FcγRIIa Ligation Induces Platelet Hypersensitivity to Thrombotic Stimuli
2012; Elsevier BV; Volume: 182; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2012.09.005
ISSN1525-2191
AutoresMark Berlacher, Joshua A Vieth, Brittany C. Heflin, S. Gay, Adam Antczak, Brian E. Tasma, Holly J. Boardman, Navinderjit Singh, Angela H. Montel, M. Bashar Kahaleh, Randall G. Worth,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoPlatelets are known for their important role in hemostasis, however their significance in other functions, including inflammation and infection, are becoming more apparent. Patients with systemic lupus erythematosus (SLE) are known to have circulating IgG complexes in their blood and are highly susceptible to thrombotic events. Because platelets express a single receptor for IgG, we tested the hypothesis that ligation of this receptor (FcγRIIa) induces platelet hypersensitivity to thrombotic stimuli. Platelets from SLE patients were considerably more sensitive to thrombin compared to healthy volunteers, and this correlated with elevated levels of surface IgG on SLE platelets. To test whether FcγRIIa ligation stimulated thrombin hypersensitivity, platelets from healthy volunteers were incubated with buffer or heat-aggregated IgG, then stimulated with increasing concentrations of thrombin. Interestingly, heat-aggregated IgG–stimulated platelets, but not buffer-treated platelets, were hypersensitive to thrombin, and hypersensitivity was blocked by an anti-FcγRIIa monoclonal antibody (mAb). Thrombin hypersensitivity was not due to changes in thrombin receptor expression (GPIbα or PAR1) but is dependent on activation of shared signaling molecules. These observations suggest that ligation of platelet FcγRIIa by IgG complexes induces a hypersensitive state whereby small changes in thrombotic stimuli may result in platelet activation and subsequent vascular complications such as transient ischemic attacks or stroke. Platelets are known for their important role in hemostasis, however their significance in other functions, including inflammation and infection, are becoming more apparent. Patients with systemic lupus erythematosus (SLE) are known to have circulating IgG complexes in their blood and are highly susceptible to thrombotic events. Because platelets express a single receptor for IgG, we tested the hypothesis that ligation of this receptor (FcγRIIa) induces platelet hypersensitivity to thrombotic stimuli. Platelets from SLE patients were considerably more sensitive to thrombin compared to healthy volunteers, and this correlated with elevated levels of surface IgG on SLE platelets. To test whether FcγRIIa ligation stimulated thrombin hypersensitivity, platelets from healthy volunteers were incubated with buffer or heat-aggregated IgG, then stimulated with increasing concentrations of thrombin. Interestingly, heat-aggregated IgG–stimulated platelets, but not buffer-treated platelets, were hypersensitive to thrombin, and hypersensitivity was blocked by an anti-FcγRIIa monoclonal antibody (mAb). Thrombin hypersensitivity was not due to changes in thrombin receptor expression (GPIbα or PAR1) but is dependent on activation of shared signaling molecules. These observations suggest that ligation of platelet FcγRIIa by IgG complexes induces a hypersensitive state whereby small changes in thrombotic stimuli may result in platelet activation and subsequent vascular complications such as transient ischemic attacks or stroke. Platelets have long been known for their role in maintenance of vascular homeostasis through aggregation and thrombus formation. More recently, platelets are being appreciated for a plethora of activities associated with vascular remodeling, host defense, and inflammation.1Smyth S.S. McEver R.P. Weyrich A.S. Morrell C.N. Hoffman M.R. Arepally G.M. French P.A. Dauerman H.L. Becker R.C. Platelet functions beyond hemostasis.J Thromb Haemost. 