Activated Protein C Levels Found In Trauma Patients Are Insufficient To Inactivate Platelet Factor Va and Produce Coagulopathy In An In Vitro Model

Abstract

Background A subset of severe trauma victims suffering from uncontrolled hemorrhage present to the emergency room with an acute coagulopathy of trauma (ACoT). This condition occurs within the first 30 min following injury, is not the result of resuscitation-associated dilutional effects, and is defined by an INR > 1.2. The mechanisms behind this coagulopathy have not been completely identified, but recent studies posit that the exuberant activation of protein C is a key contributor to ACoT. Protein C is activated through the interaction of thrombin and thrombomodulin on the endothelial surface. Activated protein C (APC) has anti-inflammatory and cytoprotective functions, in addition to regulating coagulation by inactivating factor Va (FVa). Evidence that APC is implicated in ACoT includes: 1) circulating APC levels are elevated 3-5 fold in patients with high injury severity scores (>15) and base deficit (>6); 2) high APC levels (>6 ng/ml, or 105 pM) in these patients are correlated with an increase in prothrombin time (PT) and partial thromboplastin time [Cohen et al. Ann Surg 2012; 255(2): 379-85]. FVa exists in a soluble plasma fraction and in a platelet-associated fraction (in the membrane or alpha-granules). The platelet pool of FVa is more resistant to APC degradation [Camire et al. J Biol Chem 1995; 270(35): 20794-800] and is sufficient to maintain thrombin generation even in patients with severe congenital FV-deficiencies [Duckers et al. Blood 2010; 115(4): 879-86]. We hypothesize that healthy platelets in sufficient quantity will serve as an effective promoter of hemostasis in patients with ACoT, overcoming the effects of APC. Methods and Results We studied the effects of APC on coagulation in commercially obtained FV-deficient plasma (FVdp) and platelets/plasma from healthy volunteers. Tests included prothrombin time, turbidimetric assessment of fibrin cross-linking, and thromboelastography (TEG). Prothrombin time in platelet-free plasma or FVdp subjected to exogenous APC display a dose-dependent response where clotting is only significantly delayed at APC >1 nM, much higher than the systemic concentrations found in the ACoT patients (105pM) or even in subjects treated with drotrecogin alfa, a recombinant APC product intended for the treatment of sepsis (steady-state concentration was found to be ∼790pM) [Macias et al. Clin Pharmacol Ther 2002; 72(4): 391-402]. Examining fibrin crosslinking in a static 96-well turbidimetric plate assay demonstrated that concentrations of APC of 1nM or below had no effect on the initiation time, rate, or strength of clotting in FVdp or PFP from healthy volunteers. Titration of plasma FV into FVdp illustrated that clotting time and rate (as measured by TEG) were not significantly affected at 10nM FV, less than 50% of the “normal” 23nM concentration. Clot strength was unaffected by FV depletion. To examine the contributions of platelet FVa, isolated and washed platelets were introduced in increasing amounts to FVdp and PFP; the rate of clotting and clot strength aligned between the two plasmas given equivalent numbers of platelets, emphasizing the importance of platelet FVa. At lower platelet concentrations (particularly 10,000 to 100,000 / µl), there were statistically significant differences in clotting time between FVdp and normal plasma, but these differences diminished with increasing platelet concentration. To evaluate the resistance of the platelet fraction to APC effects, 200,000 platelets / µl were suspended in FVdp and treated with a titration of APC from 1pM to 100nM. The samples were unaffected by APC up to a dose of at least 10nM as measured by TEG clotting parameters, well above the amounts found in the trauma patients or drotrecogin alfa-treated subjects. Conclusions FV-deficiency affects clotting only in cases of severe depletion (<50%). In a low shear in vitro closed system, APC does not affect coagulation except at very high levels (>1nM). Healthy platelets in sufficient quantity are capable of rescuing FV-deficiency and are resistant to APC effects. APC is unlikely to be the main driver of ACoT. Disclosures: No relevant conflicts of interest to declare.