The antifibrinolytic function of factor XIII is exclusively expressed through α₂-antiplasmin cross-linking

Abstract

Factor XIII (FXIII) generates fibrin-fibrin and fibrin-inhibitor cross-links. Our flow model, which is sensitive to cross-linking, was used to assess the effects of FXIII and the fibrinolytic inhibitor, α₂-antiplasmin (α₂AP) on fibrinolysis. Plasma model thrombi formed from FXIII or α₂AP depleted plasma lysed at strikingly similar rates, 9-fold faster than pooled normal plasma (PNP). In contrast, no change was observed on depletion of PAI-1 or thrombin activatable fibrinolysis inhibitor (TAFI). Inhibition of FXIII did not further enhance lysis of α₂AP depleted thrombi. Addition of PNP to FXIII or α₂AP depleted plasmas normalized lysis. Lysis rate was strongly inversely correlated with total cross-linked α₂AP in plasma thrombi. Reconstitution of FXIII into depleted plasma stabilized plasma thrombi and normalized γ-dimers and α-polymers formation. However, the presence of a neutralizing antibody to α₂AP abolished this stabilization. Our data show that the antifibrinolytic function of FXIII is independent of fibrin-fibrin cross-linking and is expressed exclusively through α₂AP.

Figure 1

Thrombi formed from plasma depleted of FXIII or α₂AP show comparable lysis. (A) Plasma thrombi were prepared from pooled normal plasma (PNP; ○; n = 6) or plasma depleted of FXIII (●; n = 6), α₂AP (▴; n = 9), TAFI (▵; n = 2) and PAI-1 (□; n = 2) and lysed with 1 μg/mL tissue plasminogen activator (tPA). Lysis was monitored as release of fluorescence and expressed as mean ± SEM. FXIII and α₂AP depleted plasmas lysed significantly faster (P < .005) than PNP, TAFI, and PAI-1 depleted plasma, whereas FXIII and α₂AP depleted plasma lysed at comparable rates (P = .5). (B) Plasma thrombi were prepared from PNP (●, ○; n = 6) or α₂AP depleted plasma (▴, ▵;r n = 3) in the absence (closed symbols) and presence (open symbols) of a TG inhibitor. Thrombi were lysed as described in panel A. Lysis of α₂AP depleted plasma thrombi was significantly different from PNP (P < .005) but no difference in lysis was observed on incorporation of TG inhibitor into α₂AP depleted plasma before thrombus formation (P = .5). (C-E) Plasma thrombi were prepared from PNP or mixtures of PNP with FXIII (●) or α₂AP (▴) depleted plasma, resulting in different percentages of FXIII (0, 1.5, 6, 24, 48, 100%) or α₂AP (0, 20, 40, 60, 80, 100%) relative to their plasma concentration. Lysis was recorded as described in panel A and the mean lysis rate (FU/minutes) plotted against % PNP (n = 6; C). Alternatively, thrombi were solubilized in reducing sample buffer and subjected to SDS-PAGE followed by Western blotting for α₂AP (D; n = 6). The Western blot (D) was analyzed using Image J densitometry software and the peak area of total cross-linked α₂AP was plotted against the mean lysis rate for each sample of PNP and FXIII or α₂AP depleted plasma containing different percentages of plasma (E).

Figure 2

FXIII requires α₂AP to down-regulate fibrinolysis. (A) Plasma thrombi were prepared from FXIII depleted plasma in the absence (●) and presence (○) of a neutralizing antibody to α₂AP or α₂AP depleted plasma (▴). Various concentrations of FXIII (0, 0.1, 0.3, 1 U/mL) were added before thrombus formation and thrombi were lysed with 1 μg/mL tissue plasminogen activator (tPA). Lysis was monitored as release of fluorescence and expressed as mean lysis rate per minute ± SEM (n = 4). (B) Plasma thrombi prepared as described in panel A in the presence of FXIII (1 U/mL) or neutralizing antibody to α₂AP (Mab) were solubilized in reducing sample buffer. After separating on 7.5% acrylamide gels samples were immunoblotted with antibodies to α₂AP, the α-chain or the γ-chain of fibrinogen.