Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibα and subsequent integrin αIIbβ3-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous l-β,γ-methylene ATP, a stable P2X1 agonist, as well as by ADP, confirming that in addition to ADP (via P2Y1 and P2Y12), ATP (via P2X1) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X1 activation with l-β,γ-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of α-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca2+ and VWF-glycoprotein Ibα interactions without involving αIIbβ3. Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X1 stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X1 with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X1 ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation. Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibα and subsequent integrin αIIbβ3-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous l-β,γ-methylene ATP, a stable P2X1 agonist, as well as by ADP, confirming that in addition to ADP (via P2Y1 and P2Y12), ATP (via P2X1) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X1 activation with l-β,γ-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of α-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca2+ and VWF-glycoprotein Ibα interactions without involving αIIbβ3. Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X1 stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X1 with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X1 ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation. Blood platelets are constantly subjected to hemodynamic forces imposed by the blood flow, including fluid shear stress. High shear stress is generated at sites of arterial injury where laminar blood flow is forced through a stenosis (1Kroll M.H. Hellums J.D. McIntire L.V. Schafer A.I. Moake J.L. Blood. 1996; 88: 1525-1541Crossref PubMed Google Scholar, 2Berndt M.C. Shen Y. Dopheide S.M. Gardiner E.E. Andrews R.K. Thromb. Haemostasis. 2001; 86: 178-188Crossref PubMed Scopus (238) Google Scholar). Shear stress-triggered platelet activation and subsequent aggregation, termed SIPA 1The abbreviations used are: SIPA, shear-induced platelet aggregation; meATP, methylene ATP; CaM, calmodulin; MLC, myosin light chain; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; VWF, von Willebrand factor; GP, glycoprotein; PAR, protease-activated receptor; TRAP1–6, thrombin receptor activating peptide SFFLRN.1The abbreviations used are: SIPA, shear-induced platelet aggregation; meATP, methylene ATP; CaM, calmodulin; MLC, myosin light chain; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; VWF, von Willebrand factor; GP, glycoprotein; PAR, protease-activated receptor; TRAP1–6, thrombin receptor activating peptide SFFLRN. (3Ikeda Y. Murata M. Goto S. Ann. N. Y. Acad. Sci. 1997; 811: 325-336Crossref PubMed Scopus (49) Google Scholar), may thus contribute to the pathogenesis of vascular diseases. In addition, platelets from patients with acute myocardial infarction (4Goto S. Sakai H. Goto M. Ono M. Ikeda Y. Handa S. Ruggeri Z.M. Circulation. 1999; 99: 608-613Crossref PubMed Scopus (100) Google Scholar) or cerebral ischemia (5Konstantopoulos K. Grotta J.C. Sills C. Wu K.K. Hellums J.D. Thromb. Haemostasis. 1995; 74: 1329-1334Crossref PubMed Scopus (107) Google Scholar) display enhanced SIPA in vitro. High shear stress is required for the interaction between von Willebrand factor (VWF) and platelet glycoprotein Ibα (GPIbα) (for review, see Ref. 6Ruggeri Z.M. Curr. Opin. Hematol. 