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Alterations of the platelet proteome in type I Glanzmann thrombasthenia caused by different homozygous delG frameshift mutations in ITGA2B
08 July 2016
Accepted after major revision: 09 January 2016
22 November 2017 (online)
Glanzmann thrombasthenia (GT) is one of the best characterised inherited platelet function disorders but global platelet proteome has not been determined in these patients. We investigated the proteome and function of platelets from two patients with type I GT, caused by different homozygous ITGA2b mutations, from family members and unrelated controls. The global proteome of highly purified washed platelets was quantified by liquid chromatography-mass spectrometry (LC-MS) and targeted MS-methods. Platelet function was analysed by flow cytometry, light transmission aggregometry and flow-based as-says. Platelets from GT patients showed less than 5 % relative levels of the integrin subunit αIIb and 5–9 % fibrinogen compared to controls. These patients demonstrated loss of αIIbβ3-dependent platelet function, but normal platelet granule secretion induced by physiological agonists. Platelets from heterozygous family members of a patient expressed 50–60 % of control αIIb levels which were sufficient for normal αIIbβ3-dependent platelet function. Studying type I GT as model disease we established quantitative LC-MS to detect and clearly distinguish normal platelets, platelets from GT heterozygotes and platelets from GT patients. Diminished levels of factor XIIIB chain, plasminogen and carboxypeptidase 2B were identified in thrombasthenic platelets. Additionally, GT platelets showed up to 2.5-fold increased levels of FcγRIIA and laminin-α4 chain. Elevated levels of platelet FcγRIIA was associated with increased CD63-surface expression after FcγRIIA-crosslinking in one GT-patient which might present a compensatory mechanism of platelet activation in GT. We demonstrate that quantitative LC-MS based proteomics is suitable to validate known but also to identify previously unknown protein level changes of dysfunctional platelets.
Supplementary Material to this article is available online at www.thrombosis-online.com.
- 1 George JN, Caen JP, Nurden AT. Glanzmann’s thrombasthenia: the spectrum of clinical disease. Blood 1990; 75: 1383-1395.
- 2 Nurden AT, Fiore M, Nurden P. et al. Glanzmann thrombasthenia: a review of ITGA2B and ITGB3 defects with emphasis on variants, phenotypic variability, and mouse models. Blood 2011; 118: 5996-6005.
- 3 Plow EF, McEver RP, Coller BS. et al. Related binding mechanisms for fibrinogen, fibronectin, von Willebrand factor, and thrombospondin on thrombin-stimulated human platelets. Blood 1985; 66: 724-727.
- 4 Reheman A, Gross P, Yang H. et al. Vitronectin stabilizes thrombi and vessel occlusion but plays a dual role in platelet aggregation. J Thromb Haemost 2005; 03: 875-883.
- 5 Andre P, Prasad KS, Denis CV. et al. CD40L stabilizes arterial thrombi by a beta3 integrin--dependent mechanism. Nature Med 2002; 08: 247-252.
- 6 Cox AD, Devine DV. Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa. Blood 1994; 83: 1006-1016.
- 7 Miles LA, Ginsberg MH, White JG. et al. Plasminogen interacts with human platelets through two distinct mechanisms. J Clin Invest 1986; 77: 2001-2009.
- 8 Nurden AT, Nurden P. Congenital platelet disorders and understanding of platelet function. Br J Haematol 2014; 165: 165-178.
- 9 Sandrock-Lang K, Oldenburg J, Wiegering V. et al. Characterisation of patients with Glanzmann thrombasthenia and identification of 17 novel mutations. Thromb Haemost 2015; 113: 782-791.
- 10 Nurden AT, Pillois X, Fiore M. et al. Expanding the Mutation Spectrum Affecting alphaIIbbeta3 Integrin in Glanzmann Thrombasthenia: Screening of the ITGA2B and ITGB3 Genes in a Large International Cohort. Human Mut 2015; 36: 548-561.
- 11 Buitrago L, Rendon A, Liang Y. et al. alphaIIbbeta3 variants defined by next-generation sequencing: predicting variants likely to cause Glanzmann thrombasthenia. Proc Natl Acad Sci USA 2015; 112: E1898-1907.
