Mechanisms of Cold-induced Platelet Actin Assembly
Various agonists but also chilling cause blood platelets to increase cytosolic calcium, polymerize actin, and change shape. We report that cold increases barbed end nucleation sites in octyl glucoside-permeabilized platelets by 3-fold, enabling analysis of the intermediates of this response. Although chilling does not change polyphosphoinositide (ppI) levels, a ppI-binding peptide completely inhibits cold-induced nucleation. The C terminus of N-WASp, which inhibits the Arp2/3 complex, blocks nucleation by 40%; GDPβS, N17Rac and N17Cdc42 have no effects. Some gelsolin translocates to the detergent-insoluble cytoskeleton after cooling. Chilled platelets from gelsolin-deficient mice have ∼50% fewer new actin nuclei compared with platelets from wild-type mice. EGTA completely inhibits gelsolin translocation into the cytoskeleton, and the small amount of gelsolin initially there becomes soluble. Chilling releases adducin from the detergent-resistant cytoskeleton. We conclude that platelet actin filament assembly induced by cooling involves ppI-mediated actin filament barbed end uncapping and de novo nucleation independently of surface receptors or downstream signaling intermediates besides calcium. The actin-related changes occur in platelets at temperatures below 37 °C, suggesting that the platelet may be more activable at temperatures at the body surface than at core temperature, thereby favoring superficial hemostasis over internal thrombosis. Various agonists but also chilling cause blood platelets to increase cytosolic calcium, polymerize actin, and change shape. We report that cold increases barbed end nucleation sites in octyl glucoside-permeabilized platelets by 3-fold, enabling analysis of the intermediates of this response. Although chilling does not change polyphosphoinositide (ppI) levels, a ppI-binding peptide completely inhibits cold-induced nucleation. The C terminus of N-WASp, which inhibits the Arp2/3 complex, blocks nucleation by 40%; GDPβS, N17Rac and N17Cdc42 have no effects. Some gelsolin translocates to the detergent-insoluble cytoskeleton after cooling. Chilled platelets from gelsolin-deficient mice have ∼50% fewer new actin nuclei compared with platelets from wild-type mice. EGTA completely inhibits gelsolin translocation into the cytoskeleton, and the small amount of gelsolin initially there becomes soluble. Chilling releases adducin from the detergent-resistant cytoskeleton. We conclude that platelet actin filament assembly induced by cooling involves ppI-mediated actin filament barbed end uncapping and de novo nucleation independently of surface receptors or downstream signaling intermediates besides calcium. The actin-related changes occur in platelets at temperatures below 37 °C, suggesting that the platelet may be more activable at temperatures at the body surface than at core temperature, thereby favoring superficial hemostasis over internal thrombosis. polyphosphoinositide Wiskott-Aldrich syndrome protein n-octyl-β-d-glucopyranoside 1,4-piperazinediethanesulfonic acid guanosine 5′-3-O-(thio)triphosphate guanyl-5′-yl thiophosphate fluorescein isothiocyanate 5-P2, phosphatidylinositol 4,5-bisphosphate high pressure liquid chromatography acetoxymethyl ester glutathioneS-transferase thrombin receptor anchoring peptide As the suspension medium temperature falls below 15 °C platelets abruptly change their shape from smooth discs into spiny spheres with irregular projections and aggregate (1White J. Krivit W. Blood. 1967; 30: 625-635Crossref PubMed Google Scholar, 2White J. Krumwiede M. Blood. 1973; 41: 823-832Crossref PubMed Google Scholar, 3White J. Am. J. Pathol. 1982; 108: 184PubMed Google Scholar, 4Winokur R. Hartwig J. Blood. 1995; 85: 1796-1804Crossref PubMed Google Scholar, 5Zucker M. Borrelli J. Blood. 1954; 28: 524-534Crossref Google Scholar). These events resemble platelet responses to thrombin, ADP, collagen, and other stimuli that operate optimally at 37 °C through energy-dependent signaling reactions resulting in actin remodeling. However, cooling slows these reactions and causes most cell types to round up. The response of human blood platelets to chilling has profound medical consequences. It limits the storage of over 90 milion platelet units collected worldwide per year for transfusion to 5 days at room temperature, because longer storage leads to unacceptable amounts of microbial growth. Even 5-day storage of platelets without refrigeration results in occasional septic complications following transfusion (6Chernoff A. Snyder E. Transfusion. 1992; 32: 386-390Crossref PubMed Scopus (62) Google Scholar, 7Moroff G. Holme S. George V. Heaton W. Transfusion. 