Platelet adhesion at sites of vascular injury is mediated, under high shear stress, by the reversible interaction of von Willebrand factor (vWF) with the platelet glycoprotein (GP) Ib-IX-V complex. This interaction leads to platelet translocation onto immobilized vWF. Translocation precedes platelet arrest and spreading which is mediated by the binding of vWF to the platelet integrin \bby We have measured single platelet instant velocity during translocation and changes in platelet cytosolic calcium concentration ([Ca]-4 using a computerized image analyzer apparatus to investigate the mechanism by which platelets became activated during the process of translocation. Washed platelets were loaded with FLUO-3AM (8|iM) and resuspended in reconstituted whole blood containing 400 U/ml of recombinant hirudin. Blood was then perfused at 37°C onto a surface covered with multimeric vWF at shear rates from 500 to 20000 s '. During the first phase of translocation, platelets showed a variable [Ca]i elevation with peaks lower than 100 nM. Subsequently, platelets showed either a limited [Ca]j elevation of 200-300 nM with a slow reversal to basal values (2-3 sec) or a rapid peak of elevation (500-600 nM) lasting less than 1 sec. Similar results were obtained when platelets were exposed to surfaces coated with the isolated recombinant Al domain rather than intact multimeric vWF. The two different patterns of [CaJ. increase could be seen in the same platelet at different times. The percentage of rolling platelets showing a [Ça], elevation was greater with increasing shear rates, reaching 50% at 10,000 sj. The addition of EGTA (10 mM) did not influence the [CaJ. elevation, but the addition of BAPTA-AM (10 |JM) completely blocked it. Apyrase (10 U/ml), Indomethacin (10 (iM), Bromophenacyl-Bromide (5uM), Manoalide (0.2\iM), Genistein (lOO-4M and 10 p.M) or Wortmannin (100 nM and 10|iM) did not significantly inhibit the [CaJ. elevation occurring during platelet translocation at increasing shear rates. On the contrary, the addition of PGE1 (0.15 |lM) exerted a strong inhibition effect. Our results suggest that the reversible interaction of GP Iba with the vWF Al domain is per se sufficient to induce signaling through an increase in [CaJ. elevation that is dependent on intracellular calcium mobilization and substantially independent of prostaglandin metabolism, tyrosines phosphorylation, PKC, PI 3-K and PLA2.
|Issue number||11 PART I|
|Publication status||Published - 2000|
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