Preparation of native and denuded (triplet-free) platelets
Platelets were isolated from fresh blood samples collected from
healthy volunteers after obtaining informed consent in writing in
accordance with Institutional Ethics Committee (IEC) guidelines and
permission (IEC/1072) of this institute by the procedure described by
Jennings and Philips [13]. Anticoagulant-treated blood samples (10
ml) were centrifuged at 150 g for 5 min at 25°C. Plasma in the
supernatant was mixed with one-tenth its volume of 100 mM EDTA to
prevent platelet activation and centrifuged at 230 g for 10 min at 25°C
to remove erythrocytes and again centrifuged at 4530 g for 15 min.
Supernatant was removed and the pellet was collected as platelets. This
platelet was incubated for 2 h at 25°C in 300 µl of a mixture of
specific sugars for anti-Gal and ABG (15 mM each of MαG and cellobiose)
and the supernatant containing triplets extracted from platelets was
recovered by centrifugation at 4530 g for 15 min. The pellet was washed
twice with PBS to get denuded (triplet-free) platelets. Platelets
treated as above, but with non-specific sugar MαM were used as native
(non-denuded platelets).