2003; 10: 142-149Crossref PubMed Scopus (106) Google Scholar), but the effects of shear forces on GPIbα signaling are only just beginning to be defined. Downstream effectors that have been implicated in the VWF-dependent activation of αIIbβ3 include phosphatidylinositol 3-kinase (PI3K) (7Yap C.L. Anderson K.E. Hughan S.C. Dopheide S.M. Salem H.H. Jackson S.P. Blood. 2002; 99: 151-158Crossref PubMed Scopus (106) Google Scholar), protein kinase C (PKC) (8Kroll M.H. Hellums J.D. Guo Z. Durante W. Razdan K. Hrbolich J.K. Schafer A.I. J. Biol. Chem. 1993; 268: 3520-3524Abstract Full Text PDF PubMed Google Scholar), Syk, and Src, as well as co-associated immunoreceptor tyrosine-activated motif-containing transmembrane proteins and adaptor proteins (2Berndt M.C. Shen Y. Dopheide S.M. Gardiner E.E. Andrews R.K. Thromb. Haemostasis. 2001; 86: 178-188Crossref PubMed Scopus (238) Google Scholar). A recent study of platelet adhesion to dimeric VWF A1 domain, which recognizes only GPIbα, showed that GPIbα itself can signal to activate αIIbβ3 through sequential actions of Src kinases, Ca2+ oscillations, and PI3K/PKC (9Kasirer-Friede A. Cozzi M.R. Mazzucato M. De Marco L. Ruggeri Z.M. Shattil S.J. Blood. 2004; 103: 3403-3411Crossref PubMed Scopus (157) Google Scholar). It has also been proposed that GPIb signals directly as a consequence of its association with structural cytoskeletal proteins (10Christodoulides N. Feng S. Resendiz J.C. Berndt M.C. Kroll M.H. Thromb. Res. 2001; 102: 133-142Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), among which GPIbα-bound filamin A, filamentous actin cross-linked by α-actinin, vinculin, and talin would directly link the cytoplasmic domain of GPIbα with the β3 tail of αIIbβ3 (11Feng S. Reséndiz J.C. Lu X. Kroll M.H. Blood. 2003; 102: 2122-2129Crossref PubMed Scopus (70) Google Scholar). Studies examining SIPA have suggested that VWF primarily stimulates platelet activation through an indirect pathway linked to ADP release (12Moritz M.W. Reimers R.C. Baker R.K. Sutera S.P. Joist J.H. J. Lab. Clin. Med. 1983; 101: 537-544PubMed Google Scholar, 13Moake J.L. Turner N.A. Stathopoulos N.A. Nolasco L. Hellums J.D. Blood. 1988; 71: 1366-1374Crossref PubMed Google Scholar). These studies have also shown that platelet activation initiated by VWF-GPIbα interaction requires a transmembrane Ca2+ influx independent of released ADP and VWF binding to αIIbβ3 (14Chow T.W. Hellums J.D. Moake J.L. Kroll M.H. Blood. 1992; 80: 113-120Crossref PubMed Google Scholar, 15Ikeda Y. Handa M. Kamata T. Kawano K. Kawai Y. Watanabe K Kawakami K. Sakai K. Fukuyama M. Itagaki I. Yoshioka A. Ruggeri Z.M. Thromb. Haemostasis. 1993; 69: 496-502Crossref PubMed Scopus (161) Google Scholar), which promotes dense granule secretion of ADP and activates integrin αIIbβ3 through engagement of the P2 receptors for ADP. A role for the platelet ADP receptors P2Y1 and P2Y12 (reviewed in Ref. 16Kunapuli S.P. Dorsam R.T. Kim S. Quinton T.M. Curr. Opin. Pharmacol. 2003; 3: 175-180Crossref PubMed Scopus (126) Google Scholar) in SIPA has subsequently been confirmed (17Goto S. Tamura N. Eto K. Ikeda Y. Handa S. Circulation. 2002; 105: 2531-2536Crossref PubMed Scopus (113) Google Scholar, 18Turner N.A. Moake J.L. McIntire L.V. Blood. 2001; 98: 3340-3345Crossref PubMed Scopus (130) Google Scholar). Reséndiz et al. (19Reséndiz J.C. Feng S. Ji G.A. Francis K.A. Berndt M.C. Kroll M.H. Mol. Pharmacol. 2003; 63: 639-645Crossref PubMed Scopus (49) Google Scholar) recently reported that the selective P2Y12 antagonist AR-C69931MX inhibits the shear-induced aggregation of washed platelets and showed that P2Y12 mediates shear-induced PI3K activation, a process coupled to phosphorylation of PI3K-associated Syk tyrosine kinase. In blood vessels, shear stress causes the release of high levels of nucleotides, including ATP and ADP, into the extracellular environment; these nucleotides mainly originate from endothelial cells (20Bodin P. Bailey D. Burnstock G. Br. J. Pharmacol. 