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.