2009; 7: 1759-1766Crossref PubMed Scopus (406) Google Scholar Interestingly, platelets have been reported to alter expression of Bcl-3 and IL-1β upon stimulation with thrombin and fibrinogen.2Weyrich A.S. Dixon D.A. Pabla R. Elstad M.R. McIntyre T.M. Prescott S.M. Zimmerman G.A. Signal-dependent translation of a regulatory protein, Bcl-3, in activated human platelets.Proc Natl Acad Sci U S A. 1998; 95: 5556-5561Crossref PubMed Scopus (240) Google Scholar Because platelets are anucleate, changes in expression are due solely to posttranscriptional modulation. These studies provide evidence that platelets are not simply “clotting factors,” but complex cells that have the potential to alter their response depending on the nature of stimulation. Supporting a role for platelets in inflammatory diseases, Boilard and colleagues3Boilard E. Nigrovic P.A. Larabee K. Watts G.F. Coblyn J.S. Weinblatt M.E. Massarotti E.M. Remold-O'Donnell E. Farndale R.W. Ware J. Lee D.M. Platelets amplify inflammation in arthritis via collagen-dependent microparticle production.Science. 2010; 327: 580-583Crossref PubMed Scopus (841) Google Scholar recently observed the presence of IL-1β–containing platelet microparticles in synovium of patients with rheumatoid arthritis. Furthermore, in their mouse model of collagen-induced arthritis, depletion of platelets resulted in significantly reduced pathology.3Boilard E. Nigrovic P.A. Larabee K. Watts G.F. Coblyn J.S. Weinblatt M.E. Massarotti E.M. Remold-O'Donnell E. Farndale R.W. Ware J. Lee D.M. Platelets amplify inflammation in arthritis via collagen-dependent microparticle production.Science. 2010; 327: 580-583Crossref PubMed Scopus (841) Google Scholar Our recent studies measuring soluble CD40 ligand (sCD40L) and regulated on activation normal T cell expressed and secreted (RANTES) production subsequent to IgG exposure suggest that platelets may be responsible for recruiting and stimulating other inflammatory cells.4Antczak A.J. Singh N. Gay S.R. Worth R.G. IgG-complex stimulated platelets: a source of sCD40L and RANTES in initiation of inflammatory cascade.Cell Immunol. 2010; 263: 129-133Crossref PubMed Scopus (31) Google Scholar, 5Antczak A.J. Vieth J.A. Singh N. Worth R.G. Internalization of IgG-coated targets results in activation and secretion of soluble CD40 ligand and RANTES by human platelets.Clin Vaccine Immunol. 2011; 18: 210-216Crossref PubMed Scopus (30) Google Scholar Consistent with our observations, another recent study has shown that platelets from systemic lupus erythematosus (SLE) patients express elevated levels of CD40L, due to IgG complex binding to platelets via the fragment crystallizable (Fc) region receptor FcγRIIa.6Duffau P. Seneschal J. Nicco C. Richez C. Lazaro E. Douchet I. Bordes C. Viallard J.F. Goulvestre C. Pellegrin J.L. Weil B. Moreau J.F. Batteux F. Blanco P. Platelet CD154 potentiates interferon-alpha secretion by plasmacytoid dendritic cells in systemic lupus erythematosus.Sci Transl Med. 2010; 2: 47ra63Crossref PubMed Scopus (159) Google Scholar Importantly, immune complexes collected from the serum of SLE patients were shown to activate healthy platelets resulting in expression of CD40L.6Duffau P. Seneschal J. Nicco C. Richez C. Lazaro E. Douchet I. Bordes C. Viallard J.F. Goulvestre C. Pellegrin J.L. Weil B. Moreau J.F. Batteux F. Blanco P. Platelet CD154 potentiates interferon-alpha secretion by plasmacytoid dendritic cells in systemic lupus erythematosus.Sci Transl Med. 2010; 2: 47ra63Crossref PubMed Scopus (159) Google Scholar Interestingly, platelets can respond to various stimuli by exhibiting select activation profiles such as aggregation, surface expression of CD62P (P-selectin), and release of soluble molecules such as sCD40L.4Antczak A.J. Singh N. Gay S.R. Worth R.G. IgG-complex stimulated platelets: a source of sCD40L and RANTES in initiation of inflammatory cascade.Cell Immunol. 