Protein S Is Antithrombotic In Vivo Independent of APC.
Authors: Heeb Mary J. , Marzec Ulla , Gruber Andras , Hanson Stephen R. 
Published: October 2019

Abstract
Protein S in plasma has APC-independent, direct anticoagulant activity. Preparations of purified protein S vary in this activity, depending on purification methods. Plasma and purified protein S form multimers that do not appear to differ in ability to inhibit prothrombinase activity (Heeb et al J Thromb Haemost 2006) except in the case of protein S purified using quarternary amine resins. We sought to determine if protein S purified by different methods has direct antithrombotic activity in vivo using a well-characterized baboon thrombosis model. A silicon rubber graft was placed between the femoral vein and artery and included a thrombogenic dacron segment that simulated thrombus formed under arterial flow conditions, followed by a teflon chamber of larger diameter that simulated thrombus formed under venous flow, then a region in which a propagating tail thrombus formed. 111In-labeled autologous platelets and 125I-labeled fibrinogen were infused prior to opening the graft. Protein S (2 mg/hr) or saline was infused 1 cm upstream circumferentially along the wall of the shunt for 1 hr after opening the graft. Deposition of platelets and fibrin was monitored for 2 hr. Eleven experiments were performed, using four different baboons. During the 1 hr infusion, affinity-purified recombinant and plasma-derived protein S inhibited platelet deposition by up to 62% in the arterial segment, up to 89% in the venous segment, and up to 100% in the propagating tail thrombus. Recombinant protein S purified by a different method using quaternary resins had weaker activity, consistent with in vitro clotting assay data. Protein S was more effective in experiments performed at flow rates of 40 ml/hr than in experiments performed at flow rates of 100 ml/hr. In other experiments, plasma-derived protein S was infused in the absence and presence of systemically administered antibody that blocked protein C activation (HPC4). Ex vivo blood samples showed that the ability of protein C to be activated was blocked by 91% or more by HPC4. During the 1 hr infusion, protein S suppressed at least 95% of platelet deposition in the absence of HPC4. In presence of HPC4, protein S suppressed platelet deposition by 79% in the dacron segment, and by 97% in the other segments. After protein S infusion was discontinued, a prothrombotic effect due to HPC4 was observed, in that platelet deposition occurred in the graft somewhat faster than in the saline control without protein S and HPC4, and the shunt occluded 20 min after cessation of protein S. Final fibrin deposition 2 hr after the beginning of the experiments was suppressed by protein S by 34 and 67% in the dacron segments and by 83 and 90% in the chambers, in the presence and absence of HPC4, respectively. Plasma from blood taken downstream of the shunt had modest but significant elevation of direct anticoagulant protein S activity, measured in endogenous thrombin potential assays, after local protein S infusion in the presence and absence of HPC4. APC cofactor activity was not significantly changed in any samples, but there was a trend to higher values after local protein S infusion. These experiments are the first to demonstrate that purified protein S has APC-independent, direct antithrombotic activity in vivo in primates, and suggest therapeutic potential for protein S as an antithrombotic agent.