Plasma Fibronectin In Thrombosis and Hemostasis: Exploring the Fibrin Dependent and Independent Mechanisms

Abstract Abstract 484 Background: Plasma fibronectin (pFn) is an abundant protein in the blood. It has long been suspected that pFn plays a role in thrombosis/hemostasis, but this has remained controversial. Our previous study using pFn deficient mice demonstrated that pFn supports thrombosis (PNAS. 2003; 100: 2415-9). Unexpectedly, depletion of pFn in fibrinogen (Fg) and von Willebrand factor (VWF) double deficient (Fg/VWF−/−) mice enhanced, rather than abolished, platelet aggregation and thrombus formation, revealing a functional switch of pFn in the presence and absence of Fg and VWF (Blood. 2009;113:1809-17). However, the mechanism that controls this switch is not known. Furthermore, the hemostatic function of pFn in VW disease (VWD) or afibrinogenemia is unclear. Methods: To address these questions, we bred pFn conditional knockout mice with VWF−/− or Fg−/− mice, establishing 2 new strains of mice: Fg/pFn−/− and VWF/pFn−/−. We also extended our studies of pFn in the triple knockout (TKO, Fg/VWF/pFn−/−) mice. PolyI-polyC was injected into Cre+ and Cre- mice, which resulted in the depletion of plasma pFn (>98%) and platelet pFn (>80%) in Cre+ mice but not in Cre- littermate controls. Aggregometry, a perfusion chamber system, thromboelastography (TEG), tail vein bleeding assay and intravital microscopy were used to study these mice. Results: We first observed a significantly higher mortality in TKO (25%, P<0.05) mice than their Cre- Fg/VWF−/− littermates within 1–2 weeks following the depletion of pFn. Autopsy of these mice revealed severe subcutaneous or abdominal bleeding at the sites of injection. The tail vein bleeding time in TKO mice was also prolonged (P<0.05). Using a laser injury model of intravital microscopy, we observed rapid deposition of fluorescently-labeled pFn at sites of vessel injury in Fg/VWF−/− mice prior to significant platelet deposition. This suggested pFn is a quick/efficient factor contributing to hemostasis in the absence of Fg/VWF. We further found that the mortality rate in Fg/pFn−/− mice was also higher than their Cre- Fg−/− littermates (29%, P<0.05), demonstrating that pFn is a critical hemostatic factor that prevents fatal hemorrhage in afibrinogenemic mice via a fibrin-independent mechanism. We also found that pFn supports hemostasis in VWF−/− mice, although no significant mortality difference was observed (P>0.05). The tail vein bleeding time was longer in VWF/pFn−/− mice than in Cre- VWF−/− littermates (P<0.05), and significantly smaller thrombi were observed when VWF/pFn−/− whole blood was perfused over a collagen surface under shear rate of 1800s-1 (P<0.05). This suggests that pFn may play a role in VWF deficiency (i.e. in type 3 VWD). pFn was also found to support hemostasis in a fibrin-dependent manner. We first demonstrated with TEG that fibrin clot strength was significantly stronger in Cre- littermates than in pFn−/− mice (P<0.05). Platelet aggregation in gel-filtered platelets induced by thrombin, which converts Fg to fibrin on the platelet surface, was greater in Cre- VWF−/− than VWF/pFn−/− platelets (P<0.05). Very interestingly, in keeping with our earlier observation in TKO mice, pFn also inhibited platelet aggregation when fibrin was absent. In Fg−/− mice, we found that pFn depletion enhanced gel-filtered platelet aggregation induced by both thrombin and thrombin receptor activating peptide (TRAP, AYPGKF; P<0.05). In Cre- VWF−/− mice where Fg is present, pFn depletion also enhanced gel-filtered platelet aggregation induced by TRAP (which cannot convert Fg to fibrin) (P<0.05). pFn therefore plays a dual role in platelet aggregation based on the presence of fibrin (i.e. covalently linked fibrin-pFn supports platelet aggregation, while pFn alone inhibits aggregation). Conclusion: Our data demonstrated that pFn is a critical factor for the survival of Fg−/− mice and supports hemostasis in VWF−/− mice via both fibrin-independent and dependent pathways. We clearly showed that fibrin, likely in the form of covalently-linked fibrin-pFn complexes, is required for pFn to support platelet aggregation. Through inhibition of platelet aggregation, non-fibrin-linked soluble pFn may play an important role in the prevention of excessive thrombus formation at the site of vessel injury and thus maintains blood circulation. pFn is therefore likely a crucial supportive factor in hemostasis (for afibrinogenemic and VWD patients), and an important regulator in thrombosis. Disclosures: No relevant conflicts of interest to declare.