Ultrastructural analysis of thrombin-induced interaction between human platelets and liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute

Hidenori Suzuki, Yosuke Okamura, Yasuo Ikeda, Shinji Takeoka, Makoto Handa

    Research output: Contribution to journalArticle

    7 Citations (Scopus)

    Abstract

    Background: The dodecapeptide HHLGGAKQAGDV (H12) in the carboxy-terminus of the fibrinogen γ-chain is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute. Objectives: We examined the interaction between human platelets and H12-liposomes during thrombin-induced activation using flow cytometry and electron microscopy (EM). Methods and results: After thrombin-activation, a remarkable time-dependent increase in binding of the H12-liposomes to platelets was found by flow cytometry. A large-sized swollen open canalicular system (OCS) was observed in the spheroidal platelets from 60 sec to 5 min after thrombin-activation, but intact H12-liposomes were not evident by conventional EM. Cryoultramicrotomy and immunogold staining with anti-H12 antibody were successful in identifying the liposomes; they appeared as small particles with a unit membrane around 0.2 to 0.4 μm in diameter, and gold labels representing H12 were distributed homogeneously on the surface. Abundant H12-liposomes were localized not only on the surface membrane but also in the lumen of the large-sized swollen OCS in the platelets at 60 sec after thrombin-activation. The formation of the large-sized swollen OCS was inhibited by pre-incubation with unbound H12, EDTA or anti-GPIIb/IIIa antibody. In thrombin-induced platelet aggregates we observed electron-transparent areas between adherent platelets, in which abundant H12-liposomes were distributed. Conclusions: We demonstrate morphologically that H12-liposomes bind to thrombin-activated platelets and accumulate between adherent platelets like fibrinogen, leading to large-scale aggregation.

    Original languageEnglish
    Pages (from-to)552-559
    Number of pages8
    JournalThrombosis Research
    Volume128
    Issue number6
    DOIs
    Publication statusPublished - 2011 Dec

    Fingerprint

    Liposomes
    Thrombin
    Fibrinogen
    Blood Platelets
    Electron Microscopy
    Flow Cytometry
    Cryoultramicrotomy
    Membranes
    Edetic Acid
    Gold
    Anti-Idiotypic Antibodies
    Binding Sites
    Electrons
    Staining and Labeling
    Ligands
    Antibodies

    Keywords

    • Aggregation
    • Dodecapeptide (H12)
    • Electron microscopy
    • Liposomes
    • Platelet substitute
    • Platelets

    ASJC Scopus subject areas

    • Hematology

    Cite this

    Ultrastructural analysis of thrombin-induced interaction between human platelets and liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute. / Suzuki, Hidenori; Okamura, Yosuke; Ikeda, Yasuo; Takeoka, Shinji; Handa, Makoto.

    In: Thrombosis Research, Vol. 128, No. 6, 12.2011, p. 552-559.

    Research output: Contribution to journalArticle

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    title = "Ultrastructural analysis of thrombin-induced interaction between human platelets and liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute",
    abstract = "Background: The dodecapeptide HHLGGAKQAGDV (H12) in the carboxy-terminus of the fibrinogen γ-chain is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute. Objectives: We examined the interaction between human platelets and H12-liposomes during thrombin-induced activation using flow cytometry and electron microscopy (EM). Methods and results: After thrombin-activation, a remarkable time-dependent increase in binding of the H12-liposomes to platelets was found by flow cytometry. A large-sized swollen open canalicular system (OCS) was observed in the spheroidal platelets from 60 sec to 5 min after thrombin-activation, but intact H12-liposomes were not evident by conventional EM. Cryoultramicrotomy and immunogold staining with anti-H12 antibody were successful in identifying the liposomes; they appeared as small particles with a unit membrane around 0.2 to 0.4 μm in diameter, and gold labels representing H12 were distributed homogeneously on the surface. Abundant H12-liposomes were localized not only on the surface membrane but also in the lumen of the large-sized swollen OCS in the platelets at 60 sec after thrombin-activation. The formation of the large-sized swollen OCS was inhibited by pre-incubation with unbound H12, EDTA or anti-GPIIb/IIIa antibody. In thrombin-induced platelet aggregates we observed electron-transparent areas between adherent platelets, in which abundant H12-liposomes were distributed. Conclusions: We demonstrate morphologically that H12-liposomes bind to thrombin-activated platelets and accumulate between adherent platelets like fibrinogen, leading to large-scale aggregation.",
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    author = "Hidenori Suzuki and Yosuke Okamura and Yasuo Ikeda and Shinji Takeoka and Makoto Handa",
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    T1 - Ultrastructural analysis of thrombin-induced interaction between human platelets and liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute

