Importance of Temporal Flow Gradients and Integrin αIIbβ3 Mechanotransduction for Shear Activation of Platelets
2005; Elsevier BV; Volume: 280; Issue: 15 Linguagem: Inglês
10.1074/jbc.m410235200
ISSN1083-351X
AutoresIsaac Goncalves, Warwick S. Nesbitt, Yuping Yuan, Shaun P. Jackson,
Tópico(s)Heparin-Induced Thrombocytopenia and Thrombosis
ResumoDisturbances of blood flow play an important role in promoting platelet activation and arterial thrombus formation in stenosed, injured, atherosclerotic arteries. To date, glycoprotein Ib (GPIb) has been considered the primary platelet mechanosensory receptor, responding to increased shear with enhanced adhesive and signaling function. We demonstrate here that von Willebrand factor-GPIb interaction is inefficient at inducing platelet activation even when platelets are exposed to very high wall shear stresses (60 dyn/cm2). Rapid platelet activation under flow was only observed under experimental conditions in which transiently adherent platelets were exposed to sudden accelerations in blood flow. Platelet responsiveness to temporal shear gradients was integrin αIIbβ3-dependent and occurred only on a von Willebrand factor substrate, as platelets forming integrin αIIbβ3 adhesive contacts with immobilized fibrinogen were unresponsive to sudden increases in shear. The calcium response induced by temporal shear gradients was distinct from previously identified integrin αIIbβ3 calcium responses in terms of its transient nature, its requirement for platelet co-stimulation by the P2Y1 purinergic ADP receptor, and its dependence on the influx of extracellular calcium. Our studies demonstrate a key role for temporal shear gradients in promoting platelet activation. Moreover, they define for the first time the involvement of P2Y receptors in integrin mechanotransduction. Disturbances of blood flow play an important role in promoting platelet activation and arterial thrombus formation in stenosed, injured, atherosclerotic arteries. To date, glycoprotein Ib (GPIb) has been considered the primary platelet mechanosensory receptor, responding to increased shear with enhanced adhesive and signaling function. We demonstrate here that von Willebrand factor-GPIb interaction is inefficient at inducing platelet activation even when platelets are exposed to very high wall shear stresses (60 dyn/cm2). Rapid platelet activation under flow was only observed under experimental conditions in which transiently adherent platelets were exposed to sudden accelerations in blood flow. Platelet responsiveness to temporal shear gradients was integrin αIIbβ3-dependent and occurred only on a von Willebrand factor substrate, as platelets forming integrin αIIbβ3 adhesive contacts with immobilized fibrinogen were unresponsive to sudden increases in shear. The calcium response induced by temporal shear gradients was distinct from previously identified integrin αIIbβ3 calcium responses in terms of its transient nature, its requirement for platelet co-stimulation by the P2Y1 purinergic ADP receptor, and its dependence on the influx of extracellular calcium. Our studies demonstrate a key role for temporal shear gradients in promoting platelet activation. Moreover, they define for the first time the involvement of P2Y receptors in integrin mechanotransduction. The transduction of biomechanical stimuli into biochemical signals is fundamental to the regulation of a broad range of physiological processes, including sensory perception, blood pressure regulation, bone remodeling, and maintenance of muscle mass (1Gillespie P.G. Walker R.G. Nature. 2001; 413: 194-202Crossref PubMed Scopus (527) Google Scholar, 2Davies P.F. Physiol. Rev. 1995; 75: 519-560Crossref PubMed Scopus (2308) Google Scholar, 3Lehoux S. Tedgui A. J. Biomech. 2003; 36: 631-643Crossref PubMed Scopus (266) Google Scholar). Mechanotransduction mechanisms are particularly relevant to the vasculature, where hemodynamic forces generated as a result of blood flow modulate the phenotypic characteristics of vascular (endothelial and smooth muscle cells) and circulating blood cells (platelets and leukocytes). Shear effects on vascular cells are mediated in part by transmembrane extracellular matrix receptors, principally involving integrins. Various members of the β1, β2, and β3 families of integrin receptors have been demonstrated to transduce mechanical signals that are critically linked to the development of atherosclerotic lesions (4Katsumi A. Orr A.W. Tzima E. Schwartz M.A. J. Biol. Chem. 2004; 279: 12001-12004Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar, 5Shyy J.Y. Chien S. Curr. Opin. Cell Biol. 1997; 9: 707-713Crossref PubMed Scopus (294) Google Scholar).The importance of integrin mechanotransduction in regulating the adhesive function of circulating blood cells has been less clearly defined, although it is important given the key role played by platelets and leukocytes in the atherothrombotic process. In general, rapid transduction of biomechanical stimuli is typically mediated through mechanically gated ion channels (1Gillespie P.G. Walker R.G. Nature. 