2010; 263: 129-133Crossref PubMed Scopus (31) Google Scholar These observations suggest that platelets may be important mediators of inflammatory conditions such as rheumatoid arthritis, SLE, and other autoimmune disorders. It is well known that patients with autoimmune diseases are more susceptible to platelet abnormalities such as thrombocytopenia, transient ischemic attack, and stroke.7Sanna G. D'Cruz D. Cuadrado M.J. Cerebral manifestations in the antiphospholipid (Hughes) syndrome.Rheum Dis Clin North Am. 2006; 32: 465-490Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 8Esdaile J.M. Abrahamowicz M. Grodzicky T. Li Y. Panaritis C. du Berger R. Cote R. Grover S.A. Fortin P.R. Clarke A.E. Senecal J.L. Traditional Framingham risk factors fail to fully account for accelerated atherosclerosis in systemic lupus erythematosus.Arthritis Rheum. 2001; 44: 2331-2337Crossref PubMed Scopus (966) Google Scholar, 9Miller M.H. Urowitz M.B. Gladman D.D. The significance of thrombocytopenia in systemic lupus erythematosus.Arthritis Rheum. 1983; 26: 1181-1186Crossref PubMed Scopus (49) Google Scholar, 10Iyoda M. Suzuki H. Ashikaga E. Nagai H. Kuroki A. Shibata T. Kitazawa K. Akizawa T. Elderly onset systemic lupus erythematosus (SLE) presenting with disseminated intravascular coagulation (DIC).Clin Rheumatol. 2008; : S15-S18Crossref PubMed Scopus (2) Google Scholar, 11Fernandez M. Alarcon G.S. Apte M. Andrade R.M. Vila L.M. Reveille J.D. Systemic lupus erythematosus in a multiethnic US cohort: XLIII. The significance of thrombocytopenia as a prognostic factor.Arthritis Rheum. 2007; 56: 614-621Crossref PubMed Scopus (57) Google Scholar, 12Beyan E. Beyan C. Turan M. Hematological presentation in systemic lupus erythematosus and its relationship with disease activity.Hematology. 2007; 12: 257-261Crossref PubMed Scopus (37) Google Scholar, 13Cervera R. Khamashta M.A. Font J. Sebastiani G.D. Gil A. Lavilla P. Mejia J.C. Aydintug A.O. Chwalinska-Sadowska H. de Ramon E. Fernandez-Nebro A. Galeazzi M. Valen M. Mathieu A. Houssiau F. Caro N. Alba P. Ramos-Casals M. Ingelmo M. Hughes G.R. Morbidity and mortality in systemic lupus erythematosus during a 10-year period: a comparison of early and late manifestations in a cohort of 1,000 patients.Medicine (Baltimore). 2003; 82: 299-308Crossref PubMed Scopus (1012) Google Scholar The mechanisms behind these clinical observations are unknown and may be due to a multitude of factors including activation of vascular endothelium, interaction with leukocytes, opsonization by antiplatelet antibodies, or reaction to immune complexes. Many autoimmune disorders are characterized by the presence of IgG complexes in circulation, which could bind to platelet FcγRIIa. It is possible that in addition to inducing expression of select activation markers, binding of IgG complexes to platelet FcγRIIa initiates signaling that results in platelets becoming more sensitive to other thrombotic stimuli. To this end, we tested the hypothesis that exposure to IgG complexes will induce platelets to become hypersensitive to thrombotic stimuli. Interestingly, platelets from SLE patients exhibit hypersensitivity to thrombin compared to platelets from healthy volunteers. Moreover, platelets from healthy volunteers pretreated with heat-aggregated IgG (aggIgG) before thrombin stimulation were hypersensitive to thrombin, resulting in increased expression of CD62P (P-selectin) compared to platelets pretreated with buffer alone. Patients (n = 20) who fulfilled at least four American College of Rheumatology criteria for SLE were enrolled into this study in accordance with University of Toledo Biomedical Institutional Review Board approval. Patients on platelet-modifying therapy [aspirin, thienopyridines, or glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors] were excluded from the study. Patient profiles show demographics (Table 1), SLE Disease Activity Index criteria (Table 2), and medications (Table 3). Healthy volunteers consisted of employees of the University of Toledo Health Science Campus who were free of autoimmune disease, had no active infections, and were not on any of the platelet-modifying therapies listed above.Table 1Healthy Volunteer and SLE Patient DemographicsDemographicsSLEControlNo.2020Male33Female1717Average age (range)46.5 (27–59)46 (27–55)Ethnicity Hispanic99 Caucasian44 African American77Average disease duration in years (range)14.83 (10–21)NAMean platelet count246,000/cm3∗No patients had thrombocytopenia at the time of sample collection.232,000/cm3∗No patients had thrombocytopenia at the time of sample collection.NA, not applicable.