A new platelet activation marker specific for activated integrin alpha2beta1: monoclonal antibody IAC-1

Abstract number: OC139

Schoolmeester^* A., Vanhoorelbeke† K., Lecut‡ C., Heemskerk§ J. M. W., Hoylarts† M. F., Deckmyn† H.

†Belgium; ‡France; ^*Laboratory for Thrombosis Research, IRC, Belgium; §Netherlands

Integrin alpha2beta1 is a major collagen adhesion receptor on platelets. Previous data suggested that alpha2beta1 may assume various activation states. Our aim was to study the requirements of this conformational change. To this end monoclonal antibodies (MAbs) against recombinant alpha2-I-domain were raised and the binding of alpha2-I-domain binders to resting or stimulated platelets was studied by flow cytometry. The control MAb Gi9 bound equally well to human platelets with or without activating collagen related peptides (CRP's). Mab 1D12 did neither bind to resting nor to CRP-activated platelets. MAb IAC-1 failed to bind to resting platelets (3.3%), but, interestingly, bound to 87.8% of the CRP-stimulated platelets, implying that IAC-1 can detect the activated state. Binding assays with 125 I-labelled IAC-1 confirmed the flow cytometry results. Binding of IAC-1 to platelets could also be induced by convulxin, ADP, U46619, thrombin, or VWF-ristocetin. Conversely, also the binding of FITC-labelled human collagen I to platelets stimulated with convulxin or U46619 increased. This binding was dependent on alpha2beta1 as it could be prevented by MAb Gi9. Furthermore, platelet aggregates formed on a collagen surface under flow at high shear (1000 s^-1), stained positive for both FITC-labelled IAC-1 and the activation state marker annexin-V. Platelets still adhering in the presence of an inhibitory anti-GPVI MAb were negative for both annexin-V and IAC-1. The epitope of the three different MAbs was determined in ELISA and Western blot using human/mouse chimeras (gift from D. Tuckwell) The epitope location of Gi9 was confirmed near the collagen binding site of alpha2beta1, between alpha-helix3 and alpha-helix4. Our results suggest that this area is always exposed on the surface of the integrin. The epitope of 1D12, located in the beta-C sheet, is assumed to be hidden in the receptor but available in the recombinant alpha2-I-domain. In contrast, the epitope of IAC-1, mapped at the opposite site of the collagen binding site between alpha-helix3 and the beta-C sheet, is encrypted in the resting receptor but becomes available upon platelet activation. We thus provide a new platelet activation marker, IAC-1, which is specific for the activated integrin alpha2beta1. Furthermore, IAC-1 is a highly interesting tool to unravel the mechanisms leading to alpha2beta1 activation on platelets and other cells and to determine the role of alpha2beta1 in platelet–collagen interaction.