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 platelet aggregation by acting as glycoprotein IIb-IIIa (GPIIb-IIIa) antagonists. They are cysteine-rich, Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-IIIa complex on platelet membrane with a very high affinity (Kd, 10(-7)-10(-8) M). In this study, we analyzed GPIIb-IIIa 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-IIIa. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Liu
C Z
CZ
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei.
Wang
Y W
YW
Shen
M C
MC
Huang
T F
TF
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D019483:Disintegrins; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010980:Platelet Membrane Glycoproteins / Q000032:analysis*; D013915:Thrombasthenia / Q000097:blood; D014688:Venoms / Q000737:chemistry
D000911:Antibodies, Monoclonal; D019483:Disintegrins; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D014688:Venoms; D016650:Fluorescein-5-isothiocyanate