Alloantigenic Polymorphisms of the b–Terminal Domain Impair the Function of Platelet aIIbb3 Integrin

Abstract number: OR219

Hartmut K, Sentot S

The HPA-1a/1b dimorphism (Leu33Pro) located in the PSI domain of platelet b3 integrin has been discussed as a genetic risk factor for cardiovascular disease. Recent studies showed that the Pro33 isoform (HPA-1b) could enhance adhesive functions and also modulates cell migration into extracellular matrix proteins. However, the role of the PSI domain in integrin activation is not yet clear. In contrast, crystal structure analysis strongly indicated that the b–terminal domain (b–TD) plays an important role in the regulation of aIIbb3 integrin states (active-inactive state). Mutations in this region lead to platelet functional defects in patients with Glanzmann's thrombasthenia. We, therefore, analysed the functional impact of point mutations located in the b–TD, which are responsible for the formation of platelet alloantigens (HPA-14, -11, and -8). Full-length b3 cDNA constructs encoding for the respective mutations (dLys611, His633, Cys636) were generated to establish stable CHO cell lines expressing allele-specific aIIbb3 isoforms. Conformational changes leading to fibrinogen binding of aIIbb3 were measured both in inactive and active state after treatment with dithiothreitol (DTT). After DTT treatment, the cell lines expressing Cys636 and His633 showed no/weak binding to monoclonal antibody PAC-1 and do not form cell aggregates in the presence of fibrinogen. In contrast, deletion of Lys611 did not impair both PAC-1 and fibrinogen binding. The consequence of these mutations in outside-in signalling was confirmed by phosphorylation analysis of focal adhesion kinase. In conclusions, these observations indicate that alloantigenic point mutations located in the b–TD of b3 integrin can impair platelet function. These findings allow new insights in understanding of the impact of platelet alloantigens in cardiovascular and immune mediated diseases.