2001; 413: 194-202Crossref PubMed Scopus (527) Google Scholar) and, in the case of platelets, such membrane channels may be linked to the shear-regulated binding of von Willebrand factor (vWf) 1The abbreviations used are: vWf, von Willebrand factor; GP, glycoprotein; IP3, 1,4,5-trisphosphate.1The abbreviations used are: vWf, von Willebrand factor; GP, glycoprotein; IP3, 1,4,5-trisphosphate. to its surface receptor, glycoprotein (GP) Ib/V/IX. Mechanotransduction mechanisms operating downstream of integrins are typically linked to slower adaptive responses such as cytoskeletal remodeling and gene transcription (1Gillespie P.G. Walker R.G. Nature. 2001; 413: 194-202Crossref PubMed Scopus (527) Google Scholar); however, the role of integrin mechanotransduction in regulating functional processes in rapidly responding cells such as platelets remains largely unknown.Insight into the mechanisms by which shear forces regulate platelet function requires consideration of the hemodynamic conditions operating at sites of atherothrombosis. For example, shear forces change significantly at sites of arterial stenosis, increasing dramatically at the apex of the stenosis and decreasing rapidly in the post-stenotic recirculation region (6Mailhac A. Badimon J.J. Fallon J.T. Fernandez-Ortiz A. Meyer B. Chesebro J.H. Fuster V. Badimon L. Circulation. 1994; 90: 988-996Crossref PubMed Scopus (120) Google Scholar). Flow patterns in the post-stenotic region can change from unidirectional laminar flow to disturbed flow patterns involving eddy formation, flow reversal, and shear gradients. Significantly, a major role for rapid accelerations of blood flow in promoting platelet deposition and thrombus formation has been demonstrated in vivo (7Strony J. Beaudoin A. Brands D. Adelman B. Am. J. Physiol. 1993; 265: H1787-H1796PubMed Google Scholar); furthermore, propagation of thrombi has also been established to occur downstream of the initial shear flux in the post-stenotic region (7Strony J. Beaudoin A. Brands D. Adelman B. Am. J. Physiol. 1993; 265: H1787-H1796PubMed Google Scholar). Although such disturbances of blood flow patterns have long been identified as powerful atherogenic stimuli and have been demonstrated to be important in regulating endothelial cell function (8Zarins C.K. Giddens D.P. Bharadvaj B.K. Sottiurai V.S. Mabon R.F. Glagov S. Circ. Res. 1983; 53: 502-514Crossref PubMed Scopus (1212) Google Scholar, 9Ku D.N. Giddens D.P. Zarins C.K. Glagov S. Arteriosclerosis. 1985; 5: 293-302Crossref PubMed Google Scholar), the role of such flow changes in regulating platelet function remains ill-defined.In this study we demonstrate a key role for shear gradients in inducing platelet activation. Temporal shear gradients stimulate platelet activation through a signaling mechanism involving the platelet P2Y1 purinergic receptor and integrin αIIbβ3. We demonstrate that cooperative signaling by these receptors is necessary for efficient shear activation of platelets. Moreover, we demonstrate that this mechanosensory signaling mechanism is operational over a narrow temporal shear range. Overall, our studies define for the first time an important role for P2Y receptors in integrin mechanotransduction.EXPERIMENTAL PROCEDURESMaterials—Human vWf was purified to homogeneity from a plasma cryoprecipitate according to the method of Montgomery and Zimmerman (33Montgomery R.R. Zimmerman T.S. J. Clin. Investig. 1978; 61: 1498-1507Crossref PubMed Scopus (76) Google Scholar). Human fibrinogen was purified from fresh frozen plasma according to Jakobsen and Kierulf (34Jakobsen E. Kierulf P. Thromb. Res. 1973; 3: 145-159Abstract Full Text PDF Scopus (97) Google Scholar). Ristocetin was obtained from ICN (Costa Mesa, CA). Apyrase was purified as described previously (10Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The anti-αIIbβ3 chimeric Fab fragment of the monoclonal antibody 7E3 (c7E3 Fab-abciximab) was from Eli Lilly and Centocor (Leiden, The Netherlands). All other reagents were obtained from sources described previously (10Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 11Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 12Nesbitt W.S. Kulkarni S. Giuliano S. Goncalves I. Dopheide S.M. Yap C.L. Harper I.S. