Br. J. clin. Pharmac. (1990), 30
Editorial
175
Platelet function tests in the assessment of antithrombotic agents Recognition of the role of thrombotic mechanisms in myocardial infarction and stroke has led to increasing investigation and use of antithrombotic agents in the prophylaxis and treatment of these common disorders. In addition to conventional anticoagulants which inhibit the generation of fibrin, the insoluble product of the fluid phase of coagulation, drugs which interfere with platelet function have an important role in preventing thrombotic disease. New drugs are being developed and undergoing laboratory and clinical assessment. Blood platelets are highly reactive cells with a central role in the primary haemostatic response. Furthermore, platelets are involved in pathological thrombosis, particularly in the arterial system and microvasculature, and they are likely to be important in the pathophysiology of atherosclerosis. Increased platelet reactivity ex vivo has been demonstrated in disorders associated with diffuse vascular disease, diabetes mellitus and some hyperlipidaemias. Platelets circulate as disc-shaped cells and are subject to rheological forces which lead to platelet-endothelial cell interactions. Several interdependent changes occur during activation: adhesion, shape change, aggregation and the release reaction. The interaction between platelets and exposed subendothelial elements, including collagen and fibronectin, occurs with great rapidity. Such adhesion is mediated by a plasma macromolecule derived from endothelial cells and platelets themselves -von Willebrand factor-and platelet membrane glycoprotein receptors. Platelets possess contractile elements including actin and on stimulation undergo a morphological change to spiny spheres and interact with adjacent platelets to form an aggregate. Aggregation involves the association of fibrinogen and other adhesive proteins with different membrane glycoprotein receptors exposed on the surface of adjacent platelets and occurs in response to a wide range of physiologically important agonists including adrenaline and thrombin. Aggregation is usually coupled with the release reaction in which granular components are secreted in an irreversible process; this releases ADP, serotonin and other agonists from their intra-
cellular stores, resulting in the recruitment of more platelets into the aggregate. More is now known of the mechanisms of agonist-response coupling in blood platelets. As in other secretory cells, calcium mobilisation plays a central role, as does peroxidation of arachidonic acid from membrane phospholipids to form the potent aggregator and vasoconstrictor thromboxane A2. Intracellular cyclic AMP plays a regulatory role and is partly under the control of prostaglandin I2, a powerful aggregation inhibitor and vasodilator synthesised predominantly by the cells of the vascular endothelium. Aggregation leads to exposure of phospholipid components of platelet membrane which act as potent catalysts in the fluid phase of coagulation leading to fibrin deposition. Clearly the processes of platelet activation are complex and dependent upon rheological, biochemical and cellular interactions which are not easily reproduced in vitro. The assessment of platelet function has most commonly relied upon the turbidometric method of Born (1962) in which platelets, isolated from some other cellular components of blood and suspended in plasma, are exposed to a variety of agonists in variable concentrations. Shape change and aggregation are monitored by the resulting changes in light transmission through the suspension (Born, 1962). The simultaneous measurement of thromboxane and products of release such as serotonin may provide additional information (Greaves & Preston, 1985). The effects of many drugs and their mechanisms of action have been studied in this manner. Aspirin and ticlopidine are for example particularly potent inhibitors in this type of system. However, such an approach to the assessment of drug effects on primary haemostasis has many limitations. Anticoagulated blood must be used for the preparation of platelet rich plasma. This may lead to platelet activation during the procedure, loss of platelet subpopulations and to separation of platelet responses from the activation of coagulation. These processes are interdependent in vivo. The method also provides little information on the important process of platelet adhesion, and the physiologically crucial effects of erythro-
