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    In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function

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    Authors
    Michelson, Alan D.
    Barnard, Marc R.
    Hechtman, Herbert B.
    Macgregor, Hollace
    Connolly, Raymond J.
    Loscalzo, Joseph
    Valeri, C. Robert
    UMass Chan Affiliations
    Department of Surgery
    Department of Pediatrics
    Document Type
    Journal Article
    Publication Date
    1996-10-15
    Keywords
    Animals
    Antibodies, Monoclonal
    Blood Platelets
    Blood Transfusion, Autologous
    Flow Cytometry
    Fluorescent Dyes
    Male
    Organic Chemicals
    P-Selectin
    Papio
    Platelet Activation
    Platelet Glycoprotein GPIIb-IIIa Complex
    Platelet Transfusion
    Thrombin
    Time Factors
    Life Sciences
    Medicine and Health Sciences
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    Link to Full Text
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC38152/
    Abstract
    To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.
    Source

    Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11877-82.

    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/38974
    PubMed ID
    8876231
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    • Thumbnail

      Evaluation of platelet function by flow cytometry

      Michelson, Alan D.; Barnard, Marc R.; Krueger, Lori A.; Frelinger, Andrew L. III; Furman, Mark I. (2000-07-01)
      Platelet function in whole blood can be comprehensively evaluated by flow cytometry. Flow cytometry can be used to measure platelet reactivity, circulating activated platelets, platelet-platelet aggregates, leukocyte-platelet aggregates, procoagulant platelet-derived microparticles, and calcium flux. Clinical applications of whole blood flow cytometric assays of platelet function in disease states (e.g., acute coronary syndromes, angioplasty, and stroke) may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Circulating monocyte-platelet aggregates appear to be a more sensitive marker of in vivo platelet activation than circulating P-selectin-positive platelets. Flow cytometry can also be used in the following clinical settings: monitoring of GPIIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.
    • Thumbnail

      Flow cytometry: a clinical test of platelet function

      Michelson, Alan D. (1996-06-15)
    • Thumbnail

      In vitro testing of fresh and lyophilized reconstituted human and baboon platelets

      Valeri, C. Robert; Macgregor, Hollace; Barnard, Marc R.; Summaria, L.; Michelson, Alan D.; Ragno, G. (2004-10-01)
      BACKGROUND: Studies have been performed on human fresh, liquid-preserved, and cryopreserved platelets (PLTs) to assess PLT-adhesive surface receptors, PLT membrane procoagulant activity, PLT aggregation, and thromboxane production. Lyophilization has been developed as a method to preserve PLTs. This study was performed to evaluate these measurements on human and baboon fresh and lyophilized reconstituted PLTs. STUDY DESIGN AND METHODS: In both human and baboon fresh and lyophilized PLTs, aggregation response and PLT production of thromboxane A2 were measured after stimulation, and PLT surface markers P-selectin, glycoprotein (GP) Ib, GPIIb-IIIa, and factor (F) V were measured before and after stimulation. RESULTS: Fresh PLTs responded to the dual agonists arachidonic acid and adenosine diphosphate (ADP) to aggregate and produce thromboxane A2, and in both the PLT surface markers P-selectin and GPIIb-IIIa increased and GPIb decreased after stimulation. Neither human nor baboon lyophilized reconstituted PLTs aggregated to dual agonists, and neither produced thromboxane A2, increased PLT surface markers P-selectin or GPIIb-IIIa, or decreased PLT GPIb after stimulation. Nevertheless, after recalcification the lyophilized reconstituted PLTs accumulated FV to a significantly greater degree than fresh PLTs. CONCLUSIONS: Lyophilized reconstituted PLTs exhibited modification of the PLT membrane that interfered with aggregation and thromboxane production, prevented increases in PLT P-selectin and GPIIb-IIIa and decreases in GPIb after stimulation, and increased FV accumulation after recalcification. The in vitro data suggest that lyophilized PLTs may have reduced in vivo survival. In vivo studies are needed to determine the survival and function of lyophilized PLTs.
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