Elimination of fibrin γ-chain cross-linking by FXIIIa increases pulmonary embolism arising from murine inferior vena cava thrombi

Abstract

The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and applied methods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolization without affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.

Novel venous thromboembolism mouse model to evaluate the role of complete and partial factor XIII deficiency in pulmonary embolism risk

“…This effect may be due to decreased fibrin crosslinking and/or decreased crosslinking of antifibrinolytic molecules (e.g., α 2 ‐antiplasmin) to fibrin 61 . Given differences in thrombus composition, enhanced PE in F13a −/− mice subjected to FeCl 3 ‐mediated thrombosis 11–13 or stasis‐induced VT (present study) strengthens evidence supporting a specific role of FXIII in PE. Longer‐term studies of thrombus resolution in FXIII‐deficient mice are needed to characterize the fate of VT and PE and reconcile apparent discrepancies in PE incidence between FXIII‐deficient humans and mice.…”

“…For example, compared to FXIIIsufficient mice (F13 +/+ ), FXIII-deficient mice (F13a −/− ) show increased bleeding following tail transection, but not saphenous vein puncture. 42 Moreover, mice with reduced FXIII antigen (F13a +/− or F13a −/− ) or delayed FXIII activation (Fibγ 390−396A ) have normal arterial thrombus formation and no measurable reflow events following FeCl 3 injury, 50,51 but reduced stasis-or stenosisinduced VT. 8,42 Collectively, these and other studies of FXIII function ( 8,(11)(12)(13)42,50,(52)(53)(54) and present work) suggest FXIII has nonoverlapping roles in venous and arterial thrombosis. Thus, a major aspect of the current work investigating VTE is the use of a mouse model that recapitulates key aspects of VTE in humans.…”

“…Previous studies using FeCl 3 to induce femoral vein thrombosis suggested FXIII‐deficient mice on a mixed 129Ola/CBACa background 10 have increased embolic events compared to wild‐type C57Bl/6 mice 11,12 . Furthermore, following FeCl 3 ‐induced thrombosis, mice expressing fibrinogen with mutated crosslinking sites (FGG3X) have increased PE, suggesting FXIII stabilizes thrombi via fibrin γ‐chain crosslinking 13 . In addition, compared to patients with suspected but excluded PE, patients with acute PE have lower circulating FXIII‐A subunit 14 .…”

“…11,12 Furthermore, following FeCl 3 -induced thrombosis, mice expressing fibrinogen with mutated crosslinking sites (FGG3X) have increased PE, suggesting FXIII stabilizes thrombi via fibrin γ-chain crosslinking. 13 In addition, compared to patients with suspected but excluded PE, patients with acute PE have lower circulating FXIII-A subunit. 14 However, reduced FXIII in these patients is associated with decreased fibrinogen and increased Ddimer, suggesting this relationship is due to consumption of FXIII during VTE rather than low FXIII levels prior to the event.…”

Why fibrin biomechanical properties matter for hemostasis and thrombosis

“…However, recent studies from our lab showed associations between decreased average clot (and fiber) stiffness and increased occurrence of clot rupture. 55,84 The association between these properties requires further investigation.…”