176
M. Greaves
cytes (which are rich in ADP, a platelet agonist), leucocytes and endothelium are not monitored. Some but not all of these limitations have been overcome by development of methods for the study of adhesion/aggregation in whole blood in which activation is detected by counting residual individual platelets after stirring with an agonist (Saniabadi et al., 1983), or by alteration in electrical impedance associated with platelet activation (Cardinal & Flower, 1980). These methods have also been applied to pharmacological studies. It is pertinent that the phosphodiesterase inhibitor, dipyridamole, has little or no effect on aggregation at pharmacological concentrations in platelet-rich plasma but is a significant inhibitor in whole blood preparations (Gresele et al., 1983; Heptinstall et al., 1986). Red cells must be present to demonstrate this inhibitory effect. The measurement of platelet adhesion remains a technically difficult area. Methods involving the passage of blood through columns of glass beads have been applied to drug evaluation (David et al., 1979) but have poor reproducibility and measure aggregation rather than adhesion (Greaves & Preston, 1985). Rheological factors are important and ingenious methods have been devised which use perfusion chambers containing de-endothelialised segments of mammalian blood vessel mounted on probes (Baumgartner & Haudenschild, 1972; Escolar et al., 1985). In this issue of the journal Muller and colleagues (1990) describe experiments utilising a novel approach to the in vitro assessment of platelet adhesion and aggregation in the presence of subendothelial components. When vascular endothelial cells are grown in culture they generate a subendothelial matrix which resembles that of the vascular subendothelial basal lamina and possesses the same important macromolecules including fibronectin, collagen, laminin and proteoglycans. This matrix has been shown to be capable of initiating many of the changes which occur during platelet activation at sites of endothelial injury, thus providing a convincing model for the study of platelet
subendothelial interactions ex vivo (Booyse et al., 1982; Eldor et al., 1985; Vlodavski et al., 1982). Using this system Muller et al. (1990) have confirmed the activity of aspirin as a platelet inhibitor and also suggest that dipyridamole has significant inhibitory activity. It is apparent that no test of platelet function can reflect comprehensively the complex interactions which occur during platelet (sub) endothelial interactions in vitro. A multifaceted approach is clearly needed for the assessment of platelet reactive drugs ex vivo. Further application of methodology such as that described by Muller et al. (1990) may add to our understanding of the pharmacology of anti-platelet agents, but a full assessment will still require measurement of in vivo parameters of platelet function in carefully controlled studies. The standardised skin bleeding time remains a useful in vivo test for the assessment of primary haemostasis in health and disease (Greaves & Preston, 1985). In conditions where pathological platelet activation is implicated measurement of plasma products of the release reaction, P-thromboglobulin and platelet factor 4 (Kaplan & Owen, 1981), and of the survival of isotopically labelled autologous platelets remain essential approaches for the assessment of significant pharmacological activity. Newer methods, using monoclonal antibodies directed against antigens exposed during platelet activation (Abrams et al., 1990) may also prove useful in the assessment of the ability of drugs to inhibit activation in vivo. Finally, it is noteworthy that it has proven to be extremely difficult to demonstrate clinical efficacy for dipyridamole (Anonymous, 1984), despite apparent activity in some tests of platelet function. The most sophisticated laboratory approaches to testing antiplatelet drugs will never replace the need for well-conducted clinical trials. M. GREAVES Department of Haematology, Royal Hallamshire Hospital, Glossop Road, Sheffield SJO 2JF
References Abrams, C. S., Ellison, N., Budzynski, A. N. & Shattil, S. J. (1990). Direct detection of activated platelets and platelet-derived microparticles in humans. Blood, 75, 128-138.
