1. GPIIb-IIIa was purified fromoutdated platelet concentrates using affinity
2. Briefly, human platelets suspended in Tris-buffered saline (TBS; 150
mM NaCl, 20 mM Tris-HCl, pH 7.4) containing 1 mM benzamidine (GP
buffer) were sonicated and ultracentrifuged at 100,000g for 60 min at
3. The pellet was then resuspended in GP buffer containing 1% Triton
X-100 (wt/vol) and allowed to stand on ice for 1 h.
4. The solubilized platelet membrane preparation was ultracentrifuged
again as indicated above.
5. The platelet membrane lysate was applied to the affinity column, in
which the IgG fraction of mAb to human GPIIb-IIIa was coupled to
cyanogen bromide-activated SepharoseCL-4B (Amersham Pharmacia
Biotech Inc., Piscataway, NJ).
6. After extensive washing with 0.1% Triton X-100/GP buffer containing
0.5 M LiCl, the bound GPIIb-IIIa was eluted with 0.05 M diethylamine in
0.05% Triton X-100/GP buffer (pH 11.5).
7. Fractions with a peak in the OD280 reading were collected as
GPIIb-IIIa antigen. After GPIIb-IIIa was eluted, fractions in which the
OD280 reading was below 0.1 were collected as a control preparation. 8. GPIIb-IIIa and control preparations were immediately neutralized with
solid glycine, dialyzed against TBS containing 0.05% Triton X-100,
sterilized by passage through 0.45μm pore-size syringe filters, and
9. The relative amounts of purified GPIIb-IIIa determined by Coomassie
blue staining revealed that GPIIb and IIIa represented.
10. 95% of the total stained protein. GPIIb-IIIa was also purified from
platelets of two healthy donors on a small scale.