Nj. Greco et al., CONTRIBUTIONS OF GLYCOPROTEIN-IB AND THE 7-TRANSMEMBRANE DOMAIN RECEPTOR TO INCREASES IN PLATELET CYTOPLASMIC [CA2-THROMBIN(] INDUCED BY ALPHA), Biochemistry, 35(3), 1996, pp. 906-914
The individual contributions of glycoprotein Ib (GPIb) and the seven t
ransmembrane domain receptor (STDR) to increases in platelet [Ca2+](i)
induced by alpha-thrombin or the tethered ligand peptide (TLP; SFLLRN
PNDKYEPF) have been determined in control platelets, in platelets wher
e the thrombin binding site on GPIb was blocked with the monoclonal an
tibodies TM60 and LJ-Ib10, in platelets where access of thrombin to th
e STDR was blocked by polyclonal antipeptide antibodies, and in Bernar
d-Soulier platelets which constitutively lack GPIb. Curve-fitting anal
yses (LIGAND) showed that binding of PPACK-thrombin and alpha-thrombin
to the moderate-affinity site was not detected in the best-fit model
in the presence of anti-STDR antibodies although with alpha-thrombin t
here was also decreased binding at the high-affinity site. Conversely,
TM60 blocked binding of alpha-thrombin to the high-affinity site but
also decreased binding at the moderate affinity site. Separately, eith
er TM60 or anti-TNA (150 mu g/mL) reduced thrombin (0.5 nM)-induced el
evations in [Ca2+](i) to 50% of control values, but Ca2+ elevations we
re essentially abrogated (4.2 +/- 5%) when the two were added in combi
nation. [Ca2+](i) dose-response curves for alpha-thrombin were curvili
near and were only 50% of controls in the presence of anti-GPIb or ant
i-STDR antibodies at up to 10 nM alpha-thrombin, with their greatest s
ensitivity being below 2 nM. With Bernard-Soulier platelets, changes i
n [Ca2+](i) were not detectable at less than or equal to 0.5 nM alpha-
thrombin but were also 50% of controls at 5-10 nM alpha-thrombin. [Ca2
+](i) responses to TLP (1-100 mu M) of antibody-blocked platelets were
identical to those of controls whereas responses were similar to 50%
of controls in Bernard-Soulier platelets. The rate of increase in [Ca2
+](i) in controls was twice that seen in antibody-blocked platelets an
d about 5-fold greater than in Bemard-Soulier platelets. These results
demonstrate that both GPIb and the STDR are required to ensure the op
timal rate and extent of platelet activation over a range of alpha-thr
ombin concentrations (0.3-10 nM) and that the STDR corresponds to the
previously described moderate-affinity thrombin receptor.