Antithrombotic Efficacy in the Guinea Pig of a Derivative of Human Protein C With Enhanced Activation by Thrombin

Abstract

Conversion by α-thrombin of the zymogen human protein C (HPC) to activated protein C (aPC) is an important physiologic feedback control mechanism for the coagulation cascade. Although activation of HPC by thrombomodulin-bound thrombin is relatively rapid, activation by free thrombin occurs at a significantly slower rate. Previously, we generated a “hyper-activatable” derivative of HPC (FLIN-Q3) with an increased activation rate by free α-thrombin in vitro. In this study, the antithrombotic efficacy of FLIN-Q3 was compared with both native zymogen and aPC in an arteriovenous shunt model of thrombosis in the guinea pig. Recombinant proteins were infused 15 minutes before and throughout a 15-minute period while blood was circulated from carotid to jugular through tubing that enclosed a thread on which fibrin was deposited. Parallel dose-dependent antithrombotic responses were observed. Under these non–steady-state conditions, the calculated infusion doses associated with a 50% reduction of thrombus mass were 2.7, 24, and 250 mg/kg/h for aPC, FLIN-Q3, and HPC, respectively. Thrombus weight correlated inversely with plasma concentration of aPC, measured amidolytically, from either direct infusion of aPC or that generated from the zymogens in the animal, and similarly correlated inversely with anticoagulant activity measured by whole blood aPTT. Neither zymogen form showed significant aPC activity before shunt circulation, suggesting a requirement for exposure to thrombin. After the infusion was discontinued for 15 minutes, a second period of thrombus formation in the shunt demonstrated the ability of zymogen forms of PC, unlike aPC, to provide “on-demand” anticoagulant responses to repeated thrombotic stimuli. Thus, a “hyper-activatable” PC molecule such as FLIN-Q3 may represent a superior form of anticoagulant therapy than either the native zymogen or aPC.

Tissue Factor, Blood Coagulation, and Beyond: An Overview

“…A human APC product (CTC-111) reduces venous thrombosis in mice [ 369 ]. FLIN-Q3 diminishes A-V shunt-induced thrombosis in guinea pigs [ 370 ]. hAPC attenuates rat mesenteric occlusion [ 371 ], and rhAPC inhibits arterial thrombosis in baboons [ 372 ].…”

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“…In examining the anticoagulant/antithrombotic activity of human APC in nonprimates, much higher doses of human APC were used to overcome the cross-species barrier effect. For example, the dose of human APC that produced an antithrombotic effect in a guinea pig was about 2 mg/kg per hour compared with a dose of about 0.015 mg/kg per hour in rhesus monkey [ 51 , 52 ]. Given the antithrombotic effects of APC, it also serves as an indirect inhibitor of the inflammatory activities of thrombin.…”

“…The antithrombotic activity of APC has been well established in various thrombotic models and in multiple animal species [ 9 , 52 , 61 - 65 ]. The antithrombotic activity of drotrecogin alfa (activated) was demonstrated in patients with severe sepsis by the reduction in levels of D-dimers and markers of thrombin generation (F1.2, thrombin–antithrombin complex) compared with placebo-treated patients [ 66 ].…”

“…Using intravital microscopy of the dorsal skin fold of a hamster endotoxemia model, Hoffmann and colleagues [ 24 ] demonstrated that intravenous administration of human plasma-derived APC at 24 μg/kg per hour significantly reduced endotoxin-induced leukocyte rolling and adhesion in both arterioles and venules. At this infusion rate, there is minimal anticoagulant activity of human APC in the hamster due to species specificity [ 52 ]. The study by Hoffmann and colleagues [ 24 ] strongly suggests that these anti-inflammatory properties of APC are independent of its anticoagulant activity.…”

New Anticoagulant Drugs

“…109 Alternatively, the functional activity of recombinant protein C can be enhanced by altering the extent of glycosylation 110 or by generating protein C mutants that are more readily activated by thrombin 111,112 or have longer half-lives. 113…”