∗ No patients had thrombocytopenia at the time of sample collection. Open table in a new tab Table 2SLEDAI-Qualifying Symptoms at Time of Sample CollectionSymptomPatients (%)SLEDAI scoreArthritis11 (79%)4dsDNA Antibodies3 (21%)2Low complement C3 or C40 (0%)2Antinuclear antibodies (<1:40) in 11 patients (79%).dsDNA, double-stranded DNA; SLEDAI, SLE Disease Activity Index. Open table in a new tab Table 3Current Treatments at Time of Sample CollectionTreatmentPatients (%)Mycophenolate7 (50%)Hydroxychloroquine5 (36%)Prednisone4 (29%)Methotrexate2 (14%)Celecoxib2 (14%)Adalimumab1 (7%) Open table in a new tab NA, not applicable. Antinuclear antibodies ( 90% pure as assessed by flow cytometry for platelet glycoprotein Ib alpha chain (GPIbα) (Supplemental Figure S1). aggIgG was prepared by diluting fluorescein isothiocyanate–labeled IgG from human serum (Sigma-Aldrich, St. Louis, MO) in Dulbecco's phosphate buffered saline (Thermo Fisher Scientific, Waltham, MA) to a concentration of 10 mg/mL and heating the suspension to 62°C for 20 minutes. Large aggregates were cleared by centrifugation at 10,000 × g for 10 minutes, leaving a mixture of aggIgG consisting of two to six molecules per complex as previously reported.14Worth R.G. Chien C.D. Chien P. Reilly M.P. McKenzie S.E. Schreiber A.D. Platelet FcgammaRIIA binds and internalizes IgG-containing complexes.Exp Hematol. 2006; 34: 1490-1495Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Platelets (100-μL aliquots) at a concentration of 1 × 107/mL were placed in a 37°C water bath for 30 minutes in the presence or absence of 0.5 mg/mL aggIgG. Buffer alone or buffer containing specified concentrations of thrombin, collagen, ADP, or arachidonic acid (Chronolog, Havertown, PA) was added at the indicated times, and the platelets were returned to the water bath for an additional 30 minutes. Following incubation, platelets were fixed in 2% paraformaldehyde for 1 hour at 4°C. Treatment with specific inhibitors was performed by pretreating platelets for 10 minutes at 37°C followed by the addition of aggIgG or buffer and allowing the activation assay to occur in the presence of the inhibitor for the entirety of the experiment. Inhibitors of Src kinase (PP2; Sigma-Aldrich)15Huang Z.Y. Hunter S. Kim M.K. Chien P. Worth R.G. Indik Z.K. Schreiber A.D. The monocyte Fcgamma receptors FcgammaRI/gamma and FcgammaRIIA differ in their interaction with Syk and with Src-related tyrosine kinases.J Leukoc Biol. 2004; 76: 491-499Crossref PubMed Scopus (20) Google Scholar and spleen tyrosine kinase (Syk) (BAY61-3606; Santa Cruz Biotechnology, Santa Cruz, CA)16Yamamoto N. Takeshita K. Shichijo M. Kokubo T. Sato M. Nakashima K. Ishimori M. Nagai H. Li Y.F. Yura T. Bacon K.B. The orally available spleen tyrosine kinase inhibitor 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]nicotinamide dihydrochloride (BAY 61-3606) blocks antigen-induced airway inflammation in rodents.J Pharmacol Exp Ther. 2003; 306: 1174-1181Crossref PubMed Scopus (165) Google Scholar were used at 10 μmol/L and 20 nmol/L, respectively, which is consistent with previous studies testing specificity. We also performed dose responses to determine the optimal concentration of BAY61-3606 to use in our system by detecting the ability to inhibit hypersensitivity (Supplemental Figure S2).17Pearce G. Audzevich T. Jessberger R. SYK regulates B-cell migration by phosphorylation of the F-actin interacting protein SWAP-70.Blood. 