Albumin and either of two O-glycosylated proteins are attached through anti-Gal or ABG to platelets
Proteins extracted from platelets using a mixture of 15 mM each of MαG (anti-Gal- specific) and cellobiose (ABG-specific), when subjected to alkaline polyacrylamide gel electrophoresis, showed bands with mobilities identical to those of a mixture of equal amounts of APAG and APABG which are plasma triplets of anti-Gal and of ABG respectively [1] (Fig.1 inset). The fastest and slowest moving bands were identified as albumin (HSA) and immunoglobulins respectively by electroelution, coating on polystyrene microplates and ELISA using corresponding enzyme-labeled antibodies as done earlier [1]. Bands with mobility equal to that of AOP1 or AOP2 were as heavily O-glycosylated as the latter, judging from the capacity of their microplate-coated forms to capture HRP-conjugated O-glycan-specific lectin jacalin [16] (result not shown). Platelet-bound immunoglobulins released by sugars and electroeluted as above contained anti-Gal as evidenced by binding to microplate-coated α-galactoside-bearing protein thyroglobulin and inhibition of this binding by MαG but not by cellobiose (Fig.1). Electroeluted immunoglobulins also contained ABG which bound to microplate-coated TIC and was inhibited by cellobiose but not by MαG. Only anti-Gal and ABG were eluted from platelets by the mixture of MαG and cellobiose since a mixture of guar galactomannan gel [10] and cellulose [11], respective affinity matrices for these antibodies, could capture all the proteins in the electroeluted immunoglobulin sample. Results suggested that attachment of the two O-glycoproteins and albumin to platelets is mediated by anti-Gal or ABG. The procedure for extraction of platelet-bound triplets ensured that immune complexes if any bound non-specifically to platelets [17] could not have been liberated from the platelets.