Transferrin Saturation Inversely Correlates with Platelet FunctionFunding This work was supported by grants from the Department of Clinical and Biological Sciences of Turin University (Ricerca Locale Ex-60%) to I.R. (RUSI_RILO_13, RUSI_RILO_15, RUSI_RILO_17), A.M. (MORA_RILO_16) and M.D.G. (DEGM_RILO_15).
27 August 2018
17 January 2019
12 March 2019 (eFirst)
Background The association between iron overload (IO) and risk of cardiovascular disease is controversial. Epidemiological studies have found a significant negative association of transferrin (Tf) saturation and cardiovascular events suggesting that higher body iron possibly confer a protective effect towards developing cardiovascular events. The biological mechanisms of this phenomenon are unknown.
Objective This article investigates the role of IO on platelet reactivity.
Materials and Methods This study was a prospective case–control study comparing 45 patients with IO, mostly characterized by the HFE gene mutations C282Y and/or H63D, with 32 healthy controls. We evaluated: (1) platelet aggregation in both platelet-rich plasma and whole blood, (2) platelet membrane expression of the activation marker CD62P, (3) activation of platelet signalling phosphoinositide 3-kinase /Akt and mitogen-activated protein kinase/extracellular signal-regulated kinases (Erk)-1/2 pathways, (4) a pattern of in vivo platelet activation markers, and (5) iron biomarker predictors of platelet reactivity.
Results IO patients had significantly lower platelet aggregability, expression of CD62P and phosphorylation amounts of pAkt and pErk-2 in response to agonists. Furthermore, patients with higher Tf saturation levels were characterized by lower circulating levels of sCD40L, PDGF-BB and thromboxane B2. Platelet aggregation and activation parameters inversely correlated with Tf saturation and the stepwise multivariate regression analysis underlined the role of Tf saturation in predicting platelet reactivity. We also found that in vitro platelet exposure to diferric Tf, but not to iron-depleted TF, dose-dependently inhibited platelet function in all investigated subjects.
Conclusion Tf saturation is inversely associated with platelet reactivity and this could explain, at least in part, the association of high Tf and lower risk of cardiovascular diseases in IO.
Conception and study design: I.R. and A.M. Performing platelet function studies: C.B., R.S. and I.R. Statistical analyses: I.R. and R.S. Interpretation of the data: I.R., C.B., R.S., A.G. and A.M. Drafting the article: C.B., I.R. and A.M. Revising the manuscript critically for important intellectual content: M.D.G., F.N., A.G., A.M. and I.R. All authors gave their consent to the final version of the manuscript.
* Alessandro Morotti and Isabella Russo contributed equally to this work.
- 1 Sullivan JL. Iron and the sex difference in heart disease risk. Lancet 1981; 1 (8233): 1293-1294
- 2 Das De S, Krishna S, Jethwa A. Iron status and its association with coronary heart disease: systematic review and meta-analysis of prospective studies. Atherosclerosis 2015; 238 (02) 296-303
- 3 Gill D, Del Greco M F, Walker AP, Srai SKS, Laffan MA, Minelli C. The effect of iron status on risk of coronary artery disease: a Mendelian randomization study-brief report. Arterioscler Thromb Vasc Biol 2017; 37 (09) 1788-1792
- 4 Frazer DM, Anderson GJ. The regulation of iron transport. Biofactors 2014; 40 (02) 206-214
