Co-Administration of Factor XIII with Hemostatic Agents in Hemophilia Promotes Clot Stability and Composition

Hemophilia A results from the absence, deficiency, or inhibition of factor VIII. Bleeding is treated with use of two broad classes of hemostatic agents; replacement factors (recombinant or plasma-derived human FVIII [FVIII] or factor IX) or bypassing agents (recombinant activated factor VIIa [rFVIIa], plasma-derived anti-inhibitor coagulant complex [FEIBA], or recombinant porcine FVIII [rpFVIII]). Despite treatment with hemostatic agents, some hemophilia patients experience refractory bleeding. Factor XIII-A2B2 (FXIII) is a thrombin-activated protransglutaminase. Activated FXIII (FXIIIa) crosslinks fibrin to stabilize clots and increase red blood cell retention during clot contraction, and therefore, clot weight; these functions have been specifically associated with fibrin alpha-chain crosslinking and production of high molecular weight (HMW) crosslinked fibrin species. Although pilot studies have indicated that in hemophilia co-administration of FXIII with hemostatic agents improves hemostasis, the biochemical mechanisms evoked during co-administration have yet to be elucidated. We hypothesize that co-administration of FXIII with hemostatic agents enhances hemostasis in hemophilia by accelerating FXIII activation, increasing fibrin crosslinking, and improving the composition of contracted clots. We first analyzed in vitro tissue factor-activated coagulation in FVIII-deficient plasmas (n=5) with or without buffer (normal saline with 0.75% bovine serum albumin), plasma-derived FXIII (2 U/mL), and/or recombinant human FVIII (1 U/mL). Reactions were performed at 37°C and quenched with urea/EDTA at varying time points. Clot lysates were separated on 10% Tris-glycine gels, blotted, probed for FXIII(a) and fibrin(ogen), and analyzed by densitometry. Compared to buffer, FXIII, or FVIII-treated hemophilic plasma, FVIII+FXIII co-treatment increased the formation rate and level of FXIIIa. Furthermore, compared to buffer or FXIII-treated hemophilic plasma, both FVIII-treatment and FVIII+FXIII co-treatment increased the formation rate and level of crosslinked fibrin species (gamma chain and HMW species). Notably, compared to FVIII-treatment (alone), FVIII+FXIII co-treatment increased the formation rate and level of HMW crosslinked fibrin species. Next, we analyzed clot formation in whole blood obtained from untreated FVIII-deficient (n=2) or FVIII-inhibitor (n=6) patients. Citrated whole blood was pre-incubated at 37°C for 30 minutes with or without buffer, FXIII (2 U/mL), rFVIIa (25 nM), FEIBA (1 U/mL), or rpFVIII (1 U/mL), alone and in combination with FXIII, and clot formation was triggered with tissue factor and recalcification. In thrombelastography assays, compared to buffer or FXIII-treated hemophilic whole blood, rFVIIa, FEIBA, and rpFVIII treatments alone or with FXIII co-treatment shortened the time to clot formation (clot time [R]). Compared to buffer, FXIII, FEIBA or rpFVIII-treated hemophilic whole blood, rFVIIa-treatment (alone) and co-treatment with rFVIIa+FXIII or FEIBA+FXIIII increased clot stability (area under the elastic curve). In whole blood clot contraction assays, compared to buffer, FXIII, rFVIIa, FEIBA, or rpFVIII-treated hemophilic whole blood, co-treatment with rFVIIa+FXIII, FEIBA+FXIII, or rpFVIII+FXIII significantly increased clot weight. Our data show that in hemophilic plasmas, co-administration of FXIII with hemostatic agents accelerates FXIIIa activation, resulting in accelerated and increased HMW species formation. Furthermore, in hemophilic whole blood, co-administration of FXIII with hemostatic agents improved clot biophysical characteristics and increased clot weight. Collectively, these data suggest co-administration of FXIII with conventional hemostatic agents may promote clot stability and improve the composition of contracted clots by enhancing fibrin alpha-chain crosslinking. Disclosures No relevant conflicts of interest to declare.