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. Antibody –O-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.