Recombinant albumins containing additional peptide sequences smaller than barbourin retain the ability of barbourin-albumin to inhibit platelet aggregation.
The previously described fusion protein BLAH(6) (Marques JA et al.,Thromb Haemost 2001; 86: 902-8) 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 BLAH(6) was immunogenic; and (2) if its barbourin domain could be productively replaced with smaller peptides. Purified BLAH(6) was injected into rabbits, and anti-barbourin antibodies were universally detected in plasma 28 days later; BLAH(6) was, however, equally effective in reducing platelet aggregation in both naive and pre-treated rabbits. Thrombocytopenia was not observed, and complexing BLAH(6) to alpha(IIb)beta(3) had no effect on antibody detection. The barbourin moiety of BLAH(6) was replaced with each of four sequences: Pep I (VCKGDWPC); PepII (VCRGDWPC); PepIII (bar-bourin 41-54); and PepIV (LPSPGDWR). The corresponding fusion proteins were tested for their ability to inhibit ADP-induced platelet aggregation. PepIII-LAH(6) inhibited neither rabbit nor human platelets. PepI-LAH(6) and PepIV-LAH(6) inhibited rabbit platelet aggregation as effectively as BLAH(6), but PepIV-LAH(6) did not inhibit human platelet aggregation. PepI-LAH(6) and PepIILAH(6) inhibited human platelet aggregation with IC(50)s 10- and 20-fold higher than BLAH(6). Cross-immunoprecipitation assays with human platelet lysates confirmed that all proteins and peptides interacted with the platelet integrin alpha(IIb)beta(3), but with greatly varying affinities. Our results suggest that the antiplatelet activity of BLAH(6) can be retained in albumin fusion proteins in which smaller peptides replace the barbourin domain; these proteins may be less immunogenic than BLAH(6).
Sheffield
William P
WP
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada. sheffiel@mcmaster.ca
Wilson
Brianna
B
Eltringham-Smith
Louise J
LJ
Gataiance
Sharon
S
Bhakta
Varsha
V
D000244:Adenosine Diphosphate / Q000737:chemistry; D000418:Albumins / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D003435:Crotalid Venoms / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D020128:Inhibitory Concentration 50; D008297:Male; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010843:Pichia / Q000378:metabolism; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation / Q000187:drug effects*; D010979:Platelet Function Tests; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*; D013997:Time Factors
D000418:Albumins; D003435:Crotalid Venoms; D009841:Oligonucleotides; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C068858:barbourin; D000244:Adenosine Diphosphate; D004247:DNA