Change in intracellular calcium ions during shear induced platelet aggregation

The authors have developed a method to measure intracellular calcium ion concentration ([Ca²⁺]i) during shear-induced platelet aggregation. A cone and plate viscometer was adapted for continuous recording of both light transmission and fluorescence intensity. Citrated platelet rich plasma was incubated with Indo-1AM at a concentration of 10 μm for 30 min at 37° C, then applied to the albumin density gradient to prepare washed platelets. To Indo-1AM loaded washed platelets, fibrinogen, and von Willebrand factor (vWf) were added, with 1 mM CaCl₂. Platelets were then exposed to changing shear stress (6-108 dynes/cm²) for simultaneous measurement of aggregation and [Ca²⁺]i. [Ca²⁺]i in resting platelets was estimated as approximately 100 nM. At low shear stress (10-20 dynes/cm²), [Ca²⁺]i did not change. In contrast, a marked increase in [Ca²⁺]i was observed concurrent with aggregation at high shear stress (100-108 dynes/cm²). However, no increase was seen in the presence of 1 mM EGTA. The increase was prevented by monoclonal antibodies against GPIb or vWf, which inhibited vWf binding to GPIb. A monoclonal anti-vWf antibody, which inhibited vWf binding to the GPIIb/IIIa complex, did not affect [Ca²⁺]i increase during high shear induced platelet aggregation. These results suggest that binding of vWf to GPIb may trigger Ca²⁺ influx.