Conversion by
alpha-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
alpha-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.