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IIIa specific inhibition of platelet aggregation by fibrinogen receptor-antagonists
Fibrinolysis (1996) 10, Suppl. 1, 1-58 © Pearson Professional Ltd 1996
F I B R I N O G E N A N D C A R D I O V A S C U L A R DISEASE: P A T H O P H Y S I O L O G Y 1. I n f o r m a t i o n role of fibrinogen content in f u n c t i o n i n g o f f i b r i n o l y s i s a n d haemostasis G. V. Andreenko, L. V. Podorolskaja, K. A. Nikolskaja, A. V. Savonenko, T. N. Serebrjakova
Moscow State University[BiologicalDepartment] Russia Penetration of dynamical chaos concepts into medical science has an important role in the development of our ideas about the pathology state of the organism. It was shown that emergence of pathology situation was a result of the loss of chaos properties by physiological system, that led to a sharp decrease of essential population parameter - 'norm of reaction' (Ary et al 1987). The distribution curves of indices of fibrinolysis and haemostasis in a population of normal Wistar rats (n--120) were studied. Among the variety of routine indices, Fb concentration was the single factor that had the smallest range - 'norm of reaction' (Kvar--27.1%). Fb content was extremely stable as to activity of fibrinolysis and to functional state of each studied fibrinolytic and haemostatic indices. At the same time, Fb demonstrated the highest sensitivity to pathological factorethanol consumption: higher the ethanol consumption, the nearer was the average Fb level (4.9 1.3 g/l) to upper threshold value of norm of reaction (from 4.5 to 5.6 g/l, and its range of data became smaller (44.6%). The obtained data permit to ascribe Fb to regulatory parameter, reflecting the stability of the fibrinolytic and haemostatic system. The prognostic significance of Fb may be expanded, if one takes into account its norm of reaction.
2. Flow cytometric characterization of GPllb/lIIa specific inhibition of platelet aggregation b y fibrinogen receptor-antagonists S. Barlage, K. Wittig, G. Rothe, G. Schmitz
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg Regensburg Germany Activation of platelets is a major problem in patients with thrombotic and cardiovascular diseases. Recent data suggest that the final step in platelet aggregation is the binding of fibrinogen to the activated GpIIb/IIIa membrane glycoprotein complex. Inhibition of this mechanism results in effective inhibition of aggregation. In addition to the effects on fibrinogen binding we have analyzed the effects of fibrinogen receptor antagonists, such as MK-852 on platelet activation. In a whole blood dual colour flow cytometric assay of platelet activation the expression of GMP-140 (CD62), GpIb (CD42b), and GplIb/IIIa (CD41 a) in response to various stimuli was analysed. Upon stimulation with the thrombin receptor agonist TRAP-6 or ADP, the expression of GMP-140 and GpIb was not changed by MK-852, while MK-852 alone or in combination with the two agonists induced a strong increase in GplIb/IIIa expression. Specificity of effects for RGD binding sites was shown by competition with the tetrapeptide RGDS. The direct effects of MK-852 on fibrinogen binding to the activated GplIb/IIIa complex on ADP-stimulated platelets were further characterized using a polyclonal FITC labelled © Pearson Professional Ltd 1996
fibrinogen antibody. The fibrinogen receptor antagonist dose dependently inhibited binding of fibrinogen with nanomolar potency. Our results suggest that specific effects of fibrinogen receptor antagonists can be determined at the same time as parameters of platelet activation. The assay should be of use for risk assessment as well as monitoring of antiplatelet therapy.
3. How to detect activated coagulation K. A. Bauer
Beth Israel Hospital, Boston, Massachusetts, USA For years, clinicians have sought to employ blood tests to predict thrombotic events in high risk patients. Advances in our knowledge of the biochemistry of coagulation have facilitated the development of sensitive and specific assays that are able to detect the generation of coagulation enzymes in vivo. It has, so far, not been possible to measure directly the levels of most hemostatic enzymes in vivo as they are neutralized rapidly by naturally occurring protease inhibitors or bound to cellular receptors in the locale in which they are generated. Faced with these obstacles, investigators have resorted to developing immunochemical assays for peptides that are liberated with the activation of coagulation enzymes. Studies employing activation peptide assays indicate that a biochemical imbalance between procoagulant and anticoagulant mechanisms can be detected in the blood of patients prior to developing thrombosis and that coagulation activation is suppressed by oral anticoagulant therapy. Properly designed prospective studies will be required to evaluate whether these assays will improve our ability to identify individuals destined to develop a clinically relevant hypercoagulable state. It also remains to be determined whether such assays can pinpoint patients most likely to benefit from prolonged anticoagulant therapy or provide a better means for determining the optimal amount of drug to administer. 4. Fibrinogen: how to e x p l a i n its r i s k factor status G. V. R. Born
The William Harvey Research Institute, London, UK There is convincing evidence that fibrinogen is a major risk factor for myocardial infarction and stroke. The responsible mechanism is still uncertain; it may involve one or more of four possible fibrinogen functions: as the precursor of fibrin in clotting; as an essential co-factor of platelet aggregation; as a factor in rouleaux formation of red cells; and through increasing plasma viscosity. Plasma fibrinogen is increased in cigarette smokers and diminishes very slowly in people who have stopped smoking. Most interestingly, fibrinogen and hypertension act synergistically as risk factors. The mechanism of that is also unknown. However, a possible explanation is suggested by our discovery that different pressor agents, viz. noradrenaline, adrenaline, angiotensin II, L-NAME and desoxycortisone plus salt are associated with increases in the uptake of fibrinogen, as well as of low density lipoprotein, by artery walls. Fibrinolysis (1996) 10, Suppl. 1, 1-58
