Platelet GP Ib-IX-V: not just a binding protein

HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

While it is now well accepted that platelet adhesion and subsequent glycoprotein (GP) IIb-IIIa-dependent thrombus formation at arterial flow rates is initiated through binding of von Willebrand factor (VWF) to the platelet GP Ib-IX-V complex, the molecular mechanism of how this occurs has remained one of controversy.¹  Some investigators have argued that the role of VWF/GP Ib-IX-V interaction is primarily mechanical, slowing down translocating platelets so that accessory signaling receptors and/or GP IIb-IIIa can be functionally engaged. This is consistent with VWF containing separate binding motifs, the A1 domain and an arginine-glycine-aspartate (RGD) sequence, for engagement of GP Ib-IX-V and GP IIb-IIIa, respectively. GP Ib-IX-V would thus either play no signaling role, simply positioning the VWF RGD sequence for binding and activation of GP IIb-IIIa, or would only provide a minimal signal, either through immunoreceptor tyrosine activation motif (ITAM)-associated accessory proteins such as Fc-receptor γ-chain or FcγRIIa, or via calcium influx or minor calcium release from internal stores. Up-regulation of GP IIb-IIIa function would therefore depend on subsequent dense body adenosine diphosphate (ADP) release and/or production of thromboxane A₂ (TxA2) and propagation of signals through P2Y1, P2Y12, and TxA2 receptors. In contrast, others have provided evidence using either platelets or transfected cells that GP Ib-IX-V directly acts as a classical signaling receptor, despite having neither obvious cytoplasmic catalytic sequences nor obvious binding motifs. GP Ib-IX-V has been shown to directly bind 14-3-3-ζ, filamin, and calmodulin and to coassociate with other signaling proteins such as phosphatidylinositol 3-kinase (PI 3-kinase), Src, and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase-2 (SHIP-2).² 

In this issue of Blood, Kasirer-Friede and colleagues (page 3403) have addressed this critical question of whether ligation of GP Ib-IX-V alone is sufficient to induce signaling events leading to the activation of GP IIb-IIIa. To do this, they have employed a GP Ib-specific agonist, a dimeric form of the VWF A1 domain, immobilized on plastic. Blockade of responses through ADP receptors, TxA2 receptor, and GP IIb-IIIa ensured that only the adherent monolayer was analyzed and that confounding activation events were excluded. The authors concentrated on previously identified signaling events, such as calcium flux oscillations, or signaling effectors, such as tyrosine kinases and protein kinase C (PKC)/PI 3-kinase. They also examined the relative contribution to platelet activation of ITAM-containing accessory signaling proteins by employing mouse platelets lacking both FcγRIIa receptors and the Fc-receptor γ-chain.

There are several important new findings. First, this paper unequivocally demonstrates that activation through GP Ib-IX-V alone is sufficient to lead to up-regulation of GP IIb-IIIa integrin function. In mouse platelets, ITAM-containing receptors do not play a major role in this process. Second, Src kinases, calcium flux oscillations, and PKC/PI 3-kinase are all critical in this signaling pathway, with activation of Src kinases most proximal to GP Ib-IX-V and upstream of increased cytoplasmic calcium, which derives from both outside-in flux and intracellular mobilization. ADAP, a signaling protein previously characterized as involved in regulating integrin function, was identified as a primary Src kinase-dependent tyrosine phosphorylation event after GP Ib-IX-V ligation. Blockade of both PKC and PI 3-kinase affected GP IIb-IIIa activation downstream of the calcium flux oscillations. These findings thus provide a template for how GP Ib-IX-V orchestrates platelet signaling in thrombosis, with potential new therapeutic targets.