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

[摘要] English: The importance of FVlla and the FVlla/TF complex for the initiation of not onlyhemostasis but also thrombosis is now generally accepted. It was shown thatthe blockade of coagulation at the level of FVlla provided full anti thromboticprotection without abnormal bleeding (Harker et aI, 1996), therefore FVlla is asuitable candidate for the development of novel antithrombotics.We selected inhibitors to FVlla using the technique of phage display. Arepeated selection of phages from a cyclic heptapeptide and a linear 12-merephage display library resulted in the enrichment of phages that bind to humanFVlla. We selected twelve colonies (6 from each library) that showed thestrongest binding to FVlla. The colonies from the cyclic 7-mere libraryshowed 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 toFVlla. This colony as well as one colony from the linear library showedlengthening of the prothrombin time (PT) as well as the thrombin time (TT) ina dose-dependent manner.A cyclic heptapeptide was synthesised with the corresponding sequence asthe sequence displayed on the cyclic 7-mere colony. The peptide showedlengthening of the PT and TT in a dose-dependent manner with a morepronounced effect on the PT than the TI. We also studied the effect of thispeptide on platelet adhesion on human vascular endothelial cell matrix,collagen and TF under both venous and arterial shear stresses. The peptideinhibits platelet adhesion to HMEC-1 under both shear stresses. The effecton arterial shear is however more pronounced. It does not inhibit plateletadhesion to collagen, but has a dose-dependent inhibitory effect on plateletadhesion TF at arterial shear. Kinetic analysis of the peptide showed that thispeptide is a competitive inhibitor of FVlla by altering the Km-values but not theVmax-values. The Lineweaver-Burk plot also indicates a competitiveinhibition, because the slope of the graphs increased with increasing inhibitorconcentrations. The Ki-value was determined at 0.1232 mM.In summary, this peptide inhibits thrombus formation by preventing FVlla frombinding to TF and therefore preventing the activation of FX by the FVlla/TFcomplex. This study suggests that inhibitors to FVlla provide a noveltherapeutic approach to prevent thrombosis.

[发布机构] University of the Free State