Plasma fibronectin supports hemostasis and regulates thrombosis

Abstract

Plasma fibronectin (pFn) has long been suspected to be involved in hemostasis; however, direct evidence has been lacking. Here, we demonstrated that pFn is vital to control bleeding in fibrinogen-deficient mice and in WT mice given anticoagulants. At the site of vessel injury, pFn was rapidly deposited and initiated hemostasis, even before platelet accumulation, which is considered the first wave of hemostasis. This pFn deposition was independent of fibrinogen, von Willebrand factor, β3 integrin, and platelets. Confocal and scanning electron microscopy revealed pFn integration into fibrin, which increased fibrin fiber diameter and enhanced the mechanical strength of clots, as determined by thromboelastography. Interestingly, pFn promoted platelet aggregation when linked with fibrin but inhibited this process when fibrin was absent. Therefore, pFn may gradually switch from supporting hemostasis to inhibiting thrombosis and vessel occlusion following the fibrin gradient that decreases farther from the injured endothelium. Our data indicate that pFn is a supportive factor in hemostasis, which is vital under both genetic and therapeutic conditions of coagulation deficiency. By interacting with fibrin and platelet β3 integrin, pFn plays a self-limiting regulatory role in thrombosis, suggesting pFn transfusion may be a potential therapy for bleeding disorders, particularly in association with anticoagulant therapy.

Figure 7. pFn is a self-limiting factor for hemostasis and thrombosis.

(A) Platelet accumulation and fibrin formation in WT mice in the laser-induced cremaster arterial thrombosis model. Fibrin forms at the bottom of the platelet thrombi, while at the top of the growing thrombi, fibrin is nearly undetectable. Scale bar: 10 μm. (B) Illustration of the self-limiting role of pFn for thrombosis after vascular injury. pFn deposited at the base of the hemostatic plug likely cross-links with fibrin to support platelet aggregation. The soluble pFn at the top of the thrombi is associated with platelets but not with fibrin. These pFn molecules play a solely inhibitory role, stabilizing the thrombus and suppressing excessive thrombus buildup, thus helping to form a restricted local hemostatic plug that maintains hemostasis without causing downstream ischemia. (C) Illustration of pFn promoting efficient hemostasis after severe vascular injury. After severe trauma to a vessel, a large amount of thrombin is generated from multiple sites of the injured vessel wall, so that even at the top of the growing thrombi, there is a sufficient amount of thrombin to initiate fibrin formation. Under this condition, pFn is continuously incorporated onto the top of the growing thrombus through fibrin. This fibrin cross-linked pFn promotes the formation of an occlusive thrombus and helps to stop blood loss efficiently after severe vascular injuries. Blue arrows indicate fibrin concentration change from high level (H, red) to low level (L, yellow) as the hemostatic plug extends from the vessel wall into to the vessel lumen.

Figure 6. Role of pFn in platelet aggregation and thrombus formation in the presence or absence of fibrin.

(A) Thrombin-induced (0.2 U/ml) FG^–/– pFn^–/– and FG^–/– gel-filtered platelet aggregation. (B) Thrombin-induced (0.2 U/ml) or TRAP-induced (500 μM) Vwf^–/– pFn^–/– and Vwf^–/– gel-filtered platelet aggregation. (C) Collagen-induced (20 μg/ml) Vwf^–/– pFn^–/– and Vwf^–/– gel-filtered platelet aggregation. (D) ADP-induced (20 μM) Vwf^–/– pFn^–/– and Vwf^–/– platelet aggregation in PRP. (E)Thrombin-induced (0.2 U/ml) pFn^+/+ and pFn^–/– gel-filtered platelet aggregation. All representative tracings of platelet aggregation are shown from at least 3 independent experiments. (F) Thrombus formation in a collagen-coated perfusion chamber infused with blood from FG^–/– pFn^–/– mice and their pFn^+/+ FG^–/– littermates (shear rate = 1,800 s^–1). Images were taken with a confocal microscopy after infusion for 3 minutes. Grid in the image represents 10 μm. (G) Thrombus volume measured 3 minutes after infusion. n = 5 in each group. (H) Thrombus formation in FG^–/– pFn^–/– mice and their pFn^+/+ FG^–/– littermates observed in the laser-induced cremaster arterial thrombosis model. The curves represent pFn or platelet mean fluorescent intensity, and the shaded regions represent SEM. (I) The total area under the curve (AUC) was greater in FG^–/– pFn^–/– mice than in FG^–/– mice. n = 5 in each group. Data are presented as mean ± SEM.

Figure 5. pFn controls the diameter of human fibrin fibers.

(A) Confocal images of human pFn-fibrin network formed in vitro (z projection of 11 individual slides taken at 1-μm interval across the 10-μm thickness of the clot). Human pFn or BSA was added into pFn-depleted human PPP to a final concentration of 330 μg/ml. (B) Scanning electron microscopy analysis of human pFn-fibrin network formed in vitro. The same volume of PBS and human pFn was added into pFn-depleted human PPP (final concentration 330 μg/ml). (C) Average diameter of pFn-fibrin fibers formed in vitro. (D) Average density of pFn-fibrin fibers formed in vitro. Average diameter and density were calculated by counting the fibers crossed by 2 diagonal lines in a 1-μm² square. n = 10 in each group. Scale bar: 20 μm (A); 1 μm (B).