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2965-2972Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 13Nesbitt W.S. Giuliano S. Kulkarni S. Dopheide S.M. Harper I.S. Jackson S.P. J. Cell Biol. 2003; 160: 1151-1161Crossref PubMed Scopus (141) Google Scholar).Platelet Preparation—Washed human platelets were prepared as described previously (10Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 14Moog S. Mangin P. Lenain N. Strassel C. Ravanat C. Schuhler S. Freund M. Santer M. Kahn M. Nieswandt B. Gachet C. Cazenave J.P. Lanza F. Blood. 2001; 98: 1038-1046Crossref PubMed Scopus (104) Google Scholar) with some minor modifications. Whole blood was collected into acid citrate dextrose (ACD) anticoagulant at a ratio of 6:1 (blood/ACD), to which mixture 20 units/ml Clexane was added before being allowed to incubate at 37 °C for 15 min. Separation of platelet-rich plasma and red blood cells was achieved by centrifugation at 300 × g for 16 min. In all of the subsequent steps involving centrifugation, platelets were allowed to rest at 37 °C for 10 min prior to continuation of the next step in the washing procedure. Platelets were isolated from plasma components by centrifugation at 1,700 × g for 7 min, followed by resuspension in an equal volume of platelet washing buffer (4.3 mm K2HPO4, 4.3 mm Na2HPO4, 24.3 mm NaH2PO4, 113 mm NaCl, 5.5 mmd-glucose, and 10 mm theophylline, pH 6.5). Platelets were washed by centrifugation at 1,500 × g for 7 min, followed by a final resuspension at a concentration of 3 × 108/ml in modified Tyrode's buffer (10 mm Hepes, 12 mm NaHCO3, 137 mm NaCl, 2.7 mm KCl, and 5 mm glucose, pH 7.3) containing bovine serum albumin (5 mg/ml), calcium (1 mm), and apyrase (0.02 units/ml) (ADPase activity).In Vitro Flow Studies—Flow assays were performed as described previously (10Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Briefly, glass microcapillary slides (Lomb Scientific, New South Wales, Australia) were coated with vWf (100 μg/ml) or fibrinogen (100 μg/ml) for 2 h at room temperature and then blocked with 10% heat-inactivated human serum. Washed platelets (5 × 107/ml) in Tyrode's buffer with 1 mm Ca2+ were reconstituted with washed red blood cells (50% v/v) (containing 0.02 units/ml apyrase and 1 units/ml hirudin) and perfused through the matrix-coated microcapillary tubes at wall shear rates of 3.6, 10.8, or 60 dyn/cm2 for 2 min at 37 °C. Platelet-matrix interactions were monitored for 100 frames (0.586 frames/sec) using confocal fluorescence microscopy (TCS-SP, Leica). Stationary adhesion was defined as platelet movement of less than a single cell diameter over a 30-s observation period.For temporal shear gradient experiments, washed platelets (1.5 × 108/ml) were allowed to transiently engage the vWf surface prior to the application of a shear gradient (0.9, 3.6, 10.8, or 60 dyn/cm2·s). A pre-shear time period fixed at 5 min was employed to allow platelet-vWf matrix interactions to occur. During this period, platelets in suspension settle under gravity onto the vWf surface. Although the pre-shear time window was fixed, the platelet interaction times were variable and depended on the time of initial engagement of the surface over the 5-min period. Platelet contact on the matrix was defined when platelets remained in focus on the surface of the microcapillary tube. The mean platelet interaction time reflects the amount of time that has elapsed following initial contact of a single platelet with the matrix up until the point of shear application. The mean interaction time of platelets with the vWf surface prior to the application of shear was 15 ± 13 s. These findings highlight the fact that although a 5-min pre-adhesion step was utilized in all studies, the vast majority of platelets only interacted with the matrix for a short period of time before the application of shear. Shear accelerations were generated according to published methods (15Bao X. Lu C. Frangos J.A. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 996-1003Crossref PubMed Scopus (208) Google Scholar, 16White C.R. Haidekker M. Bao X. Frangos J.A. Circulation. 2001; 103: 2508-2513Crossref PubMed Scopus (160) Google Scholar). Briefly, incremental flow rate increases were produced by a programmable Harvard PHD syringe driver operating in-line with a glass microcapillary.Analysis of Cytosolic Calcium Flux under Static and Flow Conditions—The changes in cytosolic calcium levels were monitored according to published methods (10Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12Nesbitt W.S. Kulkarni S. Giuliano S. Goncalves I. Dopheide S.M. Yap C.L. Harper I.S. