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.