Summary
Disintegrins are a group of snake venom peptides which inhibit human platelet aggregation
by acting as glycoprotein Ilb-IIIa (GPIIb-Ilia) antagonists. They are cysteine-rich,
Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-Ilia complex on platelet
membrane with a very high affinity (Kd, 10−7 ∼ 10−8 M). In this study, we analyzed GPIIb-Ilia complex on platelet membrane by flow cytometry
using fluorescein isothiocyanate (FITC)-conjugated disintegrins as probes. Of these
FITC-conjugated disintegrins, FITC-Rhodostomin is the most sensitive probe because
Rhodostomin was conjugated with more FITC molecules than Trigramin and Halysin were.
The binding fluorescence intensity of FITC-Trigramin (FITC-Tg), FITC-Halysin (FITC-Hy)
and FITC-Rhodostomin (FITC-Rn) was measured in both resting and ADP-activated platelets
of diluted human platelet-rich plasma. The binding fluorescence of FITC-disintegrins
was abolished by EDTA and 7E3, a monoclonal antibody against GPIIb-Ilia. ADP markedly
increased the fluorescence intensity of FITC-Tg and FITC-Hy bound on platelets especially
when lower doses of these probes were used, whereas it had little effect on that of
FITC-Rn. Therefore, FITC-Tg and FITC-Hy can be used for the detection of the activated
platelets as noted by a higher ratio of fluorescence intensity (approx. 2-4) between
ADP-activated and resting platelets as compared with that (approx. 1-1.3) in the case
of FITC-Rn as the probe. The platelets from three patients with Glanzmann’s thrombasthenia
were probed with FITC-disintegrins. As a result these three patients could be classified
as type I thrombasthenia based on the extremely low level of GPIIb-Ilia detected by
this method (<5% of normal value).