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2965-2972Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Briefly, washed platelets (1 × 109/ml) were loaded with Oregon Green 488 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester (1 μm), and Fura-Red/AM (1.25 μm) for 30 min at 37 °C. Dye-loaded platelets (1 × 107/ml) were then either allowed to adhere to vWf under static conditions or reconstituted with red blood cells (50% v/v) prior to perfusion through vWf, fibrinogen, or vWf/fibrinogen-coated microcapillary tubes. To examine changes in calcium flux, sequential confocal images of adherent platelets were captured at a scan rate of 0.586 frames/s for 37.5 s at the indicated time points. Real time platelet calcium flux was calculated from ratiometric fluorescence measurements and converted to intracellular calcium concentrations as described previously (10Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12Nesbitt W.S. Kulkarni S. Giuliano S. Goncalves I. Dopheide S.M. Yap C.L. Harper I.S. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2965-2972Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar).Statistical Analysis—Statistical significance of results was determined using one-way analysis of variance, and p values are indicated where appropriate (*, p < 0.05 and **p < 0.01; Figs. 2, 3, 4, 5, 6, 7). All bars (Figs. 1, 2, 3, 4, 5, 6, 7) represent mean ± S.E. unless otherwise stated.Fig. 3Rapid platelet activation is dependent on the rate of shear application. Calcium dye-loaded, washed platelets were allowed to transiently engage the vWf surface prior to the application of a defined shear stress over a 1-s time frame. A, population analysis demonstrating the distribution of calcium flux responses as a function of the magnitude of applied shear stress. Absolute shear stresses of 0.3, 0.9, 3.6, 10.8, and 60 dyn/cm2 were applied over a 1-s time frame (n = 9). B, population analysis demonstrating the proportion of vWf-adherent platelets exhibiting calcium flux responses and stationary adhesion as a function of the rate of shear stress application. An absolute shear stress of 60 dyn/cm2 was applied at rates of 60, 30, 12, 6, 3, and 2 dyn/cm2/s (see inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Platelet activation in response to temporal shear gradients is integrin αIIbβ3-dependent and specific to vWf.A, population analysis demonstrating the effects of integrin αIIbβ3 receptor antagonists (c7E3 Fab (Reopro™) and Aggrastat) (n = 3) on the distribution of calcium flux responses as a function of temporal shear gradients. The results represent the percentage of platelets undergoing a calcium response in response to a 1-s shear gradient of 60 dyn/cm2/s (n = 3). B, population analysis demonstrating the distribution of calcium flux responses in response to a shear gradient from 0 to 60 dyn/cm2 over 1 s on a fibrinogen matrix (100 μg/ml). Calcium dye-loaded, washed platelets were allowed to transiently engage the surface (5 min) prior to the application of a shear gradient.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Relationship between the initial rapid acceleration in shear relative to sustained steady-state shear in promoting platelet activation on vWf. Calcium dye-loaded, washed platelets were allowed to transiently engage the vWf surface prior to the application of a temporal shear gradient (60 dyn/cm2/s) (Δshear) or a shear pulse up to 60 dyn/cm2 for 1 s (1 s Pulse). A, single platelet Δ[Ca2+]c profiles (– – –) and concomitant shear stress (dyn/cm2) versus time (s) (——) graph. The arrow (↓) indicates the point of application of the shear gradient. B, population analysis demonstrating the distribution of calcium flux responses in response to temporal shear gradients or shear pulse. C, population analysis demonstrating the proportion of vWf-adherent platelets exhibiting sustained calcium flux in response to a rate of applied shear stress of 60 dyn/cm2/s followed immediately by a steady-state shear plateau of 0.3, 0.9, 3.6, 10.8, or 60 dyn/cm2 for a period of 2 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Platelet activation in response to temporal shear gradients is dependent on second messenger generation. Population analysis demonstrating the effects of the mechanically gated ion channel inhibitor (gadolinium) or a variety of signaling inhibitors (2-aminoethoxydiphenyl borate (APB-2) against the IP3 receptor, U73122 