2011; 117: 1574-1584Crossref PubMed Scopus (36) Google Scholar Following fixation on ice, the platelets were labeled for analysis by flow cytometry. For gating purposes, an allophycocyanin-conjugated anti-GPIbα (clone HIP1) monoclonal antibody (mAb) (BD Biosciences, San Jose, CA) was used to verify the platelet population. Other antibodies used to measure receptor expression include anti-FcγRII (clone 3D3), anti-protease activated receptor 1 (PAR1) (clone ATAP2), anti-P-selectin (CD62P) (clone AK-4), or anti-GPIIb (clone HIP8) mAbs. Isotype-matched control IgG was used as labeling controls to set negative populations for each mAb. Association of aggIgG with platelets was assessed by measuring fluorescein isothiocyanate–aggIgG fluorescence on GPIbα+ platelets. Platelets were analyzed on a FACSCalibur flow cytometer (BD Biosciences) and data were interpreted using Cell Quest (BD Biosciences) and FloJo software (Tree Star, Ashland, OR). The effective concentration of thrombin required to elicit 50% of the maximum response (EC50) was calculated using Prism 5 software (GraphPad, La Jolla, CA). Platelets (100-μL aliquots) at a concentration of 4 × 107/mL were treated as described above with PP2 (10 μmol/L), BAY61-3606 (20 nmol/L), or buffer followed by aggIgG (0.5 mg/mL). Platelets were then incubated for an hour in 40 μL of a lysis buffer containing: 0.88 g/L sodium chloride, 1.19 g/L HEPES, 0.02 g/L sodium azide, 1% triton X-100, and 1% protease inhibitor. Samples were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA). Membranes were blocked in a 3% bovine serum albumin TBS-T solution and then blotted for 2 hours with anti-Syk (Santa Cruz Biotechnology), anti-FcγRIIa (Proteintech Group, Chicago, IL), antiphosphotyrosine (Millipore), or antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ambion, Foster City, CA). The membranes were then developed using the Western Breeze kit per the manufacturer's instructions (Invitrogen). Films were then imaged using an Omega Molecular Imaging and Analysis System instrument (UltraLum, Claremont, CA). Absorbance was calculated using UltraQuant version 6.0 software (UltraLum). All statistics were calculated using Prism 5 software. Statistical significance for thrombin hypersensitivity in healthy control platelets was determined using a two-tailed, equal variance Student's t-test. Statistical significance comparing SLE patients with healthy volunteers was determined using a two-tailed, paired Student's t-test. The ability of platelets to recognize and respond to IgG complexes depends on the expression of FcγRIIa. Therefore, we determined whether FcγRIIa expression was different on platelets obtained from SLE patients (diagnosed with SLE according to American College of Rheumatology criteria as stated in Materials and Methods) versus platelets from healthy volunteers. Platelets from SLE patients and healthy volunteers (matched for age, sex, and ethnicity) were treated identically on the same day and using the same reagents. Purified platelets were analyzed by flow cytometry for continuity of size by forward versus side scatter and the exclusion of microparticles, as determined by comparing the platelet gate to beads of 0.5, 1, and 2 μm. Platelets appear as a pure population that is larger than 1 μm, constitutively express GPIbα (CD42b), and express P-selectin (CD62P) only after stimulation (Figure 1A). As shown in Figure 1B, platelet FcγRIIa expression shows considerable person-to-person variability, but no significant differences were noticed comparing samples from healthy volunteers and SLE platelets. To assess platelet phenotypes from healthy volunteers and SLE patients, we measured GPIbα expression in freshly isolated samples and found no significant differences (Figure 1C). Because previous reports showed elevated CD62P expression on the surface of SLE platelets, we assessed CD62P expression in our samples, which confirmed the previous reports (Figure 1D).6Duffau P. Seneschal J. Nicco C. Richez C. Lazaro E. Douchet I. Bordes C. Viallard J.F. Goulvestre C. Pellegrin J.L. Weil B. Moreau J.F. Batteux F. Blanco P. Platelet CD154 potentiates interferon-alpha secretion by plasmacytoid dendritic cells in systemic lupus erythematosus.Sci Transl Med. 2010; 2: 47ra63Crossref PubMed Scopus (159) Google Scholar We then stained platelets from SLE patients or healthy volunteers with PE-conjugated anti-IgG F(ab′)2 fragments to detect surface-bound IgG. Consistent with previous observations, platelets from SLE patients exhibited elevated levels of surface-associated IgG compared to platelets from healthy individuals (Figure 1E). Interestingly, CD62P levels