F I B R I N O G E N A N D C A R D I O V A S C U L A R DISEASE: P A T H O P H Y S I O L O G Y 1. I n f o r m a t i o n role of fibrinogen content in f u n c t i o n i n g o f f i b r i n o l y s i s a n d haemostasis G. V. Andreenko, L. V. Podorolskaja, K. A. Nikolskaja, A. V. Savonenko, T. N. Serebrjakova
Moscow State University[BiologicalDepartment] Russia Penetration of dynamical chaos concepts into medical science has an important role in the development of our ideas about the pathology state of the organism. It was shown that emergence of pathology situation was a result of the loss of chaos properties by physiological system, that led to a sharp decrease of essential population parameter - 'norm of reaction' (Ary et al 1987). The distribution curves of indices of fibrinolysis and haemostasis in a population of normal Wistar rats (n--120) were studied. Among the variety of routine indices, Fb concentration was the single factor that had the smallest range - 'norm of reaction' (Kvar--27.1%). Fb content was extremely stable as to activity of fibrinolysis and to functional state of each studied fibrinolytic and haemostatic indices. At the same time, Fb demonstrated the highest sensitivity to pathological factorethanol consumption: higher the ethanol consumption, the nearer was the average Fb level (4.9 1.3 g/l) to upper threshold value of norm of reaction (from 4.5 to 5.6 g/l, and its range of data became smaller (44.6%). The obtained data permit to ascribe Fb to regulatory parameter, reflecting the stability of the fibrinolytic and haemostatic system. The prognostic significance of Fb may be expanded, if one takes into account its norm of reaction.
2. Flow cytometric characterization of GPllb/lIIa specific inhibition of platelet aggregation b y fibrinogen receptor-antagonists S. Barlage, K. Wittig, G. Rothe, G. Schmitz
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg Regensburg Germany Activation of platelets is a major problem in patients with thrombotic and cardiovascular diseases. Recent data suggest that the final step in platelet aggregation is the binding of fibrinogen to the activated GpIIb/IIIa membrane glycoprotein complex. Inhibition of this mechanism results in effective inhibition of aggregation. In addition to the effects on fibrinogen binding we have analyzed the effects of fibrinogen receptor antagonists, such as MK-852 on platelet activation. In a whole blood dual colour flow cytometric assay of platelet activation the expression of GMP-140 (CD62), GpIb (CD42b), and GplIb/IIIa (CD41 a) in response to various stimuli was analysed. Upon stimulation with the thrombin receptor agonist TRAP-6 or ADP, the expression of GMP-140 and GpIb was not changed by MK-852, while MK-852 alone or in combination with the two agonists induced a strong increase in GplIb/IIIa expression. Specificity of effects for RGD binding sites was shown by competition with the tetrapeptide RGDS. The direct effects of MK-852 on fibrinogen binding to the activated GplIb/IIIa complex on ADP-stimulated platelets were further characterized using a polyclonal FITC labelled © Pearson Professional Ltd 1996
fibrinogen antibody. The fibrinogen receptor antagonist dose dependently inhibited binding of fibrinogen with nanomolar potency. Our results suggest that specific effects of fibrinogen receptor antagonists can be determined at the same time as parameters of platelet activation. The assay should be of use for risk assessment as well as monitoring of antiplatelet therapy.
3. How to detect activated coagulation K. A. Bauer
Beth Israel Hospital, Boston, Massachusetts, USA For years, clinicians have sought to employ blood tests to predict thrombotic events in high risk patients. Advances in our knowledge of the biochemistry of coagulation have facilitated the development of sensitive and specific assays that are able to detect the generation of coagulation enzymes in vivo. It has, so far, not been possible to measure directly the levels of most hemostatic enzymes in vivo as they are neutralized rapidly by naturally occurring protease inhibitors or bound to cellular receptors in the locale in which they are generated. Faced with these obstacles, investigators have resorted to developing immunochemical assays for peptides that are liberated with the activation of coagulation enzymes. Studies employing activation peptide assays indicate that a biochemical imbalance between procoagulant and anticoagulant mechanisms can be detected in the blood of patients prior to developing thrombosis and that coagulation activation is suppressed by oral anticoagulant therapy. Properly designed prospective studies will be required to evaluate whether these assays will improve our ability to identify individuals destined to develop a clinically relevant hypercoagulable state. It also remains to be determined whether such assays can pinpoint patients most likely to benefit from prolonged anticoagulant therapy or provide a better means for determining the optimal amount of drug to administer. 4. Fibrinogen: how to e x p l a i n its r i s k factor status G. V. R. Born
The William Harvey Research Institute, London, UK There is convincing evidence that fibrinogen is a major risk factor for myocardial infarction and stroke. The responsible mechanism is still uncertain; it may involve one or more of four possible fibrinogen functions: as the precursor of fibrin in clotting; as an essential co-factor of platelet aggregation; as a factor in rouleaux formation of red cells; and through increasing plasma viscosity. Plasma fibrinogen is increased in cigarette smokers and diminishes very slowly in people who have stopped smoking. Most interestingly, fibrinogen and hypertension act synergistically as risk factors. The mechanism of that is also unknown. However, a possible explanation is suggested by our discovery that different pressor agents, viz. noradrenaline, adrenaline, angiotensin II, L-NAME and desoxycortisone plus salt are associated with increases in the uptake of fibrinogen, as well as of low density lipoprotein, by artery walls. Fibrinolysis (1996) 10, Suppl. 1, 1-58