Cooperative Calcium Signaling Mediated by Glycoprotein (GP) Ib-IX-V, GPVI and Integrin α₂β₁ during Platelet Adhesion to Collagen under Flow.

We have investigated signaling mediated by GPIb-IX-V, GPVI and α₂β₁ during platelet adhesion to collagen type I either in the presence or absence of von Willebrand factor (VWF) A1A2A3 domain under flow conditions (wall shear rate 600 or 3000 s⁻¹). We used platelets labeled with FLUO3-AM and real-time videomicroscopy with high-speed image acquisition to analyze concurrently adhesion, translocation and calcium transients in single platelets interacting with the surface. All experiments were performed in the presence of integrillin (100 μg/ml) to block the possible involvement of α_IIbβ₃. Platelet adhesion and activation at 600 s⁻¹ were similar in the absence or presence of VWF A1A2A3 domain. Calcium signaling consisted of both short lasting peaks (release from intracellular stores) and long sustained waves (transmembrane flux). In our experimental condition blockade of α₂β₁ or GPVI with monoclonal antibodies reduced platelet adhesion by 50% and the proportion of adherent platelets that became activated by 20–30%. Concomitant inhibition of both receptors decreased platelet adhesion by >90%. Thus, both α₂β₁ and GPVI participate in the initial stage of platelet adhesion to collagen type I. At the higher shear rate of 3000 s⁻¹, platelet adhesion and activation was 40–50% less than at 600 s⁻¹ in the absence of VWF A1A2A3 domain; addition of the latter enhanced adhesion and activation by 12 to 14-fold, and >80% of the platelets exhibited stable adhesion during the 30 s observation period. Intracellular Ca⁺⁺ peaks in adherent platelets reached 2–3 μM. Blockade of GP Ib-IX-V inhibited both platelet adhesion and activation by at least 90%. Blockade of α₂β₁ markedly reduced the number of adhering platelets, which were mostly (>90%) translocating on the surface and exhibited only short lasting Ca⁺⁺ transients. Blockade of GPVI greatly decreased the number of adherent platelets, most of which, however, were firmly attached to the surface (<15% exhibiting translocation). Calcium transients in the activated platelet consisted of short lasting peaks and rare long lasting waves reaching concentrations <1.5–2 μM. These results suggest that, on a surface of collagen type I, α₂β₁ is required for normal platelet arrest following tethering through the VWF A1 domain-GPIb-IX-V interaction at higher shear rates, and Ca⁺⁺ signaling results from the concerted action of the two receptors reinforced by GPVI. The adhesion potential of platelets exposed to collagen, therefore, appears to depend on signaling triggered by different ligand-receptor interactions varying as a function of flow conditions.