correlated with surface IgG (Figure 1F), suggesting that IgG may be involved in activating human platelets. Because patients with SLE have a higher incidence of thrombotic disorders, we next wanted to determine whether SLE platelets exhibit hypersensitivity to thrombotic stimuli. To accomplish this, platelets from SLE patients and healthy volunteers were treated with the indicated doses of thrombin, fixed, and then stained with anti-GPIbα, as a platelet marker, and anti-CD62P, as a marker of platelet activation. CD62P is known to be stored in platelet α-granules and expressed on the surface of the granules, fusing with the plasma membrane after stimulation. As shown in Figure 2A, platelets isolated from SLE patients displayed a higher sensitivity to thrombin (as determined by activation at lower concentrations of thrombin). Data from each thrombin curve were then used to calculate the thrombin EC50 values (Figure 2B). Of note, it took a >2.5-fold increase in thrombin to activate platelets from healthy volunteers than those from SLE patients. To determine whether hypersensitivity is limited to thrombin or common to many agonists, platelets from healthy volunteers or SLE patients were exposed to ADP, collagen, or arachidonic acid. Interestingly, a slight degree of hypersensitivity was observed for ADP, but not for collagen or arachidonic acid (Figure 2C). Therefore, we used thrombin as the agonist in subsequent experiments. We next determined whether FcγRIIa ligation could induce thrombin hypersensitivity of platelets from healthy volunteers. To do this, we exposed healthy platelets to aggIgG before stimulation with thrombin. As shown previously and in Figure 3A, aggIgG does not result in significant CD62P expression during short incubation periods of 30 minutes at 37°C.4Antczak A.J. Singh N. Gay S.R. Worth R.G. IgG-complex stimulated platelets: a source of sCD40L and RANTES in initiation of inflammatory cascade.Cell Immunol. 2010; 263: 129-133Crossref PubMed Scopus (31) Google Scholar Buffer alone or buffer containing thrombin at indicated concentrations was then added to platelet samples and incubated for another 30 minutes at 37°C. We noted that platelets pretreated with aggIgG responded to lower concentrations of thrombin compared to platelets pretreated with buffer alone (Figure 3A). When EC50 values for thrombin sensitivity were calculated, we found that aggIgG induced a significant decrease in the thrombin EC50 (Figure 3B). In similar experiments, SLE platelets were treated with buffer or aggIgG and then measured for thrombin hypersensitivity. We found that exposure to aggIgG had only marginal effects on SLE platelets, presumably because platelets from SLE patients already had IgG on their surface (Figure 1E), and subsequent addition of aggIgG had only a minimal effect (Figure 3B). As a control to determine whether hypersensitivity is simply due to exposure to two different stimuli, platelets were pretreated with thrombin followed by aggIgG to determine whether increased levels of CD62P are induced on platelet surfaces. Of note, there was no appreciable difference in CD62P expression on platelets preincubated with the indicated doses of thrombin for 30 minutes at 37°C followed by addition of buffer alone or buffer containing aggIgG (Figure 3C). These data suggest that aggIgG induces platelets to become hypersensitive to thrombin. Because platelets from SLE patients showed hypersensitivity to thrombin, we next sought to determine whether serum from SLE patients could convert healthy platelets into a hypersensitive state. To do this, healthy platelets were incubated with SLE serum for 30 minutes at 37°C then stimulated with thrombin and EC50 values were calculated. SLE serum induced healthy platelets to become hypersensitive to thrombin (Figure 3D); however, the magnitude of hypersensitivity is not the same as that elicited by aggIgG. Of note, healthy serum did not alter the EC50 of SLE platelets (Figure 3D). Given that platelets express FcγRIIa as their only IgG-binding receptor, we next tested whether the hypersensitivity is dependent on