Anonymous (1984). Doubts about dipyridamole as an antithrombotic drug. Drug and Therapeutics Bulletin, 22, 25-28. Baumgartner, H. R. & Haudenschild, C. (1972),
Editorial Adhesion of platelets to subendothelium. Ann. N. Y. Acad. Sci., 201, 22-36. Booyse, F. M., Quarfoot, A. J. & Feder, A. (1982). Culture-produced subendothelium I. Platelet interaction and properties. Haemostasis, 11, 4961. Born, G. V. R. (1962). Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature, 194, 927-929. Cardinal, D. C. & Flower, R. J. (1980). The electronic aggregometer: a novel device for assessing platelet behaviour in blood. J. pharmac. Methods, 3, 135-138. David, J. L., Monfort, F., Herion, F. & Raskinet, R. (1979). Compared effects of three dose-levels of ticlopidine on platelet function in normal subjects. Thrombos. Res., 14, 35-49. Eldor, A., Vlodavski, I., Martinowicz, V., Fuks, Z. & Colter, B. S. (1985). Platelet interaction with subendothelial extracellular matrix. Plateletfibrinogen interactions are essential for platelet aggregation but not for matrix induced release reaction. Blood, 65, 1477-1483. Escolar, G., Bastida, E., Ordinas, A. S. & Castillo, R. (1985). Interactions of platelets with subendothelium in humans treated with acetylsalicylic acid and dipyridamole alone or in combination. Thrombos. Res., 40, 419-424. Greaves, M. & Preston, F. E. (1985). The laboratory investigation of acquired and congenital platelet disorders. In Blood coagulation and haemostasis:
177
A practical guide, ed. Thompson, J. M., Third Edition. London, Edinburgh: Churchill Livingtone. Gresele, P., Zoja, C., Deckmyn, H., Arnout, J., Vermylen, J. & Verstraete, M. (1983). Dipyridamole inhibits platelet aggregation in whole blood. Thrombos. Haemostas., 50, 852-856. Heptinstall, S., Fox, S., Crawford, J. & Hawkins, M. (1986). Inhibition of platelet aggregation in whole blood by dipyridamole and acetylsalicylic acid. Thrombos. Res., 42, 215-223. Kaplan, K. L. & Owen, J. (1981). Plasma levels of Bthromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood, 57, 199-202. Muller, T. H., Su, C. A. P. F., Weisenberger, H., Brickl, R., Nehmiz, G. & Eisert, W. G. (1990). Dipyridamole alone or combined with low-dose acetylsalicylic acid inhibits platelet aggregation in human whole blood ex vivo. Br. J. clin. Pharmac., 30, 179-186. Saniabadi, A. R., Lowe, G. D. O., Forbes, C. D., Prentice, C. R. M. & Barbenel, J. C. (1983). Platelet aggregation studies in whole human blood. Thrombos. Res., 30, 625-632. Vlodavski, I., Eldor, A., Hy-Am, E., Atzmon, R. & Fuks, Z. (1982). Platelet interaction with the extracellular matrix produced by cultured endothelial cells: A model to study the thrombogenicity of isolated subendothelial basal lamina. Thrombos. Res., 28, 179-191.
Editorial
175
Platelet function tests in the assessment of antithrombotic agents Recognition of the role of thrombotic mechanisms in myocardial infarction and stroke has led to increasing investigation and use of antithrombotic agents in the prophylaxis and treatment of these common disorders. In addition to conventional anticoagulants which inhibit the generation of fibrin, the insoluble product of the fluid phase of coagulation, drugs which interfere with platelet function have an important role in preventing thrombotic disease. New drugs are being developed and undergoing laboratory and clinical assessment. Blood platelets are highly reactive cells with a central role in the primary haemostatic response. Furthermore, platelets are involved in pathological thrombosis, particularly in the arterial system and microvasculature, and they are likely to be important in the pathophysiology of atherosclerosis. Increased platelet reactivity ex vivo has been demonstrated in disorders associated with diffuse vascular disease, diabetes mellitus and some hyperlipidaemias. Platelets circulate as disc-shaped cells and are subject to rheological forces which lead to platelet-endothelial cell interactions. Several interdependent changes occur during activation: adhesion, shape change, aggregation and the release reaction. The interaction between platelets and exposed subendothelial elements, including collagen and fibronectin, occurs with great rapidity. Such adhesion is mediated by a plasma macromolecule derived from endothelial cells and platelets themselves -von Willebrand factor-and platelet membrane glycoprotein receptors. Platelets possess contractile elements including actin and on stimulation undergo a morphological change to spiny spheres and interact with adjacent platelets to form an aggregate. Aggregation involves the association of fibrinogen and other adhesive proteins with different membrane glycoprotein receptors exposed on the surface of adjacent platelets and occurs in response to a wide range of physiologically important agonists including adrenaline and thrombin. Aggregation is usually coupled with the release reaction in which granular components are secreted in an irreversible process; this releases ADP, serotonin and other agonists from their intra-
