Two missense mutations identified in venous thrombosis patients impair the inhibitory function of the protein Z dependent protease inhibitor

Abstract

<jats:title>Summary</jats:title><jats:p>Protein Z-dependent protease inhibitor (ZPI) is a plasma inhibitor of factor (F)Xa and FXIa. 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 in important regions of the protein. Here we report that two of these latter three mutations, F145L and Q384R, impair the inhibitory function of ZPI in vitro. Recombinant wild-type and mutant proteins were prepared; stability in response to thermal challenge was similar. Inhibition of FXa in the presence of the cofactor protein Z was reduced 68-fold by the Q384R mutant; inhibition of FXIa by the F145L mutant was reduced two- to three-fold compared to the wild-type ZPI. An analysis of all five ZPI mutations was undertaken in a cohort of venous thrombosis patients (n=550) compared to healthy controls (n=600). Overall, there was a modest increase in incidence of these mutations in the thrombosis group (odds ratio 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 F145L and particularly the Q384R mutation impaired the function of the coagulation inhibitor ZPI; however, there was no convincing association between these mutations and venous thrombosis risk. The functional role for ZPI in vivo has yet to be clarified.</jats:p>

Single Nucleotide Variant rs2232710 in the Protein Z-Dependent Protease Inhibitor (ZPI, SERPINA10) Gene Is Not Associated with Deep Vein Thrombosis

“…However, independent replication in the two Dutch studies LETS and MEGA did not reproduce this finding. These results confirmed in a large replication cohort the lack of association of the rs2232710 variant with DVT that was previously observed by Young et al [ 20 ].…”

“…We identified two SNVs, rs2232710 in the ZPI gene and rs36117715 in the COL6A3 gene that were predicted to be damaging by SIFT prediction ( Table 1 ). The rs2232710 SNV encodes a missense change of Glutamine to Arginine at position p.Gln384Arg (Q384R) that has been previously shown to impair the inhibitory activity of the ZPI protein towards activated factor X (FXa) in vitro [ 20 ]. The rs36117715 SNV encodes Proline to Lysine amino acid change at position p.Pro2218Lys (P2218L) and its effect on the structure or function of the Col6a3 protein is unknown.…”

“…The reasons for the different prevalence of the rs2232710-C risk allele among controls and cases in DVT-Milan replication population and in the two independent Dutch cohorts LETS and MEGA as well as that of Young et al [ 20 ] may be the different recruitment criteria for patients and controls, different ancestral origin or a random effect due to the relatively small Italian replication cohort. Since other genetic risk factors such as FVL and prothrombin G20120A variants or deficiencies in natural anticoagulants (protein C, protein S and antithrombin) can influence the predisposition to develop venous thrombosis, patients carrying these defects were excluded from our analysis.…”

“…Since other genetic risk factors such as FVL and prothrombin G20120A variants or deficiencies in natural anticoagulants (protein C, protein S and antithrombin) can influence the predisposition to develop venous thrombosis, patients carrying these defects were excluded from our analysis. In the study conducted by Young et al [ 20 ], only the presence of the FVL variant was reported leaving all the other thrombophilia markers unchecked. The different patient recruitment criteria may also be a reason for the negative replication of the rs2232710 in the Dutch population where patients with provoked and unprovoked first events of deep vein thrombosis or pulmonary embolism have been included [ 34 , 35 ].…”

“…However, the combination of ZPI deficiency with the homozygous FVL variant led to a more severe thrombotic phenotype than PZ KO/FVL, implying an important role for the ZPI protein in the inhibition of activated XI [ 19 ]. Recently, two missense mutations F145L and Q384R in the ZPI gene were shown to impair the inhibitory activity of the ZPI protein in vitro , even though they were not associated with DVT in humans [ 20 ].…”