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

Abstract

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...

Coated platelets function in platelet-dependent fibrin formation via integrin α IIb β 3 and transglutaminase factor XIII

“…A role for platelet-derived FXIII was recently proposed in regulating anti-fibrinolysis by crosslinking α 2 -antiplasmin to fibrin. 38 Our present findings significantly extend this study by demonstrating a role for factor XIII earlier in the process of hemostasis and thrombosis, i.e. by mediating the formation of a star-like coat of fibrin fibers on the platelet surface.…”

“…35 Although earlier studies found tissue type transglutaminase to be present in human platelets, 8,36 more recent studies showed it could not be detected on the platelet protein level. 37,38 Interestingly, in mouse platelets, tissue type transglutaminase (Tgm2) could be detected, albeit with a 25-fold lower expression in comparison to factor XIII (F13a). 28 Our present findings point to a predominant role of factor XIII, and not of tissue type transglutaminase, in both the human and murine systems, which is in line with the recent studies.…”

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity

“…Flow-through material and subsequent low-salt (50 mM NaCl) and high-salt (1 M NaCl) washes were collected, and fractions were analyzed by western blotting as described previously 32 using horseradish peroxidase-conjugated goat anti-human FXII or horseradish peroxidase-conjugated goat anti-human plasminogen (both Enzyme Research Laboratories). …”

“…32 Pooled normal plasma that is essentially free of platelets was prepared from whole blood of 20 normal donors collected into 3.2% trisodium citrate. 33 …”

Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow

“…Interestingly, we have recently shown that platelet-derived factor XIII-A is found in the "cap" of PS-exposing platelets. 55 The presence of activated FXIII-A potentially stabilizes fibrin emanating from the platelet surface both mechanically, via cross-linking of fibrin, and against fibrinolytic degradation by cross-linking a 2 AP.55 Coated platelets express a IIb b 3 on their surface that becomes inactivated after occupation by a ligand such as fibrinogen.3,9,56 PS-exposing platelets in this study did not express the active a IIb b 3 , as demonstrated by the lack of PAC-1 staining in the "cap." Intriguingly, despite the lack of active integrin, we show that blocking a IIb b 3 and fibrin polymerization attenuates plasminogen binding to PS-exposing platelets.…”