Procedimiento desarrollado en el estudio 1. Consideraciones éticas

Endothelial cells synthesise certain substances which have inhibitory influence on haemostasis. These include—thrombomodulin, protein S, heparin- related substances, prostacycline (PGI2), and tissue plasminogen activator (tPA). Binding of thrombomodulin to thrombin causes activation of protein C. Protein C inactivates factors V and VIII:

C and is a potent inhibitor of coagulation. Protein S is a cofactor for protein C.

Deficiency of protein C or protein S is associated with tendency towards thrombosis.

Heparin-like substances on the surface of endothelial cells potentiate the action of antithrombin. Prostacycline, a prostaglandin synthesised by endothelial cells, induces vasodilatation and also inhibits platelet aggregation. Endothelial cells also synthesise tissue plasminogen activator, which converts plasminogen to plasmin, and activates fibrinolytic system.

Certain factors synthesised by endothelial cells promote haemostasis and include tissue factor, von Willebrand factor and platelet activating factor. Tissue factor or thromboplastin activates extrinsic system of coagulation. von Willebrand factor mediates adhesion of platelets to subendothelium. Platelet activating factor induces aggregation of platelets (Fig. 1.27).

Another vascular factor promoting haemostasis is vasoconstriction of small vessels following injury.

Subendothelial collagen promotes platelet adhesion and also activates factor XII (intrinsic pathway).

Platelets

Platelets are derived from cytoplasmic fragmentation of bone marrow cells called megakaryocytes. They measure 2 to 3 µ in diameter. Normal platelet count in peripheral blood is 1.5 to 4 lacs/cmm. Platelets remain viable in circulation for approximately 10 days. About one-third of the total platelets in the body are in the spleen and remainder in peripheral blood. Under light microscope, in peripheral blood smears stained with one of the Romanowsky stains, platelets appear as small, irregular with fine cytoplasmic processes. Cytoplasmic granules are often visible. These granules may

Figure 1.27: Role of blood vessels in haemostasis

Ultrastructure of Platelets

Ultrastructurally, following three zones can be distinguished : (1) Peripheral zone:

exterior coat (glycocalyx), cell membrane, open canalicular system; (2) Sol-gel zone:

microfilaments, circumferential microtubules, dense tubular system; (3) Organelle zone: alpha granules, dense granules, mitochondria, lysosomes (Fig. 1.28).

Peripheral zone: Exterior or surface coat (glycocalyx) overlies the cell membrane. It is made of proteins, glycoproteins, and mucopolysaccharides. Some of the glycoproteins are polysaccharide side chains of the integral membrane proteins while others are adsorbed from the plasma.

The cell membrane is a trilaminar membrane composed of proteins, lipids, and carbohydrates. The chief membrane lipids are phospholipids which are arranged as a bilayer; the polar head groups are oriented both externally (towards plasma) and internally (towards cytoplasm) while the fatty acid chains are oriented toward each other. Phospholipids are distributed asymmetrically in the membrane with phosphatidylinositol concentrated on the inner half of the bilayer and phosphati-dylethanolamine on the outer half. The phospholipids play an important role in prostaglandin synthesis and in platelet procoagulant activity.

An extensive open canalicular system, formed by invagination of the cell membrane, communicates with the exterior. It functions as a route through which platelet contents are secreted outside the cell.

Sol-gel zone: Microtubules provide structural support to the platelets. Microfilaments have contractile function. The dense tubular system, derived from smooth endoplasmic reticulum, is the site of pooling of calcium and formation of prostaglandin and thromboxane.

Figure 1.28: Ultrastructure of platelet

Organelle zone: Platelet organelles are alpha granules, dense granules, lysosomes and peroxisomes. Contents of platelet organelles are shown in Table 1.4.

Platelet Membrane Glycoproteins

The cell membrane contains integral membrane glycoproteins (Gp), which play an important role in haemostasis. Important platelet membrane glycoproteins and their functions are as follows:

Gp Ib-IX-V: This is a constitutively active receptor that mediates vWF-dependent adhesion of platelets to subendothelial collagen.

Gp IIb/IIIa: On activation, serves to bind fibrinogen and thus mediates aggregation.

Also receptor for vWF, fibronectin, and thrombospondin.

Gp Ia-IIa: Constitutively active receptor for collagen and mediates platelet adhesion independent of vWF.

Platelet Antigens

Platelets possess HLA antigens and platelet-specific antigens. HLA class I antigens induce alloimmunisation and cause refractoriness to platelet transfusions when platelets are obtained from random donors. The platelet-specific antigen systems are now known as human platelet antigen (HPA) systems. Platelet specific antigens play an important role in neonatal alloimmune thrombocytopaenic purpura (NATP) and in post transfusion purpura.

