The structure and function of ZPI, and the role of ZPI deficiency in venous thrombosis

Abstract

Protein Z dependent protease inhibitor (ZPI) is a plasma inhibitor of factors Xa and XIa. In an earlier study, five mutations were identified within the ZPI gene of venous thrombosis patients and healthy controls. Two of these were nonsense mutations and three were missense mutations within the D helix (F145L and S143Y) and β sheet A (Q384R). Recombinant wildtype and mutant proteins were prepared in Escherichia coli to compare the stability and kinetics. The wildtype protein efficiently inhibited Xa (in the presence of its cofactor, protein Z) and also XIa, particularly in the presence of unfractionated heparin, suggesting a physiologically important role for heparinoids in vivo. The two D helix mutations were readily expressed and had similar stability to the wildtype in response to thermal challenge, however the yield of the Q384R variant was low; inhibition of Xa in the presence of protein Z was markedly reduced with no measurable inhibition of XIa by this protein. The inhibition of XIa by the F145L mutant was reduced 2-3 fold compared to the wildtype. An analysis of all five ZPI mutations was undertaken in a cohort of venous thrombosis patients (n=550) compared to healthy controls (n=600) to correlate possible ZPI deficiency with thrombosis risk. Overall, there was a modest increase in incidence of these mutations in the thrombosis group (OR 2.0, 1.05-3.7, p=0.044) however in contrast to W324X (nonsense mutation), the Q384R missense mutation and R88X nonsense mutation were evenly distributed in patients and controls; F145L was rare. The final mutation (S143Y) was also rare and did not significantly alter ZPI function in laboratory studies. The nonsense and Q384R mutations reduced plasma ZPI in both patients and controls, although there was no difference in ZPI levels between the two groups overall. The F145L and particularly the Q384R mutation impaired the function of the coagulation inhibitor ZPI, however there was no convincing association between these functional mutations, the nonsense mutations and venous thrombosis risk. The functional role for ZPI in vivo has yet to be clarified.