    AU - Suzuki, Hidenori

    AU - Okamura, Yosuke

    AU - Ikeda, Yasuo

    AU - Takeoka, Shinji

    AU - Handa, Makoto

    PY - 2011/12

    Y1 - 2011/12

    N2 - Background: The dodecapeptide HHLGGAKQAGDV (H12) in the carboxy-terminus of the fibrinogen γ-chain is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute. Objectives: We examined the interaction between human platelets and H12-liposomes during thrombin-induced activation using flow cytometry and electron microscopy (EM). Methods and results: After thrombin-activation, a remarkable time-dependent increase in binding of the H12-liposomes to platelets was found by flow cytometry. A large-sized swollen open canalicular system (OCS) was observed in the spheroidal platelets from 60 sec to 5 min after thrombin-activation, but intact H12-liposomes were not evident by conventional EM. Cryoultramicrotomy and immunogold staining with anti-H12 antibody were successful in identifying the liposomes; they appeared as small particles with a unit membrane around 0.2 to 0.4 μm in diameter, and gold labels representing H12 were distributed homogeneously on the surface. Abundant H12-liposomes were localized not only on the surface membrane but also in the lumen of the large-sized swollen OCS in the platelets at 60 sec after thrombin-activation. The formation of the large-sized swollen OCS was inhibited by pre-incubation with unbound H12, EDTA or anti-GPIIb/IIIa antibody. In thrombin-induced platelet aggregates we observed electron-transparent areas between adherent platelets, in which abundant H12-liposomes were distributed. Conclusions: We demonstrate morphologically that H12-liposomes bind to thrombin-activated platelets and accumulate between adherent platelets like fibrinogen, leading to large-scale aggregation.

    AB - Background: The dodecapeptide HHLGGAKQAGDV (H12) in the carboxy-terminus of the fibrinogen γ-chain is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute. Objectives: We examined the interaction between human platelets and H12-liposomes during thrombin-induced activation using flow cytometry and electron microscopy (EM). Methods and results: After thrombin-activation, a remarkable time-dependent increase in binding of the H12-liposomes to platelets was found by flow cytometry. A large-sized swollen open canalicular system (OCS) was observed in the spheroidal platelets from 60 sec to 5 min after thrombin-activation, but intact H12-liposomes were not evident by conventional EM. Cryoultramicrotomy and immunogold staining with anti-H12 antibody were successful in identifying the liposomes; they appeared as small particles with a unit membrane around 0.2 to 0.4 μm in diameter, and gold labels representing H12 were distributed homogeneously on the surface. Abundant H12-liposomes were localized not only on the surface membrane but also in the lumen of the large-sized swollen OCS in the platelets at 60 sec after thrombin-activation. The formation of the large-sized swollen OCS was inhibited by pre-incubation with unbound H12, EDTA or anti-GPIIb/IIIa antibody. In thrombin-induced platelet aggregates we observed electron-transparent areas between adherent platelets, in which abundant H12-liposomes were distributed. Conclusions: We demonstrate morphologically that H12-liposomes bind to thrombin-activated platelets and accumulate between adherent platelets like fibrinogen, leading to large-scale aggregation.

    KW - Aggregation

    KW - Dodecapeptide (H12)

    KW - Electron microscopy

    KW - Liposomes

    KW - Platelet substitute

    KW - Platelets

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