Cytosolic Calcium Changes in a Process of Platelet Adhesion and Cohesion on a von Willebrand Factor-Coated Surface Under Flow Conditions

Abstract

Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca2+ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca2+]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca2+]i) to green (high [Ca2+]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s−1, no change in [Ca2+]i was observed during translocation of platelets, but [Ca2+]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca2+]i elevation. The intracellular Ca2+chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca2+]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca2+]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

Dual Biomembrane Force Probe enables single-cell mechanical analysis of signal crosstalk between multiple molecular species

“…Previous population studies using cone-and-plate viscometers 37 , bulk flow chambers 6 , 7 , 38 and microfludics devices 39 have documented the mechanical signal crosstalk between GPIb and GPIIb-IIIa. However, the control over the incoming signal is very limited in these studies.…”

Signaling events underlying thrombus formation

“…The dynamics of platelet adhesion on a purified VWF matrix are strikingly similar to those observed during thrombus development in vivo [26], in that platelets tethering to the VWF surface translocate prior to forming firm adhesion contacts [4,5]. Recent studies have provided insight into the mechanisms controlling adhesion dynamics on VWF [23,33–36] that may be relevant to the understanding of thrombus growth on more complex biological surfaces. While the importance of the VWF–GPIb interaction in supporting platelet–subendothelial and platelet–platelet interactions has been clearly delineated, the contribution of this adhesion event to the initiation of platelet activation remains less clearly defined.…”

“…Considerable controversy continues to exist with respect to whether GPIb signals. Some studies have failed to detect signaling downstream of GPIb [35], others have detected weak activating signals [23,36–42], while a few reports have demonstrated strong platelet activation by VWF [43–45]. Much of the discrepancy between different studies is most likely due to the large variations in experimental strategies used to examine GPIb signaling, including the use of different ligands (venom peptides, human or bovine VWF, recombinant VWF fragments, soluble or immobilized VWF), artificial modulators (ristocetin, botrocetin), cell types (human and mouse platelets, transfected CHO and K562 cell lines) and functional assays (suspension aggregation studies vs. flow‐based adhesion assays, static vs. shear conditions).…”

Differential integrin activity mediated by platelet collagen receptor engagement under flow conditions

“…Following platelet activation, Ca 2+ is either released from intracellular Ca 2+ stores, mainly via inositol trisphosphate (IP 3 ) receptors located on the dense tubular system, or enters from the extracellular medium via plasma membrane Ca 2+ channels. Whole blood perfusion studies have previously investigated the effect of shear forces on platelet [Ca 2+ ] i oscillations upon adhesion to collagen, VWF or fibrinogen (6,(12)(13)(14)(15)(16)(17)(18)(19)(20). Roles have been identified for GpIb/V/IX and α IIb β 3 in [Ca 2+ ] i responses via association with VWF A1 and RGD domains, respectively (12-14, 16, 17).…”