The vasodilator-stimulated phosphoprotein (VASP) is associated with actin filaments and focal adhesions, which form the interface between the cytoskeleton and the extracellular matrix. VASP is phosphorylated by both the cAMP-and cGMP-dependent protein kinases in a variety of cells, including platelets and smooth muscle cells. Since both the cAMP and cGMP signalling cascades relax smooth muscle and inhibit platelet activation, it was speculated that VASP mediates these effects by modulating actin filament dynamics and integrin activation. To study the physiological relevance of VASP in these processes, we inactivated the VASP gene in mice. Adult VASP-deficient mice had normal agonist-induced contraction, and normal cAMP-and cGMP-dependent relaxation of intestinal and vascular smooth muscle. In contrast, cAMP-and cGMP-mediated inhibition of platelet aggregation was significantly reduced in the absence of VASP. Other cAMP-and cGMPdependent effects in platelets, such as inhibition of agonist-induced increases in cytosolic calcium concentrations and granule secretion, were not dependent on the presence of VASP. Our data show that two different cyclic, nucleotide-dependent mechanisms are operating during platelet activation: a VASP-independent mechanism for inhibition of calcium mobilization and granule release and a VASP-dependent mechanism for inhibition of platelet aggregation which may involve regulation of integrin function.

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake Ctype lectin family and is thought to activate platelets by binding to platelet glycoprotein ␣ 2 ␤ 1 . We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to ␣ 2 ␤ 1 . Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor ␥ chain (Fc␥)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fc␥ pathway. Platelets from Fc␥-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including ␣ 2 ␤ 1 or GPIb for the lack of the Fc␥ pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72 SYK , p125 FAK , and PLC␥2, whereas, in comparison with collagen and convulxin, the Fc␥ subunit neither is phosphorylated nor coprecipitates with p72 SYK . This supports an independent, GPIb-and integrin-based pathway for activation of p72 SYK not involving the Fc␥ receptor.Platelet-collagen interactions are integral to primary hemostasis (1, 2). Resting platelets using several receptors adhering to subendothelium of damaged blood vessels are activated and spread to provide finally a new nonthrombogenic surface until vasculature regeneration occurs. Reversible binding between GPIb-V-IX 1 and von Willebrand factor, associated with collagen, is crucial to slow down the platelet (especially under high shear) so that it can bind more firmly via other receptors (3, 4). This mechanism strongly parallels that of the selectins in leukocyte adhesion (5). Another important receptor is the ␣ 2 ␤ 1 integrin, which is essential for anchoring the platelet to collagen in the subendothelium (6) and for linking to the platelet cytoskeleton to prevent the receptor being torn from the membrane by the forces that it has to withstand. Activation induces the release of storage granules and the expression of new receptors on the platelet surface (7) as well as changes in other receptors such as the fibrinogen receptor, ␣ IIb ␤ 3 , which is critical for spreading. Although GPIb-V-IX and ␣ 2 ␤ 1 also participate in signaling to the platelet interior (8, 9), recent studies, particularly in patients with platelet receptor deficiencies, have implicated GPVI/Fc␥ as a major collagen receptor for platelet activation (10 -12). Patients with platelets lacking any one of these receptors (GPIb-V-IX, ␣ 2 ␤ 1 , or GPVI/Fc␥) have increased bleeding times, and platelet adhesio...

Platelet function is developmentally regulated. Healthy neonates do not spontaneously bleed, but their platelets are hypo-reactive to several agonists. The mechanisms underlying immature platelet function in neonates are incompletely understood. This critical issue remains challenging for the establishment of age-specific reference ranges. In this study, we evaluated platelet reactivity of five pediatric age categories, ranging from healthy full-term neonates up to adolescents (11–18 years) in comparison to healthy adults (>18 years) by flow cytometry. We confirmed that platelet hypo-reactivity detected by fibrinogen binding, P-selectin, and CD63 surface expression was most pronounced in neonates compared to other pediatric age groups. However, maturation of platelet responsiveness varied with age, agonist, and activation marker. In contrast to TRAP and ADP, collagen-induced platelet activation was nearly absent in neonates. Granule secretion markedly remained impaired at least up to 10 years of age compared to adults. We show for the first time that neonatal platelets are deficient in thrombospondin-1, and exogenous platelet-derived thrombospondin-1 allows platelet responsiveness to collagen. Platelets from all pediatric age groups normally responded to the C-terminal thrombospondin-1 peptide RFYVVMWK. Thus, thrombospondin-1 deficiency of neonatal platelets might contribute to the relatively impaired response to collagen, and platelet-derived thrombospondin-1 may control distinct collagen-induced platelet responses.