Heterogeneity in filamentous actin content among individual human blood platelets.

The content of filamentous actin in individual platelets was measured by flow cytometry, using a fluorescent probe specific for filamentous actin (F-actin), 7-nitrobenz-2-oxa-1,3-phallacidin (NBD-phallacidin). NBD-phallacidin binding to fixed platelets was specific in that either pretreatment of platelets with unlabeled phallacidin or absorption of NBD-phallacidin by rabbit skeletal F-actin, but not globular actin (G-actin), resulted in a significant loss in the bound fluorescent probe. Mean NBD-phallacidin binding to fixed platelets varied with the agonist and paralleled the changes in F-actin reported with the DNAse I inhibition assay. (1) NBD-phallacidin binding increased with stimulation by ADP, U46619 (a prostaglandin H2 analogue), or collagen and paralleled shape change. (2) Epinephrine did not increase NBD-phallacidin binding. (3) Platelets treated at 4 degrees C contained more F-actin than did platelets kept at 37 degrees C. (4) Cytochalasin D (10 mumol/L) inhibited the increase of phallacidin binding to individual platelets stimulated by either ADP or U46619. In measurements of cytosolic free calcium concentration ([Ca2+]i) by flow cytometry in Indo-1-loaded platelets, ADP's dose-response for actin polymerization was similar to that for calcium mobilization. As shown by flow cytometry, a tail population that had a minimal increase in F-actin upon stimulation with ADP or U46619 also contained the platelets with the least forward and right angle light scattering, which are functions of platelet size and shape. When platelets treated with NBD-phallacidin were incubated with S12-murine monoclonal antibody (a marker of alpha-granule secretion detected by phycoerythrin-conjugated antimouse IgG second antibody), phallacidin fluorescence paralleled S12 binding. Thus, human blood platelets are heterogeneous in regard to actin polymerization at rest and in association with platelet activation; different degrees of phallacidin binding may identify functionally different platelet populations.

[1]  T. Pollard,et al.  Mechanism of the interaction of human platelet profilin with actin , 1991, The Journal of cell biology.

[2]  A. Michelson,et al.  Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound. , 1991, Blood.

[3]  J. Daley,et al.  Quasi-simultaneous measurement of ionized calcium and alpha-granule release in individual platelets. , 1991, The American journal of physiology.

[4]  J. Thompson,et al.  Effect of temperature on receptor-activated changes in [Ca2+]i and their determination using fluorescent probes. , 1991, The Journal of biological chemistry.

[5]  L. Corash.  Platelet Density, Heterogeneity and Platelet Ageing , 1990 .

[6]  M. Lichtman,et al.  Activation of neutrophils: measurement of actin conformational changes by flow cytometry. , 1990, Blood cells.

[7]  U. Till,et al.  Blood platelet behaviour in patients with a type I diabetes mellitus. , 2009, Experimental and clinical endocrinology.

[8]  L. Jennings,et al.  Calcium mobilization in human platelets using indo-1 and flow cytometry. , 1989, Blood.

[9]  T. Stossel,et al.  From signal to pseudopod. How cells control cytoplasmic actin assembly. , 1989, The Journal of biological chemistry.

[10]  L. Jennings,et al.  Heterogeneity of fibrinogen receptor expression on platelets activated in normal plasma with ADP: analysis by flow cytometry , 1989, British journal of haematology.

[11]  U. Till,et al.  Actin filament content in platelets—A sensitive index of cellular reactivity , 1989, Bioscience reports.

[12]  H. Arita,et al.  Possible involvement of cytoskeleton in collagen-stimulated activation of phospholipases in human platelets. , 1989, The Journal of biological chemistry.

[13]  W. Siess.  Molecular mechanisms of platelet activation. , 1989, Physiological reviews.

[14]  A. Konchuba,et al.  Light scatter and total protein signal distribution of platelets by flow cytometry as parameters of size. , 1988, The Journal of laboratory and clinical medicine.

[15]  C. R. Porter,et al.  Platelet storage: changes in cytosolic Ca2+ actin polymerization and shape. , 1988, Blood.

[16]  E. Simons,et al.  Flow cytometric measurements of cytoplasmic calcium changes in human platelets. , 1988, Cytometry.

[17]  Salzman Ew,et al.  Ionized calcium in blood platelets. , 1988 .

[18]  E. Salzman,et al.  Ionized calcium in blood platelets. , 1988, Progress in clinical and biological research.

[19]  J. Hoxie,et al.  Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry , 1987 .

[20]  J. George,et al.  Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometry. , 1987, Blood.

[21]  U. Lindberg,et al.  Platelet microfilaments and motility , 1987 .

[22]  M. Dockter,et al.  Analysis of human platelet glycoproteins IIb-IIIa and Glanzmann's thrombasthenia in whole blood by flow cytometry. , 1986, Blood.

[23]  R. Wersto,et al.  Chemotactic peptide-induced changes in neutrophil actin conformation , 1984, The Journal of cell biology.

[24]  R. McEver,et al.  A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. , 1984, The Journal of biological chemistry.

[25]  J. Daniel,et al.  Evidence for a role of myosin phosphorylation in the initiation of the platelet shape change response. , 1984, The Journal of biological chemistry.

[26]  W. Leiserson,et al.  Actin activity is high in the immunocompetent fraction of thymocytes. , 1984, Thymus.

[27]  J. Fox,et al.  Polymerization and organization of actin filaments within platelets. , 1983, Seminars in hematology.

[28]  M M Frojmovic,et al.  Human platelet size, shape, and related functions in health and disease. , 1982, Physiological reviews.

[29]  Shin Lin,et al.  Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change , 1981, Nature.

[30]  J. Fox,et al.  Inhibition of actin polymerization in blood platelets by cytochalasins , 1981, Nature.

[31]  Salzman Ew,et al.  Cyclic nucleotides in hemostasis and thrombosis. , 1980 .

[32]  U. Lindberg,et al.  Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I , 1978, Cell.

[33]  G. Born.  Observations on the change in shape of blood platelets brought about by adenosine diphosphate , 1970, The Journal of physiology.