A collaborative study to establish the 1st International Standard for factor XIII plasma

Raut, S.; Merton, R E; Rigsby, Peter; Muszbek, László; Seitz, Rainer; Ariëns, Robert A. S.; Barrowcliffe, Trevor W.; Ichinose, Akitada

doi:10.1111/j.1538-7836.2007.02684.x

Cited by 17 publications

(16 citation statements)

References 15 publications

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“…Human fibronectin (5 μg per well) was used to coat 96‐well plates (CoStar; Corning, Lowell, MA, USA). The FXIII standard was pooled normal plasma, standardized against the international standard [32], preactivated with 1 U mL −1 bovine thrombin at 37 °C for 5 min. Residual thrombin was neutralized by hirudin (2 μg mL −1 ), and samples were diluted in 0.1 m Tris (pH 7.4) and 1 m m dithiothreitol (DTT), to construct a standard curve.…”

Section: Methodsmentioning

confidence: 99%

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

Mutch

Koikkalainen

Fraser

et al. 2010

Journal of Thrombosis and Haemostasis

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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.

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Section: Methodsmentioning

confidence: 99%

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

Mutch

Koikkalainen

Fraser

et al. 2010

Journal of Thrombosis and Haemostasis

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“…The WHO First International Standard FXIII plasma (National Institute for Biological Standards and Control, Hertfordshire, UK) was used. 42 The reaction was performed in 100 mM TrisHCl [pH 7.4], 1 mM dithiothreitol, 1% BSA, 0.5 mM 5-(biotinamido) pentylamine (Pierce Thermo Fisher Scientific, Rockford, IL), and 5 mM CaCl 2 . Plates were washed with 0.01% Triton-X-100 in 100 mM Tris-HCl [pH 7.4] before adding para-nitrophenyl phosphate substrate (Sigma-Aldrich).…”

Section: Preparation Of Platelet Fractionsmentioning

confidence: 99%

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

Mitchell

Lionikiene

Fraser

et al. 2014

Blood

104

141

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Key Points• Factor XIII-A is exposed in protruding caps on the activated platelet surface.• Platelet FXIII-A exerts antifibrinolytic function by cross-linking a 2 AP to fibrin.Factor XIII (FXIII) stabilizes thrombi against fibrinolysis by cross-linking a 2-antiplasmin (a 2 AP) to fibrin. Cellular FXIII (FXIII-A) is abundant in platelets, but the extracellular functions of this pool are unclear because it is not released by classical secretion mechanisms. We examined the function of platelet FXIII-A using Chandler model thrombi formed from FXIIIdepleted plasma. Platelets stabilized FXIII-depleted thrombi in a transglutaminase-dependent manner. FXIII-A activity on activated platelets was unstable and was rapidly lost over 1 hour. Inhibiting platelet activation abrogated the ability of platelets to stabilize thrombi. Incorporating a neutralizing antibody to a 2 AP into FXIII-depleted thrombi revealed that the stabilizing effect of platelet FXIII-A on lysis was a 2 AP dependent. Platelet FXIII-A activity and antigen were associated with the cytoplasm and membrane fraction of unstimulated platelets, and these fractions were functional in stabilizing FXIII-depleted thrombi against lysis. Fluorescence confocal microscopy and flow cytometry revealed exposure of FXIII-A on activated membranes, with maximal signal detected with thrombin and collagen stimulation. FXIII-A was evident in protruding caps on the surface of phosphatidylserine-positive platelets. Our data show a functional role for platelet FXIII-A through exposure on the activated platelet membrane where it exerts antifibrinolytic function by cross-linking a 2 AP to fibrin. (Blood. 2014;124(26):3982-3990) IntroductionFactor XIII (FXIII) plays an essential role in normal hemostasis where it contributes to the regulation of fibrinolysis, 1-3 the maintenance of pregnancy, 4 wound healing, and angiogenesis. 5 The function of FXIII in hemostasis is further emphasized in its deficiency, which results in hemorrhage 6,7 and slow wound healing. 4,7 FXIII circulates in plasma as a heterotetramer (FXIII-A 2 B 2 ) where the catalytic A (FXIII-A 2 ) subunits are almost exclusively in complex with the inhibitory carrier B (FXIII-B 2 ) subunits. 8 FXIII is activated by the combined action of thrombin and Ca 21 to form the active transglutaminase (TG) FXIIIa. 9,10 FXIIIa confers stability to the fibrin matrix by cross-linking fibrin, substantially altering its rheologic properties.11,12 TG catalyzes formation of covalent e-(g-glutamyl) lysyl bonds 13 in which the lysine e-amino group is cross-linked to the glutamine g-carboxymide group. 14The cross-linking of g-chains confers a degree of rigidity to the fibrin network, which is further stabilized by the cross-linking of a-chains 15 to generate high-molecular-weight polymers. 10,16 FXIIIa also cross-links inhibitors of fibrinolysis to fibrin, further increasing its insolubility and resistance to plasmin. These inhibitors include a 2 -antiplasmin (a 2 AP), 17 thrombin activatable fibrinolysis inhibitor, 18 and plasminogen ac...

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“…Most recently, based on the standardization activity of the Factor XIII Standardization Working Party of the ISTH SSC [8], a World Health Organization reference plasma with assigned pFXIII activity and FXIII‐A 2 B 2 antigen values in International Units (1st International Standard for Factor XIII, Plasma [IS]) was established. The IS is available from the National Institute of Biological Standards and Control (Potters Bar, UK).…”

Section: Laboratory Diagnosismentioning

confidence: 99%