Ristocetin-induced platelet agglutination stimulates GPIIb/IIIa-dependent calcium influx

We found that intracellular Ca²⁺ concentration ([Ca²⁺](i)) increased during ristocetin-induced agglutination of aequorin loaded platelets resuspended in plasma. Chelation of extracellular Ca²⁺ had no effect on platelet clumping, but delayed and greatly reduced Ca²⁺ increase, indicating that it derived for the most part from Ca²⁺ influx. Nine monoclonal antibodies (MA) against glycoprotein (GP) Ib largely prevented ristocetin-induced platelet clumping and [Ca²⁺](i) increase, while three anti-GPIb MA with no effect on platelet clumping did not interfere with Ca²⁺ movement. In unstirred samples platelet agglutination was greatly reduced and [Ca²⁺](i) increase was abolished, suggesting that close platelet-to-platelet contact in addition to von Willebrand factor (vWF) binding to GPIb, is necessary for Ca²⁺ transient. Nine MA against GPIIb/IIIa, the gly-arg-gly-asp-ser (GRGDS) peptide and GPIIb/IIIa complex dissociation had no effect on platelet agglutination, but significantly reduced Ca²⁺ increase. Our results suggest that platelet clumping induced by vWF binding to GPIb is responsible for GPIIb-IIIa dependent Ca²⁺ influx.