Interactions of the platelet GP Ib-IX-V complex with immobilized von Willebrand factor under flow conditions

[摘要] Platelets play key roles in physiology, such as the arrest of bleeding following vascular injury, and in pathology, such as mural arterial thrombosis. The initial step in both processes is platelet adhesion to von Willebrand factor. The adhesion molecule on the platelet surface responsible for this interaction is the platelet glycoprotein (GP) Ib-IX-V complex. This complex consists of four subunits, and the GP Ibasubunit contains the vWf binding site. The exact region or regions of GP Ibathat are involved in the GP Ib-IX-V -- vWf interaction are unknown.The purpose of this work was to investigate the importance of several structural domains of GP Ibain the GP Ib-IX-V -- vWf interaction under high fluid shear stress. This was done by several methods which all used a parallel-plate flow chamber to create wall shear stress: an antibody blocking study of cell rolling, mutational analysis of four different domains of GP Iba , and dog/human chimeras of GP Iba .First, we evaluated the blocking effects of GP Ibaand vWf antibodies on rolling of CHO cells expressing the GP Ib-IX complex on immobilized vWf and then compared this data with the antibody blocking studies of modulator- and shear-induced platelet aggregation. Our results suggest that the mechanism of the GP Ib-IX-V -- vWf interaction in rolling is different than the mechanisms involved in shear- and modulator-induced platelet aggregation.Next, we used mutational analysis to investigate the importance of the leucine-rich repeat (LRR) region, the disulfide loop region, the anionic sulfated region, and the cytoplasmic region of GP Ibain the GP Ib-IX-V -- vWf interaction under high fluid shear stress. By comparing the interaction of the mutant cells with immobilized vWf to the interaction of the wild-type cells with the same surface, we found that all four of these regions of GP Ibaare important in the receptor-ligand interaction under fiow.Finally, using dog/human chimeras of GP Iba , we found that the N-terminal 59 residues containing the N-terminal flanking disulfide loop and the first LRR of GP Iba , may not be ligand binding sites for vWf but rather may be regulatory elements for the GP Ib-IX-V -- vWf interaction under high fluid shear stress. Using re-humanized dog/human chimeras of GP Iba , we found evidence to support the involvement of the second, third, and fourth leucine-rich repeats in the receptor-ligand interaction.

[发布机构] Rice University

[效力级别] engineering