Platelet Proteome Analysis Reveals Integrin-dependent Aggregation Defects in Patients with Myelodysplastic Syndromes

Bleeding complications are a significant clinical problem in patients with myelodysplastic syndromes even at sufficient platelet counts (>50,000/μl). However, the underlying pathology of this hemorrhagic diathesis is still unknown. Here, we analyzed the platelet proteome of patients with myelodysplastic syndromes by quantitative two-dimensional difference gel electrophoresis followed by mass spectrometric protein identification. Proteins identified with lower concentrations, such as Talin-1, Vinculin, Myosin-9, Filmain-A, and Actin play critical roles in integrin αIIbβ3 signaling and thus platelet aggregation. Despite normal agonist receptor expression, calcium flux, and granule release upon activation, the activation capacity of integrin αIIbβ3 was diminished in myelodysplastic syndrome platelets. Förster resonance energy transfer analysis showed a reduced co-localization of Talin-1 to the integrin’s β3-subunit, which is required for receptor activation and fibrinogen binding. In addition, platelet spreading on immobilized fibrinogen was incomplete, and platelet aggregation assays confirmed a general defect in integrin-dependent platelet aggregation in patients with myelodysplastic syndromes. Our data provide novel aspects on the molecular pathology of impaired platelet function in myelodysplastic syndromes and suggest a mechanism of defective integrin αIIbβ3 signaling that may contribute to the hemorrhagic diathesis observed in these patients. Bleeding complications are a significant clinical problem in patients with myelodysplastic syndromes even at sufficient platelet counts (>50,000/μl). However, the underlying pathology of this hemorrhagic diathesis is still unknown. Here, we analyzed the platelet proteome of patients with myelodysplastic syndromes by quantitative two-dimensional difference gel electrophoresis followed by mass spectrometric protein identification. Proteins identified with lower concentrations, such as Talin-1, Vinculin, Myosin-9, Filmain-A, and Actin play critical roles in integrin αIIbβ3 signaling and thus platelet aggregation. Despite normal agonist receptor expression, calcium flux, and granule release upon activation, the activation capacity of integrin αIIbβ3 was diminished in myelodysplastic syndrome platelets. Förster resonance energy transfer analysis showed a reduced co-localization of Talin-1 to the integrin’s β3-subunit, which is required for receptor activation and fibrinogen binding. In addition, platelet spreading on immobilized fibrinogen was incomplete, and platelet aggregation assays confirmed a general defect in integrin-dependent platelet aggregation in patients with myelodysplastic syndromes. Our data provide novel aspects on the molecular pathology of impaired platelet function in myelodysplastic syndromes and suggest a mechanism of defective integrin αIIbβ3 signaling that may contribute to the hemorrhagic diathesis observed in these patients. Myelodysplastic syndromes (MDS) 1The abbreviations used are:MDSmyelodysplastic syndromePEphycoerythrinAPCallophycocyaninTRAPthrombin receptor activating peptideCDcluster of differentiationMFImean fluorescence intensity. 1The abbreviations used are:MDSmyelodysplastic syndromePEphycoerythrinAPCallophycocyaninTRAPthrombin receptor activating peptideCDcluster of differentiationMFImean fluorescence intensity. are among the most frequent hematological malignancies (1Germing U. Strupp C. Kündgen A. Bowen D. Aul C. Haas R. Gattermann N. No increase in age-specific incidence of myelodysplastic syndromes.Haematologica. 2004; 89: 905-910PubMed Google Scholar). Each of the hematopoietic lineages may be affected by dysplasia and/or cytopenia, and patients suffering from this clonal bone marrow disease are affected by anemia and infectious as well as hemorrhagic complications (1Germing U. Strupp C. Kündgen A. Bowen D. Aul C. Haas R. Gattermann N. No increase in age-specific incidence of myelodysplastic syndromes.Haematologica. 