1991; 103: 1203-1205Crossref PubMed Scopus (205) Google Scholar) and platelet-dense granules (21Nakai K. Hayashi T. Nagaya S. Toyoda H. Yamamoto M. Shiku H. Ikeda Y. Nishikawa M. Life Sci. 1997; 60: 181-191PubMed Google Scholar). Although the shear-induced ATP release from endothelial cells probably involves vesicular exocytosis (22Bodin P. Burnstock G. J. Cardiovasc. Pharmacol. 2001; 38: 900-908Crossref PubMed Scopus (234) Google Scholar), the molecular mechanisms of such a release are largely unknown. In platelets, the time course of this ATP release appeared to parallel the association of myosin with the actin cytoskeleton, suggesting that these two processes are related (21Nakai K. Hayashi T. Nagaya S. Toyoda H. Yamamoto M. Shiku H. Ikeda Y. Nishikawa M. Life Sci. 1997; 60: 181-191PubMed Google Scholar). In addition, Ca2+ influx-dependent phosphorylation of myosin light chain, preceding the myosin-actin interactions, has been proposed to be an initial step during SIPA (21Nakai K. Hayashi T. Nagaya S. Toyoda H. Yamamoto M. Shiku H. Ikeda Y. Nishikawa M. Life Sci. 1997; 60: 181-191PubMed Google Scholar). ATP is the physiological agonist at P2X1, but its role in platelet activation is only starting to be unraveled. In the aggregometer, the selective P2X1 agonists α,β-meATP and l-β,γ-meATP evoke a transient Ca2+ influx accompanied by rapid and reversible platelet shape change and myosin light chain (MLC) phosphorylation, provided that measures are taken to avoid desensitization of P2X1 by ATP spontaneously released during platelet preparation (23Rolf M.G. Brearley C.A. Mahaut-Smith M.P. Thromb. Haemostasis. 2001; 85: 303-308Crossref PubMed Scopus (119) Google Scholar, 24Oury C. Toth-Zsamboki E. Thys C. Tytgat J. Vermylen J. Hoylaerts M.F. Thromb. Haemostasis. 2001; 86: 1264-1271Crossref PubMed Scopus (86) Google Scholar, 25Toth-Zsamboki E. Oury C. De Vos R. Vermylen J. Hoylaerts M.F. J. Biol. Chem. 2003; 278: 46661-46667Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Recent studies, using P2X1 knock-out mice (26Hechler B. Lenain N. Marchese P. Vial C. Heim V. Freund M. Cazenave J.P. Cattaneo M. Ruggeri Z.M. Evans R. Gachet C. J. Exp. Med. 2003; 198: 661-667Crossref PubMed Scopus (185) Google Scholar) or transgenic mice overexpressing this ion channel selectively in the megakaryocytic cell lineage (27Oury C. Kuijpers M.J.E. Toth-Zsamboki E. Bonnefoy A. Danloy S. Vreys I. Feijge M.A. De Vos R. Vermylen J. Heemskerk J.W.M. Hoylaerts M.F. Blood. 2003; 101: 3969-3976Crossref PubMed Scopus (120) Google Scholar), have reported a prominent role for P2X1 in platelet aggregation and thrombus formation in shear stress conditions. Notably, P2X1 overexpressing platelets displayed potent SIPA in conditions where wild-type platelets hardly aggregated (27Oury C. Kuijpers M.J.E. Toth-Zsamboki E. Bonnefoy A. Danloy S. Vreys I. Feijge M.A. De Vos R. Vermylen J. Heemskerk J.W.M. Hoylaerts M.F. Blood. 2003; 101: 3969-3976Crossref PubMed Scopus (120) Google Scholar). In the present study, we have investigated the role of the P2X1-mediated Ca2+ influx and the associated downstream signaling pathways in VWF-dependent shear-induced granule release and aggregation of washed human platelets. Materials—ADP, l-β,γ-meATP, the P2X1 antagonist MRS2159, the P2Y1 antagonist MRS2179, and the ectonucleotidase apyrase (grade I) were purchased from Sigma. AR-C69931MX was a gift from AstraZeneca R&D, Charnwood, UK. The P2X1 antagonist MRS2159 at the concentration used in this study was