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

Abstract

SummaryBackground: 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.

Functional factor XIII-A is exposed on the stimulated platelet surface

“…39 PNP, FXIII-depleted plasma, or a 2 AP-depleted plasma (the latter two from Affinity Biologicals, Ancaster, ON, Canada) containing fluorescein isothiocyanate (FITC)-labeled fibrinogen (43.5 mg/mL) with an FITC:fibrinogen ratio of approximately 6:1 were recalcified with CaCl 2 (10.9 mM). The loops were rotated for 90 minutes at 30 rpm.…”

“…FXIII activity assay FXIII activity was quantified using an in-house activity assay as previously described 39 with the following modifications. The WHO First International Standard FXIII plasma (National Institute for Biological Standards and Control, Hertfordshire, UK) was used.…”

“…[33][34][35] However, it is unclear how platelet FXIII-A elicits these functions because it is not released during activation 37 and does not follow classical routes of secretion in platelets or other cells. 38 We have previously shown, using thrombi formed under flow, that FXIII increases resistance to fibrinolysis, 39 specifically via crosslinking of a 2 AP to fibrin. 1 Here we show that activated platelets externalize their cytoplasmic pool of FXIII-A onto their membranes.…”

“…Initial experiments were performed with lysates prepared by freeze-thawing apheresis platelets. A nonreversible inhibitor of TG (1,3-dimethyl-2-[(2-oxopropyl) thio]imidazolium chloride) (1 mM) 39,43 or a neutralizing antibody to a 2 AP (150 mg/mL; Technoclone) 1 were incorporated prior to recalcification. Formed thrombi were washed in 0.9% (w/v) NaCl and bathed at 37°C in 10 mM Tris, 0.01% TWEEN 20 [pH 7.4] containing 1 mg/mL singlechain tissue-type plasminogen activator (Genentech, San Francisco, CA).…”

Cryoprecipitate transfusion: current perspectives

“…It also cross-links inhibitors of fibrinolysis, predominantly α2-antiplasmin to fibrin chains, which prevents premature clot lysis and contributes to clot stability. 63 Patients with trauma-induced coagulopathy have low factor XIII levels. 45 However, although it has been shown that low levels of FXIII are sufficient for mechanical clot stability in healthy patients, there is uncertainty regarding the critical levels of FXIII required in trauma.…”