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Thromb Haemost 1994; 72(06): 919-925
DOI: 10.1055/s-0038-1648984
Original Article
Schattauer GmbH Stuttgart

Analysis of Human Platelet Glycoprotein llb-llla by Fluorescein Isothiocyanate-Conjugated Disintegrins with Flow Cytometry

Authors

  • Chao-Zong Liu

    1   Pharmacological Institute and Department of Laboratory Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan

  • Yi-Wen R Wang

    1   Pharmacological Institute and Department of Laboratory Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan

  • Ming-Ching Shen

    1   Pharmacological Institute and Department of Laboratory Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan

  • Tur-Fu Huang

    1   Pharmacological Institute and Department of Laboratory Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
Further Information

Publication History

Received 21 January 1994

Accepted after resubmission 18 August 1994

Publication Date:
06 July 2018 (online)

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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).