Rac, a small guanosine triphosphate–binding protein, and p21-activated kinase are activated during platelet spreading on collagen-coated surfaces: roles of integrin α2β1

Abstract

In this study, the receptors and signals involved in collagen-induced platelet spreading were examined. It was found that platelet spreading on collagen (presenting a polygon shape with a number of filopodialike projections) was inhibited by the anti-integrin ␣ 2 antibody, suggesting the involvement of integrin ␣ 2 ␤ 1 in this process. Studies with a glutathione-Stransferase fusion protein that binds specifically to activated Rac and in vitro p21-activated kinase (PAK) kinase assays revealed that Rac and PAK were activated during this collagen-activated process. Platelet spreading on collagen-coated sur- IntroductionPlatelet adhesion to collagen fibers through specific receptors is one of the initial processes in thrombus formation. Platelets adhere to, then spread on, collagen fibers, and finally form aggregates by recruiting other platelets through glycoprotein (GP)IIb/IIIa-fibrinogen binding. Platelet spreading and aggregation appear to involve different processes. The collagen receptors responsible for each process also seem to be different. A number of candidates for platelet collagen receptors have been proposed to date, including integrin ␣ 2 ␤ 1 , GPVI, GPIV, and p65. 1 Among them, GPVI and integrin ␣ 2 ␤ 1 are accepted as collagen receptors necessary for collagen-induced platelet aggregation, because human blood platelets, which lack the surface expression of integrin ␣ 2 ␤ 1 2,3 or GPVI, 4,5 fail to form aggregates on collagen stimulation. The GPVI-mediated activation signals that lead to platelet aggregation have been extensively investigated and several signaling molecules have been characterized. [6][7][8] However, whether integrin ␣ 2 ␤ 1 mediates activation signals during platelet aggregation remains controversial.There have been several studies on the receptors responsible for platelet spreading on collagen-coated surfaces. Kehrel et al 9 reported that only 9.9% of GPIa-deficient platelets spread on type I collagen, in contrast to 82% with control platelets. Nakamura et al 10 showed that an anti-integrin ␣ 2 ␤ 1 antibody markedly inhibited platelet spreading on collagen, whereas an anti-GPIV antibody had virtually no effect. They also reported that an anti-GPVI antibody had only slight inhibitory effects on platelet spreading, although Falet et al 30 have recently shown that human platelets spread on collagen-related peptide (CRP)-coated surfaces, implying that signals from GPVI can also mediate platelet spreading. These findings suggest that integrin ␣ 2 ␤ 1 is at least one of the major receptors responsible for platelet spreading and that it elicits certain signals necessary for this spreading. However, as described above for platelet aggregation, the presence of integrin ␣ 2 ␤ 1 -mediated activation signals has been challenged by others. 1,11 Thus, we sought to characterize the integrin ␣ 2 ␤ 1 -mediated signals that lead to platelet spreading in this study. Platelets adhere to collagen, then extend projections, and finally form curtainlike extensions between the projections during the proces...

Integrin-dependent phagocytosis – spreading from microadhesion to new concepts

“…Thus, as seen for β2 during αMβ2-dependent phagocytosis (Wiedemann et al, 2006), the cytoplasmic tail of the β1 subunit is essential for invasin-induced uptake. Consistent with previous studies showing that β1 integrins signal to Rac1 during cell spreading (Hirsch et al, 2002;Pankov et al, 2005;Stewart and Nemerow, 2007;Suzuki-Inoue et al, 2001), invasin-triggered phagocytosis requires Rac1 activity (Alrutz et al, 2001;McGee et al, 2001). The similarities between the classical and phagocytic behaviours of β1 integrins is further illustrated by the fact that, when β1A-expressing cells spread onto an invasincoated surface, β1A integrins colocalise with vinculin, paxillin and FAK into structures that are essentially identical to the focal adhesions that form when cells spread onto fibronectin; comparatively, β1B-expressing cells spread poorly (Gustavsson et al, 2002;Gustavsson et al, 2004).…”

Integrin α1β1 Controls Reactive Oxygen Species Synthesis by Negatively Regulating Epidermal Growth Factor Receptor-Mediated Rac Activation

“…This small GTPase can serve as an adapter to position p67 phox for interaction with the membrane-bound cytochrome b 558 (reviewed in reference 2). Moreover, Rac and consequent ROS synthesis can be transiently activated by integrin clustering in various cell types, including fibroblasts and platelets (11,27,49,54).…”

Platelet adhesion signalling and the regulation of thrombus formation

“…The adhesion of platelets to GFOGER peptides is accompanied by the tyrosine phosphorylation of several proteins, including Src, Syk, SLP-76 and PLCγ2, which are also involved in GPVI signalling, and calcium-dependent spreading (Inoue et al, 2003). p38 MAP kinase, ILK, Rac and PAK have also been implicated downstream of α2β1 ligation (Stevens et al, 2004;Sundaresan and Farndale, 2003;Suzuki-Inoue et al, 2001). A more detailed map combining these molecules into an α2β1 signalling pathway has yet to be established, and this is an important focus for future research.…”