Fibrin activates GPVI in human and mouse platelets

Abstract

The glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet signaling receptor for collagen. Paradoxically, in a FeCl3 injury model, occlusion, but not initiation of thrombus formation, is delayed in GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that GPVI is a receptor for fibrin and speculate that this contributes to development of an occlusive thrombus. We observed a marked increase in tyrosine phosphorylation, including the FcRγ chain and Syk, in human and mouse platelets induced by thrombin in the presence of fibrinogen and the αIIbβ3 blocker eptifibatide. This was not seen in platelets stimulated by a protease activated receptor (PAR)-4 peptide, which is unable to generate fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar to that induced by activation of GPVI. Consistent with this, thrombin did not induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient mouse platelets. Mouse platelets underwent full spreading on fibrin but not fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in the absence of GPVI. Spreading on fibrin was associated with phosphatidylserine exposure (procoagulant activity), and this too was blocked in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to immobilized monomeric and polymerized fibrin. A marked increase in embolization was seen following FeCl3 injury in GPVI-deficient mice, likely contributing to the delay in occlusion in this model. These results demonstrate that GPVI is a receptor for fibrin and provide evidence that this interaction contributes to thrombus growth and stability.

Figure 1

Fibrin stimulates tyrosine phosphorylation in a GPVI-dependent manner. Platelets from (Ai) human, (Aii) mice, (Bi) αIIb-deficient mice, and (Bii) GPVI-deficient mice were stimulated with thrombin (1 U/mL) or PAR-4 peptide (150 μM) in the presence of eptifibatide and, where shown, fibrinogen (200 μg/mL) and GPRP (5 μM). Stimulations were stopped after (A) 1 or (B) 3 minutes with the addition of 2× lysis buffer. A sample of the WCL was removed, and the remaining lysate was used to IP Syk. WCLs and IPs were separated by SDS-polyacrylamide gel electrophoresis and western blotted for pTyr, Syk, and the FcRγ chain, which coprecipitates with Syk. The results are shown as representative of 3 experiments.

Figure 2

Fibrin stimulates spreading in a GPVI-dependent manner. Fibrinogen was coated onto glass coverslips and converted into fibrin by treatment with thrombin. Hirudin was used to neutralize any residual thrombin before washing and blocking. (A) Alexa-488 fibrinogen was used to visualize fibrin formation. Platelets (2 × 10⁷/mL) were allowed to spread on coated coverslips, followed by actin staining with Alexa-568 phalloidin. Scale bar, 5 μm. (B) Platelets (2 × 10⁷/mL) were allowed to spread on nonfluorescent fibrinogen or fibrin-coated coverslips, followed by actin staining with Alexa-488 phalloidin. Where shown, platelets were preincubated with dasatinib (10 μM) or hirudin (5 U/mL). Scale bar, 5 μm. (C) WT or GPVI KO platelets were allowed to spread on fibrinogen or fibrin-coated coverslips, followed by actin staining with Alexa-488 phalloidin. Scale bar, 5 μm. The results are shown as mean ± standard error of the mean (SEM) of 3 experiments. **P < .01; ***P < .001.

Figure 3

Fibrin polymerization is not essential for platelet spreading or GPVI binding. (A) Fibrinogen was coated onto glass coverslips and converted into fibrin by treatment with thrombin. GPRP (5 μM) was used to inhibit polymerization of fibrin monomers. Hirudin was used to neutralize any residual thrombin before washing and blocking. Platelets (2 × 10⁷/mL) were allowed to spread, followed by actin staining with Alexa-488 phalloidin. Scale bar, 5 μm. The results are representative of 3 experiments. (B) Fibrinogen was coated onto the wells of Nunc Maxisorp plates and converted into fibrin by treatment with thrombin. GPRP (5 μM) was used to inhibit polymerization of fibrin monomers. The ectodomain of GPVI was incubated, and following washing, adherent GPVI was detected with a HRP-conjugated antibody. The results are shown as mean ± SEM of 3 experiments performed in duplicate. *P < .05; **P < .01.

Figure 4

GPVI KO platelets have reduced PS exposure and reduced thrombus stability. (A) WT or GPVI-deficient platelets were allowed to spread on fibrinogen or fibrin-coated coverslips, followed by incubation with FITC-Annexin V. Actin was counterstained with Alexa-568 phalloidin to count total number of adherent platelets (data not shown). Scale bar, 20 μm. The results are shown as mean ± SEM of 3 experiments. (B) Representative fluorescent images before and after injury. Asterisk indicates vessel occlusion. Embolization rate was determined by counting embolized thrombus fragments (size > 10 µm) after an initial thrombus had formed (thrombus size ≥ 10 µm). Observation period: 40 minutes or until vessel occlusion. The results are shown as mean ± standard deviation of ≥6 mice. ***P < .001.