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

Abstract

The individual contributions of glycoprotein Ib (GPIb) and the seven transmembrane domain receptor (STDR) to increases in platelet [Ca2+]i induced by alpha-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 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 there 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, either TM60 or anti-TNA (150 micrograms/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 alpha-thrombin were curvilinear and were only 50% of controls in the presence of anti-GPIb or anti-STDR antibodies at up to 10 nM alpha-thrombin, with their greatest sensitivity being below 2 nM. With Bernard-Soulier platelets, changes in [Ca2+]i were not detectable at < or = 0.5 nM alpha-thrombin but were also 50% of controls at 5-10 nM alpha-thrombin. [Ca2+]i responses to TLP (1-100 microM) of antibody-blocked platelets were identical to those of controls whereas responses were approximately 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 alpha-thrombin concentrations (0.3-10 nM) and that the STDR corresponds to the previously described moderate-affinity thrombin receptor.

Human Factor XII Binding to the Glycoprotein Ib-IX-V Complex Inhibits Thrombin-induced Platelet Aggregation

“…Thrombin binds to PAR1 and cleaves the peptide bond between Arg 41 and Ser 42 (10). The peptide LLRNPNDKYEPF represents amino acids 44 -55 of PAR1, and an antibody to this peptide, anti-STDR, inhibits thrombin binding to PAR1 (11) Anti-STDR failed to block factor XII binding (Fig. 5).…”

“…Specific targeting of either receptor should modulate the effect of thrombin on platelets without inhibiting thrombin-induced proteolysis or the effects of other agonists for platelets including ADP, collagen, and thromboxane A2. Greco et al (11) have presented evidence that both GP Ib and PAR1 are required to ensure optimal rate and extent of platelet responses, such as an increase in intracellular Ca 2ϩ , at a range of thrombin concentrations of 0.3-10 nM. Recently a third human protein, PAR4 (12), has been shown to be involved in thrombin activation of platelets at high thrombin concentrations, e.g.…”

Novel structurally altered P2X1 receptor is preferentially activated by adenosine diphosphate in platelets and megakaryocytic cells

“…Flow cytometry 36 confirmed the surface expression of a P 2X1 receptor(s) using both the anti-hP 2X1 antibody (described above) and an anti-rat P 2X1 antibody (kindly provided by Dr Julian Barden, University of Sydney, Australia) directed against an identical amino acid sequence in the human receptor. Both Dami cells and CMK 11-5 cells demonstrated binding of both antibodies (data not shown).…”

Sequential activation of p38 and ERK pathways by cGMP-dependent protein kinase leading to activation of the platelet integrin αIIbβ3

“…[6][7][8][9][10] GPIb-IX also binds thrombin and is important in thrombin-induced platelet activation. [11][12][13][14][15] The signaling mechanism of GPIb-IX-mediated integrin activation is not fully understood but may involve coordination of multiple signaling pathways. 9,[16][17][18][19] We recently have shown that ligand binding to GPIb-IX induces elevation of intracellular cGMP and activation of cGMP-dependent protein kinase (protein kinase G, PKG), 20 which leads to activation of extracellular signal-regulated kinase (ERK) and activation of ␣ IIb ␤ 3 .…”