Abstract:
Protein Z dependent protease inhibitor (ZPI) is a human plasma serpin that inhibits factors Xa and XIa in the coagulation pathway. The hypothesis that a deficiency of this protein could cause thrombosis is supported by studies in KO mice which showed that combined homozygous factor V Leiden and homozygous ZPI deficiency causes lethal thrombosis. The association is not so well established in clinical studies. We have identified three point mutations in a thrombosis population which on molecular modelling could potentially alter ZPI function. The aim of this study was to examine the effect of these point mutations on ZPI activity. Recombinant wtZPI and ZPI mutants with S122Y, F124L or Q363R point mutations were expressed in E. coli. The wtZPI and S122Y and F124L mutants had characteristics similar to those described for the native protein; the circular dichroism showed an unfolding transition on thermal denaturation typical of a serpin with Tm of 58.8oC. Heparin affinity was similar in all proteins. The rZPI Q363R yielded a large insoluble fraction but sufficient protein was available for kinetic studies. Discussion: The Q363R variant had a 40 fold reduction in inhibition of fXa in the presence of protein Z. Mutations in antitrypsin and thyroxine binding globulin in this region (strand 5A) are associated with both altered stability and inhibitory function consistent with our findings. The S122Y mutation had inhibitory activity similar to wtZPI and the F124L mutation showed near normal fXa inhibition but a 4 fold reduction in fXIa inhibition. Both F124L and S122Y are located in the D helix which is critical for protein binding and folding notably in antithrombin. S122Y has similar stability and function to wtZPI suggesting that an exposed position within the D helix is necessary to alter inhibitory function. The F124L and Q363R mutations reduce the inhibitory efficiency of ZPI and therefore maybe associated with a type 2 pattern of deficiency in vivo.