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

BACKGROUND: Venous thrombosis (VT) and pulmonary embolism (PE), collectively venous thromboembolism (VTE), cause high mortality and morbidity. Factor XIII (FXIII) crosslinks fibrin to enhance thrombus stability and consequently may influence PE risk. Elucidating mechanisms contributing to PE is limited by a lack of models that recapitulate human PE characteristics. OBJECTIVE: We aimed to develop a mouse model that permits embolization of red blood cell (RBC)- and fibrin-rich VT and determine the contribution of FXIII to PE risk. METHODS AND RESULTS: In a thrombin-infusion PE model, F13a+/+ , F13a+/- , and F13a-/-  mice had similar incidence of microthrombi in the lungs; however, thrombi were small, with low RBC content (≤7%), unlike human PEs (~70%). To identify a model producing PE consistent with histological characteristics of human PE, we compared mouse femoral vein electrolytic injury, femoral vein FeCl3 injury, and infrarenal vena cava (IVC) stasis models of VT. Electrolytic and FeCl3  models produced small thrombi with few RBCs (5% and 4%, respectively), whereas IVC stasis produced large thrombi with higher RBC content (68%) that was similar to human PEs. After IVC stasis and ligature removal (de-ligation) to permit thrombus embolization, compared to F13a+/+ mice, F13a+/-  and F13a-/-  mice had similar and increased PE incidence, respectively. CONCLUSIONS: Compared to thrombin infusion-, electrolytic injury-, and FeCl3 -based models, IVC stasis produces thrombi that are more histologically similar to human thrombi. IVC stasis followed by de-ligation permits embolization of existing RBC- and fibrin-rich thrombi. Complete FXIII deficiency increases PE incidence, but partial deficiency does not.

Factor XIII deficiency does not prevent FeCl3-induced carotid artery thrombus formation in mice.

BACKGROUND: The compositions of venous (red blood cell-rich) and arterial (platelet-rich) thrombi are mediated by distinct pathophysiologic processes; however, fibrin is a major structural component of both. The transglutaminase factor XIII (FXIII) stabilizes fibrin against mechanical and biochemical disruption and promotes red blood cell retention in contracted venous thrombi. Previous studies have shown factor XIII (FXIII) inhibition decreases whole blood clot mass and therefore, may be a therapeutic target for reducing venous thrombosis. The role of FXIII in arterial thrombogenesis is less studied, and the particular contribution of platelet FXIII remains unresolved. OBJECTIVE: To determine whether FXIII reduction prevents experimental arterial thrombogenesis. METHODS: Using wild-type mice and mice with genetically imposed deficiency in FXIII, we measured thrombus formation and stability following ferric chloride-induced arterial thrombosis. We also determined the impact of FXIII on the mass of contracted platelet-rich plasma clots. RESULTS: Following vessel injury, F13a+/+ , F13a+/- , and F13a-/- mice developed occlusive arterial thrombi. FXIII deficiency did not significantly reduce the incidence or prolong the time to occlusion. FXIII deficiency also did not alter the timing of reflow events or decrease platelet-rich clot mass. CONCLUSIONS: FXIII does not significantly alter the underlying pathophysiology of experimental arterial thrombus formation.

The factor XIII-A Val34Leu polymorphism decreases whole blood clot mass at high fibrinogen concentrations.

BACKGROUND: Factor XIII (FXIII) promotes fibrin crosslinking and red blood cell (RBC) retention in clots. The FXIII-A polymorphism, Val34Leu, is associated with protection against venous thrombosis. This effect is hypothesized to result from fibrinogen concentration-dependent changes in fibrin structure. Effects of the FXIII-A Val34Leu polymorphism in whole blood clots have not been investigated. AIM: Characterize effects of FXIII-A Val34Leu polymorphism and fibrinogen on whole blood clots. METHODS: We isolated platelet-poor plasmas from human donors (FXIIIVal/Val , FXIIIVal/Leu , FXIIILeu/Leu ), reconstituted plasmas with platelets and RBCs, and triggered clotting. We assessed contributions of gender, age, clotting times, thrombin generation, FXIII activity, FXIII-A Val34Leu polymorphism, and fibrinogen to clot mass. We also reconstituted FXIII-depleted plasma with platelets, RBCs, and purified FXIIIVal/Val or FXIIILeu/Leu , varied fibrinogen, and characterized effects on clot mass. RESULTS: Clot mass was associated with age, fibrinogen, prothrombin time, and thrombin generation. Clots reconstituted with plasmas from individuals with FXIII-AVal/Val and FXIII-AVal/Leu did not differ in mass from clots with FXIII-ALeu/Leu . However, clots containing a 34Val allele demonstrated a fibrinogen concentration-dependent increase in mass, whereas clots with homozygous 34Leu did not. In plasmas with high fibrinogen, mass was higher for clots with 34Val alleles compared with clots with homozygous 34Leu. In clots reconstituted with purified FXIII, increasing fibrinogen enhanced clot mass in the presence of 34Val, but decreased mass in the presence of 34Leu. CONCLUSIONS: FXIII 34Leu mitigates the effect of elevated fibrinogen on whole blood clot mass. The Val34Leu polymorphism may protect against venous thrombosis by reducing clot mass.

