Genome-Wide Association Study Links Receptor Tyrosine Kinase Inhibitor Sprouty 2 to Thrombocytopenia after Coronary Artery Bypass SurgeryFunding Funding for this study was provided by: the Duke Anesthesiology Developing Research Excellence in Anesthesia Management (DREAM) Award (to Dr. Kertai); the National Institutes of Health grants 1R56HL126891–01 (to Dr. Karhausen), HL075273 and HL092071 (to Dr. Podgoreanu), HL096978, HL108280 and HL109971 (to Dr. Mathew), HL095987 (to Dr. Shah) and HL101621 (to Dr. Kraus); the American Heart Association grants 15SDG25080046 (to Dr. Karhausen), 9951185U (to Dr. Mathew) and 0120492U (to Dr. Podgoreanu); and a Duke School of Medicine Health Scholar award (to Dr. Karhausen). Logistics support was provided by CATHGEN. The authors are solely responsible for the design and conduct of this study, all study analyses and drafting and editing of the manuscript and its final contents.
16 December 2017
08 June 2018
13 August 2018 (eFirst)
Introduction Thrombocytopenia after cardiac surgery independently predicts stroke, acute kidney injury and death. To understand the underlying risks and mechanisms, we analysed genetic variations associated with thrombocytopenia in patients undergoing coronary artery bypass grafting (CABG) surgery.
Materials and Methods Study subjects underwent isolated on-pump CABG surgery at Duke University Medical Center. Post-operative thrombocytopenia was defined as platelet count < 100 × 109/L. Using a logistic regression model adjusted for clinical risk factors, we performed a genome-wide association study in a discovery cohort (n = 860) and validated significant findings in a replication cohort (n = 296). Protein expression was assessed in isolated platelets by immunoblot.
Results A total of 63 single-nucleotide polymorphisms met a priori discovery thresholds for replication, but only 1 (rs9574547) in the intergenic region upstream of sprouty 2 (SPRY2) met nominal significance in the replication cohort. The minor allele of rs9574547 was associated with a lower risk for thrombocytopenia (discovery cohort, odds ratio, 0.45, 95% confidence interval, 0.30–0.67, p = 9.76 × 10−5) with the overall association confirmed by meta-analysis (meta-p = 7.88 × 10−6). Immunoblotting demonstrated expression of SPRY2 and its dynamic regulation during platelet activation. Treatment with a functional SPRY2 peptide blunted platelet extracellular signal-regulated kinase (ERK) phosphorylation after agonist stimulation.
Conclusion We identified the association of a genetic polymorphism in the intergenic region of SPRY2 with a decreased incidence of thrombocytopenia after CABG surgery. Because SPRY2—an endogenous receptor tyrosine kinase inhibitor—is present in platelets and modulates essential signalling pathways, these findings support a role for SPRY2 as a novel modulator of platelet responses after cardiac surgery.
* The first two and last two authors contributed equally to this work.
** Members of the Duke Perioperative Genetics and Safety Outcomes (PEGASUS) Investigative Team are acknowledged in the Acknowledgment section.
- 1 Paparella D, Yau TM, Young E. Cardiopulmonary bypass induced inflammation: pathophysiology and treatment. An update. Eur J Cardiothorac Surg 2002; 21 (02) 232-244
- 2 Williamson DR, Lesur O, Tétrault JP, Nault V, Pilon D. Thrombocytopenia in the critically ill: prevalence, incidence, risk factors, and clinical outcomes. Can J Anaesth 2013; 60 (07) 641-651
- 3 Kertai MD, Zhou S, Karhausen JA. , et al. Platelet counts, acute kidney injury, and mortality after coronary artery bypass grafting surgery. Anesthesiology 2016; 124 (02) 339-352
- 4 Karhausen JA, Smeltz AM, Akushevich I. , et al. Platelet counts and postoperative stroke after coronary artery bypass grafting surgery. Anesth Analg 2017; 125 (04) 1129-1139
- 5 Weerasinghe A, Taylor KM. The platelet in cardiopulmonary bypass. Ann Thorac Surg 1998; 66 (06) 2145-2152
- 6 Lapchak PH, Kannan L, Ioannou A. , et al. Platelets orchestrate remote tissue damage after mesenteric ischemia-reperfusion. Am J Physiol Gastrointest Liver Physiol 2012; 302 (08) G888-G897
- 7 Singbartl K, Forlow SB, Ley K. Platelet, but not endothelial, P-selectin is critical for neutrophil-mediated acute postischemic renal failure. FASEB J 2001; 15 (13) 2337-2344
- 8 Mangano DT. ; Multicenter Study of Perioperative Ischemia Research Group. Aspirin and mortality from coronary bypass surgery. N Engl J Med 2002; 347 (17) 1309-1317
- 9 Verma S, Goodman SG, Mehta SR. , et al. Should dual antiplatelet therapy be used in patients following coronary artery bypass surgery? A meta-analysis of randomized controlled trials. BMC Surg 2015; 15: 112
