Figure legends
Fig. 1. Anti-Gal- and ABG-specific sugars release two O-glycosylated proteins, albumin and the antibodies from platelets.Inset: Electrophoresis of platelet-bound triplet extracted by mixture of sugars (section 3.2) (a; 50 µg) and mixture of APAG and APABG from plasma (b; 25 µg each) in 6 % polyacrylamide gel using Tris-glycine pH 8.3 buffer. Gels were stained with Coomassie Brilliant blue G-250. Bands in gel (a) suspected (*) to be antibody were electroeluted, dialyzed against PBS1 and 100 ng in 200 µl PBST was treated with anti-Gal- specific sugar MαG, ABG-specific sugar cellobiose (both 25 mM) or no sugar for 2 h at 4°C before adding to polystyrene microwells coated with Tg or TIC (1µg per well). After 2 h incubation at 4°C and washing wells were treated with anti-immunoglobulin-HRP (1.5 µg/ml in PBS), washed and again treated with OPD as described under ‘methods. Immunoglobulins in electroeluted samples were expressed in terms of optical density (OD) response at 490 nm in the ELISA protocol.**: P value < 0.0001 for inhibition using specific sugars. Mean ± SD of 6 platelet samples.
Fig. 2. Anti-Gal and ABG, each linked to albumin via O-glycosylated protein (triplets) are released from platelets by antibody-specific sugars. a. Either sugar releases both triplets.Platelet samples prepared as described in ‘methods’ were diluted to 0.25 million cells per µl in PBS and 300 µl incubated for 2 h at 25 °C with non-specific sugar MαM or ABG-specific sugar cellobiose (Cb) or anti-Gal- specific sugar MαG ( 25 mM each ). Following centrifugation at 4500 g for 15 min at 25 °C 250 µl of the supernatant was dialyzed against PBS to remove sugars and 100X dilution of the dialysate (200 µl) added to microwell-coated (1 µg per well) guar galactomannan ( to capture anti-Gal complexes) or yeast glycoproteins (to capture ABG complexes ). After incubation for 2 h at 4 °C wells were washed with PBST and probed with anti-albumin-HRP (3.75 µg antibody per ml). ***: P value < 0.0001 for elution of anti-Gal-containing triplets, ABG-containing triplets and O-glycoprotein-linked albumin by specific sugar versus non-specific sugar. Mean ± SD of 7 blood samples.b. Specific sugars release free albumin from triplets. Plasma diluted with PBS to 1 mg protein per ml was treated with cellobiose and methyl α-D-galactoside (25 mM each), incubated for 3 hours at 40 C and again for 3 hours after addition of AOP1-FITC or AOP2–FITC to final concentration of 1 mg per ml. After DGUC as described total fluorescence in middle (400 µl) and bottom (300 µl) layers were measured. *** : P value < 0.0001 for increase in middle layer and decrease in bottom layer of added AOP1-FITC or AOP2-FITC brought about by treatment of plasma with antibody-specific sugars. Mean ± SD of response in 6 plasma samples.
Fig. 3.Platelet-bound and plasma triplets are identical. De novo plasma triplet samples were prepared by adding 600 ng anti-Gal in 200 µl PBS (pre-treated for 2 h with 25 mM anti-Gal specific sugar MαG or non-specific sugar MαM) to a pre-incubated (2 h at 4°C) mixture of AOP1, AOP2 and FITC-labeled albumin (200 ng each in 200 µl PBS) and the mixture incubated again for 2h at 4°C. Denuded or native platelet suspension (0.25 million per µl in PBS; 300µl) was mixed with i) glycoprotein-free anti-Gal-FITC (isolated from FITC-labeled plasma APAG1 ;600 ng in 400 µl PBS) pre-incubated for 2 h at 4 °C with 25 mM MαG or MαM or ii) de novo plasma triplets described above (400 µl ) . The mixture was incubated for 2h at 4°C and centrifuged at 4500 g for 15 min to remove supernatant. Pellet washed twice with PBS was re-suspended in 320 µl PBS. Fluorescence was measured using 300 µl suspension at 485/528 nm. ***: P value < 0.0001 for fluorescence attached to denuded platelets treated with labeled anti-Gal or triplet, treated in advance with specific vs non-specific sugar. Mean ± SD of 6 different blood samples. AOP = AOP1+AOP2.Inset : From platelets harvested from 6 ml blood a fraction (320 µl washed platelet suspension ;0.25 million cells per µl) was treated with a mixture of MαG and cellobiose (15 mM each), mixture centrifuged at 4500 g and supernatant (250 µl) dialyzed against PBS. This and cell free plasma of the same donor (supernatant after removing all cells by centrifugation at 5000 g for 15 min) were diluted 100 X in PBST and 200 µl added to microwell-coated TIM or TIC (1 µg per well) and probed using anti-albumin-HRP (3.75 µg antibody per ml) to estimate triplets of anti-Gal and ABG respectively. Anti-albumin response of APAG and APABG (100 ng in 200 µl PBST) captured on microwell-coated TIM or TIC (1µg per well) under same conditions was used as standard. Mean ± SD of 8 different blood samples.
Fig.4. AntibodyO-glycoprotein–albumin triplet and anti-Gal bind to O-glycosylated membrane glycoprotein(s) on triplet-free platelet surface.  a. Denuded platelets (0.25 million cells per µl; 250 µl) were incubated with 50 ng jacalin or heat-inactivated jacalin for  1 h at 4°C followed by removal of supernatant by centrifugation at 4500 g for 15 min. Platelets in pellet were washed twice with PBS and incubated  at 25 °C for 3h in either 400 µl PBS containing 600 ng glycoprotein-free anti-Gal-FITC or 400 µl de novo triplet reconstituted from components of platelet-derived triplets, among which albumin alone had been labeled in advance with FITC. After washing thrice in PBS by centrifugation at 4500 g the pellet was re-suspended in 320 µl PBS and cell-bound fluorescence measured as for Fig.3 protocol.   ***: P value <0.0001 for difference between cells pretreated with jacalin and those treated with inactive jacalin in bound fluorescence due to FITC-labeled anti-Gal or triplet. Mean ± SD of 6 blood samples. AOP = AOP1+AOP2. b. Denuded platelets (0.25 million cells /µl; 350 µl) were incubated with 1µM fibrinogen (F) at 25 °C for 2h. After removing supernatant the pellet was washed twice with PBS and again incubated in 300 µl PBS for 4 h at  25 °C with 2 µg of triplet released from platelets using sugars (Fig. 3 inset). From supernatant collected after centrifugation at 4500 g for 15 min, 200 µl was added to wells coated with both TIM and TIC ( 0.5 µg each). After 2h incubation at 40 C and washing, bound triplet was assayed using anti-albumin-HRP as probe as described earlier.