- 12 Nurden AT. Glanzmann thrombasthenia. Orphanet J Rare Dis 2006; 01: 10.
- 13 Burkhart JM, Vaudel M, Gambaryan S. et al. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood 2012; 120: e73-82.
- 14 Vannier C, Behnisch W, Bartsch I. et al. Novel homozygous mutation (c. 175delG) in platelet glycoprotein ITGA2B gene as cause of Glanzmann’s thrombasthenia type I. Klin Padiatr 2010; 222: 150-153.
- 15 Lahav J, Jurk K, Hess O. et al. Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalysed disulfide exchange. Blood 2002; 100: 2472-2478.
- 16 Beck F, Geiger J, Gambaryan S. et al. Time-resolved characterisation of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways. Blood 2014; 123: e1-e10.
- 17 Burkhart JM, Schumbrutzki C, Wortelkamp S. et al. Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics. J Proteomics 2012; 75: 1454-1462.
- 18 Vaudel M, Burkhart JM, Zahedi RP. et al. PeptideShaker enables reanalysis of MS-derived proteomics data sets. Nature Biotechnol 2015; 33: 22-24.
- 19 Jurk K, Schulz AS, Kehrel BE. et al. Novel integrin-dependent platelet malfunction in siblings with leukocyte adhesion deficiency-III (LAD-III) caused by a point mutation in FERMT3. Thromb Haemost 2010; 103: 1053-1064.
- 20 Harrison P, Wilbourn B, Debili N. et al. Uptake of plasma fibrinogen into the alpha granules of human megakaryocytes and platelets. J Clin Invest 1989; 84: 1320-1324.
- 21 Handagama P, Scarborough RM, Shuman MA. et al. Endocytosis of fibrinogen into megakaryocyte and platelet alpha-granules is mediated by alpha IIb beta 3 (glycoprotein IIb-IIIa). Blood 1993; 82: 135-138.
- 22 Bury L, Falcinelli E, Chiasserini D. et al. Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia. Haematologica 2016; 101: 46-56.
- 23 Disdier M, Legrand C, Bouillot C. et al. Quantitation of platelet fibrinogen and thrombospondin in Glanzmann’s thrombasthenia by electroimmunoassay. Thrombosis Res 1989; 53: 521-533.
- 24 Burkhart JM, Gambaryan S, Watson SP. et al. What can proteomics tell us about platelets? Circulation Res. 2014; 114: 1204-1219.
- 25 Rosenberg N, Hauschner H, Peretz H. et al. A 13-bp deletion in alpha(IIb) gene is a founder mutation that predominates in Palestinian-Arab patients with Glanzmann thrombasthenia. J Thromb Haemost 2005; 03: 2764-2772.
- 26 Wagner CL, Mascelli MA, Neblock DS. et al. Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets. Blood 1996; 88: 907-914.
- 27 Phillips DR, Agin PP. Platelet membrane defects in Glanzmann’s thrombasthenia. Evidence for decreased amounts of two major glycoproteins. J Clin Invest 1977; 60: 535-545.
- 28 Clemetson KJ, Capitanio A, Pareti FI. et al. Additional platelet membrane glycoprotein abnormalities in Glanzmann’s thrombasthenia: A comparison with normals by high resolution two-dimensional polyacrylamide gel electrophoresis. Thrombosis Res 1980; 18: 797-806.
- 29 Kehrel B, Wierwille S, Clemetson KJ. et al. Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not. Blood 1998; 91: 491-499.
- 30 Sandrock-Lang K, Wentzell R, Santoso S. et al. Inherited platelet disorders. Hamostaseologie 2016; 36: 178-186.
- 31 Cramer EM, Debili N, Martin JF. et al. Uncoordinated expression of fibrinogen compared with thrombospondin and von Willebrand factor in maturing human megakaryocytes. Blood 1989; 73: 1123-1129.
- 32 Wencel-Drake JD, Painter RG, Zimmerman TS. et al. Ultrastructural localisation of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section. Blood 1985; 65: 929-938.
- 33 Preissner KT, Holzhuter S, Justus C. et al. Identification of and partial characterisation of platelet vitronectin: evidence for complex formation with platelet-derived plasminogen activator inhibitor-1. Blood 1989; 74: 1989-1996.