Role of Platelets in Haemostasis

Activation of platelets refers to adhesion, aggregation, and release reaction of platelets which occurs after platelet stimulation (i.e. after vascular damage).

Adhesion: This means binding of platelets to nonendothelial surfaces, particularly subendothelium which is uncovered following vascular injury. von Willebrand factor (vWF) mediates adhesion of platelets to subendothelium via GpIb on the surface of platelets (Fig. 1.29). Congenital absence of glycoprotein receptor GpIb

(Bernard-Table 1.4: Platelet organelles and their contents Alpha granules

• Platelet-specific proteins: platelet factor-4, β thromboglobulin, platelet-derived growth factor, thrombospondin

• Coagulation system proteins: fibrinogen, factor V, von Willebrand factor, high molecular weight kininogen

• Fibrinolytic system proteins: α2-antiplasmin, plasminogen, PAI-1

• Others: fibronectin, albumin

Dense granules

• Anions: ADP, ATP, GTP, GDP

• Cations: calcium, serotonin

Soulier syndrome) or of von Willebrand factor in plasma (von Willebrand’s disease) causes defective platelet adhesion and bleeding disorder.

Platelets normally circulate as round to oval disc-like structures. With activation, platelets undergo shape change, i.e. they become more spherical and form pseudopodia.

This shape change is due to reorganisation of microtubules and contraction of actomyosin of microfilaments.

Release reaction (secretion): Immediately after adhesion and shape change, process of release reaction or secretion begins. In this process, contents of platelet organelles are released to the exterior. ADP released from dense granules promotes platelet aggregation. Platelet factor 4 released from alpha granules neutralises the anticoagulant activity of heparin while platelet-derived growth factor stimulates proliferation of vascular smooth muscle cells and skin fibroblasts and plays a role in wound healing.

Activated platelets also synthesise and secrete thromboxane A2 (TxA2) (Fig. 1.30).

Platelet agonists such as ADP, epinephrine, and low-dose thrombin bind to their specific receptors on platelet surface, and activate phospholipase enzymes, which release arachidonic acid from membrane phospholipids. Arachidonic acid is converted to cyclic endoperoxides PGG2 and PGH2 by the enzyme cyclo-oxygenase. These are then converted to thromboxane A2 by thromboxane synthetase. Thromboxane A2 has a very short half-life and is degraded into thromboxane B2 which is biologically inactive. TxA2 causes shape change and stimulates release reaction from alpha and dense granules.

TxA2 also induces aggregation of other platelets and local vasoconstriction.

Aggregation: This may be defined as binding of platelets to each other. ADP released from platelets or from damaged cells binds to specific receptors on platelet surface.

This causes inhibition of adenyl cyclase and reduction in the level of cyclic AMP in platelets. A configurational change in the membrane occurs so that receptors for fibrinogen (GpIIb and IIIa) become exposed on the surface. Binding of fibrinogen

Figure 1.29: Platelet adhesion to subendothelial collagen. GpIb receptor on platelets and von Willebrand factor are necessary for attachment of platelets to subendothelial collagen

Figure 1.30: Synthesis of thromboxane A2. Modes of action of certain antiplatelet drugs are also shown

Figure 1.31: Platelet aggregation. This requires binding of fibrinogen molecules to GpIIb/IIIa receptors on platelets

molecules to GPIIb/IIIa receptors on adjacent platelets causes platelet aggregation (Fig. 1.31). The activated platelets release ADP and TxA2 and so a self-sustaining reaction is generated leading to the formation of a platelet plug. Thrombin generated from activation of coagulation system is a potent platelet-aggregating agent and also converts fibrinogen to fibrin. Fibrin and aggregated mass of platelets at the site of injury constitute the haemostatic plug.

Platelet procoagulant activity: When platelets are activated, negatively charged phospholipids (phosphotidylserine and phosphatidylinositol) located in the inner half of the lipid bilayer become exposed on the outer surface. These phospholipids play active role in coagulation by providing surface for interaction of some coagulation factors. Critical coagulation reactions for which activated platelets provide a negatively charged phospholipid (PL) surface are shown in Figure 1.32.

Platelets may play a role in the activation of F XII in the presence of ADP and kallikrein. Platelets also can directly activate F XI independent of F XII. This may explain the absence of bleeding diathesis in persons with F XII deficiency.

In addition platelets also secrete calcium, FV, fibrinogen, and FXII and contribute to the coagulation system.