Phage display selection of peptide inhibitors of FVIIa and their functional characterisation

English: The importance of FVlla and the FVlla/TF complex for the initiation of not only hemostasis but also thrombosis is now generally accepted. It was shown that the blockade of coagulation at the level of FVlla provided full anti thrombotic protection without abnormal bleeding (Harker et aI, 1996), therefore FVlla is a suitable candidate for the development of novel antithrombotics. We selected inhibitors to FVlla using the technique of phage display. A repeated selection of phages from a cyclic heptapeptide and a linear 12-mere phage display library resulted in the enrichment of phages that bind to human FVlla. We selected twelve colonies (6 from each library) that showed the strongest binding to FVlla. The colonies from the cyclic 7-mere library showed a higher affinity binding for FVlla than the colonies from the linear 12- mere library. TF also prevents the binding of one of the cyclic colonies to FVlla. This colony as well as one colony from the linear library showed lengthening of the prothrombin time (PT) as well as the thrombin time (TT) in a dose-dependent manner. A cyclic heptapeptide was synthesised with the corresponding sequence as the sequence displayed on the cyclic 7-mere colony. The peptide showed lengthening of the PT and TT in a dose-dependent manner with a more pronounced effect on the PT than the TI. We also studied the effect of this peptide on platelet adhesion on human vascular endothelial cell matrix, collagen and TF under both venous and arterial shear stresses. The peptide inhibits platelet adhesion to HMEC-1 under both shear stresses. The effect on arterial shear is however more pronounced. It does not inhibit platelet adhesion to collagen, but has a dose-dependent inhibitory effect on platelet adhesion TF at arterial shear. Kinetic analysis of the peptide showed that this peptide is a competitive inhibitor of FVlla by altering the Km-values but not the Vmax-values. The Lineweaver-Burk plot also indicates a competitive inhibition, because the slope of the graphs increased with increasing inhibitor concentrations. The Ki-value was determined at 0.1232 mM. In summary, this peptide inhibits thrombus formation by preventing FVlla from binding to TF and therefore preventing the activation of FX by the FVlla/TF complex. This study suggests that inhibitors to FVlla provide a novel therapeutic approach to prevent thrombosis.