Dominant negative anticoagulant effects of mouse recombinant meizothrombin precursor R157A/R268A delay thrombus formation in a mouse injured carotid artery

Abstract number: OC413

Shim K., Zhu H., Westfield L. A., Sadler J. E.

Washington University School of Medicine, USA

Compared to thrombin, human meizothrombin (mZa), a naturally occurring unstable intermediate, has enhanced anti-coagulant function and decreased procoagulant activity. A variety of evidence suggests that mZa may have dominant anti-coagulant effects in vivo. To test this in the mouse, a putative mouse recombinant meizothrombin precursor, R157A/R268A, was expressed in BHK cells. The protein was purified using Q-Fast Sepharose chromatography and active mZa was obtained by digestion with bead-conjugated Echis carinatus venom. Thrombin (IIa) was prepared by a standard method. Although the major autocleavage site was mutated, mZa was still converted slowly to meizothrombin des F1 (mZa des F1) like species. All enzymes were quantitated by active site titration with PPACK. The mZa, mZa des F-1 and IIa had similar activity toward S2238 with Km values of 3.00 ± 0.4 µM, 3.09 ± 0.7 µM, and 2.43 ± 0.3 µM, respectively. Mouse mZa and mZa des F1 had reduced fibrinogen clotting activity of 25–30% compared to IIa. In contrast, mZa and mZa des F1 had normal thrombomodulin-mediated-protein C activation activity; mZa showed a stronger dependency on Ca⁺⁺ , and activity was nearly abolished in the absence of thrombomodulin. These results are consistent with those of the corresponding human proteins. To assess the effects of mouse mZa in vivo, we developed a model in which a recombinant precursor of relatively stable mouse mZa is given intravenously to mice heterozygous for a prothrombin null allele. These mice have about 50% of normal plasma prothrombin, and approximately normal prothrombin levels were restored by infusion of 50 µg of recombinant proteins, as shown by Western blot analysis. In this model, prothrombin R157A/268 A prolonged the time-to-occlusion after FeCl3 induced carotid artery injury (11.8 min, n = 7, compared 5.3 min, n = 3 for WT protein, P < 0.05) and these effects were dose dependent (up to 50 µg). Thus a mouse meizothrombin precursor, R157A/R268A, has dominant anti-coagulant effects in vivo, probably through enhanced activation of protein C.