1994; 34: 317-321Crossref PubMed Scopus (31) Google Scholar). Unactivated discoid platelets have a unique submembrane coil of microtubules. Some microtubules depolymerize at low temperatures, and the microtubule coils of platelets dissolve in the cold (1White J. Krivit W. Blood. 1967; 30: 625-635Crossref PubMed Google Scholar, 8White J. Am. J. Pathol. 1968; 53: 281-291PubMed Google Scholar). At reduced temperatures, cells also become less able to maintain energy-dependent low cytosolic calcium levels, and chilled platelets have increased cytosolic calcium concentrations (4Winokur R. Hartwig J. Blood. 1995; 85: 1796-1804Crossref PubMed Google Scholar). Since calcium-dependent severing of the actin scaffolding that maintains the discoid shape of the resting platelet is an early step in normal platelet activation, this calcium rise is presumably a mediator of cold-induced platelet activation. In addition to these clues to possible mechanisms underlying cold activation of platelets, recent work has implicated phosphoinositide-mediated actin assembly (9Hartwig J. Bokoch G. Carpenter C. Janmey P. Taylor L. Toker A. Stossel T. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (613) Google Scholar, 10Tolias K. Hartwig J. Ishihara H. Shibisaki Y. Erickson J. Cantley L. Carpenter C. Curr. Biol. 2000; 10: 153-156Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Platelet stimulation through the PAR-1 receptor activates the Rho GTPase Rac leading to the synthesis of polyphosphoinositides (ppIs).1 Studies in permeabilized platelets indicated that these lipids induce actin assembly by producing actin nucleation sites equivalent to actin filament fast growing barbed ends. Since these ppIs release barbed end capping proteins such as gelsolin from actin filaments in permeabilized platelets, uncapping of preexisting actin filaments is one pathway proposed from ppIs to actin assembly and is amplifiable by actin filament severing and capping, which increase the number of ends. A second pathway involves ppIs and Cdc42-activated unfolding of Wiskott-Aldrich syndrome protein (WASp) family proteins, which then bind the Arp2/3 complex, resulting in branching barbed end nucleation at the cell cortex that leads to cell movement (11Derry J. Ochs H. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (839) Google Scholar, 12Kolluri R. Tolias K. Carpenter C. Rosen F. Kirchhausen T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5615-5618Crossref PubMed Scopus (189) Google Scholar, 13Symons M. Derry J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. 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Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar) and link signaling pathways to cell motility. Although ppI turnover would predictably diminish in the cold, if degradation declined more than synthesis, net ppI levels might increase, leading to actin assembly. A more plausible explanation is that temperature influences the structure of these lipids. The physical chemistry of lipid presentation affects lipid-protein interactions in general and gelsolin actin binding in particular (21Janmey P. Stossel T. J. Biol. Chem. 1989; 264: 4825-4831Abstract Full Text PDF PubMed Google Scholar). Tablin et al. (22Tablin F. Oliver A. Walker N. Crowe L. Crowe J. J. Cell. Physiol. 1996; 168: 305-313Crossref PubMed Scopus (128) Google Scholar) have proposed for lipid changes at the temperature for cold-induced platelet activation. We that chilled platelets actin from barbed end nuclei (4Winokur R. Hartwig J. Blood. 1995; 85: 1796-1804Crossref PubMed Google Scholar). In this that chilling activates barbed end assembly independently of synthesis and GTPase activation. We that ppIs work through actin filament barbed end uncapping and nucleation in chilled We also that chilling of platelets activates gelsolin to the actin response. from actin and with as J. J. Cell Biol. 1992; PubMed Scopus Google Scholar). The peptide of gelsolin P. J. P. P. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The of the Arp2/3 by of by The C. Stossel J. PubMed Scopus Google Scholar). of and of from the end of N-WASp, by and Kirschner of Cell The and by G. Bokoch proteins in and at °C in concentrations of at 37 °C and following through and with from normal human by into of as J. M. J. Cell Biol. PubMed Scopus Google Scholar). by of blood at for and platelets from the proteins by J. M. J. Cell Biol. PubMed Scopus Google Scholar) through a small from wild-type and gelsolin W. A. Hartwig J. T. 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Chem. 1989; 264: 4825-4831Abstract Full Text PDF PubMed Google Scholar) and to the chilled and permeabilized round to the of a in The with in for the platelets permeabilized for a of chilled in for 5 in an In platelets, the to 37 °C for in a for with a of in The platelets in a and a with a for and with a and with a with a platelets and from by at for in the of The platelets for at 37 °C with of in the medium from the platelets by over a as The platelets at temperatures for or and of ppIs and by as (9Hartwig J. Bokoch G. Carpenter C. Janmey P. Taylor L. Toker A. Stossel T. