Bennett J S, Shattil S J, Power J W, Gartner T K
Hematology-Oncology Section, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104.
J Biol Chem. 1988 Sep 15;263(26):12948-53.
The platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) recognizes peptides containing the amino acid sequence Arg-Gly-Asp, a sequence present at two locations in the alpha chain of fibrinogen. GPIIb-IIIa also interacts with peptides containing the carboxyl-terminal 10-15 residues of the fibrinogen gamma chain. We found that the alpha chain tetrapeptide, Arg-Gly-Asp-Ser (RGDS), and the gamma chain peptide, Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (LGGAKQAG-DV), each inhibited fibrinogen binding to ADP-stimulated platelets with Ki values of 15.6 +/- 2.7 and 46.2 +/- 8.2 microM, respectively. Furthermore, the inhibitory effect of the peptides was additive, indicating that they interact with GPIIb-IIIa in a mutually exclusive manner. Mutually exclusive binding suggests that either the alpha and gamma chain peptides bind to identical or overlapping sites on the GPIIb-IIIa complex or that one peptide induces a change in the complex that excludes the other. To differentiate between these possibilities, we compared the ability of RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and PAC-1, to ADP-stimulated platelets. A2A9 and PAC-1 appear to bind to different sites on GPIIb-IIIa because A2A9 binds to both stimulated and unstimulated platelets while PAC-1 only binds to stimulated platelets. RGDS specifically inhibited fibrinogen and PAC-1 binding with nearly identical Ki values of 15.6 +/- 2.7 and 20.2 +/- 3.5 microM, respectively. In contrast, LGGAKQAGDV had a differential effect on fibrinogen and PAC-1 binding, inhibiting PAC-1 binding with a Ki of 116.1 +/- 12.9 microM and fibrinogen binding with a Ki of 46.2 +/- 8.2 microM (p less than 0.005). Furthermore, while RGDS had no effect on the binding of the monoclonal antibody A2A9, LGGAKQAGDV was a partial inhibitor of A2A9 binding to activated platelets. These results suggest that the bindings sites for RGDS and LGGAKQAGDV are spatially distinct. They also suggest that ligand-induced changes in GPIIb-IIIa conformation are likely to be responsible for the mutually exclusive nature of alpha and gamma chain peptide binding.
血小板膜糖蛋白IIb-IIIa复合物（GPIIb-IIIa）识别含有氨基酸序列精氨酸-甘氨酸-天冬氨酸（Arg-Gly-Asp，RGD）的肽段，该序列存在于纤维蛋白原α链的两个位置。GPIIb-IIIa还与含有纤维蛋白原γ链羧基末端10-15个残基的肽段相互作用。我们发现，α链四肽Arg-Gly-Asp-Ser（RGDS）和γ链肽Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val（LGGAKQAG-DV），各自抑制纤维蛋白原与ADP刺激的血小板的结合，其抑制常数（Ki）值分别为15.6±2.7和46.2±8.2微摩尔。此外，这些肽段的抑制作用是相加的，表明它们以互斥的方式与GPIIb-IIIa相互作用。互斥结合表明，α链和γ链肽段要么结合到GPIIb-IIIa复合物上相同或重叠的位点，要么一种肽段诱导复合物发生变化从而排斥另一种肽段。为了区分这些可能性，我们比较了RGDS和LGGAKQAGDV抑制纤维蛋白原以及两种GPIIb-IIIa复合物特异性单克隆抗体A2A9和PAC-1与ADP刺激的血小板结合的能力。A2A9和PAC-1似乎结合到GPIIb-IIIa上不同的位点，因为A2A9能结合刺激和未刺激的血小板，而PAC-1仅结合刺激的血小板。RGDS特异性抑制纤维蛋白原和PAC-1的结合，其抑制常数（Ki）值几乎相同，分别为15.6±2.7和20.2±3.5微摩尔。相反，LGGAKQAGDV对纤维蛋白原和PAC-1的结合有不同的影响，抑制PAC-1结合的Ki为116.1±12.9微摩尔，抑制纤维蛋白原结合的Ki为46.2±8.2微摩尔（p<0.005）。此外，虽然RGDS对单克隆抗体A2A9的结合没有影响，但LGGAKQAGDV是A2A9与活化血小板结合的部分抑制剂。这些结果表明，RGDS和LGGAKQAGDV的结合位点在空间上是不同的。它们还表明，配体诱导的GPIIb-IIIa构象变化可能是α链和γ链肽段结合互斥性质的原因。