cellular stores, resulting in the recruitment of more platelets into the aggregate. More is now known of the mechanisms of agonist-response coupling in blood platelets. As in other secretory cells, calcium mobilisation plays a central role, as does peroxidation of arachidonic acid from membrane phospholipids to form the potent aggregator and vasoconstrictor thromboxane A2. Intracellular cyclic AMP plays a regulatory role and is partly under the control of prostaglandin I2, a powerful aggregation inhibitor and vasodilator synthesised predominantly by the cells of the vascular endothelium. Aggregation leads to exposure of phospholipid components of platelet membrane which act as potent catalysts in the fluid phase of coagulation leading to fibrin deposition. Clearly the processes of platelet activation are complex and dependent upon rheological, biochemical and cellular interactions which are not easily reproduced in vitro. The assessment of platelet function has most commonly relied upon the turbidometric method of Born (1962) in which platelets, isolated from some other cellular components of blood and suspended in plasma, are exposed to a variety of agonists in variable concentrations. Shape change and aggregation are monitored by the resulting changes in light transmission through the suspension (Born, 1962). The simultaneous measurement of thromboxane and products of release such as serotonin may provide additional information (Greaves & Preston, 1985). The effects of many drugs and their mechanisms of action have been studied in this manner. Aspirin and ticlopidine are for example particularly potent inhibitors in this type of system. However, such an approach to the assessment of drug effects on primary haemostasis has many limitations. Anticoagulated blood must be used for the preparation of platelet rich plasma. This may lead to platelet activation during the procedure, loss of platelet subpopulations and to separation of platelet responses from the activation of coagulation. These processes are interdependent in vivo. The method also provides little information on the important process of platelet adhesion, and the physiologically crucial effects of erythro-
176
M. Greaves
cytes (which are rich in ADP, a platelet agonist), leucocytes and endothelium are not monitored. Some but not all of these limitations have been overcome by development of methods for the study of adhesion/aggregation in whole blood in which activation is detected by counting residual individual platelets after stirring with an agonist (Saniabadi et al., 1983), or by alteration in electrical impedance associated with platelet activation (Cardinal & Flower, 1980). These methods have also been applied to pharmacological studies. It is pertinent that the phosphodiesterase inhibitor, dipyridamole, has little or no effect on aggregation at pharmacological concentrations in platelet-rich plasma but is a significant inhibitor in whole blood preparations (Gresele et al., 1983; Heptinstall et al., 1986). Red cells must be present to demonstrate this inhibitory effect. The measurement of platelet adhesion remains a technically difficult area. Methods involving the passage of blood through columns of glass beads have been applied to drug evaluation (David et al., 1979) but have poor reproducibility and measure aggregation rather than adhesion (Greaves & Preston, 1985). Rheological factors are important and ingenious methods have been devised which use perfusion chambers containing de-endothelialised segments of mammalian blood vessel mounted on probes (Baumgartner & Haudenschild, 1972; Escolar et al., 1985). In this issue of the journal Muller and colleagues (1990) describe experiments utilising a novel approach to the in vitro assessment of platelet adhesion and aggregation in the presence of subendothelial components. When vascular endothelial cells are grown in culture they generate a subendothelial matrix which resembles that of the vascular subendothelial basal lamina and possesses the same important macromolecules including fibronectin, collagen, laminin and proteoglycans. This matrix has been shown to be capable of initiating many of the changes which occur during platelet activation at sites of endothelial injury, thus providing a convincing model for the study of platelet
subendothelial interactions ex vivo (Booyse et al., 1982; Eldor et al., 1985; Vlodavski et al., 1982). Using this system Muller et al. (1990) have confirmed the activity of aspirin as a platelet inhibitor and also suggest that dipyridamole has significant inhibitory activity. It is apparent that no test of platelet function can reflect comprehensively the complex interactions which occur during platelet (sub) endothelial interactions in vitro. A multifaceted approach is clearly needed for the assessment of platelet reactive drugs ex vivo. Further application of methodology such as that described by Muller et al. (1990) may add to our understanding of the pharmacology of anti-platelet agents, but a full assessment will still require measurement of in vivo parameters of platelet function in carefully controlled studies. The standardised skin bleeding time remains a useful in vivo test for the assessment of primary haemostasis in health and disease (Greaves & Preston, 1985). In conditions where pathological platelet activation is implicated measurement of plasma products of the release reaction, P-thromboglobulin and platelet factor 4 (Kaplan & Owen, 1981), and of the survival of isotopically labelled autologous platelets remain essential approaches for the assessment of significant pharmacological activity. Newer methods, using monoclonal antibodies directed against antigens exposed during platelet activation (Abrams et al., 1990) may also prove useful in the assessment of the ability of drugs to inhibit activation in vivo. Finally, it is noteworthy that it has proven to be extremely difficult to demonstrate clinical efficacy for dipyridamole (Anonymous, 1984), despite apparent activity in some tests of platelet function. The most sophisticated laboratory approaches to testing antiplatelet drugs will never replace the need for well-conducted clinical trials. M. GREAVES Department of Haematology, Royal Hallamshire Hospital, Glossop Road, Sheffield SJO 2JF
References Abrams, C. S., Ellison, N., Budzynski, A. N. & Shattil, S. J. (1990). Direct detection of activated platelets and platelet-derived microparticles in humans. Blood, 75, 128-138.