2004; 89: 905-910PubMed Google Scholar, 2van Lom K. Houtsmuller A.B. van Putten W.L. Slater R.M. Löwenberg B. Cytogenetic clonality analysis of megakaryocytes in myelodysplastic syndrome by dual-color fluorescence in situ hybridization and confocal laser scanning microscopy.Genes Chromosomes Cancer. 1999; 25: 332-338Crossref PubMed Scopus (18) Google Scholar). Thrombocytopenia itself is prevalent in up to 65% of patients with MDS at the time of diagnosis (3Kantarjian H. Giles F. List A. Lyons R. Sekeres M.A. Pierce S. Deuson R. Leveque J. The incidence and impact of thrombocytopenia in myelodysplastic syndromes.Cancer. 2007; 109: 1705-1714Crossref PubMed Scopus (195) Google Scholar). Recently, a detailed study of the clinical features of 2900 patients with MDS reported that even among patients with platelet counts of >50,000/μl, 19% had signs of bleeding at the time of diagnosis (4Neukirchen J. Blum S. Kuendgen A. Strupp C. Aivado M. Haas R. Aul C. Gattermann N. Germing U. Platelet counts and haemorrhagic diathesis in patients with myelodysplastic syndromes.Eur. J. Haematol. 2009; 83: 477-482Crossref PubMed Scopus (55) Google Scholar). Taken together, these data suggest a platelet count-independent functional platelet defect in MDS, which is supported by sporadic reports indicating insufficient platelet aggregation (3Kantarjian H. Giles F. List A. Lyons R. Sekeres M.A. Pierce S. Deuson R. Leveque J. The incidence and impact of thrombocytopenia in myelodysplastic syndromes.Cancer. 2007; 109: 1705-1714Crossref PubMed Scopus (195) Google Scholar, 5Pamphilon D.H. Aparicio S.R. Roberts B.E. Menys V.C. Tate G. Davies J.A. The myelodysplastic syndromes-a study of haemostatic function and platelet ultrastructure.Scand. J. Haematol. 1984; 33: 486-491Crossref PubMed Scopus (22) Google Scholar, 6Mittelman M. Zeidman A. Platelet function in the myelodysplastic syndromes.Int. J. Hematol. 2000; 71: 95-98PubMed Google Scholar, 7Zeidman A. Sokolover N. Fradin Z. Cohen A. Redlich O. Mittelman M. Platelet function and its clinical significance in the myelodysplastic syndromes.Hematol. J. 2004; 5: 234-238Crossref PubMed Scopus (31) Google Scholar, 8Girtovitis F.I. Ntaios G. Papadopoulos A. Ioannidis G. Makris P.E. Defective platelet aggregation in myelodysplastic syndromes.Acta Haematol. 2007; 118: 117-122Crossref PubMed Scopus (26) Google Scholar). The existence of such a functional platelet defect may very well contribute to the high rate of hemorrhagic complications observed in patients with MDS. myelodysplastic syndrome phycoerythrin allophycocyanin thrombin receptor activating peptide cluster of differentiation mean fluorescence intensity. myelodysplastic syndrome phycoerythrin allophycocyanin thrombin receptor activating peptide cluster of differentiation mean fluorescence intensity. Platelet aggregation following a vascular injury or other stimuli is a highly organized process to ensure optimal efficacy. Exposure to collagen, von Willebrand factor, or soluble platelet agonists such as ADP, thrombin, or thromboxane A2 at the site of injury attracts platelets and initiates platelet adhesion. This activates and recruits additional platelets from the blood to finally cover the injury (9Jennings L.K. Role of platelets in atherothrombosis.Am. J. Cardiol. 2009; 103: 4A-10AAbstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 10Li Z. Delaney M.K. O'Brien K.A. Du X. Signaling during platelet adhesion and activation.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 2341-2349Crossref PubMed Scopus (587) Google Scholar). The fibrinogen receptor (integrin αIIbβ3) plays an essential role in this process (10Li Z. Delaney M.K. O'Brien K.A. Du X. Signaling during platelet adhesion and activation.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 2341-2349Crossref PubMed Scopus (587) Google Scholar, 11Knezevic I. Leisner T.M. Lam S.C. Direct binding of the platelet integrin αIIbβ3 (GPIIb-IIIa) to talin. Evidence that interaction is mediated through the cytoplasmic domains of both αIIb and β3.J. Biol. Chem. 1996; 271: 16416-16421Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 12Nieswandt B. Moser M. Pleines I. Varga-Szabo D. Monkley S. Critchley D. Fässler R. Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo.J. Exp. Med. 2007; 204: 3113-3118Crossref PubMed Scopus (198) Google Scholar, 13Petrich B.G. Marchese P. Ruggeri Z.M. Spiess S. Weichert R.A. Ye F. Tiedt R. Skoda R.C. Monkley S.J. Critchley D.R. Ginsberg M.H. Talin is required for integrin-mediated platelet function in hemostasis and thrombosis.J. Exp. Med. 2007; 204: 3103-3111Crossref PubMed Scopus (219) Google Scholar). On resting platelets, integrin αIIbβ3 resides in a low affinity state and depends on intrinsic signals for activation and function (14Vinogradova O. Velyvis A. Velyviene A. Hu B. Haas T. Plow E. Qin J. A structural mechanism of integrin α(IIb)β(3) “inside-out” activation as regulated by its cytoplasmic face.Cell. 2002; 110: 587-597Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). The final step in this activation process is the co-localization of Talin-1 from the cytoplasm to the β-subunit of the integrin (12Nieswandt B. Moser M. Pleines I. Varga-Szabo D. Monkley S. Critchley D. Fässler R. Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo.J. Exp. Med. 2007; 204: 3113-3118Crossref PubMed Scopus (198) Google Scholar, 13Petrich B.G. Marchese P. Ruggeri Z.M. Spiess S. Weichert R.A. Ye F. Tiedt R. Skoda R.C. Monkley S.J. Critchley D.R. Ginsberg M.H. Talin is required for integrin-mediated platelet function in hemostasis and thrombosis.J. Exp. Med. 2007; 204: 3103-3111Crossref PubMed Scopus (219) Google Scholar, 15Ye F. Hu G. Taylor D. Ratnikov B. Bobkov A.A. McLean M.A. Sligar S.G. Taylor K.A. Ginsberg M.H. Recreation of the terminal events in physiological integrin activation.J. Cell Biol. 2010; 188: 157-173Crossref PubMed Scopus (206) Google Scholar, 16Petrich B.G. Fogelstrand P. Partridge A.W. Yousefi N. Ablooglu A.J. Shattil S.J. Ginsberg M.H. The antithrombotic potential of selective blockade of talin-dependent integrin αIIbβ3 (platelet GPIIb-IIIa) activation.J. Clin. Invest. 2007; 117: 2250-2259Crossref PubMed Scopus (106) Google Scholar). Once activated, αIIbβ3 is linked to the actin cytoskeleton by several linker proteins, which is required for platelet aggregation and spreading (12Nieswandt B. Moser M. Pleines I. Varga-Szabo D. Monkley S. Critchley D. Fässler R. Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo.J. Exp. Med. 2007; 204: 3113-3118Crossref PubMed Scopus (198) Google Scholar, 17Widell S. Hast R. Balloon-like platelets in myelodysplastic syndromes-a feature of dysmegakaryopoiesis?.Leuk. Res. 1987; 11: 747-752Crossref PubMed Scopus (9) Google Scholar, 18Seri M. Cusano R. Gangarossa S. Caridi G. Bordo D. Lo Nigro C. Ghiggeri G.M. Ravazzolo R. Savino M. Del Vecchio M. d'Apolito M. Iolascon A. Zelante L.L. Savoia A. Balduini C.L. Noris P. Magrini U. Belletti S. Heath K.E. Babcock M. Glucksman M.J. Aliprandis E. Bizzaro N. Desnick R.J. Martignetti J.A. Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome 2000; PubMed Scopus Google Scholar). a of Talin-1, Vinculin, and to be in platelets for sufficient integrin αIIbβ3 function and Despite that a functional platelet defect may in MDS, data are to the hemorrhagic diathesis in patients with sufficient platelet In this we a analysis of the platelet proteome of MDS patients and MDS platelet aggregation. that the critical integrin αIIbβ3 is impaired and that a of essential for its function a reduced in MDS. The MDS patients this study had a of and a platelet of and platelet or with platelet that used in and for detailed and to the was to a by the of the to a of the blood was for at to from by a at and in (12Nieswandt B. Moser M. Pleines I. Varga-Szabo D. Monkley S. Critchley D. Fässler R. Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo.J. Exp. Med. 2007; 204: 3113-3118Crossref PubMed Scopus (198) Google Scholar). The of the for and was by of MDS patients and normal and and The protein of the was by protein to the of the with was as by the of was with and with for two-dimensional of protein of was with of protein of the with MDS with and with or gel and to by was on a as S. J. H. B. S. S. C. F. D. of binding on the protein in by PubMed Scopus Google Scholar). The and was a on an as S. J. H. B. S. S. C. F. D. of binding on the protein in by PubMed Scopus Google Scholar). a and protein and by for of the in and the of normal Each of the patients an the gel gel and the in protein by the following the was at of its in at of the patients with the normal and this in was significant of from the a and and the as S. J. H. B. S. S. C. F. D. of binding on the protein in by PubMed Scopus Google and to a analyzed in an mass and mass from the by on identified to that be was by and with the following and mass and molecular mass data by or of a protein we used the following The and molecular mass from the two-dimensional with the data of the in molecular mass or the with the was Proteins as identified with a of on at and signaling analysis was by The platelet of the MDS patients and normal by a and with followed by used and and was used as by platelets from MDS patients and normal with thrombin and for at in the with and to phycoerythrin and allophycocyanin was a with and the was in to the the energy at and at the of the The increase in the at was to co-localization of and the for sufficient to blood of blood from MDS patients and normal was in with with for at in the and analyzed a used thrombin peptide and used and and and from with or calcium of the platelets of MDS patients and normal was upon activation with or thrombin blood in and with for at in the of MDS patients and normal was to a at the of and blood was and with ADP, collagen, or was on a platelet aggregation with fibrinogen and with in used as platelets of MDS patients and normal at a of with thrombin, and analyzed by and mean used for are in the and are used the to the of significance No for to for of for the of at the of interaction was used for the of the aggregation defect on clinical the molecular pathology of platelet in MDS, we a analysis of the MDS platelet two-dimensional difference gel electrophoresis for a quantitative of platelet from MDS patients and normal a of protein of which MDS and normal platelets the protein had a lower in MDS platelets. identified of the protein protein identified from protein be identified from indicating of these The of these in had in the of the Vinculin, Myosin-9, protein and protein and with lower in MDS, are in integrin αIIbβ3 signaling and function for mass and peptide The of Talin-1, Myosin-9, and was confirmed by a of Talin-1 in MDS platelets with platelets as well as of Vinculin, of and of Myosin-9, and analysis of the identified with in MDS integrin signaling and both critical for platelet to be the most affected A and with in of cytoplasmic cytoplasmic protein protein protein and protein is to the in the of the identified the the mean of MDS and platelet in a of platelet from MDS patients and The platelet from MDS patients and the of Talin-1, Vinculin, and in MDS and analysis identified from two-dimensional difference gel electrophoresis analysis of integrin the in the MDS platelet in and the in The is to the in the of the identified the the mean of MDS and platelet The platelet protein in MDS an integrin signaling as a potential of defective platelet function in MDS. the of several and other platelet and and in the of MDS and normal platelets we the very step in platelet activation following agonist as we at the in to from and MDS and the calcium is critical for granule and we analyzed granule release upon platelet activation Our analysis difference in the of to the platelet in MDS platelets with indicating a normal activation of the MDS platelets. In the activation signaling of integrin αIIbβ3 was a for platelet activation and aggregation. this Talin-1 and to from the cytoplasm to the and to domains of the integrin we that the energy transfer Talin-1 and integrin αIIbβ3 upon activation with thrombin was reduced in MDS platelets with normal which insufficient activation of integrin αIIbβ3 the of this receptor to from its low affinity to an high affinity we binding to this receptor by the site of integrin αIIbβ3 that is in its high affinity state D. J. Shattil S.J. Plow Ginsberg M.H. of the affinity of integrin αIIbβ3 (GPIIb-IIIa) by the cytoplasmic