Anti-Gal and its triplets recognize the O-glycosylated region of their platelet surface glycoprotein receptor
Jacalin was used to block the O-glycosylated regions of proteins on the surface of denuded platelets. FITC-labeled anti-Gal and FITC-labeled de novo triplet (reconstituted using anti-Gal, AOP1, AOP2 and FITC-labeled albumin) were prepared from components of sugar-extracted triplets of platelets. Both could bind to denuded platelets unless the latter were treated in advance with jacalin (Fig.4a). Heat-inactivated jacalin did not effect this blocking of re-binding of anti-Gal or triplets. Results showed that STPS that underlie the O-glycans in O-glycoproteins on platelet surface and therefore get blocked upon jacalin occupation of the O-glycans, are true ligands for anti-Gal since terminal α-linked galactose, the monosaccharide ligand for this antibody  is absent in humans and O-glycans per se are not ligands for anti-Gal [19], unlike for jacalin. Jacalin-mediated blocking of access of anti-Gal to STPS had been shown in the case of O-glycoproteins in lipoprotein(a) [20] and in plasma AOP1 and AOP2 [1]. STPS underlying the O-glycans had been confirmed as anti-Gal and ABG ligands since de-O-glycosylation of AOP1, AOP2 and Lp(a) without destruction of peptide sequences did only enhance the binding of the antibodies to them [1,20]. Since ABG shares the STPS specificity of anti-Gal the above result suggested platelet surface O-glycosylated protein (s) as receptors for unoccupied binding sites of anti-Gal- and ABG-derived triplets.
Binding of triplets to denuded platelets was also assessed by determining the percentage remaining unbound in the supernatant out of a limited quantity of the former added to denuded platelets in suspension. Prior treatment of denuded platelets with fibrinogen (1 µM) resulted in complete blocking of triplet binding to denuded platelets (Fig.4b). Interference by fibrinogen in the assay of unbound triplet was ruled out. Since fibrinogen recognizes the GPIIb/IIIa integrin present on platelet surface [4,21], the result pointed to GPIIb/IIIa as possible receptor for anti-Gal and ABG antibodies present in triplets. In support to this contention, GPIIb/IIIa is the extra-ordinarily dominant O-glycosylated protein on platelet membrane [21-23]. Requirement of O-glycosylated site for binding of anti-Gal or triplet to platelets (Figs. 4a) also supports the above assumption. Though plasma fibrinogen concentration (8-9 µM) far exceeds the concentration used in the above experiment for blocking triplet attachment, circulating platelets still bear triplets showing that while fibrinogen and triplets may use the same receptor(s) to bind to denuded platelets competitive displacement of bound triplets by fibrinogen does not seem to take place appreciably.