PLATELET RECEPTORS FOR THE STREPTOCOCCUS-SANGUIS ADHESIN AND AGGREGATION-ASSOCIATED ANTIGENS ARE DISTINGUISHED BY ANTI-IDIOTYPICAL MONOCLONAL-ANTIBODIES

Platelets aggregate in response to an adhesin and the platelet aggrega tion-associated protein (PAAP) expressed on the cell surfaces of certa in strains of Streptococcus sanguis. We sought to identify the corresp onding PAAP receptor and accessory adhesin binding sites on platelets. Since the adhesin(s) of S. sanguis for platelets has not been charact erized, an anti-idiotype (anti-id) murine monoclonal antibody (MAb2) s trategy was developed, First, MAb1s that distinguished the adhesin and PAAP antigens on the surface of S. sanguis I 133-79 were selected, Fa b fragments of MAb1,2 (immunoglobulin G2b [IgG2b]; 70 pmol) reacted wi th 5 x 10(7) cells of S. sanguis to completely inhibit the aggregation of human platelets in plasma, Under similar conditions, MAb1.1 (IgG1) inhibited the adhesion of S. sanguis cells to platelets by a maximum of 34% with a comparatively small effect on platelet aggregation, Toge ther, these two MAb1s inhibited S. sanguis-platelet adhesion by 63%. I n Western immunoblots, both MAb1s reacted with S. sanguis 133-79 87- a nd 150-kDa surface proteins and MAb1.2 also reacted with purified type I collagen. The hybridomas producing MAb1.1 and MAb1.2 were then inje cted into BALB/c mice, Enlarged spleens were harvested, and a panel of MAb2 hybridomas was prepared. To identify anti-ids against the specif ic MAb1s, the MAb2 panel was screened by enzyme-linked immunosorbent a ssay for reaction with rabbit polyclonal IgG antibodies against the 87 - and 150-kDa antigens, The reactions between the specific rabbit anti bodies and anti-ids were inhibited by the 87- and 150-kDa antigens, Wh en preincubated with platelets, MAb2.1 (counterpart of MAb1.1) inhibit ed adhesion to platelets maximally by 46% and MAb2.2 (anti-MAb1.2) inh ibited adhesion to platelets maximally by 35%. Together, both MAb2s in hibited the adhesion of S. sanguis to platelets by 81%. MAb2.2 also in hibited induction of platelet aggregation, MAb2.2 immunoprecipitated a biotinylated platelet membrane antigen of 170 kDa (unreduced); MAb2.1 precipitated membrane antigens of 175- and 230-kDa (unreduced). There fore, platelet binding sites and the receptor for the S. sanguis adhes in and PAAP, respectively, are distinguished by the anti-id MAb2s.