- 5 Steere AN, Byrne SL, Chasteen ND, Mason AB. Kinetics of iron release from transferrin bound to the transferrin receptor at endosomal pH. Biochim Biophys Acta 2012; 1820 (03) 326-333
- 6 Brissot P, Pietrangelo A, Adams PC, de Graaff B, McLaren CE, Loréal O. Haemochromatosis. Nat Rev Dis Primers 2018; 4: 18016
- 7 Gupta R, Musallam KM, Taher AT, Rivella S. Ineffective erythropoiesis: anemia and iron overload. Hematol Oncol Clin North Am 2018; 32 (02) 213-221
- 8 Feder JN, Gnirke A, Thomas W. , et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996; 13 (04) 399-408
- 9 Beutler E, Gelbart T, West C. , et al. Mutation analysis in hereditary hemochromatosis. Blood Cells Mol Dis 1996; 22 (02) 187-194
- 10 Crownover BK, Covey CJ. Hereditary hemochromatosis. Am Fam Physician 2013; 87 (03) 183-190
- 11 Bardou-Jacquet E, Ben Ali Z, Beaumont-Epinette MP, Loreal O, Jouanolle AM, Brissot P. Non-HFE hemochromatosis: pathophysiological and diagnostic aspects. Clin Res Hepatol Gastroenterol 2014; 38 (02) 143-154
- 12 Franco RF, Zago MA, Trip MD. , et al. Prevalence of hereditary haemochromatosis in premature atherosclerotic vascular disease. Br J Haematol 1998; 102 (05) 1172-1175
- 13 Ekblom K, Marklund SL, Jansson JH, Hallmans G, Weinehall L, Hultdin J. Iron stores and HFE genotypes are not related to increased risk of first-time myocardial infarction: a prospective nested case-referent study. Int J Cardiol 2011; 150 (02) 169-172
- 14 Xu XR, Zhang D, Oswald BE. , et al. Platelets are versatile cells: new discoveries in hemostasis, thrombosis, immune responses, tumor metastasis and beyond. Crit Rev Clin Lab Sci 2016; 53 (06) 409-430
- 15 Berger M, Brass LF. Severe thrombocytopenia in iron deficiency anemia. Am J Hematol 1987; 24 (04) 425-428
- 16 Kellar KL, Bridges NB, Monroe MC, Bell BA, Callaway CS, Evatt BL. Maintenance of normal platelet mass in anemic Belgrade rats and their response to iron. Exp Hematol 1990; 18 (09) 979-984
- 17 Soff GA, Levin J. Thrombocytopenia associated with repletion of iron in iron-deficiency anemia. Am J Med Sci 1988; 295 (01) 35-39
- 18 Yang M, Collis CS, Kelly M, Diplock AT, Rice-Evans C. Do iron and vitamin C co-supplementation influence platelet function or LDL oxidizability in healthy volunteers?. Eur J Clin Nutr 1999; 53 (05) 367-374
- 19 Loo M, Beguin Y. The effect of recombinant human erythropoietin on platelet counts is strongly modulated by the adequacy of iron supply. Blood 1999; 93 (10) 3286-3293
- 20 Calişkan U, Oner AF, Kabakuş N, Koç H. Diminished platelet aggregation in patients with iron deficiency anemia. Clin Appl Thromb Hemost 1999; 5 (03) 161-163
- 21 Kürekçi AE, Atay AA, Sarící SU, Zeybek C, Köseoğlu V, Ozcan O. Effect of iron therapy on the whole blood platelet aggregation in infants with iron deficiency anemia. Thromb Res 2000; 97 (05) 281-285
- 22 Hannuksela J, Parkkila S, Waheed A. , et al. Human platelets express hemochromatosis protein (HFE) and transferrin receptor 2. Eur J Haematol 2003; 70 (04) 201-206
- 23 Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194: 927-929
- 24 Woulfe DS. Akt signaling in platelets and thrombosis. Expert Rev Hematol 2010; 3 (01) 81-91
- 25 Li Z, Zhang G, Feil R, Han J, Du X. Sequential activation of p38 and ERK pathways by cGMP-dependent protein kinase leading to activation of the platelet integrin alphaIIb β3. Blood 2006; 107 (03) 965-972
- 26 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
- 27 Feder JN, Penny DM, Irrinki A. , et al. The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. Proc Natl Acad Sci U S A 1998; 95 (04) 1472-1477