verified to selectively inhibit a P2X1-mediated platelet shape change without affecting platelet shape change and aggregation induced by ADP. The calmodulin inhibitor W-7, the MLC kinase inhibitor ML-7, the Rho kinase inhibitor Y-27632, and the PKC inhibitors GF109203X, Go6976, and Go6983 were purchased from Calbiochem. Human VWF was purified from plasma cryoprecipitate by gel filtration on a Sepharose 4B-CL column. Tirofiban (Aggrastat) was obtained from Merck Sharp and Dohme. The murine-neutralizing anti-VWF monoclonal antibody AJvW-2 was from Ajinomoto Co., Inc. (Kawasaki, Japan) (28Kageyama S. Yamamoto H. Nagano M. Arisaka H. Kayahara T. Yoshimoto R. Br. J. Pharmacol. 1997; 122: 165-171Crossref PubMed Scopus (68) Google Scholar), and the neutralizing monoclonal anti-GPIbα antibody G19H10 was raised in our laboratory. ADP and l-β,γ-meATP were purified by high pressure liquid chromatography on an Adsorbosphere HS C18, 7 μm, 250 × 4.6-mm column (Alltech) as described by Oury et al. (24Oury C. Toth-Zsamboki E. Thys C. Tytgat J. Vermylen J. Hoylaerts M.F. Thromb. Haemostasis. 2001; 86: 1264-1271Crossref PubMed Scopus (86) Google Scholar). Preparation of Washed Human Platelets—Washed human platelets (2.5–3.5 × 105 platelets/μl) were prepared as described previously (29Oury C. Toth-Zsamboki E. Vermylen J. Hoylaerts M.F. Blood. 2002; 100: 2499-2505Crossref PubMed Scopus (90) Google Scholar). Apyrase (0.5 units/ml) was added to the blood sample and platelet resuspension buffer when indicated. Platelet preparations were free of red blood cells, as validated via microscopy and blood cell counting (CellDyn 1300, Abbott, Abbott Park, IL). Shear-induced Platelet Aggregation, ATP Secretion, and P-selectin Expression—Shear-induced aggregations of washed human platelets were performed in an annular ring-shaped viscometer generating laminar flow (Ravenfield viscometer; Heywood, Lancashire, UK) at 37 °Cin the presence of 2 mm CaCl2 and 10 μg/ml human soluble VWF unless otherwise indicated. A shear rate of 9000 s-1 was used, which corresponds to a shear stress of 80 dynes/cm2. The threshold shear rate causing platelet aggregation ranged between 3000 s-1 and 5000 s-1. At defined time points, platelet samples were collected and fixed in 1% paraformaldehyde; the percentage of platelet aggregation was calculated by comparing single platelet counts before and after shearing. Shear-induced P-selectin surface translocation on tirofiban-treated washed platelets was detected by flow cytometry with the phycoerythrin-conjugated anti-P-selectin CD62P antibody (BD Biosciences). For ATP secretion assays, the reactions were stopped with an ice-cold Hepes buffer (20 mm Hepes, pH 7.4, 150 mm NaCl, 5 mm EDTA). After immediate centrifugation at 4 °C, ATP was measured in the supernatants by the addition of a luciferase/luciferin reagent (Chrono-Lume, Kordia) in a microplate luminometer LB96V (Berthold Technologies, Vilvoorde, Belgium). At least five independent experiments were performed on platelets from different individuals. The data are represented as the mean ± S.D. Statistical analysis of the data was done using nonpaired Student's t tests. Immunoblotting Analyses—Platelets (0.3-ml platelet suspensions) were lysed in SDS sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mm dithiothreitol, 0.1% bromphenol blue). Sample aliquots were loaded on SDS-PAGE (12.5%) and subjected to Western blotting. Thr-18/Ser-19 MLC phosphorylation was detected with the anti-phospho-MLC polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) according to the instructions of the manufacturers. Electron Microscopy—Washed platelets were immediately fixed overnight at 4 °C in 