ANALYSIS OF HUMAN PLATELET GLYCOPROTEIN IIB-IIIA BY FLUORESCEIN ISOTHIOCYANATE-CONJUGATED DISINTEGRINS WITH FLOW-CYTOMETRY

Disintegrins are a group of snake venom peptides which inhibit human p latelet aggregation by acting as glycoprotein IIb-IIIa (GPIIb-IIIa) an tagonists. They are cysteine-rich Arg-Gly-Asp (RGD)-containing peptide s, and bind to GPIIb-IIIa complex on platelet membrane with a very hig h affinity (Kd, 10(-7) similar to 10(-8) M). In this study, we analyze d GPIIb-IIIa complex on platelet membrane by flow cytometry using fluo rescein isothiocyanate (FITC)-conjugated disintegrins as probes. Of th ese FITC-conjugated disintegrins, FITC-Rhodostomin is the most sensiti ve probe because Rhodostomin was conjugated with more FITC molecules t han 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-d isintegrins was abolished by EDTA and 7E3, a monoclonal antibody again st GPIIb-IIIa. ADP markedly increased the fluorescence intensity of FI TC-Tg and FITC-Hy bound on platelets especially when lower doses of th ese 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 a ctivated platelets as noted by a higher ratio of fluorescence intensit y (approx. 2-4) between ADP-activated and resting platelets as compare d with that(approx. 1-1.3) in the case of FITC-Rn as the probe. The pl atelets from three patients with Glanzmann's thrombasthenia were probe d with FITC-disintegrins. As a result these three patients could be cl assified as type I thrombasthenia based on the extremely low level of GPIIb-IIIa detected by this method (<5% of normal value).