Figure 4. pFn enhances the mechanical strength of the fibrin clot and controls the diameter of fibrin fibers in mice.

(A–C) Representative TEG tracings and MA measured by TEG in (A) whole blood and (B) PPP. (C) Adding back purified pFn partially rescued the MA of pFn^–/– blood. n = 5 in each group. (D) Confocal images of mouse pFn-fibrin network formed in vitro (z projection of 11 individual slides taken at 1-μm interval across the 10-μm thickness of the clot). Mouse pFn or BSA was added into pFn^–/– mouse PPP to a final concentration of 330 μg/ml. (E) Scanning electron microscopy analysis of mouse pFn-fibrin network formed in vitro. The same volume of PBS and mouse pFn was added into pFn^–/– mouse PPP (final concentration 330 μg/ml). (H) Scanning electron microscopy analysis of pFn^+/+ and pFn^–/– mouse pFn-fibrin network formed in vitro. pFn-fibrin network formation was initiated with 0.5 U/ml murine thrombin and 30 mM CaCl₂. (F and I) Average diameter of pFn-fibrin fibers formed in vitro. (G and J) Average density of pFn-fibrin fibers formed in vitro. Average diameter and density were calculated by counting the fibers crossed by 2 diagonal lines in a 1-μm² square. n = 9 in each group. Scale bar: 20 μm (D); 1 μm (E and H).

Figure 3. pFn deposition on the injured vessel wall is independent of Fg and β3 integrin and persists after heparin treatment.

(A) pFn deposition and platelet accumulation in FG^–/– mice in the laser-induced cremaster arterial thrombosis model. Images were taken at 1 minute after injury. (B) 3D reconstruction of pFn deposition and initial platelet accumulation in mesenteric arteries and veins 5 minutes after FeCl₃ injury in FG^–/– mice. (C) pFn deposition and platelet accumulation in Itgβ3^–/– mice in the laser-induced cremaster arterial thrombosis model. Images were taken at 1 minute after injury. (D) 3D reconstruction of pFn deposition and initial platelet accumulation in a mesenteric artery 5 minutes after FeCl₃ injury in a Itgβ3^–/– mouse. (E) Fluorescently labeled pFn is detectable at the injury site 60 minutes after laser injury in WT mice. (F) 3D reconstruction of pFn deposition and initial platelet accumulation in mesenteric arteries and veins 5 minutes after FeCl₃ injury in WT mice treated with heparin. Scale bar: 10 μm (A, C, and E); 50 μm (B, D, and F). All images shown are representative of at least 5 independent experiments.

Figure 2. pFn deposition is a hemostatic event preceding platelet accumulation.

(A) pFn deposition and platelet accumulation in WT mice in the laser-induced cremaster arterial thrombosis model. pFn deposition at the injury site was apparent 10 seconds after laser injury, prior to significant platelet accumulation. (B) No deposition of fluorescently labeled BSA was observed. (C) pFn deposition and platelet accumulation at the site of injury of WT mice in the cremaster arterial thrombosis model were measured by fluorescence intensity. The curves represent pFn or platelet mean fluorescent intensity, and the shaded regions represent SEM (n = 5 in each group). (D) 3D reconstruction of pFn deposition and initial platelet accumulation in mesenteric arteries and veins 5 minutes after FeCl₃ injury in WT mice. The images shown are the longitudinal sections of the vessels, viewed from the inside of the lumen or from the outside the vessel wall. (E) pFn deposition and platelet accumulation in FG^–/– Vwf^–/– mice in the laser-induced cremaster arterial thrombosis model. (F) Measurement of fluorescent intensity of pFn deposition and platelet accumulation at the site of injury of FG^–/– Vwf^–/– mice in the cremaster arterial thrombosis model (n = 5 in each group). (G) 3D reconstruction of pFn deposition and initial platelet accumulation in mesenteric arteries and veins 5 minutes after FeCl₃ injury in FG^–/– Vwf^–/– mice. Scale bar: 10 μm (A, B, and E); 50 μm (D and G). All images shown are representative of at least 5 independent experiments.

Figure 1. pFn is a key hemostatic factor in deficiencies of Fg and blood coagulation.

(A) Representative images of abdominal and subcutaneous bleeding in TKO mice. Red arrows show the bleeding site. (B) Tail bleeding time in WT, FG^–/– Vwf^–/–, and TKO mice. (C) Tail bleeding time in FG^–/– and FG^–/– pFn^–/– mice. (D) Tail bleeding time in FG^–/– pFn^–/– mice transfused with 200 μl PBS, 1.5 mg/ml purified pFn, or BSA. (E) Tail bleeding time in pFn^–/– mice and their WT (pFn^+/+) littermates treated with i.p. injection of PBS, 20 U heparin, or 0.5 mg/kg recombinant hirudin (r-hirudin). Tail bleeding assay was performed 30 minutes after the injection. (F) Tail bleeding time in WT mice given high-dose heparin (200 U) and then transfused with PBS or pFn. For all tail bleeding assays, 2 mm of the tip of the tail was severed. Bleeding time assay was terminated at 20 or 30 minutes, as indicated, and mice that bled beyond this end point were counted as 20 or 30 minutes, respectively. Data are presented as mean ± SEM. Individual symbols represent individual mice.