2.5% (w/v) glutaraldehyde, 0.1 m phosphate buffer, pH 7.2. After centrifugation at 800 × g for 10 min, a condensed pellet of platelets was formed. After fixation in 1% OsO4 (w/v), 0.1 m phosphate buffer, pH 7.2, and dehydration in a graded series of ethanol, the pellets were embedded in epoxy resin. Ultrathin sections were cut and stained with uranyl acetate and lead citrate before examination with a Zeiss EM 10 electron microscope (Oberkochen, Germany). VWF-GPIbα Interactions and Extracellular Ca2+ in SIPA—When suspensions of washed human platelets were subjected to uniform high shear stress (80 dynes/cm2 = 9000 s-1 shear rate) in the presence of soluble human VWF in an annular ring-shaped viscometer, aggregation rapidly occurred, reaching a plateau after 3 min (Fig. 1A). In contrast, a shear rate of 1000 s-1 did not cause platelet aggregation (Fig. 1A). The inclusion of the VWF A1 domain-blocking antibody AJvW-2, neutralizing anti-GPIbα antibody G19H10, or the αIIbβ3 antagonist tirofiban (Fig. 1B) confirmed the findings by others (30Ikeda Y. Handa M. Kawano K. Kamata T. Murata M. Araki Y. Anbo H. Kawai Y. Watanabe K. Itagaki I. Sakai K. Ruggeri Z.M. J. Clin. Investig. 1991; 87: 1234-1240Crossref PubMed Scopus (531) Google Scholar) that SIPA requires VWF interactions with GPIbα and the activation of αIIbβ3. We also confirmed that SIPA does not occur in the absence of added extracellular Ca2+ (14Chow T.W. Hellums J.D. Moake J.L. Kroll M.H. Blood. 1992; 80: 113-120Crossref PubMed Google Scholar, 15Ikeda Y. Handa M. Kamata T. Kawano K. Kawai Y. Watanabe K Kawakami K. Sakai K. Fukuyama M. Itagaki I. Yoshioka A. Ruggeri Z.M. Thromb. Haemostasis. 1993; 69: 496-502Crossref PubMed Scopus (161) Google Scholar) (Fig. 1B). Platelet aggregation was accompanied by gradually increasing shear stress-dependent ATP release (Fig. 1A). ATP release was initiated as early as 10 s after exposure to shear stress. When measured at the maximal aggregation time point, this release clearly relied on VWF-GPIbα signaling, as it was at least partly inhibited by AJvW-2 and G19H10 (Fig. 1B). Omitting the addition of extracellular Ca2+ also reduced the amount of released ATP, although only partly (Fig. 1B). The combined absence of VWF and extracellular Ca2+ almost abolished platelet degranulation (Fig. 1B). In contrast, αIIbβ3 antagonism had no effect on the degree of platelet degranulation (Fig. 1B). Therefore, both VWF-GPIbα signaling and Ca2+ contribute to platelet release under shear stress, a process that is independent of αIIbβ3 outside-in signals. MLC Kinase (but Not PKCs) Controls the SIPA-induced Platelet Degranulation—Platelet granule release that is induced by most agonists requires an increase of intracellular Ca2+ and PKC activation (reviewed in Ref. 31Flaumenhaft R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1152-1160Crossref PubMed Scopus (125) Google Scholar). Whether or not PKC plays a role in SIPA is controversial (8Kroll M.H. Hellums J.D. Guo Z. Durante W. Razdan K. Hrbolich J.K. Schafer A.I. J. Biol. Chem. 1993; 268: 3520-3524Abstract Full Text PDF PubMed Google Scholar, 32Oda A. Yokoyama K. Murata M. Tokuhira M. Nakamura K. Handa M. Watanabe K. Ikeda Y. Thromb. Haemostasis. 