Some Physiology

Circulating 'resting' platelets do not stick to healthy endothelium or each other but if a vessel wall is

19 A religious sect that is opposed to blood transfusion on a scriptural basis.

breached they react at the site by four steps: attachment, spreading, secretion and aggregation.

1. Exposure of constituents of the subendothelial matrix most notably collagen initiates platelet attachment which is stabilised by von Willebrand factor.

2. Shape change of the attached platelets, spreading along the fibrils permits multiple tight contacts with the matrix and there is simultaneous release of thromboxane-A2 (TXA2) and adenosine diphosphate (ADP) which recruit additional platelets.

3. Agonists in the microenvironment also trigger secretion of the contents of intracellular storage granules which activate circulating platelets and vasoconstriction (including proteins, enzymes, enzyme inhibitors, vasoactive and other peptides and agents that participate in the coagulation process) and translocation of negatively charged phospholipids to the outer surface of the plasma membrane providing a binding site for coagulation proteins (an activity known as 'platelet factor 3').

4. These platelets interact with each other and aggregate through binding of fibrinogen or fibrin to the surface through glycoprotein (GP) Ilb/IIIa (integrin o.||bP3) to form an effective plug to seal the injured vessel which is stabilised by cross linked fibrin

The system that enables platelets to distinguish between healthy and damaged endothelium is shown in simplified form in Figure 28.3. It is a continuation of, and should be studied in conjunction with, the general diagram for eicosanoids on page 281.

Platelet mechanisms

The mechanism which transforms a freely circulating resting platelet (surrounded by fibrinogen and buffeted in the circulation) into an adherent platelet has been a frequent target for drug development. Platelet aggregation does not occur as long as the resting conformation of GP Ilb/IIIa is maintained and several external and internal factors dampen activation signals.

1. Cyclic AMP plays a key role. High concentrations of intraplatelet cyclic AMP inhibit platelet adhesion, aggregation and the release of active substances (see above), and low concentrations of cyclic AMP have the opposite effect.

2. The quantity of cyclic AMP within platelets is under enzymatic control, for it is formed by the action of adenylate cyclase and degraded by phosphodiesterase.

3. Platelet adenylate cyclase formation in turn is stimulated by prostacyclin (from the endothelium, also called PGI2) and inhibited by thromboxane-A2 (from within platelets, also called TXA2). Hence the action of thromboxane-A2 lowers cyclic AMP concentration and promotes platelet adhesion; prostacyclin raises cyclic AMP concentration and prevents platelet adhesion.

4. Prostacyclin and thromboxane-A2 are derived from arachidonic acid which is a constituent of cell walls, both platelet and endothelial. Cyclo-oxygenase (COX, PGH synthase), an enzyme present in cells at both sites, converts arachidonic acid to cyclic endoperoxides which are further metabolised by prostacyclin synthase to prostacyclin in the endothelium and by thromboxane synthase to thromboxane-A2 in platelets. Thus prostacyclin is principally

Fig. 28.3 Prostacyclin, thromboxane and the formation of platelet cyclic AMP

formed in the endothelium whereas thromboxane-A2 is formed mainly in platelets.

5. These differences in the prostaglandins synthesised in endothelium and platelets are important. Intact vascular endothelium does not activate platelets because of the high concentration of prostacyclin in the intima. Subintimal tissues contain little prostacyclin and platelets, under the influence of thromboxane-A2, immediately adhere and aggregate at any breach in the intima. Atheromatous plaques do not generate prostacyclin—which explains platelet adhesion and thrombosis at these sites.

6. Endothelial cells also produce nitric oxide which raises cyclic GMP levels in platelets to inhibit activation and have on their surface ecto-ADPase (CD39) that metabolises secreted ADP before it can cause platelet activation.

Inhibitors or activators of platelet aggregation act directly or indirectly by altering the rate of formation or degradation of platelet cyclic AMP. Local concentrations of these substances determine whether the platelet adhesion/aggregation process will occur.

DRUGSTHAT INHIBIT PLATELET ACTIVITY (ANTIPLATELET DRUGS)

(See also Myocardial infarction Ch. 23)

Aspirin (acetylsalicylic acid) acetylates and thus inactivates COX, the enzyme responsible for the first step in the formation of prostaglandins, the conversion of arachidonic acid to prostaglandin H2. It follows from the diagram on page 281 (Fig. 15.1) that aspirin can prevent formation of both thromboxane-A2 (TXA2) and prostacyclin (PGI2). Acylation of COX is irreversible and, as the platelet is unable to synthesise new enzyme, COX activity is irreversibly lost for its lifetime (8-10 d). Therapeutic interest in the antithrombotic effect of aspirin has centred on separating its actions on thromboxane-A2 and prostacyclin formation, and this can be achieved by using a low dose. Thus 75-100 mg/d by mouth is sufficient to abolish synthesis of thromboxane-A2 without significant impairment of prostacyclin formation, i.e. amounts substantially below the 2.4 g/d used to control pain and inflammation. Low-dose aspirin is yet not without risk:

some 13% of episodes of peptic ulcer bleeds in people over 60 years can be attributed to prophylactic asprin (use in the community about 8%).20

Dipyridamole reversibly inhibits platelet phosphodiesterase (see Fig. 28.3) and in consequence cyclic AMP concentration is increased and platelet (thrombotic) reactivity reduced; evidence also suggests that its antithrombotic effect may derive from release of prostaglandin precursors by vascular endothelium. Dipyridamole is extensively bound to plasma proteins and has a t'/2 of 12 h.

