P.A. Castaldi

Thrombin Interactions with Platelet Membrane Proteinsa

Thrombin is among the most potent of the physiological stimuli for platelets. In response to thrombin, platelets change shape from discoid to spherical, secrete the contents of their storage organelles, and providing extracellular calcium ions are present, aggregate. Despite intensive efforts during the past 10-20 years, the mechanism by which a-thrombin stimulates platelets is an essentially unresolved problem in hemostasis. One difficulty in understanding the mechanism of thrombin interaction with platelets is that the reaction contains elements consistent with both a catalytic event (receptor processing) and a more classical receptor-agonist equilibrium.’ In addition, there is evidence to suggest that there may be more than one type of thrombin receptor on platelets. The potent thrombin inhibitor, hirudin, rapidly dissociates bound thrombin from platelets.’ If hirudin is added during the lag period between thrombin addition and the onset of dense body secretion, no secretion occurs.’ If hirudin is added after this lag, the subsequent platelet secretion and aggregation responses are unaffe~ted.’.~ By contrast, hirudin added at this point blocks lysosomal secretion and the mobilization of arachidonic acid, indicating a difference in the requirement for receptor occupancy with these reactions. A similar conclusion can be derived from the analysis of the interaction between the dysthrombin, thrombin quick I, and platelets.’ There is considerable evidence that the activation of platelets by thrombin involves the interaction of platelets with the thrombin active site. First, the catalytic active site of a-thrombin is essential for the physiological platelet response inasmuch as active-site-blocked a-thrombin does not stimulate platelets.68 Second, proteases whose cleavage specificity overlaps that of a-thrombin, trypsin? papain,’ thrombocytin,’” and a-clostripain,” activate platelets, but only when catalytically active, whereas proteases with dissimilar specificity such as plasmin and a-chymotrypsin do not.’ Third, a competitive inhibitor of a-thrombin, p-(p-nitrophenoxypropoxy)-benzamidine benzenesulfonate inhibits platelet stimulation with a y essentially identical to that

Factor VIII/von Willebrand Factor has potent lectin activity

Highly-purified plasma and platelet Factor VIII/von Willebrand Factor had potent lectin activity when measured in a haemagglutination assay. This lectin activity was inhibited by monoclonal and heterologous antibodies to Factor VIII/von Willebrand Factor as well as by hexosamines, mannose and net-positively charged amino acids.

Evidence for a platelet membrane defect in the myeloproliferative syndromes

Bleeding and abnormal platelet aggregation occur in patients with myeloproliferative disorders. In this study, twenty patients were examined, some sequentially, and a proportion found to have defective aggregation toward adrenaline, adenosine diphosphate (ADP), and collagen. In these seven patients, the abnormality in platelet response with defective collagen-induced [14C]serotonin release correlated with poor collagen-stimulated thromboxane B2 (TXB2) production. In contrast, five of these patients showed a normal threshold aggregation response to arachidonic acid. The combined results suggest that in these patients, there is a defect between receptor-stimulus coupling and the mobilization of arachidonic acid from membrane phospholipid.

Characterization of calcium-dependent binding of endogenous factor VIII/von Willebrand factor to surface activated platelets.

Activation of washed platelets in the presence of EDTA with either 1 U/ml of alpha-thrombin or 2 microM calcium ionophore (A23187) caused the release of one-third to one-half of the platelet factor VIII/von Willebrand factor (FVIII/vWF) into the supernatant. When calcium was present in excess, only 10% of the platelet FVIII/vWF was detected free in the supernatant, regardless of whether calcium was present before stimulation or added to the platelets after thrombin activation. Release of [14C]serotonin and beta-thromboglobulin were not affected by divalent cations indicating that reduced supernatant levels of FVIII/vWF in the presence of calcium were not due to differential release, but were probably due to a calcium-dependent association of released FVIII/vWF with the platelet surface. The presence or absence of intact glycoprotein Ib on the platelet surface made no significant difference to the observed FVIII/vWF partition. Platelets from a patient with Glanzmanns thrombasthenia, however, failed to show a calcium effect with respect to released FVIII/vWF. The combined results suggest that as well as the ristocetin-dependent, divalent cation-independent binding of FVIII/vWF to glycoprotein Ib, there is a divalent cation-dependent binding of FVIII/vWF to the activated platelet surface which is mediated via the glycoprotein IIb/IIIa complex.

Platelet thrombospondin haemagglutinin activity is due to aggregate formation

Thrombospondin released from human blood platelets by thrombin activation formed high molecular weight aggregates which co-eluted with haemagglutinin activity on Sepharose 4B gel filtration. Thrombospondin aggregation was mediated by intermolecular disulphide bridges. The aggregates consisted of a series of oligomers ranging from a dimer to polymeric forms with Mr congruent to 40 X 10(6). Native monomeric thrombospondin obtained by a modified procedure was deficient in haemagglutination activity but inhibited haemagglutination induced by aggregated thrombospondin.

Medical research: goals, problems and impacts*

The essential object of research in any field could be summarized as the process of discovery of new information, that is, processes or mechanisms leading to the technical innovations necessary for the insertion of the new knowledge into some aspect of life. This implies an essential link between the initial invention or discovery and the subsequent process of innovation or dissemination of the new information, the two steps being interdependent. Thus ‘discoveries’ result not only from the ideas, skills and energy of the researcher but also need someone’s technical capacities to produce methods and processes which allow society to adopt and benefit from them. A third link in this chain is also essential: like the biblical seed that must fall on fertile soil, so the products of discovery or the new ideas that emerge must fall on a receptive public or community before they can flourish and enrich our lives. They must be seen to fill a need that has been created by society or is perceived by the consumers. Research must be sold in the marketplace, and neglect of this third link has catastrophic consequences for research people and for the whole community. There is no point in making brilliant new discoveries, in inventing new processes or generating new ideas and theories if means of exploiting them are lacking or the society to which they are offered is indifferent to their possibilities. Scientists, medical or otherwise, need the conviction of the worth of innovation and of educating the public and its administrators to their own needs. Society, and especially its decision makers, must appreciate the value of research and understand the implications of indifference to creativity. Without doubt this indifference is one of the most serious deficiencies of our society and one that scientists must help to correct. Leaders in science, politics and philosophy often speak out in this country but to a pitiably small audience of ABC listeners or readers of papers like Quadrant. Australia needs

Predisposing factors to arterial thromboembolism: Can we detect those at risk?

Risk factors in populations at risk of atherosclerosis and thrombosis are known to include environmental, dietary, metabolic and disease associations that may be altered or manipulated in individual patients. Less is known or established about the importance of changes in platelet or coagulation behaviour that may contribute to ongoing vascular disease or to acute occlusive episodes. Current research tends to suggest an important role for platelets in the genesis of atheroma, especially since the discovery of platelet growth factor which may have mutagenic properties on vessel smooth muscle cells. Additional roles are suspected for platelet and vessel prostaglandins and current work is devoted to discovering how these important mediators and inhibitors of platelet activity may contribute to thrombosis. Coagulation and platelet function studies have been performed in patients with arterial thrombotic disease. Many of these have been investigated at the time of presentation. Global tests of coagulation and fibrinolysis have not shown any change, but a significant enhancement of the threshold response to Adenosine Diphosphate has been found and many of these patients had enhanced release of platelet β thromboglobulin. Radioimmunoassay of thromboxane B 2 , showed increased synthesis after collagen stimulation in many patients with ongoing thrombosis. These results suggest that enhanced thromboxane synthesis and increased responsiveness to ADP are important associations of thrombosis. It remains to be determined whether these changes have predictive value.