- 34 Maynard DM, Heijnen HF, Horne MK. et al. Proteomic analysis of platelet alpha-granules using mass spectrometry. J Thromb Haemost 2007; 05: 1945-1955.
- 35 Paul JI, Schwarzbauer JE, Tamkun JW. et al. Cell-type-specific fibronectin subunits generated by alternative splicing. J Biol Chem 1986; 261: 12258-12265.
- 36 Schick PK, Walker J, Profeta B. et al. Synthesis and secretion of von Willebrand factor and fibronectin in megakaryocytes at different phases of maturation. Arterioscl Thromb Vasc Biol 1997; 17: 797-801.
- 37 Seiffert D, Schleef RR. Two functionally distinct pools of vitronectin (Vn) in the blood circulation: identification of a heparin-binding competent population of Vn within platelet alpha-granules. Blood 1996; 88: 552-560.
- 38 Hill SA, Shaughnessy SG, Joshua P. et al. Differential mechanisms targeting type 1 plasminogen activator inhibitor and vitronectin into the storage granules of a human megakaryocytic cell line. Blood 1996; 87: 5061-5073.
- 39 Coller BS, Seligsohn U, West SM. et al. Platelet fibrinogen and vitronectin in Glanzmann thrombasthenia: evidence consistent with specific roles for glycoprotein IIb/IIIA and alpha v beta 3 integrins in platelet protein trafficking. Blood 1991; 78: 2603-2610.
- 40 Muszbek L, Bereczky Z, Bagoly Z. et al. Factor XIII: a coagulation factor with multiple plasmatic and cellular functions. Physiol Rev 2011; 91: 931-972.
- 41 Marx G, Korner G, Mou X. et al. Packaging zinc, fibrinogen, and factor XIII in platelet alpha-granules. J Cell Physiol 1993; 156: 437-442.
- 42 Byrnes JR, Wilson C, Boutelle AM. et al. The interaction between fibrinogen and zymogen FXIII-A2B2 is mediated by fibrinogen residues gamma 390-396 and the FXIII-B subunits. Blood 2016; 128: 1969-1978.
- 43 Veljkovic DK, Rivard GE, Diamandis M. et al. Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation. Blood 2009; 113: 1535-1542.
- 44 Lucas MA, Fretto LJ, McKee PA. The binding of human plasminogen to fibrin and fibrinogen. J Biol Chem 1983; 258: 4249-4256.
- 45 Whyte CS, Swieringa F, Mastenbroek TG. et al. Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow. Blood 2015; 125: 2568-2578.
- 46 Mosnier LO, Buijtenhuijs P, Marx PF. et al. Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets. Blood 2003; 101: 4844-4846.
- 47 Schadinger SL, Lin JH, Garand M. et al. Secretion and antifibrinolytic function of thrombin-activatable fibrinolysis inhibitor from human platelets. J Thromb Haemost 2010; 08: 2523-2529.
- 48 Tan AK, Eaton DL. Activation and characterisation of procarboxypeptidase B from human plasma. Biochemistry 1995; 34: 5811-5816.
- 49 An SSA, Suh I. Binding of Thrombin Activatable Fibrinolysis Inhibitor (TAFI) to Plasminogen May Play a Role in the Fibrinolytic Pathway. B Korean Chem Soc 2008; 29: 2209-2214.
- 50 Arman M, Krauel K. Human platelet IgG Fc receptor FcRIIA in immunity and thrombosis. J Thromb Haemost 2015; 13: 893-908.
- 51 Boylan B, Gao C, Rathore V. et al. Identification of FcgammaRIIa as the ITAM-bearing receptor mediating alphaIIbbeta3 outside-in integrin signaling in human platelets. Blood 2008; 112: 2780-2786.
- 52 Pook M, Tamming L, Padari K. et al. Platelets store laminins 411/421 and 511/521 in compartments distinct from alpha- or dense granules and secrete these proteins via microvesicles. J Thromb Haemost 2014; 12: 519-527.
- 53 Sakurai Y, Fitch-Tewfik JL, Qiu Y. et al. Platelet geometry sensing spatially regulates alpha-granule secretion to enable matrix self-deposition. Blood 2015; 126: 531-538.