Fig. 5. Jacalin binds predominantly to a 116 kDa protein subunit on platelet surface.
Platelet membrane proteins were isolated, separated by electrophoresis and trans-blotted to PVDF membrane as described in ‘methods’. Strips were probed with amido black (1), concanavalin A-HRP (150 µg per ml lectin; 2), PNA-HRP (75 µg per ml; 3), PNA-HRP after neuraminidase treatment ( 100 X dilution in PBS of 1.5 U per ml; 1 h; 370C) of strip (4), sugar control (25 mM lactose) of lane 4 (5), and jacalin-HRP (0.75 µg per ml; 6). Stained bands were detected by 4-chloronaphthol (1 mg in 0.4 ml methanol mixed with 1.6 ml PBS containing 0.05% H2 O2 ). Positions of standard proteins with their molecular weight in kDa are indicated on the right.
Fig. 6.Triplet-free platelets are more aggregable and remarkably so on adding ADP; jacalin blocks ADP binding to platelets. Suspensions in PBS of native or denuded platelets prepared as described under ‘methods’ (0.25 million cells per µl; 250 µl) were incubated with or without 50 ng of active or heat-inactivated jacalin for 1 h at 4 °C. After removing supernatant and three washings with PBS the pellet was dispersed in 220 µl PBS with or without 20 µM ADP and absorbance at 405 nm measured after 2 min. Mean ± SD of absorbance values using 10 blood samples.
Fig.7 . Amyloid-β binds to triplets bound to platelets.Denuded or native platelets (methods) in 320 µl PBS (0.25 million cells per µl) were incubated for 3 h with 75 ng fluorescent labeled amyloid β at 25°C. Following centrifugation at 4500 g for 15 min at 25 °C fluorescence in supernatant was measured at 485/528 nm using 300 µl aliquot. The pellet was washed twice with PBS and incubated for 1 h at 25 °C with a mixture of 15 mM each of anti-Gal- and ABG-specific sugars (MαG and cellobiose) in 350 µl PBS and the triplet-bound amyloid β now released with specific sugars was measured using 300 µl of supernatant as above. ***: P value <0.0001 for the difference between native and denuded platelets in amyloid β released by antibody-specific sugars. Mean ± SD of 9 blood samples.