against phospholipase C, PD98058 and SB 203580 against mitogen-activated protein kinase, Y27632 against Rho kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) against Src, and LY294002 and wortmannin against phosphatidylinositol 3-kinase) on shear gradient (Δshear)-induced calcium flux. Calcium dye-loaded, washed platelets were incubated with the inhibitors for 10 min and allowed to transiently engage the vWf surface prior to the application of a shear gradient (60 dyn/cm2/s). The results represent the mean percentage of adherent platelets ± S.E. undergoing a calcium response in response to a shear gradient from 0 to 60 dyn/cm2/s (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Role of ADP in potentiating platelet activation in response to temporal shear gradients. Calcium dye-loaded platelets were treated with either the P2Y1 and P2Y12 receptor antagonists A3P5PS (250 μm) and AR-C69931MX (10 μm) or 1 mm EGTA and 1 mm Mg2+ for 10 min prior to the assay and then allowed to transiently engage the vWf surface prior to the application of a shear stress gradient of 60 dyn/cm2/s. The arrow (↓) shows the point of shear gradient application. A, population analysis demonstrating the effects of the P2Y1 and P2Y12 receptor antagonists A3P5PS and AR-C69931MX, respectively, or 1 mm EGTA/Mg2+ on shear gradient-induced calcium flux. Results represent the percentage of platelets undergoing a calcium response in response to a shear gradient between 0 and 60 dyn/cm2/s (n = 3). B, analysis of the platelets that form firm adhesion contacts with the vWf substrate and undergo a sustained integrin αIIbβ3-dependent oscillatory calcium response after a period of time independent of the shear gradient. Effect of A3P5PS, AR-C69931MX, and EGTA/Mg2+ on sustained calcium signaling in vWf-adherent platelets following exposure to temporal shear gradients (60 dyn/cm2/s). C, effect of A3P5PS, AR-C69931MX, and EGTA/Mg2+ on sustained calcium signaling in vWf-adherent platelets following exposure to temporal shear gradients (60 dyn/cm2/s). The calcium profiles are of selected single platelet recordings to highlight the distinct nature of the transient and sustained calcium responses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 1High shear forces per se do not enhance the signaling function of GPIb. Calcium dye-loaded, washed platelets reconstituted with packed red blood cells (50% v/v) were perfused through vWf-coated (100 μg/ml) microcapillary tubes under steady-state flow conditions (wall shear stresses of 3.6, 10.8, or 60 dyn/cm2). A, the proportion of platelets exhibiting sustained calcium signaling was analyzed and expressed as a percentage of totally adherent platelets (translocating with stable adhesion). B, the proportion of platelets forming stationary adhesion contacts was quantified and expressed as a percentage of totally adherent platelets (translocating with stable adhesion). C, calcium dyeloaded platelets pretreated with the integrin αIIbβ3 receptor antagonist c7E3 Fab were perfused through vWf-coated microcapillary tubes in the presence of ristocetin (1 mg/ml) at wall shear stresses of 0, 3.6, 10.8, or 60 dyn/cm2. The percentage of platelets undergoing a calcium response was quantified and expressed as a percentage of totally adherent platelets. The frequency of calcium spikes was quantified and expressed as the number of spikes per 100 frames, where one frame equals 0.58 s.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSTo gain insight into the mechanisms by which shear forces stimulate platelet activation, we employed an in vitro flow-based adhesion assay that enables simultaneous analysis of platelet adhesion and activation (by monitoring cytosolic calcium flux) on a von Willebrand factor substrate (10Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). With this assay, changes in cytosolic calcium flux correlate closely with platelet activation and the development of stationary adhesion contacts (12Nesbitt W.S. Kulkarni S. Giuliano S. Goncalves I. Dopheide S.M. Yap C.L. Harper I.S. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2965-2972Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Consistent with previous findings (18Kroll M.H. Hellums J.D. McIntire L.V. Schafer A.I. Moake J.L. Blood. 1996; 88: 1525-1541Crossref PubMed Google Scholar, 19Moake J.L. Turner N.A. Stathopoulos N.A. Nolasco L. Hellums J.D. Blood. 