Modulating recombinant clotting factor X to improve clot-dissolution

Abstract

Introduction

Heart attacks and strokes are the leading causes of death worldwide, most often caused by clots that block the flow of blood. The favoured clot-dissolving (i.e. thrombolytic) drug is a recombinant (r) version of tissue plasminogen activator (tPA). The high rtPA dose required for clot lysis causes clinical hemorrhage in up to 6% of patients, resulting in part from systemic, rather than clot-localized, enzyme activity. The Pryzdial lab has discovered a thrombolytic function for the plasma protein, clotting factor X (FX), which acts non-enzymatically to accelerate tPA. Here we present a recombinant variant of FX (rFXic) with two key characteristics: an inhibitory (i) mutation that blocks the intrinsic clotting function, and a cleavage-resistant (c) mutation for increased half-life of tPA-accelerating function in plasma. We hypothesize that rFXic is thrombolytic and has superior safety compared to rtPA.

Methods

Wild type (rFXwt), single mutant (rFXi and rFXc), and double mutant (rFXic) FX were produced in HEK 293 cells and purified via conformational affinity chromatography. Their plasmin-cleavage profile and prothrombin clotting times functionally confirmed the successful insertion of mutations. Calcium-dependent binding to anionic phospholipid was tested to evaluate proper post-translational modification and clot-localizing function of the γ-carboxyglutamic acid (Gla)-domain, which is known to enable binding of FX to anionic phospholipid-containing membrane and fibrin. Acceleration of rtPA activity was evaluated using a plasmin-selective chromogenic substrate. In a mouse model of carotid artery occlusion, Doppler ultrasound recordings of blood flow were used to measure the ability of rFXic to affect clot dissolution.

Results

Compared to rFXwt, which was cleaved by plasmin into the predicted rFXβ and FXγ forms of FX, proteolysis of rFXic was limited to production of rFXβ. This is expected to stabilize thrombolytic activity in plasma. In contrast to rFXwt, rFXic had undetectable clotting activity in reconstituted FX-deficient plasma. Neither mutation impacted calcium-dependent binding to anionic phospholipid. In vitro, rtPA generated 10-fold more plasmin in the presence of rFXic than rFXwt, indicative of thrombolytic acceleration by the former. In mouse models of thrombosis, rFXic decreased the thrombolytic dose of rtPA by at least 50% as an adjunctive therapeutic but did not promote thrombolysis without rtPA.

Conclusion

These data support the hypothesis that rFXic has thrombolytic activity in combination with rtPA. By lowering the therapeutic dose of rtPA, rFXic could be used as an adjunctive therapeutic to reduce the bleeding risk of thrombolysis. Next, we will assess the quantitative efficacy of rFXic in vivo and therapeutic safety ex vivo, and anticipate advocating for rFXic as both an effective and safer alternative to monotherapeutic rtPA.