Haemostasis

The haemostatic mechanism consists of 4 components:

1. • 1. Prostacyclin A product of prostaglandins which prevents platelet aggregation and causes vasodilation (PGE2 as a dilator of arterial vessels and NO as a dilator of smooth muscle cells)
     2. Heparin Sulphate Has a weak antithrombin (AT) action, promoting the action of antithrombin
     3. Tissue plasminogen activator Activates plasminogen, a plasma protein which is converted to an enzyme, "Plasmin", which dissolves fibrin and other plasma proteins.
     4. von Willebrand factor (vWF) A large polymeric protein which is a part of factor VIII that enables platelets (through a receptor on the surface) to adhere to collagen and the basement membrane upon damage to the endothelial layer.
        
        
2. Platelets
   
   

   • Platelets contain granules, lysosomes, mitochondria, glycogen, microtubules and microfilaments
   • Platelets are approx. 2um in diameter, highly refractile, disk-shaped
   • Platelets have no nucleus (ie. they are not a cell) and survive for approx. 10 days
   • ¹/₃ of our platelets are produced in the spleen
   • The platelet membrane consists of a bilipid (often referred to as platelet factor 3) membrane that contains antigens important in transfusions reactions.
       The membrane contains receptor sites for ADP, vWF and fibrinogen.
   • The sol-gel zone consists of microtubules (allowing platelet contraction) and microfilaments (allowing pseudopodia production)
   • The organelle zone contains alpha (containing platelet derived growth factors (PDGF) plus factors I, V and VIII) and dense (due to calcium, ADP/ATP and serotonin components) granules
   • The tubular system is an open canalicular system linking the interior of the platelet to the exterior. The system contains calcium and is a site for prostaglandin synthesis (eg thromboxane A₂).
       Aspirin stops prostaglandin synthesis by inhibiting the enzyme cyclooxygenase therefore preventing platelet aggregation
   • After adhesion and aggregation, platelets disintegrate and liberate platelet factors 1, 5, 6, 8 and 9 which correspond to plasma clotting factors V, I, X, VIII and XIII, respectively. Consequently, the local concentration in clotting factors is elevated, so that platelets support plasma clotting. Platelet factor 3 (a phospholipid from the platelet membrane that acts as an important link between thrombocytic and plasma clotting)
   • The formation of the platelet plug is referred to as primary haemostasis. The time taken for this plug to form (Bleeding Time - BT) gives a non-specific indication of:
     
     • The state of the vascular endothelium
     • The number fo platelets in the circulation
     • The platelets are functioning correctly (can release granules and produce pseudopodia)
     • Demonstrates the presence of vWF
       
       
3. Plasma proteins
   • 1972, the year of the Munich Olympic games, may be useful in remebering the protines (Factor X=1, 9=IX, 7=VII, 2=II-thanks to Dr Agathe Beitz)!!
   • All coagulation factors are made in the liver, except for vWF
   • The coagulation mechanism, spearheaed by the plasma proteins, makes the plug firm/stable
   • Biochemical functions of coagulation factors
     
     

     Serine Protease	Cofactors	Transamidase	Substrate
     XIIa	HMWK	Factor VIII	Fibrinogen (I)
     XIa	VIII
     Kallikrein	V
     IXa	Platelet Factor 3
     VIIa
     Xa
     Thrombin (IIa)

     a-activated coagulation factor; VIII & IX - haemophiliac factors; VIII &V - labile factors

   • Vitamin K dependent coagulation factors (fat soluble vitamins found in leafy vegetables and from normal flora in the gastrointestinal tract)
       The oral anticoagulant, warfarin, acts here by blocking the reduction of oxidised (inactive) vitamin K.

     • Factor II
     • Factor VII
     • Factor IX
     • Factor X
     • Protein C
     • Protein S
   • Other names given to these proteins include:

     • Vitamin K dependent coagulation factors = prothrombin complex.
     • PIVKA - protein induced in vitamin K absence
     • PPSB - prothrombin = F II, proconvertin = F VII, Stuart-Prower-Factor = F X, and antihemophilic globulin B = F IX.
   • There are two pathways to achieve clotting:
     The Intrinsic Pathway and the Extrinsic Pathway
     1. Intrinsic Pathway
        • All factors occur from within the circulation
          
        • in vivo, the p'way is triggered by exposure of "contact factors" to collagen or basement membrane at the site of injury or a foreign substance such as a prosthetic device
          
        • in vitro, the p'way is triggered by exposure of "contact factors" to glass (non-wettable surfaces such as plastic or siliconized glass markedly decrease activation times)
          
        • Contact factors include XII, XI, KMWK and prekallikrein which results in formation of XIa - without the need for Ca²⁺
          
        • Ca²⁺ and platelet factor 3 are required as cofactors for all remaining steps in the cascade
          
          
        
     2. Extrinsic Pathway
        • Requires tissue thromboplastin to be released from damaged cells (outside the circulation)
        • Ca²⁺ and platelet factor 3 are required as cofactors for all remaining steps in the cascade
          
          

     
   • Progressive activation and magnification of the system results in a cascade effect.
   • X, V, II & I are common factors to both systems with X able to be activated by either p'way
   • XIII is not involved until after fibrin formation, stabilising the fibrin. Thrombin and Ca²⁺ act on XIII to induce fibrin to form a stable plug.
   • The intrinsic pathway is slower than the extrinsic pathway - due to the extra (contact) factors needed for activation in the intrinsic pathway.
   • The coagulation processes occur ON the aggregated platelets (providing the Ca² and phospholipid)
   • Xa feeds back to activate VIII and V
   • XIIa is cleaved into fragments by the action of XIa, kallikrein, kininogen and plasmin. These fragments activate XI-XIa, prekallikrein-kallikrein and plasminogen-plasmin
     
     
4. Fibrinolysis
   
   

   • Fibrinolysis works in a steady state with haemostasis
   • Damaged endothelium releases tissue plasminogen activator as well as plasminogen - both of which are adsorbed to the fibrin surface
   • The fibrin clot is dissolved through the action of plasminogen activators:
     • Tissue plasminogen activator (from damaged endothelial cells)
     • Leucocytes
     • Urokinase (in urine)
     • Streptokinase (external agent used in therapy)
   • These factors can act upon naturally circulating plasminogen to catalyse its formation to plasmin, an enzyme which degrades fibrin.
     • Plasmin also dissolves: Fibrinogen, Factor V and Factor VIII
   • Excessive fibrinolysis however, will lead to bleeding

   Inhibitors of Coagulation

   1. Antithrombin III
      • Major inhibitor of thrombin (II) but it also inhibits activated factors such as XII, XI and VII
      • Heparin combines with antithrombin III to produce a major inhibitory effect
   2. Protein C and Protein S
      • Vitamin K dependent proteins (blocking synthesis of vitamin K halts the function of C & S
      • When thrombin and thrombomodulin (from damaged endothelial cells) combine, protein C is activated.
      • Protein C inactivates V and VIII (labile factors) - protein S acting as a cofactor

   Inhibitors of Fibrinolysis

   1. alpha ₂ anti-plasmin
      • The main inhibitor of plasmin
      • While plasmin is adsorbed to fibrin, it is protected from the action of anti-plasmin