- 28 D'Alessio F, Hentze MW, Muckenthaler MU. The hemochromatosis proteins HFE, TfR2, and HJV form a membrane-associated protein complex for hepcidin regulation. J Hepatol 2012; 57 (05) 1052-1060
- 29 Estevez B, Du X. New concepts and mechanisms of platelet activation signaling. Physiology (Bethesda) 2017; 32 (02) 162-177
- 30 Adam F, Kauskot A, Rosa J-P, Bryckaert M. Mitogen-activated protein kinases in hemostasis and thrombosis. J Thromb Haemost 2008; 6 (12) 2007-2016
- 31 Börsch-Haubold AG, Kramer RM, Watson SP. Inhibition of mitogen-activated protein kinase does not impair primary activation of human platelets. Biochem J 1996; 318 (Pt 1): 207-212
- 32 Edwards CQ, Griffen LM, Ajioka RS, Kushner JP. Screening for hemochromatosis: phenotype versus genotype. Semin Hematol 1998; 35 (01) 72-76
- 33 Franchini M, Targher G, Montagnana M, Lippi G. Iron and thrombosis. Ann Hematol 2008; 87 (03) 167-173
- 34 Lynch S, Soslau G. Iron levels found in hemochromatosis patients inhibit γ-thrombin-induced platelet aggregation. Platelets 2012; 23 (08) 611-616
- 35 Praticó D, Pasin M, Barry OP. , et al. Iron-dependent human platelet activation and hydroxyl radical formation: involvement of protein kinase C. Circulation 1999; 99 (24) 3118-3124
- 36 Kleven MD, Jue S, Enns CA. Transferrin receptors TfR1 and TfR2 bind transferrin through differing mechanisms. Biochemistry 2018; 57 (09) 1552-1559
- 37 Machlus KR, Italiano Jr JE. The incredible journey: from megakaryocyte development to platelet formation. J Cell Biol 2013; 201 (06) 785-796
- 38 Heijnen HF, Debili N, Vainchencker W, Breton-Gorius J, Geuze HJ, Sixma JJ. Multivesicular bodies are an intermediate stage in the formation of platelet alpha-granules. Blood 1998; 91 (07) 2313-2325
- 39 Lindemann S, Krämer B, Seizer P, Gawaz M. Platelets, inflammation and atherosclerosis. J Thromb Haemost 2007; 5 (Suppl. 01) 203-211
- 40 Italiano Jr JE, Richardson JL, Patel-Hett S. , et al. Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet α granules and differentially released. Blood 2008; 111 (03) 1227-1233
- 41 Vishnevetsky D, Kiyanista VA, Gandhi PJ. CD40 ligand: a novel target in the fight against cardiovascular disease. Ann Pharmacother 2004; 38 (09) 1500-1508
- 42 Kingsley K, Huff JL, Rust WL. , et al. ERK1/2 mediates PDGF-BB stimulated vascular smooth muscle cell proliferation and migration on laminin-5. Biochem Biophys Res Commun 2002; 293 (03) 1000-1006
- 43 Depalma RG, Hayes VW, Chow BK, Shamayeva G, May PE, Zacharski LR. Ferritin levels, inflammatory biomarkers, and mortality in peripheral arterial disease: a substudy of the Iron (Fe) and Atherosclerosis Study (FeAST) Trial. J Vasc Surg 2010; 51 (06) 1498-1503
- 44 Auer J, Rammer M, Berent R, Weber T, Lassnig E, Eber B. Body iron stores and coronary atherosclerosis assessed by coronary angiography. Nutr Metab Cardiovasc Dis 2002; 12 (05) 285-290
- 45 Waalen J, Felitti V, Gelbart T, Ho NJ, Beutler E. Prevalence of coronary heart disease associated with HFE mutations in adults attending a health appraisal center. Am J Med 2002; 113 (06) 472-479
- 46 Zacharski LR, Chow BK, Howes PS. , et al. Reduction of iron stores and cardiovascular outcomes in patients with peripheral arterial disease: a randomized controlled trial. JAMA 2007; 297 (06) 603-610
- 47 Miller M, Hutchins GM. Hemochromatosis, multiorgan hemosiderosis, and coronary artery disease. JAMA 1994; 272 (03) 231-233