A Schoolmeester

Principal Role of Glycoprotein VI in α2β1 and αIIbβ3 Activation During Collagen-Induced Thrombus Formation

Objective—High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant activity. We studied regulation of &agr;2&bgr;1 and &agr;IIb&bgr;3 integrin activation during thrombus formation on collagen. Methods and Results—Blockade of glycoprotein (GP) VI by 9O12 antibody or of P2Y purinergic receptors permitted platelet adhesion but reduced aggregate formation, fibrinogen binding, and activation of &agr;2&bgr;1 and &agr;IIb&bgr;3, as detected with antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of these integrins. Combined blockade of GPVI and P2Y receptors and thromboxane formation abolished integrin activation but still allowed adhesion of morphologically unstimulated, nonprocoagulant platelets. Exogenous ADP partly restored the suppressive effect of GPVI blockade on integrin &agr;2&bgr;1 and &agr;IIb&bgr;3 activation. Adhesion was fully inhibited only with simultaneous blocking of GPVI and &agr;2&bgr;1, indicating that the integrin can support platelet–collagen binding in the absence of its activation. Blockade or absence of GPIb&agr; only moderately influenced integrin activation and adhesion unless GPVI was inhibited. Conclusions—GPVI- and autocrine-released ADP induce affinity changes of &agr;2&bgr;1 and &agr;IIb&bgr;3 during thrombus formation on collagen under flow. These integrin changes are dispensable for adhesion but strengthen platelet–collagen interactions and thereby collagen-induced platelet activation.

Inhibition of platelet adhesion to collagen as a new target for antithrombotic drugs.

Platelet adhesion to a damaged blood vessel is the initial trigger for arterial hemostasis and thrombosis. Platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between collagen within the damaged vessel wall and the platelet receptor glycoprotein Ib/V/IX (GPIb), an interaction especially important under high shear conditions[1]. This reversible adhesion allows platelets to roll over the damaged area, which is then followed by a firm adhesion mediated by the collagen receptors (alpha(2)beta(1), GPVI, ) in addition[2] resulting in platelet activation. This leads to the conformational activation of the platelet alpha(IIb)beta3 receptor, fibrinogen binding and finally to platelet aggregation. Over the past decades, modulation of platelet function has been a strategy for the control of cardiovascular disease. Lately, drugs have been developed that target the fibrinogen receptor alphaIIbbeta3 or the ADP receptor and many of these promising compounds have been tested in clinical trials. However the development of products that interfere with the first step of hemostasis, i.e. the platelet adhesion, has lagged behind. In this review we want to discuss (i) the in vivo studies that were performed with compounds that target proteins involved in different adhesion steps i.e. the VWF-GPIb-axis, the collagen-VWF axis and the collagen-collagen receptor axis and (ii) the possible advantages these putative new drugs could have over the current antiplatelet agents.

Platelet integrin α 2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display

Antibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs). This system was validated using the I-domain of alpha2 integrin as a model. The alpha2beta1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoprotein VI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunised with a DNA vaccine in which the alpha2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT). Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four alpha2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet alpha2beta1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splice-variant specific antibodies that recognise native protein.