HD1, a thrombin- and prothrombin-binding DNA aptamer, inhibits thrombin generation by attenuating prothrombin activation and thrombin feedback reactions

 

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Summary

HD1, a DNA aptamer, binds exosite 1 on thrombin and blocks its clotting activity. Because HD1 also binds prothrombin and inhibits its activation by prothrombinase, we hypothesised that HD1 would be a more potent inhibitor of coagulation than other exosite 1-directed ligands, such as Hir^54–65(SO₃ ^-). Supporting this concept, the effect of HD1 on the prothrombin time and activated partial thromboplastin time was twofold greater than that of Hir^54–65(SO₃ ^-) even though both agents inhibited thrombin-mediated factor (F) V and FVIII activation to a similar extent. In thrombin generation assays, HD1 (a) delayed the lag time, (b) reduced peak thrombin concentration, and (c) decreased endogenous thrombin potential to a greater extent than Hir^54–65(SO₃ ^-). To eliminate thrombin feedback, studies were repeated in FV- and/or FVIII-deficient plasma supplemented with FVa and/or FVIIIa. Only HD1 prolonged the lag time in FV- and FVIII-deficient plasma supplemented with FVa and FVIIIa. In contrast, HD1 and Hir^54–65(SO₃ ^-) inhibited the lag time in FVIII-deficient plasma supplemented with FVIIIa and in normal plasma. The more potent anticoagulant properties of HD1, therefore, reflect its capacity to attenuate FV activation by thrombin and inhibit prothrombinase assembly. These findings identify prothrombin as a potential target for new anticoagulants.

^* Recipient of the Heart and Stroke Foundation of Ontario/J. Fraser Mustard Chair in Cardiovascular Research and Canada Research Chair (Tier 1) in Thrombosis at McMaster University.

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