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• W2161678162 endingPage "321" @default.
• W2161678162 startingPage "312" @default.
• W2161678162 abstract "The binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb-IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg-Gly-Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site-directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z-domain by site-specific proteolysis using a mutant subtilisin BPN' and purified by reverse-phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb-IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet-rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90-fold, 2-fold, and > 200-fold, respectively. The conservative D51E mutant was still > 100-fold less potent whereas R49K had a minor effect (1.8-fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb-IIIa." @default.
• W2161678162 created "2016-06-24" @default.
• W2161678162 creator A5076479466 @default.
• W2161678162 creator A5081878988 @default.
• W2161678162 creator A5088134239 @default.
• W2161678162 date "1993-03-01" @default.
• W2161678162 modified "2023-09-25" @default.
• W2161678162 title "Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: Analysis by site-directed mutagenesis" @default.
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• W2161678162 doi "https://doi.org/10.1002/prot.340150308" @default.
• W2161678162 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8456099" @default.
• W2161678162 hasPublicationYear "1993" @default.
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