1988; 71: 1366-1374Crossref PubMed Google Scholar), exposure of platelets in suspension to progressive increases in shear resulted in a corresponding increase in platelet aggregation (data not shown). Similarly, increasing shear induced a corresponding increase in the number of platelets tethering to von Willebrand factor (20Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar) (data not shown). However, there was no positive relationship between increasing shear and the subsequent cytosolic calcium flux and concomitant stationary adhesion formation (Fig. 1, A and B). In fact, increases in steady-state shear reduced the proportion of platelets forming firm adhesion contacts, resulting in an increased number of rolling cells (Fig. 1B).It is generally assumed that GPIb is the principal mechano-receptor on the surface of platelets, responding to increases in shear with enhanced adhesive and signaling function (21Yuan Y. Kulkarni S. Ulsemer P. Cranmer S.L. Yap C.L. Nesbitt W.S. Harper I. Mistry N. Dopheide S.M. Hughan S.C. Williamson D. de la Salle C. Salem H.H. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 36241-36251Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 22Ruggeri Z.M. Dent J.A. Saldivar E. Blood. 1999; 94: 172-178Crossref PubMed Google Scholar). However, analysis of the effects of steady-state increases in blood flow on GPIb-dependent calcium flux revealed no significant shear-dependent increase in GPIb signaling (data not shown). Even when platelets were artificially anchored to the matrix through vWf-GPIb adhesive bonds by perfusing platelets in the presence of ristocetin (1 mg/ml), no shear-dependent increase in cytosolic calcium flux was observed (Fig. 1C). In control studies we confirmed that this lack of increase in calcium was not due to saturation of the calcium indicator dyes, as thrombin stimulation of these platelets resulted in a further 2-fold increase in calcium flux (data not shown). These studies suggest that high shear forces per se do not lead to a corresponding increase in GPIb-dependent calcium flux.Analysis of thrombus development in vivo has demonstrated a major role for rapid accelerations of blood flow in promoting platelet deposition and thrombus growth (7Strony J. Beaudoin A. Brands D. Adelman B. Am. J. Physiol. 1993; 265: H1787-H1796PubMed Google Scholar). Shear forces in converging inflow segments of stenosed arteries can transiently increase up to 100-fold at the apex of stenosed arteries (6Mailhac A. Badimon J.J. Fallon J.T. Fernandez-Ortiz A. Meyer B. Chesebro J.H. Fuster V. Badimon L. Circulation. 1994; 90: 988-996Crossref PubMed Scopus (120) Google Scholar). Significantly, the only experimental conditions in which platelets were rapidly activated by shear forces were when transiently adherent platelets were exposed to rapid increases in shear (Fig. 2A). In these experiments, a pre-shear adhesion period of 5 min was employed to enable the development of platelet-vWf adhesion contacts, as described under "Experimental Procedures." Notably, platelet activation and calcium signaling were not observed during this pre-shear adhesion time, with the majority of platelet activation occurring only after ∼15 min of static adhesion. Following 5 min of pre-shear adhesion, accelerations in blood flow induced three distinct platelet responses (Fig. 2B), allowing us to divide the platelets into three groups. The first group included platelets that displayed a transient Δ[Ca2+]c spike (mean peak, 412 ± 23 nm) followed by sustained base line-to-peak oscillations ranging from 50 to 800 nm (see i. Trans-Sustained in Fig. 2B). These cells formed stable adhesion contacts with the matrix and expressed active integrin αIIbβ3 on the cell surface (Fig. 2B, PAC-1 immunofluorescence). The second group exhibited a single Δ[Ca2+]c spike (mean peak, 440 ± 45 nm) that coincided with the formation of transient stationary adhesion contacts (see ii. Transient in Fig. 2B). The third subclass of platelets displayed minimal Δ[Ca2+]c in response to rapid accelerations in blood flow and exhibited rapid translocation on the vWf surface (see iii. No Response in Fig. 2B). It should be noted that a small subset of platelets (∼5%) became activated post-induction of the shear gradients. These platelets translocated on the vWf matrix and formed firm adhesion contacts after a variable time period (From 20–40 s) (see Fig. 7). The development of stationary adhesion contacts in these platelets always coincided with the development of a sustained integrin αIIbβ3-dependent oscillatory calcium response. Of the ∼55% of platelets responding instantaneously to shear gradients, the transient and sustained calcium responses were not mutually dependent, with ∼35% of platelets exhibiti
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