Anonymous (1984). Doubts about dipyridamole as an antithrombotic drug. Drug and Therapeutics Bulletin, 22, 25-28. Baumgartner, H. R. & Haudenschild, C. (1972),
Editorial Adhesion of platelets to subendothelium. Ann. N. Y. Acad. Sci., 201, 22-36. Booyse, F. M., Quarfoot, A. J. & Feder, A. (1982). Culture-produced subendothelium I. Platelet interaction and properties. Haemostasis, 11, 4961. Born, G. V. R. (1962). Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature, 194, 927-929. Cardinal, D. C. & Flower, R. J. (1980). The electronic aggregometer: a novel device for assessing platelet behaviour in blood. J. pharmac. Methods, 3, 135-138. David, J. L., Monfort, F., Herion, F. & Raskinet, R. (1979). Compared effects of three dose-levels of ticlopidine on platelet function in normal subjects. Thrombos. Res., 14, 35-49. Eldor, A., Vlodavski, I., Martinowicz, V., Fuks, Z. & Colter, B. S. (1985). Platelet interaction with subendothelial extracellular matrix. Plateletfibrinogen interactions are essential for platelet aggregation but not for matrix induced release reaction. Blood, 65, 1477-1483. Escolar, G., Bastida, E., Ordinas, A. S. & Castillo, R. (1985). Interactions of platelets with subendothelium in humans treated with acetylsalicylic acid and dipyridamole alone or in combination. Thrombos. Res., 40, 419-424. Greaves, M. & Preston, F. E. (1985). The laboratory investigation of acquired and congenital platelet disorders. In Blood coagulation and haemostasis:
177
A practical guide, ed. Thompson, J. M., Third Edition. London, Edinburgh: Churchill Livingtone. Gresele, P., Zoja, C., Deckmyn, H., Arnout, J., Vermylen, J. & Verstraete, M. (1983). Dipyridamole inhibits platelet aggregation in whole blood. Thrombos. Haemostas., 50, 852-856. Heptinstall, S., Fox, S., Crawford, J. & Hawkins, M. (1986). Inhibition of platelet aggregation in whole blood by dipyridamole and acetylsalicylic acid. Thrombos. Res., 42, 215-223. Kaplan, K. L. & Owen, J. (1981). Plasma levels of Bthromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood, 57, 199-202. Muller, T. H., Su, C. A. P. F., Weisenberger, H., Brickl, R., Nehmiz, G. & Eisert, W. G. (1990). Dipyridamole alone or combined with low-dose acetylsalicylic acid inhibits platelet aggregation in human whole blood ex vivo. Br. J. clin. Pharmac., 30, 179-186. Saniabadi, A. R., Lowe, G. D. O., Forbes, C. D., Prentice, C. R. M. & Barbenel, J. C. (1983). Platelet aggregation studies in whole human blood. Thrombos. Res., 30, 625-632. Vlodavski, I., Eldor, A., Hy-Am, E., Atzmon, R. & Fuks, Z. (1982). Platelet interaction with the extracellular matrix produced by cultured endothelial cells: A model to study the thrombogenicity of isolated subendothelial basal lamina. Thrombos. Res., 28, 179-191.
