Factor XIII cotreatment with hemostatic agents in hemophilia A increases fibrin α-chain crosslinking

Abstract

Essentials Factor XIII (FXIII)-mediated fibrin crosslinking is delayed in hemophilia. We determined effects of FXIII cotreatment with hemostatic agents on clot parameters. FXIII cotreatment accelerated FXIII activation and crosslinking of fibrin and α₂ -antiplasmin. These data provide biochemical rationale for FXIII cotreatment in hemophilia.

Summary: Background Hemophilia A results from the absence, deficiency or inhibition of factor VIII. Bleeding is treated with hemostatic agents (FVIII, recombinant activated FVII [rFVIIa], anti-inhibitor coagulation complex [FEIBA], or recombinant porcine FVIII [rpFVIII]). Despite treatment, some patients have prolonged bleeding. FXIII-A₂ B₂ (FXIII) is a protransglutaminase. During clot contraction, thrombin-activated FXIII (FXIIIa) crosslinks fibrin and α₂ -antiplasmin, which promotes red blood cell retention and increases clot stability and weight. We hypothesized that FXIII cotreatment in hemophilia would accelerate FXIII activation, leading to increased fibrin crosslinking. Methods FVIII-deficient plasma and whole blood were clotted with or without hemostatic agents (FVIII, rFVIIa, FEIBA, or recombinant B-domain-deleted porcine FVIII [rpFVIII]) and/or FXIII. The effects on FXIII activation, thrombin generation, fibrin and α₂ -antiplasmin crosslinking, clot formation and clot weight were measured by western blotting, calibrated automated thrombography, thromboelastography, and clot contraction assays. Results As compared with FVIII-treated hemophilic plasma, FVIII + FXIII cotreatment accelerated FXIIIa formation without increasing thrombin generation. As compared with buffer-treated or FXIII-treated hemophilic plasma, FVIII treatment and FVIII + FXIII cotreatment increased the generation and amount of crosslinked fibrin, including α-chain-rich high molecular weight species and crosslinked α₂ -antiplasmin. In the presence of FVIII inhibitors, as compared with hemostatic treatments (rFVIIa, FEIBA, or rpFVIII) alone, FXIII cotreatment increased whole blood clot weight. Conclusion In hemophilia A plasma and whole blood, FXIII cotreatment with hemostatic agents accelerated FXIIIa formation, increased the generation and amount of fibrin α-chain crosslinked species, accelerated α₂ -antiplasmin crosslinking, and increased clot weight. FXIII cotreatment with hemostatic therapy may augment hemostasis through increased crosslinking of fibrin and α₂ -antiplasmin.

Keywords: factor XIII; fibrin; hemophilia; hemostasis; α2-antiplasmin.

Fig. 1

As compared with FVIII treatment alone, FXIII cotreatment accelerates the maximum rate of FXIII activation. (A) Representative western blots for FXIII-A after tissue factor-initiated clotting in recalcified hemophilic plasma treated with buffer (HEPES-buffered saline), 2 IU mL⁻¹ FXIII, 1 IU mL⁻¹ FVIII, or FVIII + FXIII (final concentrations of 1 IU mL⁻¹ and 2 IU mL⁻¹, respectively). Cleavage of activation peptide(s) results in the formation of FXIII-A′ (lower band). (B) Quantification of FXIII activation (FXIII-A′) over time relative to FXIII-A′ loading control; n = 3–7 samples per time point, mean ± standard error of the mean (SEM), arbitrary units (AU). Two-way ANOVA was used to compare time and treatment. (C) Maximum rate of FXIII activation (mean ± SEM, AU min⁻¹) for n = 7 samples. Treatments were compared by the use of repeated measures ANOVA with the Holm–Sidak multiple comparisons test.

Fig. 2

As compared with FVIII treatment alone, FXIII cotreatment increases fibrin crosslinking. (A) Representative western blots for fibrin (ogen) after tissue factor-initiated clotting in recalcified hemophilic plasma treated as in Fig. 1. (B) Quantification of γ–γ dimer normalized to Bβ + β-chain. (C) Maximum rate of γ–γ formation (mean ± standard error of the mean [SEM], n = 7, arbitrary units [AU] min⁻¹). (D) Quantification of high molecular weight (HMW) species normalized to Bβ + β-chain. (E) Maximum rate of HMW species formation (mean ± SEM, n = 7, AU min⁻¹). For (B) and (D), two-way ANOVA with the Holm–Sidak multiple comparisons test was used to compare time and treatment. *P < 0.05 versus buffer-treated, FXIII-treated and FVIII-treated samples. For (C) and (E), treatments were compared by the use of repeated measures ANOVA with the Holm–Sidak multiple comparisons test.

Fig. 3

As compared with FVIII treatment alone, FXIII cotreatment accelerates and increases α₂-antiplasmin crosslinking. (A) Representative western blots for α₂-antiplasmin crosslinking in recalcified hemophilic plasma treated as in Fig. 1. (B) Quantification of crosslinked α₂-antiplasmin normalized to total α₂-antiplasmin at t = 0 min (mean ± standard error of the mean [SEM], n = 7). Symbols are: buffer, closed circles; + FXIII, half-filled boxes; + FVIII, open triangles; + FVIII + FXIII, closed inverted triangles. *P < 0.05 versus buffer-treated, FXIII-treated and FVIII-treated samples. Two-way ANOVA with the Holm–Sidak multiple comparisons test was used to compare time and treatment. (C) Maximum rate of α₂-antiplasmin crosslinking (mean ± SEM, n = 7, arbitrary units [AU] min⁻¹). Treatments were compared by the use of repeated measures ANOVA with the Holm–Sidak multiple comparisons test.

Fig. 4

As compared with hemostatic therapies alone, FXIII cotreatment increases whole blood clot weight. Hemophilic whole blood was treated with protein buffer (0.75% w/v bovine serum albumin in 0.9% sodium chloride solution), FXIII (2 IU mL⁻¹) or (A) recombinant activated FVII (rFVIIa) (25 nM), (B) plasma-derived anti-inhibitor coagulant complex (FEIBA) (1 IU mL⁻¹), or (C) recombinant B-domain-deleted porcine FVIII (rpFVIII) (1 IU mL⁻¹), with or without FXIII, and clotted with tissue factor and recalcification. Contracted clots were weighed. Dashed lines indicate ranges of clot weights for n = 4 non-hemophilic donors. Bars show the mean ± standard error of them mean for n = 5–8 individual hemophilic donors per treatment. Treatments were compared by the use of repeated measures ANOVA with the Holm–Sidak multiple comparisons test.