Ticlopidine is a thienopyridine derivative that inhibits ADP-dependent platelet aggregation. It is converted to its active form by metabolism by the liver and the t1/, of the parent drug is 40 h. Ticlopidine is more effective than aspirin in reducing stroke in patients with transient ischaemic attacks (TIA) but aspirin is safer and less expensive. It is also effective in reducing the risk of the combined outcome of stroke, myocardial infarction (MI) or vascular death in patients with thromboembolic stroke, decreasing vascular death and MI in patients with unstable angina, reducing acute occlusion of coronary bypass grafts and improving walking distance and decreasing vascular complications in patients with peripheral vascular disease. It may be used to prevent stroke in patients who are intolerant of aspirin. Neutropenia is the most serious adverse effect (risk 2.4%) and is greatest in the first 12 weeks of therapy; leucocyte counts should be checked every 2 weeks during this period. Diarrhoea and other gastrointestinal symptoms may be induced in a third of patients.

Clopidogrel is also a thienopyridine derivative which is also more effective than aspirin for the prevention of ischaemic stroke, MI or vascular death in patients at high risk but it is not associated with neutropenia. It is more expensive than aspirin though safer than ticlodipine.

Epoprostenol (prostacyclin) may be given to prevent platelet loss during renal dialysis, with or without heparin; it is infused i.v. and s.c (t'/2 3 min). It is a potent vasodilator.

20 Weil J et al 1995 Prophylactic aspirin and risk of peptic ulcer bleeding. British Medical Journal 310: 827-830.

Glycoprotein (GP) Ilb-IIIa antagonists. The platelet glycoprotein Ilb-IIIa complex is the predominant platelet integrin,21 a molecule restricted to megakaryocytes and platelets which mediates platelet aggregation via the binding of adhesive proteins such as fibrinogen and von Willebrand factor (vWF). Where there is hereditary absence of the GP Ilb-IIIa complex (Glanzmann's thrombasthenia) platelets are incapable of aggregation by all physiological agonists. GP Ilb-IIIa antagonists have been developed as antiplatelet agents and administered intravenously, they inhibit the final common pathway of platelet aggregation: binding of fibrinogen or vWF to the GP Ilb-IIIa complex. They are more complete inhibitors than either aspirin or clopidogrel which inhibit only the cyclo-oxygenase or ADP pathway respectively. GP Ilb-IIIa antagonists also have an anticoagulant effect through inhibition of prothrombin binding to the complex and inhibition of procoagulant platelet-derived microparticle formation. Platelet aggregation is inhibited in a dose-dependent manner.

Abciximab is a human-murine chimeric monoclonal antibody Fab fragment that binds to the GP Ilb-IIIa complex with high affinity and slow dissociation rate. After i.v. administration it is cleared rapidly from plasma {Xl/2 20 min). Abciximab (0.25 mg/kg bolus then 0.125 microgram/kg/min infusion for 12 h) produces immediate and profound inhibition of platelet activity that lasts for 12-36 h after termination of the infusion. This reduces the risk of death, MI or need for urgent coronary artery bypass grafting after percutaneous coronary angioplasty and benefit is maintained up to 3 years. The dose causes and maintains blockade of > 80% receptors, causing > 80% reduction in aggregation. Patients also receive aspirin and heparin and if a coronary stent has been inserted, either clopidogrel or ticlodipine. Abciximab is also effective in refractory unstable angina prior to percutaneous coronary intervention. It has a potential role in combination with low dose thrombolysis in acute myocardial infarction and as a single agent in stroke.

21 Integrins are cell surface adhesion receptors consisting of non-covalently associated alpha- and beta- subunits, now redesignated integrin ocI|bP3.

Eptifibatide is a cyclic heptapeptide based upon the Lys-Gly-Asp sequence. Tirofiban and lamifiban are nonpeptide mimetics. All three are competitive inhibitors of the GPIIb-IIIa complex with lower affinities and higher dissociation rates than abciximab and short plasma t1/, (2-2.5 h). Platelet aggregation returns to normal 30 min to 4 h after discontinuation. Eptifibatide and tirofiban are effective in acute coronary syndromes. Lamifiban is undergoing clinical development.

Adverse effects. Haemorrhage occurs but is less of a problem with low doses of heparin; it remains a particular risk in patients treated after failed fibrinolytic therapy for acute myocardial infarction. Platelet transfusion after cessation of abciximab is necessary for refractory or life threatening bleeding. After transfusion, the antibody redistributes to the transfused platelets, reduces the mean level of receptor blockade and improves platelet function. Thrombocytopenia may occur from 1 hour to days after commencing treatment in up to 1% of patients. This necessitates platelet counts at 2-4 hours and then daily; if severe, therapy must be stopped and, if necessary, platelets transfused. EDTA-induced pseudothrombocytopenia has been reported and a low platelet count should prompt examination of a blood film for agglutination before therapy is stopped.

Other drugs

Dazoxiben, an inhibitor of thromboxane-A2 but not of prostacyclin synthesis, is being evaluated in cardiovascular disease.

Dextrans, particularly of MW 70 000 (dextran 70), alter platelet function and prolong the bleeding time. Dextrans differ from the other antiplatelet drugs which tend to be used for arterial thrombosis; dextran 70 reduces the incidence of postoperative venous thromboembolism if it is given during or just after surgery. The dose should not exceed 10% of the estimated blood volume. They are rarely used.

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