1995; 74: 736-742Crossref PubMed Scopus (46) Google Scholar). In our experimental conditions, the nonselective PKC inhibitor GF109203X, which blocks both conventional (α, βI, βII, γ) and novel (δ, θ, η, ϵ) PKC isoforms, failed to inhibit either platelet aggregation or release produced by shear stress (Fig. 2A), although it prevented such responses to collagen (2 μg/ml) or to the PAR-1-activating peptide TRAP1–6 (1 μm) under stirring conditions (Fig. 2B). Similarly, the PKC α/β-specific inhibitor Go6976 did not affect SIPA and associated platelet release (Fig. 2A), although shear stress-independent collagen-induced release and aggregation were fully blocked (Fig. 2B). In agreement with previous reports that PKC α/β isoforms do not play a major role downstream of PAR-1 (33Murugappan S. Tuluc F. Dorsam R.T. Shankar H. Kunapuli S.P. J. Biol. Chem. 2004; 279: 2360-2367Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), Go6976 had a minimal inhibitory effect on TRAP1–6-induced granule release (Fig. 2B). Finally, to determine whether other PKC isoforms could be involved in platelet release accompanying SIPA, we used Go6983, which blocks the platelet-expressed atypical PKC ζ isoform in addition to PKC α, βI, βII, γ, and δ. This inhibitor had no effect on shear-induced platelet release or aggregation, although it diminished TRAP1–6-induced ATP release by about 50% without affecting platelet aggregation (Fig. 2B). These results indicate that shear-induced platelet dense granule release and aggregation are dependent on signaling pathways distinct from those activated downstream of the collagen receptor GPVI or PAR-1. They also suggest that the known conventional, novel, and atypical PKC isoforms do not play a major role in platelet degranulation during SIPA. In addition to PKC, GPVI-mediated platelet dense granule release also depends on Ca2+-sensitive MLC kinase activation (25Toth-Zsamboki E. Oury C. De Vos R. Vermylen J. Hoylaerts M.F. J. Biol. Chem. 2003; 278: 46661-46667Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Inhibiting this kinase with ML-7 abolished both the ATP release and platelet aggregation induced by collagen (2 μg/ml) (Fig. 2B). As shown in Fig. 2A, ML-7 reduced the shear-induced ATP release and platelet aggregation by about 30%, indicating a role for MLC kinase in SIPA and associated platelet degranulation. Secreted ADP and ATP in VWF-dependent Shear-induced Platelet Release and Aggregation—Under stirring conditions in an aggregometer, MLC kinase is activated downstream of the P2X1-mediated Ca2+ influx, playing an essential role during cytoskeletal rearrangements evoked by selective agonists (α,β-meATP and l-β,γ-meATP) of this ion channel (25Toth-Zsamboki E. Oury C. De Vos R. Vermylen J. Hoylaerts M.F. J. Biol. Chem. 2003; 278: 46661-46667Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The activation of MLC kinase can also result from phospholipase C-dependent Ca2+ mobilization from internal stores downstream of the Gq protein-coupled P2Y1 receptor or of GPVI (34Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Crossref PubMed Google Scholar, 35Paul B.Z. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1999; 274: 28293-28300Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). In agreement with earlier reports (17Goto S. Tamura N. Eto K. Ikeda Y. Handa S. Circulation. 2002; 105: 2531-2536Crossref PubMed Scopus (113) Google Scholar, 18Turner N.A. Moake J.L. McIntire L.V. Blood. 2001; 98: 3340-3345Crossref PubMed Scopus (130) Google Scholar, 19Reséndiz J.C. Feng S. Ji G.A. Francis K.A. Berndt M.C. Kroll M.H. Mol. Pharmacol. 2003; 63: 639-645Crossref PubMed Scopus (49) Google Scholar), the selective antagonists of P2Y1 (MRS2179, 10 μm) and P2Y12 (AR-C69931MX, 1 μm) inhibited SIPA by more than 50% (Fig. 3). In previous studies of platelet aggregation performed under stirring conditions in an aggregometer, a high concentration of the ATP/ADP scavenger apyrase always was required to demonstrate P2X1 function, as this ion channel is rapidly desensitized by ATP released during platelet preparation (23Rolf M.G. Brearley C.A. Mahaut-Smith M.P. Thromb. Haemostasis. 