Distinct [Ca⁺⁺]_i elevations mediated by GPIbα or α_IIbβ₃ in platelets interacting with immobilized VWF.

Platelets loaded with Fluo-3 AM were suspended at a count of 5 × 10⁶/mL with homologous washed red cells in a buffer containing 2 mM Ca⁺⁺ and 1 mM Mg⁺⁺ (the same results were obtained with 2 mM Mg⁺⁺). The suspension was perfused over immobilized multimeric VWF or A1VWF at the indicated shear rates for 90 seconds, after which the [Ca⁺⁺]_i of all surface-interacting platelets was monitored in real time for the next 30 seconds during translocation or stationary adhesion. In some experiments, the anti-α_IIbβ₃ monoclonal antibody LJ-CP8 was added at the final concentration of 100 μg/mL. Experiments with A1VWF were performed at the shear rate of 2000 seconds⁻¹ to obtain comparable numbers of surface-tethered platelets under all conditions. Arrows indicate typical γ Ca⁺⁺ peaks, which were present only in untreated platelets interacting with VWF. Comparable results were obtained in 4 different experiments.