- 10 Kunicki TJ, Williams SA, Nugent DJ. Genetic variants that affect platelet function. Curr Opin Hematol 2012; 19 (05) 371-379
- 11 Petersen R, Lambourne JJ, Javierre BM. , et al. Platelet function is modified by common sequence variation in megakaryocyte super enhancers. Nat Commun 2017; 8: 16058
- 12 Shameer K, Denny JC, Ding K. , et al. A genome- and phenome-wide association study to identify genetic variants influencing platelet count and volume and their pleiotropic effects. Hum Genet 2014; 133 (01) 95-109
- 13 Johnson AD, Yanek LR, Chen MH. , et al. Genome-wide meta-analyses identifies seven loci associated with platelet aggregation in response to agonists. Nat Genet 2010; 42 (07) 608-613
- 14 Astle WJ, Elding H, Jiang T. , et al. The allelic landscape of human blood cell trait variation and links to common complex disease. Cell 2016; 167 (05) 1415-1429.e19
- 15 Eicher JD, Chami N, Kacprowski T. , et al; Global Lipids Genetics Consortium; CARDIoGRAM Exome Consortium; Myocardial Infarction Genetics Consortium. Platelet-related variants identified by Exomechip meta-analysis in 157,293 individuals. Am J Hum Genet 2016; 99 (01) 40-55
- 16 Morawski W, Sanak M, Cisowski M. , et al. Prediction of the excessive perioperative bleeding in patients undergoing coronary artery bypass grafting: role of aspirin and platelet glycoprotein IIIa polymorphism. J Thorac Cardiovasc Surg 2005; 130 (03) 791-796
- 17 Welsby IJ, Podgoreanu MV, Phillips-Bute B. , et al; Perioperative Genetics and Safety Outcomes Study (PEGASUS) Investigative Team. Genetic factors contribute to bleeding after cardiac surgery. J Thromb Haemost 2005; 3 (06) 1206-1212
- 18 Rinder CS, Mathew JP, Rinder HM. , et al; Multicenter Study of Perioperative Ischemia Research Group. Platelet PlA2 polymorphism and platelet activation are associated with increased troponin I release after cardiopulmonary bypass. Anesthesiology 2002; 97 (05) 1118-1122
- 19 Muehlschlegel JD, Perry TE, Liu KY. , et al. Polymorphism in the protease-activated receptor-4 gene region associates with platelet activation and perioperative myocardial injury. Am J Hematol 2012; 87 (02) 161-166
- 20 Little J, Higgins JP, Ioannidis JP. , et al. Strengthening the reporting of genetic association studies (STREGA): an extension of the STROBE Statement. Hum Genet 2009; 125 (02) 131-151
- 21 Kertai MD, Li YW, Li YJ. , et al; Duke Perioperative Genetics and Safety Outcomes (PEGASUS) Investigative Team. G protein-coupled receptor kinase 5 gene polymorphisms are associated with postoperative atrial fibrillation after coronary artery bypass grafting in patients receiving β-blockers. Circ Cardiovasc Genet 2014; 7 (05) 625-633
- 22 Kertai MD, Li YJ, Ji Y. , et al; Duke Perioperative Genetics and Safety Outcomes (PEGASUS) Investigative Team. Genome-wide association study of new-onset atrial fibrillation after coronary artery bypass grafting surgery. Am Heart J 2015; 170 (03) 580-90.e28
- 23 American Society of Anesthesiologists Task Force on Perioperative Blood Transfusion and Adjuvant Therapies. Practice guidelines for perioperative blood transfusion and adjuvant therapies: an updated report by the American Society of Anesthesiologists Task Force on Perioperative Blood Transfusion and Adjuvant Therapies. Anesthesiology 2006; 105 (01) 198-208
- 24 Purcell S, Neale B, Todd-Brown K. , et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007; 81 (03) 559-575
- 25 Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, Reich D. Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 2006; 38 (08) 904-909
- 26 Marchini J, Howie B, Myers S, McVean G, Donnelly P. A new multipoint method for genome-wide association studies by imputation of genotypes. Nat Genet 2007; 39 (07) 906-913
- 27 Hanafusa H, Torii S, Yasunaga T, Nishida E. Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway. Nat Cell Biol 2002; 4 (11) 850-858
- 28 Comerford KM, Leonard MO, Karhausen J, Carey R, Colgan SP, Taylor CT. Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia. Proc Natl Acad Sci U S A 2003; 100 (03) 986-991
- 29 Schwarz G. Estimating the dimension of a model. Ann Stat 1978; 6 (02) 461-464
- 30 Pruim RJ, Welch RP, Sanna S. , et al. LocusZoom: regional visualization of genome-wide association scan results. Bioinformatics 2010; 26 (18) 2336-2337
- 31 Wei Y, Tejera P, Wang Z. , et al. A missense genetic variant in LRRC16A/CARMIL1 improves acute respiratory distress syndrome survival by attenuating platelet count decline. Am J Respir Crit Care Med 2017; 195 (10) 1353-1361
- 32 Chen MH, Yanek LR, Backman JD. , et al. Exome-chip meta-analysis identifies association between variation in ANKRD26 and platelet aggregation. Platelets 2017; DOI: 10.1080/09537104.2017.