2001; 85: 303-308Crossref PubMed Scopus (119) Google Scholar, 24Oury C. Toth-Zsamboki E. Thys C. Tytgat J. Vermylen J. Hoylaerts M.F. Thromb. Haemostasis. 2001; 86: 1264-1271Crossref PubMed Scopus (86) Google Scholar, 25Toth-Zsamboki E. Oury C. De Vos R. Vermylen J. Hoylaerts M.F. J. Biol. Chem. 2003; 278: 46661-46667Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In sharp contrast, the P2X1 antagonist MRS2159 (10 μm) reduced SIPA by about 40% even when apyrase was omitted (Fig. 3). Thus, the three platelet P2 receptors contribute to SIPA through secreted ADP and ATP. The effect of combined P2X1 and P2Y1 antagonism on SIPA was not significantly different from that of P2Y1 antagonism only (Fig. 3), suggesting that these receptors share common signaling pathways. Neither the antagonism of P2X1, P2Y1, nor their combination could inhibit the remaining SIPA observed in the presence of the P2Y12 antagonist (data not shown). ATP secretion assays performed in the presence of the selective P2 receptor antagonists revealed a contribution of P2Y1, P2Y12, and P2X1 to VWF-dependent platelet degranulation (measured after 3 min) (Fig. 3). With the exception of P2X1 antagonism, the effects of P2Y1 and P2Y12 antagonism on secretion did not fully correlate with their ability to inhibit platelet aggregation (Fig. 3), which is in agreement with the interpretation that these receptors participate in other processes leading to SIPA besides secretion regulation. P2X1 Enhances Degranulation and SIPA via Calmodulin-dependent MLC Kinase Activation—To further examine the contribution of individual nucleotides to SIPA, we have used platelets of a Hermansky-Pudlak patient lacking dense granules and secreting almost no ATP in response to supraoptimal doses of collagen (36Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (159) Google Scholar). These platelets displayed severely impaired SIPA comparable with SIPA observed in the presence of P2Y1 or P2Y12 receptor antagonists (Fig. 4A). Interestingly, platelet aggregation could be largely restored by the addition of not only ADP but also of the selective P2X1 agonist l-β,γ-meATP immediately prior to shearing, which supports the central roles for both ATP and ADP secreted during SIPA. We pursued this aspect of SIPA using normal platelets but in the presence of the ectonucleotidase apyrase, which degrades secreted ATP and ADP. The addition of apyrase (0.5 units/ml) to the platelet resuspension buffer reduced the level of SIPA to 70% of the control (Fig. 4B). In this condition, the selective P2Y1 and P2X1 receptor antagonists MRS2179 and MRS2159 no longer inhibited the remaining SIPA (not shown), indicating that the reduction of aggregation by apyrase is due to the absent activation of P2Y1 and P2X1 signaling (Fig. 4A). In contrast, the P2Y12 antagonist still reduced the aggregation of apyrase-treated platelets from 26.3 ± 9.5% to 17.1 ± 3.2%, indicating the presence of sufficient remaining secreted ADP to activate this receptor. The addition of the selective apyrase-resistant P2X1 agonist l-β,γ-meATP to this set-up just prior to shearing fully restored the aggregation of apyrase-treated platelets (Fig. 4B). The resulting aggregation still depended on VWF and αIIbβ3 activation, as shown by its full inhibition by AJvW-2 and tirofiban (Fig. 4B). Therefore, this experimental approach enabled us to study the P2X1-driven signaling pathways in more detail during SIPA. We found that the enhanced SIPA was the result of CaM and MLC kinase activation, as W-7 and ML-7 inhibited the l-β,γ-meATP amplification (Fig. 4B). Notably, these inhibitors had no effect on the SIPA of apyrase-treated platelets