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This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin ␣ IIb ␤ 3 . These include the following: 1) examining the sufficiency of GPIb/IX and integrin ␣ IIb ␤ 3 to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin ␣ IIb ␤ 3 activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin ␣ IIb ␤ 3 activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin ␣ IIb ␤ 3 , we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf, and integrin ␣ IIb ␤ 3 mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin ␣ IIb ␤ 3 . Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin ␣ IIb ␤ 3 activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150 -1800 s ؊1 ). Analysis of the key second messengers linking the vWf-GPIb interaction to integrin ␣ IIb ␤ 3 activation demonstrated that the first step in the activation process involves calcium release from internal stores, whereas transmembrane calcium influx is a secondary event potentiating integrin ␣ IIb ␤ 3 activation.

Platelet adhesion to sites of vascular injury is initiated by the binding of the platelet glycoprotein (GP) Ib-V-IX complex to matrix-bound von Willebrand factor (vWf). This receptor-ligand interaction is characterizedby a rapid on-off rate that enables efficient platelet tethering and rolling under conditions of rapid blood flow. We demonstrate here that platelets adhering to immobilized vWf under flow conditions undergo rapid morphological conversion from flat discs to spiny spheres during surface translocation. Studies of Glanzmann thrombasthenic platelets (lacking integrin ␣ IIb ␤ 3 ) and Chinese hamster ovary (CHO) cells transfected with GPIb/IX (CHO-Ib/IX) confirmed that vWf binding to GPIb/IX was sufficient to induce actin polymerization and cytoskeletal reorganization independent of integrin ␣ IIb ␤ 3 . vWf-induced cytoskeletal reorganization occurred independently of several well characterized signaling processes linked to platelet activation, including calcium influx, prostaglandin metabolism, protein tyrosine phosphorylation, activation of protein kinase C or phosphatidylinositol 3-kinase but was critically dependent on the mobilization of intracellular calcium. Studies of Oregon Green 488 1,2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N,N-tetraacetic acid tetraacetoxymethyl ester-loaded platelets and CHO-Ib/IX cells demonstrated that these cells mobilize intracellular calcium in a shear-dependent manner during surface translocation on vWf. Taken together, these studies suggest that the vWf-GPIb interaction stimulates actin polymerization and cytoskeletal reorganization in rolling platelets via a shear-sensitive signaling pathway linked to intracellular calcium mobilization.The ability of platelets to adhere to subendothelial matrix proteins and to other activated platelets at sites of vascular injury is essential for the arrest of bleeding and for subsequent vascular repair. The first step in the hemostatic process involves the binding of the platelet adhesion receptor, GPIb/V/IX, to the vascular adhesive protein, vWf.1 Under conditions of rapid blood flow this receptor-ligand interaction is indispensable for tethering platelets to the injured vessel wall as a prerequisite step for integrin-mediated cell arrest (1, 2). This multi-step adhesion mechanism is remarkably similar to that utilized by leukocytes to adhere to post-capillary venules in vivo. Rolling of leukocytes is mediated by one or more selectin family members, whereas irreversible cell adhesion requires activation of ␤ 2 integrins (3, 4).vWf is a unique adhesive ligand in that it has the ability to support both the initial transient phase of platelet adhesion as well as integrin ␣ IIb ␤ 3 -mediated cell arrest. The A1 domain of vWf contains the binding site for GPIb␣, whereas the C1 domain peptide sequence Arg-Gly-Asp (RGD) binds integrin ␣ IIb ␤ 3 (platelet GPIIb/IIIa) (5). Bond formation between vWf and GPIb is rapid, reversible, and inherently resistant to detachment by high shear forces. This latter property of the vWf-GPIb interaction is...

The interaction of the glycoprotein (GP) Ib-V-IX receptor complex with the membrane skeleton of platelets is dependent on a specific interaction between the cytoplasmic tail of GPIb␣ and filamin-1. This interaction has been proposed to regulate key aspects of platelet function, including the ligand binding of GPIb-V-IX and the ability of the cells to sustain adhesion to von Willebrand factor (vWf) under high shear. In this study we have examined sequences in the GPIb␣ intracellular domain necessary for interaction of the receptor with filamin-1. We have identified two adjacent sequences involving amino acids 557-568 and 569 -579 of the GPIb␣ cytoplasmic domain that are critical for normal association between the receptor complex and filamin-1. Under flow conditions, Chinese hamster ovary (CHO) cells expressing these two mutant receptors exhibited an increase in translocation velocity that was associated with increased cell detachment from the vWf matrix at high shear. The shear-dependent acceleration in velocity of mutant ⌬557-568 and ⌬569 -579 CHO cells was associated with a critical defect in receptor anchorage, evident from significant extraction of GPIb-IX from the CHO cell membrane at high shear. These studies define a critical role for amino acids within the 557-579 sequence of GPIb␣ for interaction with filamin-1.

The glycoprotein Ib-V-IX (GPIb-V-IX) complex interacts with subendothelial von Willebrand factor (VWF) to ensure recruitment of platelets at sites of vascular injury, a process that culminates in integrin ␣ IIb ␤ 3 -dependent stable adhesion and spreading. Interaction of the 14-3-3 adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIb␣ subunit has been implicated in the control of ␣ IIb ␤ 3 activation and cell spreading. In this study, we have examined potentially novel 14-3-3 binding sites by expressing mutant forms of GPIb␣ in Chinese-hamster-ovary (CHO) cells. Analysis of a series of neighboring 11-12 residue deletions identified a critical role for the 580-LVAGRRPSALS-590 sequence in promoting GPIb␣-14-3-3 interaction. Development of a phosphospecific antibody demonstrated high levels of phosphorylation of the Ser587 and Ser590 residues in resting platelets (which became dephosphorylated during platelet spreading on VWF), and peptides containing these phosphorylated residues effectively displaced 14-3-3 from GPIb␣. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIb␣-14-3-3 binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3 binding site within the cytoplasmic tail of GPIb␣ that has an important functional role in regulating integrin-dependent cell spreading. IntroductionThe 14-3-3 protein family consists of ubiquitous homodimeric or heterodimeric intracellular adaptor proteins that take part in the signaling pathways of numerous biologic responses. 1 There have been 5 isoforms identified in human platelets, the ⑀ and isoforms being weakly expressed relative to the more abundant ␥, , and ␤ isoforms. 2 The crystal structure of 14-3-3 revealed the association of 2 monomers, each composed of a bundle of 9 antiparallel helices, to form a large negatively charged groove that could be implicated in interactions with other proteins. 3 The 14-3-3 proteins bind to specific phosphoserine-containing motifs, 4 and their dimeric nature allows them to act as intramolecular and intermolecular phosphorylation-dependent bridges. 5 The functions of the different isoforms in platelets are, however, still poorly understood.In a seminal study, it was reported that 14-3-3 interacted with the platelet von Willebrand factor (VWF) receptor, the glycoprotein Ib-V-IX (GPIb-V-IX) complex. 6 A binding site for 14-3-3 was found at the C-terminus of GPIb␣ within a serine-rich 606-SGHSL-610 sequence bearing similarities to phosphoserine containing 14-3-3 binding motifs. 1,7 Further work showed that the serine at position 609 was predominantly phosphorylated in resting platelets and that phosphorylation was required for 14-3-3 binding. 8 Glutathione S-transferase (GST)-14-3-3 pull-down and immunoprecipitation studies of GPIb-IX-transfected cells pointed to the existence of a separate binding or regulatory site in the GPIb␣ 570-590 ...