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.