Contributions of Glycoprotein Ib and the Seven Transmembrane Domain Receptor to Increases in Platelet Cytoplasmic [Ca2+] Induced by ![]()
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- 作者：Nicholas J. Greco ; Narendra N. Tandon ; Glen D. Jones ; Robyn Kornhauser ; Barrington Jackson ; Naomasa Yamamoto ; Kenjiro Tanoue ; and G. A. Jamieson
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：January 23, 1996
- 年：1996
- 卷：35
- 期：3
- 页码：906 - 914
- 全文大小：459K
- 年卷期：v.35,no.3(January 23, 1996)
- ISSN：1520-4995
文摘
The individual contributions of glycoprotein Ib (GPIb)and the seven transmembrane domainreceptor (STDR) to increases in platelet[Ca2+]i induced by 
rs/alpha.gif" BORDER=0>-thrombin or thetethered ligand peptide(TLP; SFLLRNPNDKYEPF) have been determined in control platelets, inplatelets where the thrombinbinding site on GPIb was blocked with the monoclonal antibodies TM60and LJ-Ib10, in platelets whereaccess of thrombin to the STDR was blocked by polyclonal antipeptideantibodies, and in Bernard-Soulierplatelets which constitutively lack GPIb. Curve-fitting analyses(LIGAND) showed that binding of PPACK-thrombin and rs/alpha.gif" BORDER=0>-thrombin to the moderate-affinity site was not detectedin the best-fit model in the presenceof anti-STDR antibodies although with rs/alpha.gif" BORDER=0>-thrombin there was alsodecreased binding at the high-affinitysite. Conversely, TM60 blocked binding of rs/alpha.gif" BORDER=0>-thrombin to thehigh-affinity site but also decreased bindingat the moderate affinity site. Separately, either TM60 or anti-TNA(150 
r.gif">g/mL) reduced thrombin (0.5nM)-induced elevations in [Ca2+]i to 50%of control values, but Ca2+ elevations were essentiallyabrogated(4.2 ± 5%) when the two were added in combination.[Ca2+]i dose-response curves forrs/alpha.gif" BORDER=0>-thrombin werecurvilinear and were only 50% of controls in the presence of anti-GPIbor anti-STDR antibodies at up to10 nM rs/alpha.gif" BORDER=0>-thrombin, with their greatest sensitivity being below 2 nM.With Bernard-Soulier platelets,changes in [Ca2+]i were not detectable at
0.5 nM rs/alpha.gif" BORDER=0>-thrombin but were also 50% of controls at5-10nM rs/alpha.gif" BORDER=0>-thrombin. [Ca2+]i responses toTLP (1-100 r.gif">M) of antibody-blocked platelets were identicaltothose of controls whereas responses were ~50% of controls inBernard-Soulier platelets. The rate ofincrease in [Ca2+]i in controls was twicethat seen in antibody-blocked platelets and about 5-foldgreaterthan in Bernard-Soulier platelets. These results demonstrate thatboth GPIb and the STDR are requiredto ensure the optimal rate and extent of platelet activation over arange of rs/alpha.gif" BORDER=0>-thrombin concentrations(0.3-10 nM) and that the STDR corresponds to the previously describedmoderate-affinity thrombinreceptor.
rs/alpha.gif" BORDER=0>-thrombin or thetethered ligand peptide(TLP; SFLLRNPNDKYEPF) have been determined in control platelets, inplatelets where the thrombinbinding site on GPIb was blocked with the monoclonal antibodies TM60and LJ-Ib10, in platelets whereaccess of thrombin to the STDR was blocked by polyclonal antipeptideantibodies, and in Bernard-Soulierplatelets which constitutively lack GPIb. Curve-fitting analyses(LIGAND) showed that binding of PPACK-thrombin and rs/alpha.gif" BORDER=0>-thrombin to the moderate-affinity site was not detectedin the best-fit model in the presenceof anti-STDR antibodies although with rs/alpha.gif" BORDER=0>-thrombin there was alsodecreased binding at the high-affinitysite. Conversely, TM60 blocked binding of rs/alpha.gif" BORDER=0>-thrombin to thehigh-affinity site but also decreased bindingat the moderate affinity site. Separately, either TM60 or anti-TNA(150 r.gif">g/mL) reduced thrombin (0.5nM)-induced elevations in [Ca2+]i to 50%of control values, but Ca2+ elevations were essentiallyabrogated(4.2 ± 5%) when the two were added in combination.[Ca2+]i dose-response curves forrs/alpha.gif" BORDER=0>-thrombin werecurvilinear and were only 50% of controls in the presence of anti-GPIbor anti-STDR antibodies at up to10 nM rs/alpha.gif" BORDER=0>-thrombin, with their greatest sensitivity being below 2 nM.With Bernard-Soulier platelets,changes in [Ca2+]i were not detectable at0.5 nM rs/alpha.gif" BORDER=0>-thrombin but were also 50% of controls at5-10nM rs/alpha.gif" BORDER=0>-thrombin. [Ca2+]i responses toTLP (1-100 r.gif">M) of antibody-blocked platelets were identicaltothose of controls whereas responses were ~50% of controls inBernard-Soulier platelets. The rate ofincrease in [Ca2+]i in controls was twicethat seen in antibody-blocked platelets and about 5-foldgreaterthan in Bernard-Soulier platelets. These results demonstrate thatboth GPIb and the STDR are requiredto ensure the optimal rate and extent of platelet activation over arange of rs/alpha.gif" BORDER=0>-thrombin concentrations(0.3-10 nM) and that the STDR corresponds to the previously describedmoderate-affinity thrombinreceptor.