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...

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.

No abstract

We have investigated the role of adenosine diphosphate (ADP) receptors in the adhesion, activation, and aggregation of platelets perfused over immobilized von Willebrand factor (VWF) under high shear stress. Blocking P2Y(1) prevented stable platelet adhesion and aggregation, indicative of a complete inhibition of alpha(IIb)beta(3) activation, and decreased the duration of transient arrests from 5.9 seconds +/- 2.8 seconds to 1.2 seconds +/- 0.8 seconds; in contrast, blocking P2Y(12) inhibited only the formation of larger aggregates. Moreover, blocking P2Y(1) decreased the proportion of platelets showing early intracytoplasmic Ca(++) elevations (alpha/beta peaks) from 20.6% +/- 1.6% to 14.6% +/- 1.5% (P< .01), and the corresponding peak ion concentration from 1543 nM +/- 312 nM to 1037 nM +/- 322 nM (P < .05); it also abolished the Ca(++) elevations seen in firmly attached platelets (gamma peaks). Blocking P2Y(12) had no effect on these parameters, and did not enhance the effect of inhibiting P2Y(1). Inhibition of phospholipase C had similar consequences as the blocking of P2Y(1), whereas inhibition of Src family kinases abolished both type alpha/beta and gamma Ca(++) oscillations, although the former effect required a higher inhibitor concentration. Our results demonstrate that, under elevated shear stress conditions, ADP signaling through P2Y(1) may contribute to the initial stages of platelet adhesion and activation mediated by immobilized VWF, and through P2Y(12) to sustained thrombus formation.