Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation

Abstract

Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in hemostasis is still unknown. It is reported that GPV knockout mice had a decreased tendency to form arterial occluding thrombi in an intravital thrombosis model and abnormal platelet interaction with the subendothelium. In vitro, GPV-deficient platelets exhibited defective adhesion to a collagen type I-coated surface under flow or static conditions. Aggregation studies demonstrated a decreased response of the GPV-deficient platelets to collagen, reflected by an increased lag phase and reduced amplitude of aggregation. Responses to adenosine diphosphate, arachidonic acid, and the thromboxane analog U46619 were normal but were enhanced to low thrombin concentrations. The defect of GPV null platelets made them more sensitive to inhibition by the anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in aspirin-or apyrase-treated platelets. Moreover, an mAb (V.3) against the extracellular domain of human GPV selectively inhibited collagen-induced aggregation in human or rat platelets. V.3 injected in rats as a bolus decreased the ex vivo collagen aggregation response without affecting the platelet count. Finally, surface plasmon resonance studies demonstrated binding of recombinant soluble GPV on a collagen-coupled matrix. In conclusion, GPV binds to collagen and appears to be required for normal platelet responses to this agonist. IntroductionPlatelets play a central role in hemostasis through their ability to adhere to a damaged vessel wall and to aggregate in response to agonists such as thrombin, collagen, or adenosine diphosphate (ADP), 1 and these properties are known to be mediated by cell surface glycoproteins. 2,3 However, the exact functions of numerous platelet glycoproteins that have been characterized biochemically remain unknown. Glycoprotein V (GPV, Mr 82 kd) is one of the most abundant glycoproteins at the surface of blood platelets and has long been identified, 4-6 but its functional role is still subject to speculation. GPV is noncovalently linked to the GPIb-IX von Willebrand factor (vWF) receptor on the platelet surface. 7,8 This type I transmembrane protein has a large extracellular domain comprising 15 Leu-rich motifs followed by a thrombin cleavage site. 9,10 The specific release of a soluble 69-kd extracellular domain fragment (GPVf1) by thrombin has led to its proposal as a thrombin receptor, 6 whereas cloning of the gene in rat and mouse revealed a well-conserved thrombin cleavage site. 11 Studies in transfected cells have shown that GPV is required for efficient thrombin binding, possibly through a direct interaction with GPIb␣. 12,13 The release of a soluble fragment by thrombin has been used to develop a specific enzyme-linked immunosorbent assay for GPV that is being tested as a means of monitoring platelet activation under conditions of clinical thrombosis. 14 The role of GPV a...

Proplatelet formation is selectively inhibited by collagen type I through Syk-independent GPVI signaling

“…In addition, LAIR1 has been described as an early megakaryocyte inhibitory receptor for collagen, with reduced expression on mature megakaryocytes and no expression on platelets (Steevels et al, 2010). DDR1 has most recently been reported to be expressed on early megakaryocytes and to modulate their motility on collagen I (Abbonante et al, 2013) and, finally, GPV on platelets can also bind to collagen (Moog et al, 2001). Using blocking antibodies for collagen receptors on human megakaryocytes derived from cord blood CD34+ cells, Sabri and colleagues have shown that integrin α2β1 is essential for stress fiber formation by MAPK (ERK1/2) and Rho GTPase activation, whereas GPVI plays a role in maintaining ERK signaling.…”

Integrin αIIbβ3-dependent Calcium Signals Regulate Platelet-Fibrinogen Interactions under Flow

“…Platelet Preparation-Washed human and murine platelets were prepared as described previously (26,30). For adhesion studies, washed platelets were resuspended in modified Tyrode's buffer (10 mM Hepes, 12 mM NaHCO 3 , pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5 mM glucose).…”

Signaling Role for Phospholipase Cγ2 in Platelet Glycoprotein Ibα Calcium Flux and Cytoskeletal Reorganization

“…Aspirinated platelets were obtained by treating cells with 1 mM aspirin for 15 min, prior to final suspension in Tyrode's buffer. Washed mouse platelets were prepared according to the method described by Moog et al (27). Mouse blood was taken from the abdominal aorta of anesthetized animals and collected in an acid citrate dextrose anticoagulant.…”