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Kv1.3 is a voltage-gated K

Platelet activation is traditionally quantified using turbidimetric aggregometry, which reflects integrin αIIbβ3 activity, an important determinant of platelet function during pathophysiological thrombus formation. However, aggregometry does not recreate the shear conditions prevailing during thrombosis in vivo. Here we describe novel whole-frame analysis of real-time video microscopy to quantify platelet adhesion receptor activity under shear in parallel-plate flow chambers. We demonstrate that the rate of change of surface coverage (designated Platelet Population Mobility, PM) quantifies platelet mobility. On collagen, PM falls exponentially to a low level, corresponding to firm platelet adhesion, while on other substrates, PM remains high. Different receptor-specific thrombogenic surfaces reveal that the PM time constant reflects real-time changes in integrins αIIbβ3 and α2β1 activity. This ensemble kinetic analysis has the potential to provide valuable diagnostic information about platelet thrombus formation with both academic and clinical applications.

Human Domain Antibodies (dAbs) that bind to and inhibit the function of platelet glycoprotein VI (GPVI) have been isolated from phage display libraries and their efficacy demonstrated using in vitro models of platelet activation. Here we describe the properties of one such antibody, BLO8-1, which has been shown to specifically inhibit the binding of recombinant human GPVI to cross-linked collagen related peptide (CRP-XL) in vitro. BLO8-1 specifically binds to the platelet cell surface and prevents CRP-XL induced platelet aggregation in platelet-rich plasma, as well as inhibiting thrombus formation in whole blood under arterial shear conditions. Using a series of mutant GPVI molecules, BLO8-1 was shown to recognize an epitope within the collagen binding domain of GPVI, therefore the anti-thrombotic effect of this dAb is predicted to be due to direct blocking of the collagen-GPVI interaction. These data, together with the desirable properties of Domain Antibodies, show that dAbs could potentially be used to generate novel biopharmaceuticals with anti-thrombotic properties.

Equine platelet aggregation was stimulated by collagen fibres or platelet-activating factor. The action of both ligands was blocked by forskolin or prostaglandin E(1) agents which are known to activate adenylate cyclase. Equine platelet membranes were found to contain adenylate cyclase activity which was inhibited in dose-dependent fashion by both collagen and platelet-activating factor. Platelet-activating factor-induced inhibition was antagonised by WEB2086.

Platelets in an advanced stage of activation change from coagulation-inactive to coagulation-promoting cells. This procoagulant response is characterised by exposure of aminophospholipids, such as phosphatidylserine, to the platelet surface and by formation of microvesicles. Under specific conditions, when both signalling and adhesive platelet receptors are occupied, collagen and also thrombin are able to trigger this response. Thus, platelets express high coagulation-promoting activity only after interacting with multiple receptors.