Factor XIIIA-expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking.

Lung cancer is the leading cause of cancer-related deaths worldwide, and lung squamous carcinomas (LUSC) represent about 30% of cases. Molecular aberrations in lung adenocarcinomas have allowed for effective targeted treatments, but corresponding therapeutic advances in LUSC have not materialized. However, immune checkpoint inhibitors in sub-populations of LUSC patients have led to exciting responses. Using computational analyses of The Cancer Genome Atlas, we identified a subset of LUSC tumors characterized by dense infiltration of inflammatory monocytes (IMs) and poor survival. With novel, immunocompetent metastasis models, we demonstrated that tumor cell derived CCL2-mediated recruitment of IMs is necessary and sufficient for LUSC metastasis. Pharmacologic inhibition of IM recruitment had substantial anti-metastatic effects. Notably, we show that IMs highly express Factor XIIIA, which promotes fibrin cross-linking to create a scaffold for LUSC cell invasion and metastases. Consistently, human LUSC samples containing extensive cross-linked fibrin in the microenvironment correlated with poor survival.

Factor XIII in plasma, but not in platelets, mediates red blood cell retention in clots and venous thrombus size in mice.

The transglutaminase factor XIII (FXIII) stabilizes clots against mechanical and biochemical disruption and is essential for hemostasis. In vitro and in vivo models of venous thrombosis demonstrate that FXIII mediates clot size by promoting red blood cell (RBC) retention. However, the key source of FXIII and whether FXIII activity can be reduced to suppress thrombosis without imposing deleterious hemostatic consequences are 2 critical unresolved questions. FXIII is present in multiple compartments, including plasma (FXIIIplasma) as a heterotetramer of A2 and B2 subunits and platelets (FXIIIplt) as an A2 homodimer. We determined the role of the FXIII compartment and level in clot contraction, composition, and size in vitro and using in vivo models of hemostasis and venous thrombosis. Reducing overall FXIII levels decreased whole blood clot weight but did not alter thrombin generation or contraction of platelet-rich plasma clots. In reconstituted platelet-rich plasma and whole blood clot contraction assays, FXIIIplasma, but not FXIIIplt, produced high-molecular-weight fibrin crosslinks, promoted RBC retention, and increased clot weights. Genetically imposed reduction of FXIII delayed FXIII activation and fibrin crosslinking, suggesting FXIII levels mediate the kinetics of FXIII activation and activity and that the timing of these processes is a critical determinant of RBC retention during clot formation and contraction. A 50% reduction in FXIIIplasma produced significantly smaller venous thrombi but did not increase bleeding in tail transection or saphenous vein puncture models in vivo. Collectively, these findings suggest that partial FXIII reduction may be a therapeutic strategy for reducing venous thrombosis.

Gene Expression Profiling in Mouse Embryonic Stem Cells Reveals Glycogen Synthase Kinase-3-Dependent Targets of Phosphatidylinositol 3-Kinase and Wnt/ß-Catenin Signaling Pathways.

Glycogen synthase kinase-3 (Gsk-3) activity is an important regulator of numerous signal transduction pathways. Gsk-3 activity is the sum of two largely redundant proteins, Gsk-3α and Gsk-3ß, and in general, Gsk-3 is a negative regulator of cellular signaling. Genetic deletion of both Gsk-3α and Gsk-3ß in mouse embryonic stem cells (ESCs) has previously been shown to lead to the constitutive activation of the Wnt/ß-catenin signaling pathway. However, in addition to Wnt signaling, all Gsk-3-regulated pathways, such as insulin signaling, are also affected simultaneously in Gsk-3α(-) (/) (-); Gsk-3ß(-) (/) (-)ESCs. In an effort to better understand how specific signaling pathways contribute to the global pattern of gene expression in Gsk-3α(-) (/) (-); Gsk-3ß(-) (/) (-)ESCs, we compared the gene expression profiles in Gsk-3α(-) (/) (-); Gsk-3ß(-) (/) (-) ESCs to mouse ESCs in which either Wnt/ß-catenin signaling or phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling are constitutively active. Our results show that Wnt signaling has a greater effect on up-regulated genes in the Gsk-3α(-) (/) (-); Gsk-3ß(-) (/) (-)ESCs, whereas PI3K-dependent insulin signaling is more responsible for the down-regulation of genes in the same cells. These data show the importance of Gsk-3 activity on gene expression in mouse ESCs, and that these effects are due to the combined effects of multiple signaling pathways.