without additional stimulation by l-β,γ-meATP (data not shown), indicating that the activation of these pathways exclusively depends on P2X1 potentiation. The fact that W-7 inhibited the l-β,γ-meATP-driven SIPA to a larger extent than ML-7 may indicate the existence of an additional CaM-dependent pathway activated downstream of P2X1. Thus, exogenous P2X1 activation selectively triggers CaM-dependent pathways contributing to SIPA. Moreover, in the presence of the P2Y12 receptor antagonist AR-C69931MX, the l-β,γ-meATP-driven SIPA was reduced to the same level (16.2 ± 3.4%) as that achieved during inhibition of apyrase-treated platelets themselves, confirming a central role for ADP also in the l-β,γ-meATP-driven SIPA. P2X1 Activation Promotes Shear-induced Platelet Granule Centralization and Release—We investigated whether P2X1-mediated MLC kinase activation would be instrumental in platelet degranulation to explain the enhanced SIPA of Hermansky-Pudlak patient platelets or of apyrase-treated platelets in the presence of l-β,γ-meATP. Flow cytometry analysis of P-selectin expression (as a marker of α-granule release) was done on the surface of apyrase-treated (0.5 units/ml) and tirofiban-treated platelets exposed to VWF and shear for 5 min in the presence or absence of l-β,γ-meATP. Fig. 5 shows a significant increase of the percentage of platelets expressing P-selectin following their pretreatment with l-β,γ-meATP. P-selectin expression was, in turn, reduced to the initial level in the presence of the MLC kinase inhibitor ML-7 (Fig. 5). Therefore, during SIPA, the P2X1-mediated MLC kinase activation can reinforce the shear-indu
In vivo, translocation of inhaled nanoparticles to the circulation has been demonstrated. However, the interaction of nanoparticles with the lung epithelium is not understood. In this study, we investigated, in vitro, the translocation of nano-sized quantum dots (QDs; 25 pmol/ml) through a tight monolayer of primary isolated rat alveolar epithelial cells. The influence of surface charge on translocation was examined using nonfunctionalized QDs, amine-QDs, and carboxyl-QDs. The interaction between nanoparticles and the lung epithelium was monitored by repeatedly measuring the transepithelial electrical resistance (TEER) and by examining the cell layer with confocal microscopy. The effect of oxidative stress was tested by incubating the cells with tert-butyl hydroperoxide (t-BOOH; 75 μM or 1 or 10 mM); the antioxidant N-acetyl-l-cysteine was also used to assess the role of particle-mediated oxidative stress. No translocation through a tight monolayer of primary rat alveolar epithelial cells was observed for any of the different types of QDs. In general, an increase in TEER was found after incubation with QDs. A condition of low oxidative stress did not enhance translocation. In contrast, conditions of high stress (1 or 10 mM t-BOOH or due to QDs toxicity) with disruption of the cell layer, as shown in a decreased TEER, resulted in substantial translocation. In conclusion, no translocation of QDs was found through a tight monolayer of primary rat alveolar epithelial cells, regardless of the QDs surface charge. QDs did not impair the barrier function of the epithelial cells. In conditions with disruption of the cell-cell barrier, translocation was demonstrated.
Abstract The male Wolffian tumor is an extremely rare case in male patients. Here, we report a patient with such malignancy and successful radical surgical treatment at 15-year follow-up. The clinicopathological, immunohistochemical, and ultrastructural features are described. The differential diagnosis of this tumor in a male patient is discussed.
