Purification and partial characterization of a 65-kDa platelet aggregation-associated protein antigen from the...

Cells of Streptococcus sanguis express a collagen-like immunodeterminant (class II antigen) on their cell walls that induces aggregation of platelets in plasma. These platelet aggregation-associated proteins (PAAPs) are recovered in cell-free preparations obtained from cells of S. sanguis after 5 min of sonic or limited trypsin treatment. Pretreatment of platelet-rich plasma with these soluble preparations selectively inhibits platelet aggregation in response to S. sanguis cells. A PAAP antigen was isolated and purified from minimal tryptic digests of S. sanguis cells using (i) immunoaffinity chromatography or (ii) gel filtration and ion-exchange chromatography. A monospecific rabbit antiserum was prepared against PAAP (from procedure ii) and used to verify identity with PAAP fragments in different preparations. Criteria of purity included single precipitins in immunoelectrophoresis and crossed immunoelectrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western immunoblot, and COOH (Lys)- and NH2 (Pro)-terminal analyses. The 65-kDa (p65) antigen isolated by immunoaffinity chromatography had 50-fold greater specific inhibitory activity in S. sanguis-induced PRP aggregation than the original tryptic digest and about 1.4 times that recovered by sequential column chromatography. Amino acids of the p65 PAAP fragment constituted 89.5% of the total dry weight, with glycine, lysine, and glutamic acid predominant. Lesser amounts of proline were also noted. Monosaccharides, including glucose and galactose, comprised 4.0% of the total. A platelet interactive determinant of p65 was localized to a 23-kDa tryptic fragment after further trypsin treatment. Amino acids of this 23-kDa fragment constituted 99.8% of the total dry weight. In their native state on the cell wall of platelet-interactive strains of S. sanguis, platelet aggregation-associated proteins are probably assembled on fibrils as polyvalent agonists.