to plasmin → ↑ fibrinolysis
  • Inhibition of fibrinolysis: eg tranexamic acid inhibits activation of plasminogen

Kerr / Bianca 2016

Mechanism of action of Thrombolytics

“Thrombolytic agent” → drug that enhance body’s fibrinolytic system by acting on
plasminogen activators, which ↑ conversion of plasminogen into plasmin

• Streptokinase
  • Binds non-covalently to plasminogen to form a “streptokinaseplasminogen activator complex” → ↑ conversion of plasminogen to plasmin → ↑ fibrinolysis
  • It is NOT fibrin-specific → causes systemic fibrinolysis
• Urokinase:
  • similar to streptokinase
• Alteplase (rt-PA):
  • Glycoprotein that is activated when bound to fibrin (clot) only → then selectively converts fibrin-bound plasminogen to plasmin to cause fibrinolysis
  • It is fibrin-specific → causes less systemic fibrinolysis (cf. streptokinase)

Kerr / Bianca 2016

Answer by Bianca, Kerr