Triplets having anti-Gal or ABG linked to albumin through
O-glycosylated protein as bridge is released from platelets by specific
sugars; either sugar releases triplets of both antibodies
Triplet immune complexes in plasma, formed by simultaneous binding
of anti-Gal or ABG on one side and albumin on another to AOP1 or AOP2,
were assayed by capturing them on polystyrene plate-coated antibody
ligand through the unused binding sites still present on the antibodies
and determining the albumin on the other end of the bound triplet, using
HRP-labeled anti-albumin antibody as probe [1]. Protein mixture
released from platelets by antibody-specific sugars MαG or cellobiose,
dialyzed to remove sugar and assayed by the above protocol, was found to
contain significantly more albumin associated directly or indirectly
with anti-Gal or ABG than did the same dilution of proteins released by
the non-specific sugar MαM (Fig.2a). Presence of O-glycoprotein-bound
albumin in the proteins released by specific sugars from platelets was
confirmed by capturing them on microwell-coated jacalin and probing
bound proteins using HRP-labeled anti-albumin [1]. Since albumin has
no direct association with either of these two antibodies and
O-glycoproteins identical in mobility and O-glycan content with those of
AOP1 and AOP2 of plasma triplets were present in the released proteins,
results indicated that proteins released by antibody-specific sugars
from platelets contained O-glycoprotein molecules that bridged between
anti-Gal or ABG on one side and albumin on the other to form triplets of
same structure as that of plasma anti-Gal/ABG-AOP1/AOP2-albumin triplets
[1]. Since these triplets possessed binding sites mandatorily left
free in their antibodies and enabled their binding to ligand-bearing
matrices and cells, the above results suggested that platelet membranes
carried receptor molecules that possessed ligands for anti-Gal and ABG
and could capture triplets utilizing the free binding sites on
antibodies in the latter. Notably, glucose (15 mM) released nearly as
much triplets from platelets as the same concentration of MαG or
cellobiose did (Fig.2a). Since this level of serum glucose in
circulation is often reached in diabetics this result suggests that the
consequences of depriving platelets of their triplets accompany
diabetes.
Though anti-Gal and ABG share affinity for STPS their specificities
towards small sugars are distinct and different whether isolated from
plasma triplets [1] or from platelet-bound triplets (Fig.1).
Nevertheless the same amounts of anti-Gal triplets and ABG triplets were
released by either ABG-specific sugar or anti-Gal-specific sugar
(Fig.2a). One possible reason for this phenomenon is that a small sugar
specific to one antibody could occupy all binding sites of the latter,
resulting in detachment of its triplet from platelet as well as release
of albumin-bound O-glycoproteins from the triplet antibody. If the
latter event resulted in temporary destabilization of the
O-glycoprotein-albumin bondage as well, free O-glycoproteins would be
freshly generated. The latter, unlike their albumin complexes, resemble
low molecular weight antibody-specific sugars in that they occupy all
available binding sites of either antibody without steric hindrance
[1], and could liberate triplets of the other antibody as well.
To verify the above course of events involving small sugar-mediated
release of free albumin and O-glycoproteins from triplets, the
differential distribution of AOP1-FITC or AOP2-FITC, added to
sugar-treated and untreated plasma, was examined. Following DGUC of 1.1
ml KBr-treated plasma as described, undissociated triplets are found
predominantly in the bottom 300 µl owing mostly to the presence of
immunoglobulins [1]. However, in the case of plasma treated with
antibody-specific sugar, the albumin-AOP1and albumin-AOP2 complexes
liberated from triplets migrated from antibody-rich bottom layer to the
antibody-free and albumin-rich middle layer (400 µl), apparently due to
the high buoyancy of albumin, though free AOP1/AOP2 occupied mostly the
bottom layer under these conditions [1]. Results in Fig.2b show that
the majority of AOP1-FITC and AOP2-FITC added to PBS or untreated plasma
remains in the bottom 300 µl of the 1.1 ml sample subjected to DGUC.
However AOP1-FITC and AOP2-FITC added to plasma treated in advance with
anti-Gal- and ABG-specific sugars before DGUC segregated mostly to the
middle layer showing that fresh albumin ready to combine with free
AOP1/AOP2 or their FITC derivatives was liberated in this case. Since
liberation of fresh free albumin is also accompanied by release of free
AOP1 and AOP2 and the latter are capable of dissociating neighbouring
triplets of both antibodies this result supported the explanation given
above for results in Fig.2. Colligative effect of any sugar molecule per
se as reason for altered distribution upon DGUC of FITC-labeled
AOP1/AOP2 added to sugar-treated plasma had been ruled out using
non-specific sugars [1]. O-Glycoprotein-free albumin already present
in plasma before sugar treatment seemed not to combine with added
FITC-labeled AOP1 or AOP2 (Fig.2b). A possible reason is that the
albumin molecules not involved in triplet formation is likely to have
been engaged by one or more of dozens of other albumin-binding
biomolecules, unlike the nascent albumin liberated from triplets by
antibody-specific sugar.