Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking

Abstract

Key Points• In the absence of FXIIIa activity, red blood cells are extruded from clots during clot contraction.• Factor XIIIa promotes red blood cell retention in contracting clots by crosslinking fibrin a-chains.Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in a 2 -antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks g-chain crosslinking sites, but not in clots that lack a-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block a-, but not g-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin a-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size. (Blood. 2015;126(16):1940-1948

Fibrinogen splice variation and cross-linking: Effects on fibrin structure/function and role of fibrinogen γ′ as thrombomobulin II

“…Inhibition of FXIII cross-linking did not affect the loss of red blood cells in clots made of fibrin -251, whereas clots made of fibrin -3X and wild-type both showed a reduction of clot weight by around 60%. These data indicate that FXIII-dependent retention of red blood cells is mediated by the cross-linking of fibrin -chains [32].…”

“…Byrnes et al to study the effects of -and -chains cross-linking on red blood cell retention within the clot [32]. Inhibition of FXIII cross-linking did not affect the loss of red blood cells in clots made of fibrin -251, whereas clots made of fibrin -3X and wild-type both showed a reduction of clot weight by around 60%.…”

Ranking reactive glutamines in the fibrinogen αC region that are targeted by blood coagulant factor XIII

“…FXIII binding to fibrinogen g390-396 and subsequent crosslinking of the a chain helps mediate red blood cell retention in venous thrombi. 28,29 The aC region of fibrinogen contributes its own critical roles in the assembly and properties of clot structure. 8,[30][31][32][33] This region has also been directly correlated with adhesion events.…”

Limiting prothrombin activation to meizothrombin is compatible with survival but significantly alters hemostasis in mice

“…[25][26][27] To directly determine whether fII MZ supports clot retraction, we initiated coagulation with thromboplastin in both whole blood and PRP (platelet counts adjusted to 2. Figure 3A-B,E-F).…”