FcγRIIa. Thrombin hypersensitivity mediated by aggIgG was blocked by incubating platelets with the FcγRIIa-blocking mAb IV.3, but not control IgG, before exposing platelets to aggIgG (Figure 4A). IV.3 also inhibited aggIgG binding (Figure 4B), whereas control antibody did not. Platelet activation by thrombin takes place by the tethering of free thrombin to GPIbα, which brings thrombin in close proximity to PAR1 and PAR4. Thrombin then cleaves PAR1 or PAR4 leading to activation of a G-protein–related signaling pathway. To determine the mechanism behind thrombin hypersensitivity, we first measured GPIbα and PAR1 expression after exposure to aggIgG. Interestingly, no difference in expression of either GPIbα (Figure 5A) or PAR1 (Figure 5B) was observed. Because FcγRIIa has been shown to signal through Syk, we examined the amounts of Syk in platelets from healthy volunteers or SLE patients using Western blot analysis. No difference in Syk expression was observed in platelets from healthy volunteers or SLE patients (Figure 5C). We next examined intracellular signaling pathways. Platelets with prothrombotic phenotypes have been observed in certain diseases such as essential thrombocythemia and polycythemia vera.18Shukla S.D. Kansra S.V. Reddy M.A. Shukla S.M. Klachko D.M. Sturek M. Platelets from diabetic pigs exhibit hypersensitivity to thrombin.Comp Med. 2008; 58: 481-484PubMed Google Scholar, 19Randi M.L. Brunati A.M. Scapin M. Frasson M. Deana R. Magrin E. Fabris F. Donella-Deana A. Src tyrosine kinase preactivation is associated with platelet hypersensitivity in essential thrombocythemia and polycythemia vera.Blood. 2010; 115: 667-676Crossref PubMed Scopus (9) Google Scholar In both of these diseases, constitutive activation of Src kinase has been reported. Src is also used by FcγRIIa. Therefore, we incubated platelets with PP2, a potent inhibitor of Src.15Huang Z.Y. Hunter S. Kim M.K. Chien P. Worth R.G. Indik Z.K. Schreiber A.D. The monocyte Fcgamma receptors FcgammaRI/gamma and FcgammaRIIA differ in their interaction with Syk and with Src-related tyrosine kinases.J Leukoc Biol. 2004; 76: 491-499Crossref PubMed Scopus (20) Google Scholar, 20Clark E.A. Brugge J.S. Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets.Mol Cell Biol. 1993; 13: 1863-1871Crossref PubMed Scopus (198) Google Scholar, 21Huang Z.Y. Barreda D.R. Worth R.G. Indik Z.K. Kim M.K. Chien P. Schreiber A.D. Differential kinase requirements in human and mouse Fc-gamma receptor phagocytosis and endocytosis.J Leukoc Biol. 2006; 80: 1553-1562Crossref PubMed Scopus (63) Google Scholar Not surprisingly, pretreatment of platelets with PP2 inhibited both thrombin-induced activation and hypersensitivity (Figure 6A). Because FcγRIIa has also been shown to be dependent on signaling via Syk, we pretreated platelets with BAY61-3606 to inhibit Syk activity and then incubated with aggIgG followed with thrombin. Inhibition of Syk signaling completely abolished aggIgG-induced thrombin hypersensitivity but had no effect on thrombin activation (Figure 6B). These data suggest that ligation of FcγRIIa with aggIgG initiates a Syk-dependent signaling cascade that is able to potentiate subsequent signals initiated by thrombin. Finally, as inhibition of Syk was able to abolish FcγRIIa-induced hypersensitivity, we assessed the levels of tyrosine phosphorylation of platelet lysates from healthy volunteers stimulated with buffer or aggIgG in the absence or presence of PP2 or Bay61-3606. We observed increased tyrosine phosphorylation of proteins isolated from platelets stimulated with aggIgG compared to buffer-treated platelets (Figure 6C, lane 1 vs 4). We also observed that increased tyrosine phosphorylation was diminished on pretreatment with either inhibitor (Figure 6C, lane 4 vs 5 and 6). Based on the observation that aggIgG-stimulated healthy platelets show increased tyrosine phosphorylation, we tested SLE platelet lysates for increased phosphotyrosine levels (Figure 6D). We observed that platelets from SLE patients had significantly increased levels of tyrosin
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