of PubMed Scopus Google Scholar). with as well as with agonist binding was reduced in MDS platelets and that in MDS platelets a lower of integrin αIIbβ3 was to from its to its of integrin such as a structural defect at the site we its binding following activation through which is from signaling Here, we MDS and normal platelets of the receptor analysis of MDS platelet MDS platelets a significant in following and insufficient integrin αIIbβ3 activation and platelets from MDS and normal with to binding potential functional in MDS platelets, we analyzed signaling of the affected integrin platelets of MDS patients and normal to immobilized with thrombin, MDS platelets showed a spreading defect as to for up to the impaired signaling of integrin αIIbβ3 which is in with the reduced of Talin-1, Vinculin, Myosin-9, and MDS and normal platelets to the immobilized fibrinogen (integrin of the MDS platelets with of platelets The impaired signaling of integrin αIIbβ3 a general defect in integrin-dependent platelet aggregation in MDS. However, is an essential for aggregation, aggregation and aggregation Platelet and of on and J. Platelet Scholar, M.A. platelet and Google Scholar). we the of MDS platelets to in to agonists In with the observed defective integrin αIIbβ3 receptor activation in MDS, we an insufficient aggregation in to agonists and interaction of the of the aggregation defect on clinical that platelet had the with of the patients with platelets an aggregation defect with of patients with platelet counts that aggregation capacity the and patients with showed a lower incidence of the aggregation defect with the other patients with had a incidence with aggregation with high patients the platelet aggregation defect in MDS. with normal platelets, MDS platelets showed a reduced aggregation in to and and following and was reduced and of platelet aggregation capacity of MDS patients that in patients with lower platelet counts defective aggregation in to and is Platelet count-independent are a significant clinical problem in patients with MDS (3Kantarjian H. Giles F. List A. Lyons R. Sekeres M.A. Pierce S. Deuson R. Leveque J. The incidence and impact of thrombocytopenia in myelodysplastic syndromes.Cancer. 2007; 109: 1705-1714Crossref PubMed Scopus (195) Google Scholar, J. Blum S. Kuendgen A. Strupp C. Aivado M. Haas R. Aul C. Gattermann N. Germing U. Platelet counts and haemorrhagic diathesis in patients with myelodysplastic syndromes.Eur. J. Haematol. 2009; 83: 477-482Crossref PubMed Scopus (55) Google Scholar). The of a functional platelet defect may bleeding complications in patients with low platelet counts and in general increase the rate during in MDS patients. hemostasis in MDS is and a Once platelets from most of the required for quantitative platelet proteome analysis is an to that may contribute to functional platelet Our analysis of the MDS and platelet that the of with showed lower in MDS. This that functional platelet in MDS may result from of critical for platelet This was supported by analysis that showed that the of these lower are essential for integrin function of integrin αIIbβ3 is critical for platelet aggregation and hemostasis B.G. Marchese P. Ruggeri Z.M. Spiess S. Weichert R.A. Ye F. Tiedt R. Skoda R.C. Monkley S.J. Critchley D.R. Ginsberg M.H. Talin is required for integrin-mediated platelet function in hemostasis and thrombosis.J. Exp. Med. 2007; 204: 3103-3111Crossref PubMed Scopus (219) Google Scholar, R.A. fibrinogen binding to platelets and platelet PubMed Google Scholar). the with lower in MDS and both binding to the of integrin which is to as the final step for integrin activation (14Vinogradova O. Velyvis A. Velyviene A. Hu B. Haas T. Plow E. Qin J. 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Res. 1987; 11: 747-752Crossref PubMed Scopus (9) Google observed in the of MDS patients observed the in patients with platelet counts and the aggregation defect with platelet and signs of in analysis a lower platelet had the on the aggregation the platelet suggest aggregation in these patients thus indicating the existence of a functional and for with