- 33 Guy GR, Jackson RA, Yusoff P, Chow SY. Sprouty proteins: modified modulators, matchmakers or missing links?. J Endocrinol 2009; 203 (02) 191-202
- 34 Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2010; 141 (07) 1117-1134
- 35 Lee RH, Bergmeier W. Platelet immunoreceptor tyrosine-based activation motif (ITAM) and hemITAM signaling and vascular integrity in inflammation and development. J Thromb Haemost 2016; 14 (04) 645-654
- 36 Gould WR, Baxi SM, Schroeder R. , et al. Gas6 receptors Axl, Sky and Mer enhance platelet activation and regulate thrombotic responses. J Thromb Haemost 2005; 3 (04) 733-741
- 37 Boulaftali Y, Hess PR, Getz TM. , et al. Platelet ITAM signaling is critical for vascular integrity in inflammation. J Clin Invest 2013; 123 (02) 908-916
- 38 Angelillo-Scherrer A, Burnier L, Flores N. , et al. Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy. J Clin Invest 2005; 115 (02) 237-246
- 39 Gratacap MP, Martin V, Valéra MC. , et al. The new tyrosine-kinase inhibitor and anticancer drug dasatinib reversibly affects platelet activation in vitro and in vivo. Blood 2009; 114 (09) 1884-1892
- 40 Metharom P, Berndt MC, Baker RI, Andrews RK. Current state and novel approaches of antiplatelet therapy. Arterioscler Thromb Vasc Biol 2015; 35 (06) 1327-1338
- 41 Dütting S, Vögtle T, Morowski M. , et al. Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets. Circ Res 2014; 114 (03) 444-453
- 42 Delehanty LL, Mogass M, Gonias SL, Racke FK, Johnstone B, Goldfarb AN. Stromal inhibition of megakaryocytic differentiation is associated with blockade of sustained Rap1 activation. Blood 2003; 101 (05) 1744-1751
- 43 Edwin F, Anderson K, Patel TB. HECT domain-containing E3 ubiquitin ligase Nedd4 interacts with and ubiquitinates Sprouty2. J Biol Chem 2010; 285 (01) 255-264
- 44 Nadeau RJ, Toher JL, Yang X, Kovalenko D, Friesel R. Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2. J Cell Biochem 2007; 100 (01) 151-160
- 45 Rosado JA, Sage SO. Role of the ERK pathway in the activation of store-mediated calcium entry in human platelets. J Biol Chem 2001; 276 (19) 15659-15665
- 46 Li Z, Xi X, Du X. A mitogen-activated protein kinase-dependent signaling pathway in the activation of platelet integrin alpha IIbbeta3. J Biol Chem 2001; 276 (45) 42226-42232
- 47 Flevaris P, Li Z, Zhang G, Zheng Y, Liu J, Du X. Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signaling pathway. Blood 2009; 113 (04) 893-901
- 48 Hangauer MJ, Vaughn IW, McManus MT. Pervasive transcription of the human genome produces thousands of previously unidentified long intergenic noncoding RNAs. PLoS Genet 2013; 9 (06) e1003569
- 49 Yang X, Gong Y, Friesel R. Spry1 is expressed in hemangioblasts and negatively regulates primitive hematopoiesis and endothelial cell function. PLoS One 2011; 6 (04) e18374
- 50 Robless PA, Okonko D, Lintott P, Mansfield AO, Mikhailidis DP, Stansby GP. Increased platelet aggregation and activation in peripheral arterial disease. Eur J Vasc Endovasc Surg 2003; 25 (01) 16-22
- 51 George R, Bhatt A, Narayani J, Thulaseedharan JV, Sivadasanpillai H, Tharakan JA. Enhanced P-selectin expression on platelet-a marker of platelet activation, in young patients with angiographically proven coronary artery disease. Mol Cell Biochem 2016; 419 (1-2): 125-133
- 52 Tan KT, Tayebjee MH, Macfadyen RJ, Lip GY, Blann AD. Elevated platelet microparticles in stable coronary artery disease are unrelated to disease severity or to indices of inflammation. Platelets 2005; 16 (06) 368-371
- 53 Gkaliagkousi E, Passacquale G, Douma S, Zamboulis C, Ferro A. Platelet activation in essential hypertension: implications for antiplatelet treatment. Am J Hypertens 2010; 23 (03) 229-236
- 54 Ferroni P, Basili S, Falco A, Davì G. Platelet activation in type 2 diabetes mellitus. J Thromb Haemost 2004; 2 (08) 1282-1291