Coagulation factor XIIIa is inactivated by plasmin

Abstract

Coagulation factor XIIIa (FXIIIa) is a transglutaminase that covalently cross-links fibrin and other proteins to fibrin to stabilize blood clots and reduce blood loss. A clear mechanism to describe the physiological inactivation of FXIIIa has been elusive. Here, we show that plasmin can cleave FXIIIa in purified systems and in blood. Whereas zymogen FXIII was not readily cleaved by plasmin, FXIIIa was rapidly cleaved and inactivated by plasmin in solution (catalytic efficiency = 8.3 × 10(3) M(-1)s(-1)). The primary cleavage site identified by mass spectrometry was between K468 and Q469. Both plasma- and platelet-derived FXIIIa were susceptible to plasmin-mediated degradation. Inactivation of FXIIIa occurred during clot lysis and was enhanced both in plasma deficient in fibrinogen and in plasma treated with therapeutic levels of tissue plasminogen activator. These results indicate that FXIIIa activity can be modulated by fibrinolytic enzymes, and suggest that changes in fibrinolytic activity may influence cross-linking of blood proteins.

Figure 1

FXIII-A₂* is cleaved and inactivated by plasmin. FXIII (100 nM), with or without prior activation by thrombin (400 nM), was mixed with varying concentrations of plasmin (100 pM to 100 nM) for 3 hours, and analyzed by western blot against FXIII A and B subunits. (A) Blot against the A subunit of pFXIII-A₂*, with thrombin activation. (B) Blot against the A subunit of pFXIII-A₂B₂. (C) Blot against the B subunit of pFXIII-A₂*, with thrombin activation. (D) Blot against the B subunit of pFXIII-A₂B₂. (E) Time course of cleavage of FXIII-A₂* by plasmin (90 nM). (F) Transglutaminase activity (left axis) of FXIII-A₂* after incubation with plasmin (90 nM) and the relative amount of intact FXIII-A₂* (right axis), determined by quantifying the intensity of the band at 83 kDa. FXIII-A* was calculated as a percentage of total signal in the lane using densitometry. The error bars represent standard error of the mean (SEM). n = 3 for all experiments. Thr, thrombin.

Figure 2

Plasmin cleaves FXIII-A₂* at multiple sites. (A) FXIII-A₂* cleavage sites were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI/TOF) mass spectrometry after purified FXIIIa (10 μM) was incubated with plasmin (2.7 μM) for 2 hours. Surface-exposed sites are represented with black bars, and the primary cleavage site with a red bar. The frequency of detection of cut sites is indicated beside the respective bars (n = 2). (B) A reported structure of FXIII-A₂*, showing the surface-exposed K468-Q469 cleavage site (red) and the catalytic cysteine (green). The distance between the cleavage site and the catalytic cysteine is 18 Å. (C) A reported structure of FXIII-A₂, showing K468-Q469 (red) and the activation peptide (blue).

Figure 3

The rate of inactivation of FXIII-A₂* can occur on a physiologically relevant timescale. (A) Lineweaver-Burk plot used to generate kinetic parameters for FXIII-A₂*, with data sets represented by different colors. The data point circled in pink is derived from the slope of the initial velocity in panel B (pink line). (B) The generation of FXIII-A₂* cleavage products by plasmin (300 nM) at a representative FXIII-A₂* concentration, determined using an ammonia release assay. (C) Standard curves of ammonia generation over time using various [FXIII-A₂*]. (D) Ammonia generation over time, with varying times of FXIII-A₂* digestion with plasmin. n = 3 for all experiments. Abs, absorbance.

Figure 4

Plasmin degrades plasma- and platelet-derived FXIII-A*. Western blots against the FXIII A subunit. (A) Endogenous FXIII-A₂* from human plasma after adding plasmin (3 µM) for various times. (B) FXIII-A₂* from plasma with plasmin (3 µM) and α2-antiplasmin (5 µM) or TXA (7.5 mM). (C-D) Endogenous FXIII-A₂/FXIII-A₂* from platelets (PLT), 1 hour (C) and 16 hours (D) after exposure to thrombin, and incubating with various concentrations of plasmin. Samples contain combinations of Innovin (I), plasmin (P), α2-antiplasmin (A), and TXA (X). n = 3 for all experiments.

Figure 5

Endogenous plasminogen is activated by tPA to degrade endogenous FXIIIa. Plasma was analyzed by western blot against FXIII-A, after addition of tPA and TXA. (A-B) Time-dependent degradation of endogenous (A) FXIII-A₂* and (B) fibrin(ogen) in normal plasma. (C) The relative amount of intact FXIII-A* from panel A using densitometry, calculated as a percentage of total signal in the lane. *P < .05. (D) Time course for plasminogen-deficient plasma. (E) Time-dependent degradation of FXIIIa in fibrinogen-deficient plasma after adding tPA. (F) Degradation of endogenous FXIII-A₂*, but not FXIII-A₂, in whole-blood clots with tPA. Samples contain combinations of hirudin (H), Innovin (I), tPA (2 µM) (T), TXA (7.5 mM) (X), and α2-antiplasmin (4 µM) (A). n = 3 for all experiments. FDP, fibrin degradation products; MW, molecular weight.

Figure 6

Plasmin-mediated inactivation of FXIII-A₂* does not occur during normal clot formation, but does occur during fibrinolysis and thrombolytic conditions. (A-B) Clot formation in normal plasma with tPA (200 pM) and, in some cases, TXA (7.5 mM). Western blots against (A) FXIII-A and (B) fibrin(ogen) (n = 3). (C-E) TEG analyses of clot formation and cross-linking of exogenous (purified) fibrin in plasma. Schematics on the left show timelines of the procedures and characteristic shear elastic moduli (G, dashed lines), with shaded areas indicating time periods analyzed with TEG. Charts on the right show measured moduli of fibrin clots, a direct indicator of FXIII-A₂* activity and fibrin structure. Control samples contain exogenous FXIIIa or T101. (C) Moduli of clots from plasminogen-deficient, normal, and α2-antiplasmin–deficient plasma formed in the presence of tPA (200 pM). (D) Moduli of exogenous fibrin (indicator of residual FXIIIa activity), added following clot lysis. Exogenous fibrinogen (1.4 mg/mL) and TXA were added 1 or 3 hours after clot lysis by tPA (800 nM). (E) Moduli of exogenous fibrin that was added during clot formation under thrombolytic conditions. TXA and then fibrinogen were added 4 minutes after clotting was initiated in the presence of tPA (50 nM or for α2-antiplasmin–deficient plasma, 5 nM). Samples contain combinations of Innovin (I), tPA (T), and TXA (X). **P < .01, ***P < .001. n = 3 for all experiments. TF, tissue factor.