A major role for Scar/WAVE-1 downstream of GPVI in platelets

Abstract

Background: The small GTPase Rac1 plays a critical role in lamellipodia assembly in platelets on matrix proteins in the absence or presence of G protein-coupled receptor (GPCR) agonists. Rac mediates actin assembly via Scar/WAVE, a family of scaffolding proteins that direct actin reorganization by relaying signals from Rac to the Arp2/3 complex.

Objective: To evaluate the role of Scar/WAVE-1 in mediating platelet activation and cytoskeletal reorganization.

Methods and results: Using specific antibodies, we demonstrate that murine platelets, like human platelets, express Scar/WAVE-1 and Scar/WAVE-2. Lamellipodia formation in Scar/WAVE-1(-/-) platelets is markedly inhibited on immobilized collagen-related peptide (CRP) and on laminin, both of which signal through the collagen receptor GPVI. In contrast, lamellipodia formation on collagen, which requires release of the GPCR agonists ADP and thromboxane A(2), is not altered. Immobilized fibrinogen supports limited formation of lamellipodia in murine platelets, which is not altered in Scar/WAVE-1(-/-) platelets. As with Rac1(-/-) platelets, Scar/WAVE-1(-/-) platelets exhibit a marked inhibition of aggregation in response to CRP, whereas the response to the GPCR agonist thrombin is not altered. Platelet aggregation on immobilized collagen under shear, which is dependent on signaling by matrix and GPCR agonists, was unaltered in the absence of Scar/WAVE-1.

Conclusion: This study demonstrates a major role for Scar/WAVE-1 in mediating platelet cytoskeletal reorganization and aggregate formation downstream of activation by GPVI but not by GPCR agonists.

Fig. 1

Scar/WAVE isoforms are expressed in human and murine platelets. Equal amounts of human (A and C) and murine wild-type and Scar/WAVE-1^-/- (B and D) platelet lysates were analyzed for Scar/WAVE expression using Scar/WAVE-1 or Scar/WAVE-2 antibodies. Lysates from Cos-7 cells transfected with a vector containing (A) Scar/WAVE-1 or (C) Scar/WAVE-2 were included as a positive control.

Fig. 2

Role of Scar/WAVE-1 in α_IIbβ₃-mediated murine platelet spreading. Purified wild-type (WT) and Scar/WAVE-1^-/- murine platelets (2 × 10⁷/ml) were placed on coverslips coated with fibrinogen with or without 1 U/ml thrombin for 45 min and imaged using DIC microscopy. In separate experiments, platelets were treated with 10 μM ADP. Note the elongated filopodia extended by a portion of the Scar/WAVE-1^-/- platelets on fibrinogen under non-stimulated conditions as indicated by the arrows. Results are representative of at least 3 experiments.

Fig. 3

Role of Scar/WAVE-1 in GPVI-murine platelet spreading. Purified wild-type (WT) and Scar/WAVE-1^-/- murine platelets (2 × 10⁷/ml) were placed on coverslips coated with collagen, laminin or collagen-related peptide (CRP) for 45 min and imaged using DIC microscopy. Results are representative of at least 3 experiments.

Fig. 4

Role of Scar/WAVE-1 in platelet aggregation to GPVI and G-protein coupled agonists. Washed platelets (2 × 10⁸/ml) from wild-type (WT) and Scar/WAVE-1^-/- mice were stimulated with A) CRP or B) thrombin (thr) at the indicated concentrations, and the change in optical density indicative of aggregation recorded. One representative experiment of three separate trials is depicted.

Fig. 5

Role of Scar/WAVE-1 in platelet P-selectin exposure. Washed platelets (2 × 10⁷/ml) from wild-type (black bars) and Scar/WAVE-1^-/- (white bars) stimulated with 10 μg/ml CRP, 0.1 U/ml thrombin (thr) or 30 μg/ml collagen for 15 min at 37°C. The degree of FITC-conjugated anti-P-selectin mAb was recorded via FACS analysis in arbitrary units (au). Values are mean ± SEM from 3 experiments. * P < 0.01 with respect to wild-type.

Fig. 6

Role of Scar/WAVE-1 in platelet adhesion and aggregate stability on collagen under flow. Mouse blood anticoagulated with PPACK and heparin was perfused through a collagen-coated microslide at a shear rate of 1000 s^-1 for 4 min followed by modified Tyrode buffer for 3 min to remove non-adherent cells. Subsequently, slides were visualized using phase-contrast microscopy. A) Representative images of platelet adhesion from wild-type (left panel) and Scar/WAVE-1^-/- (right panel) mice are shown. Direction of flow is from left to right. Images are representative of 3 experiments. B) Platelet adhesion results are expressed as mean ± SEM of surface area covered by platelets.