Contributions of Glycoprotein Ib and the Seven Transmembrane Domain Receptor to Increases in Platelet Cytoplasmic [Ca2+] Induced by α-Thrombin

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Abstract
The individual contributions of glycoprotein Ib (GPIb) and the seven transmembrane domain receptor (STDR) to increases in platelet [Ca2+]i induced by α-thrombin or the tethered ligand peptide (TLP; SFLLRNPNDKYEPF) have been determined in control platelets, in platelets where the thrombin binding site on GPIb was blocked with the monoclonal antibodies TM60 and LJ-Ib10, in platelets where access of thrombin to the STDR was blocked by polyclonal antipeptide antibodies, and in Bernard-Soulier platelets which constitutively lack GPIb. Curve-fitting analyses (LIGAND) showed that binding of PPACK-thrombin and α-thrombin to the moderate-affinity site was not detected in the best-fit model in the presence of anti-STDR antibodies although with α-thrombin there was also decreased binding at the high-affinity site. Conversely, TM60 blocked binding of α-thrombin to the high-affinity site but also decreased binding at the moderate affinity site. Separately, either TM60 or anti-TNA (150 μg/mL) reduced thrombin (0.5 nM)-induced elevations in [Ca2+]i to 50% of control values, but Ca2+ elevations were essentially abrogated (4.2 ± 5%) when the two were added in combination. [Ca2+]i dose-response curves for α-thrombin were curvilinear and were only 50% of controls in the presence of anti-GPIb or anti-STDR antibodies at up to 10 nM α-thrombin, with their greatest sensitivity being below 2 nM. With Bernard-Soulier platelets, changes in [Ca2+]i were not detectable at ≤0.5 nM α-thrombin but were also 50% of controls at 5−10 nM α-thrombin. [Ca2+]i responses to TLP (1−100 μM) of antibody-blocked platelets were identical to those of controls whereas responses were ∼50% of controls in Bernard-Soulier platelets. The rate of increase in [Ca2+]i in controls was twice that seen in antibody-blocked platelets and about 5-fold greater than in Bernard-Soulier platelets. These results demonstrate that both GPIb and the STDR are required to ensure the optimal rate and extent of platelet activation over a range of α-thrombin concentrations (0.3−10 nM) and that the STDR corresponds to the previously described moderate-affinity thrombin receptor.