The role of platelets

Following damage to a blood vessel there is immediate vasoconstriction to slow blood flow and reduce the risk of exsanguination. The break in the endothelial cell barrier leads to the recruitment of platelets from the circulation to form an occlusive plug. Platelets interact both with the vessel subendothelial matrix (platelet ‘adhesion’) and with each other (platelet ‘aggregation’) (Fig 6.1). The first step in this process, adhesion, does not require platelet metabolic activity. It does, however, lead to the ‘activation’ of platelets.

Platelets are small disc-shaped particles produced in megakaryocyte cytoplasm which have a lifespan of around 10 days. They have no nucleus and no capacity for DNA biosynthesis but do have a complex infrastructure. Pores in the trilaminar platelet membrane connect with an open canalicular system allowing transport of agonists in and discharge of secretions out. The membrane receptors for agonists include:

In the platelet cytoplasm are organelles including alpha granules (containing fibrinogen, vWF, thrombospondin and other proteins) and dense granules (containing small molecules such as ADP and calcium).

Platelet activation follows stimulation by agonists such as ADP and thromboxane A₂ interacting with surface receptors, or by direct contact with the vessel wall subendothelial matrix. Platelets convert from a compact disc to a sphere, surface receptors become activated, and cytoplasmic granules secrete their contents. The net effect is the mediation and reinforcement of aggregation and adhesion, and the promotion of further activation. Other circulating platelets adhere to the initial layer and a loose platelet plug is formed.

In addition to the formation of a physical barrier at the site of injury, platelets have a procoagulant action. The coagulation sequence described below completes much more rapidly in the presence of platelets. Following activation, platelets rearrange their membrane phospholipids and shed vesicles from their surface. The platelet surface and vesicles reveal binding sites for coagulation proteins leading to the creation of coagulation complexes (e.g. the ‘prothrombinase complex’) which accelerate formation of factor Xa and thrombin.

Coagulation

Although often loosely used to encompass all aspects of clot formation, the term ‘coagulation’ more specifically refers to the mechanism directly leading to the conversion of the soluble plasma protein fibrinogen to the insoluble rigid polymer fibrin. The formation of the stable haemostatic plug composed of enmeshed fibrin and platelets is the culmination of a complex biochemical cascade involving circulating coagulation factors. This system allows extreme amplification with a robust thrombus arising from the initial stimulus of tissue injury. Most activated coagulation factors are proteolytic enzymes (serine proteases) which in the presence of cofactors cleave other factors in an ordered sequence. Thus, prothrombin (factor II), factor VII, factor IX and factor X are proenzymes which are converted to their active enzyme form (denoted by the letter ‘a’) by cleavage of one or two peptide bonds. Factors V and VIII are procofactors which are converted to the active cofactors (Va and VIIIa) also by cleavage of peptide bonds. The blood clotting proenzymes prothrombin and factors VII, IX and X require vitamin K for their activation (see pp. 76, 77).

The coagulation cascade, leading to the generation of thrombin and the formation of a fibrin thrombus, is classically divided into two parts: the intrinsic and extrinsic pathways (Table 6.1).

In the intrinsic pathway factor XII is activated by exposed collagen and other negatively charged components of the subendothelium. Activation of factor XII leads to the sequential activation of factors XI, IX, VIII (as cofactor), X and prothrombin. In the extrinsic pathway tissue factor complexes with factor VII with sequential activation of factors VII, X and prothrombin. Both intrinsic and extrinsic pathways terminate in the final common pathway where activated factor X, in association with the cofactor factor Va in the presence of phospholipid and calcium, converts prothrombin into thrombin. Thrombin in turn converts fibrinogen to fibrin by splitting the fibrinopeptides A and B from the centre domain to form fibrin monomers. These monomers combine spontaneously into dimers which assemble to form the fibrin polymer. Factor XIII crosslinks the fibrin polymer to consolidate the thrombus. The conventional division into two pathways is useful in the interpretation of in vitro laboratory tests of haemostasis. The prothrombin time (PT) is a simple measure of the function of the extrinsic pathway and the activated partial thromboplastin time (APTT) monitors the intrinsic pathway (p. 20). However, the physiological pathways at work in vivo are not so simply defined (see Fig 6.2). It seems that the intrinsic pathway is rarely relevant to coagulation in vivo – patients with hereditary deficiency of factor XII have a prolonged APTT but no bleeding disorder. The crucial protein in the initiation of blood coagulation is tissue factor, an integral membrane protein expressed on non-vascular cells. When a blood vessel is damaged, circulating factor VII comes into contact with tissue factor. The tissue factor/factor VIIa complex activates not only factor X (the extrinsic pathway) but also factor IX.

Fibrinolysis

Once damaged endothelium is repaired the fibrin thrombus must be removed to restore normal blood flow. Thrombus removal is facilitated by a fibrin-splitting serine protease, plasmin. The fibrinolytic system is shown schematically in Figure 6.3. Release of tissue plasminogen activator (t-PA) from endothelial cells leads to conversion of the proenzyme plasminogen into plasmin. t-PA is most active when bound to fibrin, thus maximising its action at the site of the thrombus. Plasmin has the capacity to digest fibrin in addition to fibrinogen and a number of other proteins. Digestion of a cross-linked thrombus by plasmin leads to the formation of ‘degradation products’ which themselves act as anticoagulants. Fibrinolysis is under strict control; circulating plasmin is inactivated by the protease inhibitor α₂-antiplasmin.