IntroductionPlatelets adhere at sites of vascular injury to prevent hemorrhage 1 but may also form occluding arterial thrombi that cause disease. 2,3 In vessels in which rapid blood flow creates high wall-shear rates, such as arterioles in the normal circulation or atherosclerotic arteries with restricted lumen, platelet thrombus formation depends on von Willebrand factor (VWF) immobilized on extracellular matrix components, particularly collagens. 4 Binding of glycoprotein Ib␣ (GPIb␣), a constituent of the GPIb-IX-V complex, to the VWF A1 domain (A1VWF) initiates platelet tethering to these surfaces but by itself can only support translocation with stopand-go motion. 5 Once tethered, however, platelets rapidly achieve irreversible adhesion mediated by different integrins, including ␣ IIb ␤ 3 bound to the Arg-Gly-Asp (RGD) motif in the VWF C1 domain. 4,5 Activated ␣ IIb ␤ 3 serves also to immobilize on the surface of adherent platelets the plasma proteins, mainly VWF and fibrinogen, that mediate the recruitment of additional platelets into the forming thrombus. 6 Platelet activation, necessary to promote the ligand-binding function of ␣ IIb ␤ 3 , is coupled to the interactions that establish initial platelet-surface contacts, as shown by the fact that VWF binding to GPIb␣ leads to aggregation. 7,8 Sustained elevations of intracellular calcium concentration ([Ca ϩϩ ] i ), a marker of activation, occur in association with shear-induced platelet aggregation dependent on VWF and GPIb␣ 9 and may be the consequence of a transmembrane ion flux. 10 Oscillations of [Ca ϩϩ ] i have also been observed to accompany platelet adhesion to VWF, but this finding has been given discordant interpretations. 11,12 Some results 13 suggest that the VWF-GPIb␣ interaction may induce transient elevations (spikes) of [Ca ϩϩ ] i that activate ␣ IIb ␤ 3 in an initially reversible manner and influence the dynamic aspect of platelet-surface contacts before stable adhesion is established. This is in contrast to the idea that transient tethering to immobilized VWF depends only on GPIb␣, whereas activation of ␣ IIb ␤ 3 , like that of other integrins on leukocytes, 14 leads to irreversible adhesion. 4,5 Moreover, it has been proposed that phosphatidylinositol 3-kinase (PI3-K) plays an essential role in the activation of ␣ IIb ␤ 3 required for stable platelet adhesion. 15 To evaluate these conclusions, we concurrently analyzed the instantaneous velocity and [Ca ϩϩ ] i in single platelets interacting with immobilized VWF. We identified a sequence of distinct cytosolic Ca ϩϩ elevations associated with a 2-step process For personal use only. on May 10, 2018. by guest www.bloodjournal.org From of ␣ IIb ␤ 3 activation. The first signal involves release from intracellular stores and always precedes stationary adhesion. The second signal, which is coupled to adenosine diphosphate (ADP)-receptor stimulation and is inhibited by wortmannin, follows stationary adhesion but precedes the initiation of platelet aggregation on the surface. These results challeng...

IntroductionThe initial adhesion of platelets to the extracellular matrix of injured blood vessels is mediated at high shear rates by von Willebrand factor (VWF) interaction with glycoprotein (GP) Ib-IX-V. 1 Additionally, engagement of GP Ib-IX-V by VWF is thought to contribute to stable platelet adhesion by generating intracellular signals necessary for activation of ␣IIb␤3. Indeed, ␣IIb␤3 activation and platelet thrombus formation have been observed under a number of experimental conditions following platelet interaction with VWF. [2][3][4][5][6] Furthermore, specific biochemical responses have been documented under the same conditions, including induction of Ca 2ϩ fluxes and activation of tyrosine, serine-threonine, and lipid kinases. 7,8 Consequently, GP Ib-IX-V may function as a signaling receptor and an adhesion receptor.GP Ib-IX-V is a complex of 4 transmembrane polypeptides. 9-11 Although the cytoplasmic tail of each subunit lacks a catalytic domain, each may interact directly or indirectly with proteins that can transmit intracellular signals. For example, the cytoplasmic tail of GP Ib␣ can interact directly with filamin, GP Ib␣ and Ib␤ with 14-3-3-, and GP Ib␤ and GP V with calmodulin. 12-15 GP Ib-IX-V can be coimmunoprecipitated from platelets with signaling molecules, including Src family kinases, 16 phosphatidylinositol 3-kinase (PI 3-kinase) 17 and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase-2 (SHIP-2). 18 Furthermore, VWF-dependent platelet activation may require localization of GP Ib-IX-V to lipid rafts, membrane structures implicated in cellular signaling. 19 Although there is good evidence for a functional link between GP Ib-IX-V and ␣IIb␤3, 2 critical questions remain: Is GP Ib-IX-V itself capable of transducing signals in platelets, and, if so, to what extent do these signals participate in the activation of ␣IIb␤3? Several factors have conspired to make it difficult to answer these questions. First, a subpopulation of GP Ib-IX-V in platelets may be associated with immunoreceptor tyrosine activation motif (ITAM)-bearing proteins that can signal in their own right, including the Fc␥RIIA receptor and the FcR ␥-chain. 20,21 Second, platelets express numerous receptors for soluble and matrix-associated agonists, and some of the agonists (eg, adenosine diphosphate [ADP], thromboxane A 2 ) are released by adherent platelets. 22 Third, VWF not only interacts with GP Ib-IX-V through its A1 domain but also with ␣IIb␤3 through its C1 domain. 1 Thus, outside-in signals stimulated by VWF binding to ␣IIb␤3 can confound analysis of GP Ib-IX-V signaling. 23 Finally, studies of GP Ib-IX-V signaling under static conditions have frequently utilized nonphysiological mediators, such as botrocetin or ristocetin, to promote VWF binding to GP Ib-IX-V, complicating data interpretation further.Thus, despite the publication of many important studies on the molecular contributors to signaling responses downstream of GP Ib-IX-V, most to date have failed to consistently employ conditions to avoi...

The interaction between von Willebrand factor (vWF) and the platelet membrane glycoprotein (GP) Ib-IX-V complex is essential for platelet adhesion at sites of vascular injury under high shear stress flow conditions. Moreover, GP Ib-IX-V may contribute to the mechanisms of platelet activation through its high affinity binding of alpha-thrombin. There are two distinct but partially overlapping regions of GP Ib alpha thought to be involved in interacting with vWF (residues 251-279) and alpha-thrombin (residues 271-284); they share three tyrosine residues (positions 276, 278, and 279) that have recently been shown to be sulfated (Dong, J., Li, C. Q., and Lopez, J.A. (1994) Biochemistry 33, 13946-13953). To define the functional role of these three residues, we have introduced selected mutations in a soluble recombinant GP Ib alpha fragment (corresponding to the sequence 1-302 of the mature protein) that binds vWF and alpha-thrombin with the same attributes as intact GP Ib-IX-V complex. Fragments containing a single Tyr-->Phe substitution either at position 276 or 278 or 279 exhibited normal interaction with vWF but markedly reduced or absent binding of alpha-thrombin. GP Ib alpha fragment with normal sequence but synthesized under sulfate-free conditions also failed to bind alpha-thrombin and, in addition, had markedly reduced interaction with vWF. The simultaneous substitution of three neighboring Asp residues with Asn at positions 272, 274, and 277, a multiple mutation that may impair Tyr sulfation, also resulted in loss of binding of both ligands. These results define distinct structural features of GP Ib alpha selectively involved in supporting the interaction with vWF or alpha-thrombin.