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (613) Google Scholar, J. S. T. Janmey P. Cantley L. Stossel T. Toker A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Curr. Opin. 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PubMed Scopus Google Scholar) The by the addition of of the of The for 5 by and in and then with and with an or platelets and at in and permeabilized with in the of by cell analysis a A of events for that most platelets discoid at 37 °C after with and that these permeabilized platelets change shape chilled Chilled platelets have their and that after to actin filament barbed that the assembly of suggesting that platelets cooling but become We this by cells with after or not by barbed end nucleation sites in chilled platelets, an increase of over cells at 37 Chilling of platelets for at °C by to the of barbed that the permeabilized cells of their response to of filament to barbed in platelets, an that to the barbed end and inhibits assembly this actin assembly from platelets not In cooling and of permeabilized platelets barbed end and of this through the addition of to into The addition of a peptide from the ppI-binding of gelsolin to platelets barbed actin filament end permeabilized platelets chilled for 5 peptide barbed by and barbed end following chilling platelets have actin filaments barbed ends. is with that of the peptide inhibits barbed end nucleation in permeabilized platelets and (9Hartwig J. 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Cell Biol. 2000; PubMed Scopus Google Scholar). with of the peptide the of from the surface of chilled permeabilized platelets The addition of to platelets actin nucleation in a without These that ppIs in the actin assembly that the shape of the platelet in the the ppI changes the chilling platelets, with to the at temperatures for that cooling not the of and ppIs in platelet at actin assembly and shape change of platelets to °C does not in the synthesis or degradation of Platelet to with from resting and chilled The of ppI by of phosphatidylinositol and phosphatidylinositol and of phosphatidylinositol and in platelets at temperatures for the temperature of cold-induced actin platelets at temperatures for 5 As the temperature the barbed end number per platelet increased from at 37 °C to at or below °C of barbed per The platelet increased by at temperatures of barbed end and to increase the temperature to Tablin et al. (22Tablin F. Oliver A. Walker N. Crowe L. Crowe J. J. Cell. 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The in a the in platelets and in platelets with The increases from in resting cells to after 5 of cooling and then to after of In the to the of cooling in platelets with that gelsolin is in the actin response induced by chilling from platelets from gelsolin mice. that gelsolin platelets small from their discoid shape compared with wild-type The most shape change is the of the discs into a and platelets their discoid and and in to human platelets platelets less barbed chilling compared with gelsolin that cold activation a increase of barbed end in wild-type platelets, gelsolin platelets have a increase in barbed ends. of filament is to the barbed in the permeabilized platelets, actin assembly not platelets with thrombin for at 37 °C increased the number of actin nuclei by in wild-type and in the gelsolin Chilling also the barbed end capping protein adducin from the platelet actin cytoskeleton In resting platelets, of the adducin is to the to from the platelet actin cytoskeleton after and the becomes after The in the in the cytoskeleton of The amount of adducin to the the cold activation in resting cells to after The of a barbed end capping protein that actin filament assembly by capping the of filaments K. 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As platelets by cold, their to to 5 °C, platelets to Although barbed end nucleation in platelets induced by to be at 37 °C, barbed end nucleation and actin assembly in platelets at These results that at °C nucleation from the of nucleation and the normal signaling It also that the of platelets to temperature may to a of and signaling events a response at temperatures below 37 °C, such as that in and at that platelet actin filament assembly induced by cooling ppIs to the uncapping of actin filaments by gelsolin and adducin and de novo actin nucleation by the Arp2/3 However, the signaling pathways from receptors and small The to a of cooling leads to platelet shape changes and actin assembly and their to temperature may to a storage of We and for and for to the