Model thrombi formed under flow reveal the role of factor XIII-mediated cross-linking in resistance to fibrinolysis

Abstract

Background: Activated factor XIII (FXIIIa), a transglutaminase, introduces fibrin-fibrin and fibrin-inhibitor cross-links, resulting in more mechanically stable clots. The impact of cross-linking on resistance to fibrinolysis has proved challenging to evaluate quantitatively.

Methods: We used a whole blood model thrombus system to characterize the role of cross-linking in resistance to fibrinolytic degradation. Model thrombi, which mimic arterial thrombi formed in vivo, were prepared with incorporated fluorescently labeled fibrinogen, in order to allow quantification of fibrinolysis as released fluorescence units per minute.

Results: A site-specific inhibitor of transglutaminases, added to blood from normal donors, yielded model thrombi that lysed more easily, either spontaneously or by plasminogen activators. This was observed both in the cell/platelet-rich head and fibrin-rich tail. Model thrombi from an FXIII-deficient patient lysed more quickly than normal thrombi; replacement therapy with FXIII concentrate normalized lysis. In vitro addition of purified FXIII to the patient's preprophylaxis blood, but not to normal control blood, resulted in more stable thrombi, indicating no further efficacy of supraphysiologic FXIII. However, addition of tissue transglutaminase, which is synthesized by endothelial cells, generated thrombi that were more resistant to fibrinolysis; this may stabilize mural thrombi in vivo.

Conclusions: Model thrombi formed under flow, even those prepared as plasma 'thrombi', reveal the effect of FXIII on fibrinolysis. Although very low levels of FXIII are known to produce mechanical clot stability, and to achieve γ-dimerization, they appear to be suboptimal in conferring full resistance to fibrinolysis.

Fig. 1

Inhibition of cross-linking in thrombi. Thrombi were prepared in the absence (○) or presence (•) of transglutaminase (TG) inhibitor and bathed in fluid containing tissue-type plasminogen activator (t-PA; 1 μg mL⁻¹, n = 5), urokinase-type plasminogen activator (u-PA; 1 μg mL⁻¹, n = 4) or buffer alone (spontaneous, n = 8). Lysis was monitored as release of fluorescence and expressed as mean ± standard error of the mean. Differences in lysis rate with addition of TG inhibitor were significant (P < 0.01) in all cases.

Fig. 2

Cross-linking in heads and tails of thrombi. Thrombi were prepared in the absence (○) or presence (•) of transglutaminase (TG) inhibitor. The thrombi were bisected into the platelet-rich head and fibrin-rich tail before being lysed separately with tissue-type plasminogen activator (t-PA; 1 μg mL⁻¹). Lysis was monitored as release of fluorescence, and results are expressed as mean lysis (%) ± standard error of the mean (n = 4) relative to values obtained when thrombi were lysed to completion with additional plasminogen and t-PA, as described in Materials and methods. Differences in percentage lysis with addition of TG inhibitor were significant (P < 0.005) for both heads and tails.

Fig. 3

The role of plasma FXIII in plasma clots and thrombus stabilization. (A) Plasma clots were formed with pooled normal plasma (PNP), or mixtures of PNP with FXIII-deficient plasma to give different concentrations of FXIII relative to normal (1.5–100%). Clots were also prepared from the plasma of an FXIII-deficient patient that was collected pre-transfusion on four separate occasions (A–D). After 30 min, clots were harvested and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis before staining for protein with Coomassie Brilliant Blue R. (B) Blood was collected from the patient pre-transfusion and post-transfusion with FXIII (Fibrogammin P) on four separate occasions [as in (A)]. Thrombi were formed in the absence (open symbols) or presence (closed symbols) of transglutaminase (TG) inhibitor, and lysed with tissue-type plasminogen activator (1 μg mL⁻¹). Lysis was monitored as release of fluorescence, and results are expressed as mean ± standard error of the mean (n = 4). Differences in lysis rate with addition of TG inhibitor were significant (P < 0.005) both pre-treatment and post-treatment.

Fig. 4

Cross-linking stabilizes plasma ‘thrombi’ and alters the pattern of fibrin degradation products. (A) Plasma from normal individuals was used to form ‘thrombi’, in the absence (○) and the presence (•) of transglutaminase (TG) inhibitor, and these were compared with plasma ‘thrombi’ from the FXIII-deficient patient (Δ). Lysis with tissue-type plasminogen activator (1 μg mL⁻¹) was recorded as release of fluorescence over time, and results are expressed as mean ± standard error of the mean (n = 3). The difference in lysis rate upon addition of TG inhibitor to plasma ‘thrombi’ was significant (P < 0.005), as was the difference between the normal control and the patient (P < 0.005). (B) After the final time point (4 h), samples were taken from the bathing fluid of the model thrombi formed in the absence (1) and presence (2) of TG inhibitor or from the FXIII-deficient patient (3). Samples were separated on 4–12% polyacrylamide gels before staining in Coomassie Brilliant Blue R or transferring to nitrocellulose and staining with an antibody to the γ-chain of fibrinogen.

Fig. 5

Additional stabilization of normal and FXIII-deficient blood. Thrombi were formed from the whole blood of normal subjects or from the patient deficient in FXIII with the addition of FXIII or transglutaminase 2 (TG2). Thrombi were lysed with tissue-type plasminogen activator (1 μg mL⁻¹), and lysis was recorded as release of fluorescence. The normal control data in the top and bottom panels are mean value ± standard error of the mean of 19 different subjects. The difference in lysis with addition of FXIII (1 U mL⁻¹ Fibrogammin P; •, ▴) was not significant in normal subjects (P = 0.15, n = 3) but was significant in thrombi from FXIII-deficient blood (P < 0.001, n = 3). Addition of 1 U mL⁻¹ (•), 2 U mL⁻¹ (▴) and 4 U mL⁻¹ (▪) TG2 (n = 3) resulted in significantly less lysis (P < 0.005) relative to the normal control and each concentration tested.