Traumatic bleeding is a leading cause of death and disability, without targeted interventions available outside blood component resuscitation and tranexamic acid. Acute traumatic coagulopathy (ATC) in association with bleeding occurs in ~30-50% of patients and carries a high mortality risk. Recent evidence suggests a role of activated protein C (APC) in the development of ATC, whereby excessive generation of APC leads to rapid depletion of Factor(F) V, FVIII and hyperfibrinolysis causing depletion of fibrinogen.
Here, we investigated to what extent targeted intervention with an engineered activated FV variant (superFVa), resistant to inactivation by APC, can reduce traumatic bleeding and prevent or normalize hemostasis parameters indicative of ATC, a process thought to be different from disseminated intravascular coagulation (DIC). SuperFVa was engineered to withstand cleavage by APC through mutations of 3 APC cleavage sites (Arg506/306/679Gln), with additional stability provided by introduction of a disulfide bond (Cys609-Cys1691) between the A2 and A3 domains.
Materials and Methods
First, an established trauma/shock model for ATC (Chesebro et al. 2009) was used. Here, trauma is induced by laparotomy and shock is induced by blood withdrawal (~500ul) to reduce and maintain the mean arterial pressure [MAP] from 75±5 to 35±5 mmHg for 1 hour. SuperFVa (0.4-0.8 mg/kg) was administered by continuous infusion 30 minutes after trauma/shock induction. Second, a liver laceration model was used where ATC develops as the consequence of severe bleeding. SuperFVa was administered either as bolus (0.4-0.8mg/kg) before injury or as continuous infusion (0.8mg/kg) started 30 minutes after injury. Bleeding, APTT clotting times, clotting factor activity levels (FII, FV, FVIII, FX, fibrinogen) and Thrombin-Antithrombin (TAT) complexes were assessed to determine the extent of ATC at various time points.
In the trauma/shock model, only mice subjected to both trauma and shock developed ATC, evident from a significant APTT prolongation (25.4±0.2 to 36.1±0.2 seconds), associated with a severe reduction of FV (14%) and FVIII( 6%), a partial reduction of fibrinogen (44%), and no effect on FX (89%) and FII (89%). Administration of superFVa normalized the APTT (27.1±0.2 seconds) despite the persistence of shock, returned FV and FVIII levels to their normal range, but had no effect on fibrinogen. TAT levels increased significantly upon trauma/shock (1522 ±125 ng/mL) and were reduced by superFVa administration (511±125 ng/mL), suggesting improved hemostasis.
After liver laceration severe bleeding occurred within 60 minutes, causing shock (MAP decreased to 35±5), selective reductions of FV, FVIII, and fibrinogen levels, and prolongation of the APTT clotting time, indicating the development of ATC. SuperFVa prophylaxis prior to liver laceration reduced blood loss significantly (non-treated mice 0.54±0.01 vs. pre-treated mice 0.34±0.01 mL, p<0.0001), prevented APTT prolongation and depletion of FV and FVIII. Thus, prophylactic administration of superFVa prevented the development of ATC.
Most bleeding after liver laceration occurred in the first 15-30 minutes (0.44±0.01mL at 30 minutes) and ATC became evident at 30 min (APTT prolongation to 32.63±0.2 seconds; partial depletion (~50%) of FV and FVIII). To determine effects of superFVa on the reversal of ongoing ATC, superFVa intervention was started therefore at the 30-minute time point. superFVa blunted additional blood loss after 30 minutes, normalized prolonged APTT clotting times and reversed FV and FVIII levels to their normal range. Reversal of ATC by superFVa was associated with a reduction of TAT levels (646.5±125 ng/mL) that were increased by ATC (1577.0 ±125 ng/mL).
SuperFVa restored hemostasis in two mouse models of ATC where coagulopathy was induced by either trauma/shock or by severe bleeding. Improved hemostasis in ATC by superFVa was achieved by correction of APTT clotting times and normalization of depleted FV and FVIII levels and was associated with a reduction of elevated TAT levels. Because superFVa rescued traumatic bleeding to prevent the development of ATC and was also efficacious for the reversal of ongoing ATC, targeted interception of ATC by superFVa therefore may be an attractive strategy to reduce complications associated with ATC.
Mosnier:The Scripps Research Institute: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. von Drygalski:University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy; Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder.
Asterisk with author names denotes non-ASH members.