Structure of platelets:

• Platelets are disc shaped structures with smooth surface.
• They have several openings on the surface resembling holes in the sponge.
• Peripheral zone consists of 
  • Outer fluffy surface coat- Contains glycocalyx (glycoproteins, proteins and mucopolysaccharides)
  • Phospholipid bilayer- Consists of phosphatidyl serine
  • Integral proteins- such as GlycoproteinIb (receptor for vWF), IIa, IIIa (Receptor for fibrinogen), IX
• Structural zone (which consists of supporting structure)
  • Microtubules- Consist of tubulin dimmers, which maintain the discoid shape
  • Cytoskeletal network- Consist of actin, and various actin binding proteins
  • Cytoplasmic meshwork- Consisting of actin and myosin
• Organellar zone (Responsible for storage and secretion of various substances)
  • Granules
    • Dense bodies- Consist of ATP, ADP, other nucleotides, serotonin and calcium
    • Alpha granules- Consist of various hemostatic proteins such as fibrinogen, factor V, vWF, plasminogen activator inhibitor, alpha 2 antiplasmin, 
    • Lysozymes- Consist of enzymes
  • Mitochondria- 4-5 mitochondria are present per platelet
  • Glycogen- It is broken down to glucose 1 phosphate
• Membrane system- Consist of 
  • Surface connected open canalicular system
  • Dense tubular system which originates from rough ER
  • Fused system- It connects above two systems.

Figure 1.6.1 - Platelets in peripheral smear

3 phases of hemostasis:

• Vascular: Mediated by the release of locally vasoactive agents that result in vasoconstriction at the site of injury and reduced blood flow.
• Platelet: Aggregation of platelets to form platelet plug
• Plasma: Includes initiation, propagation and stabilization of clot.

Function of platelets:

• Wherever there is endothelial injury, platelets attach to exposed subendothelial collagen, which occurs with the help of von Willebrand factor (vWF connects Glycoprotein Ib/IIb on platelet surface to collagen)
• Upon binding of vWF with Glycoprotein Ib/IIb, enzymes (phospholipase C, A2 and adenyl cyclase) in the membrane of platelets are activated.
• Activated enzymes cleave specific membrane phospholipids, leading to release of specific 2^nd messengers such as ionositoltriphosphate. Phospholipase hydrolyses arachadonic acid to form prostaglandins. Cyclooxygenase acts on arachadonic acid to form thromboxane A2.
• ADP and serotonin released from activated platelets induce recruitment of additional platelets.
• Inositoltriphosphate stimulates release of calcium from dense tubular system.
• Calcium causes changes in proteins in structural zones, because of which there is change in shape of platelets, with formation of numerous pseudopods. Platelets can fit together in a “jig-saw puzzle effect”.
• There are 2 phases in aggregation of platelets
  • Primary- Loose adherence
  • Secondary- Firm adherence after release of ADP.
• Following adhesion of platelets, there is initiation of coagulation system by these mechanisms
  • Attachment of fibrinogen to glycoprotein IIb/IIIa
  • Change in membrane surface of platelets (exposing negatively charged phospholipids) which allows coagulation factors to bind to it.
  • Activated platelets recruit blood borne tissue factor
  • They also synthesize tissue factor
  • Formation of procoagulantmicrovesicles
  • Exposure of activated Factor V which is present within the platelets
• Thrombin generated during coagulation mechanism further activates platelets.

Coagulation pathway

Figure - Coagulation and fibrinolytic pathway

• Classification of factors needed in coagulation pathway
  • Zymogens
    • Structurally all are similar and their gene structures are also similar. But they have highly specific functions, due to surface loops that are not homologous.
    • All are synthesized in liver
    • All are precursors of serine proteases that must be proteolytically activated to express their enzymatic activity.
    • They are classified into 2 types
      • Vitamin K dependent factors (Need gamma carboxylation at amino terminal of glutamic acid)- They include Factors 2, 7, 9, 10, and protein C
      • Non Vitamin K dependent- They include factors 11, 12, prekallikrien, 13, thrombin activable fibrinolysis inhibitor
  • Cofactors
    • Soluble- factor 5, 8, vWF, protein S, protein Z, high molecular weight kininogen
    • Cellular- tissue factor, thrombomodulin
    • Structural proteins- Fibrinogen
    • Inhibitors- Antithrombin, tissue factor pathway inhibitor, protein Z dependent protease inhibitor
• Blood coagulation occurs when the enzyme thrombin is generated and proteolyses soluble, plasma fibrinogen, forming the insoluble fibrin polymer or clot.
• Initiation phase:
  • Tissue factor is both necessary and sufficient to initiate coagulation in vivo.
  • Tissue factor- VIIa complex (extrinsic tenase complex) activates Factor X to form Xa.
  • Xa along with Factor Va, which is released from platelets, forms prothrombinase.
  • Prothrombinase converts prothrombin to thrombin.
  • Thrombin amplifies the coagulation loop by cleaving factor VIII from vWF, activating Factor VIII and Factor XI. This results in further release Factor V.
  • Production of small amount of thrombin is sufficient to initiate events, that “prime” the clotting system for subsequent burst of thrombin generation.
• Propagation phase:
  • Factor IXa (which is formed by action of Factor XIa or TF-FVIIa complex on Factor IX) binds to Factor VIIIa to form potent intrinsic tenase complex.
  • Large amount of Factor X is converted to Factor Xa, ultimately leading to formation of large amount of thrombin.
  • Large amount of thrombin, converts fibrinogen to fibrin, resulting in clot formation
• Stabilization phase
  • Thrombin also activates Factor XIII, which cross links the fibrin and forms a stable clot.
  • Thrombin activable fibrinolysis inhibitor also helps in this process. It removes lysine residues from the fibrin clot, thereby limiting plasmin binding.

Feedback inhibition of coagulation

• Excess of thrombin at the margin of clot binds to thrombomodulin (a receptor expressed on the surface of intact endothelium). Upon this binding thrombin acquires anticoagulant property and converts protein C to activated protein C.
• Activated Protein C, together with Protein S inactivate Factors Va and VIIIa
• Tissue factor pathway inhibitor, a protein produced from endothelium, inhibits VIIa/TF complex and Factor Xa.
• Antithrombin released at the margins of endothelial injury binds to thrombin and inactivates it. Antithrombin also inactivates Factor Xa.

Fibrinolysis

• Starts once the vessel injury is healed.
• Tissue plasminogen activator is activated when it binds to the lysine residues of fibrin. TPA activity is inhibited by plasminogen activator inhibitor 1.
• Tissue plasminogen activator and urokinase convert plasminogen to plasmin.
• Annexin A2 also augments formation of plasmin
• Plasmin causes degradation of fibrin via interaction with lysine and argenine residues.

Functions of fibrin degradation products

• Inhibition of platelet function
• Potentiation of hypotensive effects of bradykinin
• Chemotaxis
• Immune modulation

Role of endothelium in coagulation

• Antithrombotic properties
  • Antiplatelet: Intactness, Production of nitric oxide and prostaglandins, synthesis of adenosine diphosphatase
  • Anticoagulant- Membrane associated heparin like molecules activate antithrombin 3, production of thrombomodulin and tissue factor pathway inhibitor
  • Fibrinolytic- Production of tissue type plasminogen activator
• Prothrombotic
  • Platelets- Production of vWF
  • Procoagulant- Production of tissue factor
  • Antifibrinolytic- Production of inhibitors of plasminogen activator