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William P. Sheffield, Brianna Wilson, Louise J. Eltringham-Smith, Sharon Gataiance, Varsha Bhakta
Recombinant albumins containing additional peptide sequences smaller than barbourin retain the ability of barbourin-albumin to inhibit platelet aggregation
The previously described fusion protein BLAH6 (Marques JA et al.,Thromb Haemost 2001; 86: 9028) is a recombinant protein that combines the small disintegrin barbourin with hexahistidine-tagged rabbit serumalbumin (RSA) produced in Pichia pastoris yeast. We sought to determine: (1) if BLAH6 was immunogenic; and (2) if its barbourin domain could be productively replaced with smaller peptides. Purified BLAH6 was injected into rabbits, and anti-barbourin antibodies were universally detected in plasma 28 days later; BLAH6 was, however, equally effective in reducing platelet aggregation in both nave and pre-treated rabbits. Thrombocytopenia was not observed, and complexing BLAH6 to IIb3 had no effect on antibody detection. The barbourin moiety of BLAH6 was replaced with each of four sequences: Pep I (VCKGDWPC); PepII (VCRGDWPC); PepIII (bar-bourin 4154); and PepIV (LPSPGDWR). The corresponding fusion proteins were tested for their ability to inhibit ADP-induced platelet aggregation. PepIII-LAH6 inhibited neither rabbit nor human platelets. PepI-LAH6 and PepIV-LAH6 inhibited rabbit platelet aggregation as effectively as BLAH6, but PepIV-LAH6 did not inhibit human platelet aggregation. PepI-LAH6 and PepIILAH6 inhibited human platelet aggregation with IC50s 10- and 20-fold higher than BLAH6. Cross-immunoprecipitation assays with human platelet lysates confirmed that all proteins and peptides interacted with the platelet integrin IIb3, but with greatly varying affinities. Our results suggest that the antiplatelet activity of BLAH6 can be retained in albumin fusion proteins in which smaller peptides replace the barbourin domain; these proteins may be less immunogenic than BLAH6.
Thrombosis and Haemostasis, Schattauer
Print ISSN: 0340-6245
Volume: 93, 05/2005
Pages: 914 - 921