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Thromb Haemost 2022; 122(01): 080-091
DOI: 10.1055/a-1497-9573
Coagulation and Fibrinolysis

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex

Laura Delvasto-Núñez
1   Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
2   Department of Hematology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
,
Dorina Roem
1   Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
,
Kamran Bakhtiari
3   Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
,
Gerard van Mierlo
1   Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
,
Joost C. M. Meijers
3   Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
4   Department of Experimental Vascular Medicine, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands
,
Ilse Jongerius  ‡   *
1   Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
5   Department of Pediatric Immunology, Rheumatology and Infectious Diseases, Emma Children's Hospital, Amsterdam UMC, Amsterdam, the Netherlands
,
Sacha S. Zeerleder‡   *
1   Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
2   Department of Hematology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
6   Department of Hematology and Central Hematology Laboratory, Inselspital – Bern University Hospital, University of Bern, Bern, Switzerland
7   Department for BioMedical Research, University of Bern, Bern, Switzerland
› Author Affiliations Funding This study was supported by research funding from Trombosestichting Nederland to S.S.Z. (grant number 201604) and by a Product and Process Development grant obtained (in competition) from Sanquin Blood Supply Foundation (PPOC19–24/L2467) to I.J.
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Abstract

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl3) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.

Keywords

coagulation - red blood cells - iron - hemolysis - intrinsic tenase complex

Author Contributions

L.D.N., J.C.M.M., I.J., and S.S.Z. conceptualized the study and contributed to the design and execution of the research. I.J. and S.S.Z. obtained funding for the study. L.D.N., D.R., and K.B. performed the experiments and analyzed the data; L.D.N., D.R., K.B., G.v.M., J.C.M.M., I.J., and S.S.Z. interpreted the data and designed the experiments. L.D.N., J.C.M.M., I.J., and S.S.Z. wrote the initial draft of the manuscript and all authors critically commented on the manuscript.


* These authors contributed equally.


Supplementary Material



Publication History

Received: 04 November 2020

Accepted: 26 April 2021

Accepted Manuscript online:
03 May 2021

Article published online:
27 September 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Ataga KI. Hypercoagulability and thrombotic complications in hemolytic anemias. Haematologica 2009; 94 (11) 1481-1484
    CrossrefPubMedGoogle Scholar
  • 2 Brodsky RA. Complement in hemolytic anemia. Hematology (Am Soc Hematol Educ Program) 2015; 2015: 385-391
    CrossrefPubMedGoogle Scholar
  • 3 Rother RP, Bell L, Hillmen P, Gladwin MT. The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease. JAMA 2005; 293 (13) 1653-1662
    CrossrefPubMedGoogle Scholar
  • 4 Mendonça R, Silveira AA, Conran N. Red cell DAMPs and inflammation. Inflamm Res 2016; 65 (09) 665-678
    CrossrefPubMedGoogle Scholar
  • 5 Barcellini W, Fattizzo B, Zaninoni A. et al. Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients. Blood 2014; 124 (19) 2930-2936
    CrossrefPubMedGoogle Scholar
  • 6 Hillmen P, Muus P, Dührsen U. et al. Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal nocturnal hemoglobinuria. Blood 2007; 110 (12) 4123-4128
    CrossrefPubMedGoogle Scholar
  • 7 Oikonomopoulou K, Ricklin D, Ward PA, Lambris JD. Interactions between coagulation and complement--their role in inflammation. Semin Immunopathol 2012; 34 (01) 151-165
    CrossrefPubMedGoogle Scholar
  • 8 Wouters D, Zeerleder S. Complement inhibitors to treat IgM-mediated autoimmune hemolysis. Haematologica 2015; 100 (11) 1388-1395
    CrossrefPubMedGoogle Scholar
  • 9 Schaer DJ, Buehler PW, Alayash AI, Belcher JD, Vercellotti GM. Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood 2013; 121 (08) 1276-1284
    CrossrefPubMedGoogle Scholar
  • 10 Gozzelino R, Jeney V, Soares MP. Mechanisms of cell protection by heme oxygenase-1. Annu Rev Pharmacol Toxicol 2010; 50: 323-354
    CrossrefPubMedGoogle Scholar
  • 11 Parkkinen J, von Bonsdorff L, Ebeling F, Sahlstedt L. Function and therapeutic development of apotransferrin. Vox Sang 2002; 83 (Suppl. 01) 321-326
    CrossrefPubMedGoogle Scholar
  • 12 Knovich MA, Storey JA, Coffman LG, Torti SV, Torti FM. Ferritin for the clinician. Blood Rev 2009; 23 (03) 95-104
    CrossrefPubMedGoogle Scholar
  • 13 Muller-Eberhard U, Javid J, Liem HH, Hanstein A, Hanna M. Plasma concentrations of hemopexin, haptoglobin and heme in patients with various hemolytic diseases. Blood 1968; 32 (05) 811-815
    CrossrefPubMedGoogle Scholar
  • 14 Santiago RP, Guarda CC, Figueiredo CVB. et al. Serum haptoglobin and hemopexin levels are depleted in pediatric sickle cell disease patients. Blood Cells Mol Dis 2018; 72: 34-36
    CrossrefPubMedGoogle Scholar
  • 15 Fibach E, Rachmilewitz EA. Iron overload in hematological disorders. Presse Med 2017; 46 (12 Pt 2): e296-e305
    CrossrefPubMedGoogle Scholar
  • 16 Fibach E, Dana M. Oxidative stress in paroxysmal nocturnal hemoglobinuria and other conditions of complement-mediated hemolysis. Free Radic Biol Med 2015; 88 (Pt A): 63-69
    CrossrefPubMedGoogle Scholar
  • 17 Alaarg A, Schiffelers RM, van Solinge WW, van Wijk R. Red blood cell vesiculation in hereditary hemolytic anemia. Front Physiol 2013; 4: 365
    CrossrefPubMedGoogle Scholar
  • 18 Hugel B, Socié G, Vu T. et al. Elevated levels of circulating procoagulant microparticles in patients with paroxysmal nocturnal hemoglobinuria and aplastic anemia. Blood 1999; 93 (10) 3451-3456
    CrossrefPubMedGoogle Scholar
  • 19 Connor J, Pak CC, Schroit AJ. Exposure of phosphatidylserine in the outer leaflet of human red blood cells. Relationship to cell density, cell age, and clearance by mononuclear cells. J Biol Chem 1994; 269 (04) 2399-2404
    CrossrefPubMedGoogle Scholar
  • 20 Kozuma Y, Sawahata Y, Takei Y, Chiba S, Ninomiya H. Procoagulant properties of microparticles released from red blood cells in paroxysmal nocturnal haemoglobinuria. Br J Haematol 2011; 152 (05) 631-639
    CrossrefPubMedGoogle Scholar
  • 21 Ninomiya H, Kawashima Y, Hasegawa Y, Nagasawa T. Complement-induced procoagulant alteration of red blood cell membranes with microvesicle formation in paroxysmal nocturnal haemoglobinuria (PNH): implication for thrombogenesis in PNH. Br J Haematol 1999; 106 (01) 224-231
    CrossrefPubMedGoogle Scholar
  • 22 Brummel-Ziedins KE, Vossen CY, Butenas S, Mann KG, Rosendaal FR. Thrombin generation profiles in deep venous thrombosis. J Thromb Haemost 2005; 3 (11) 2497-2505
    CrossrefPubMedGoogle Scholar
  • 23 Smith SA, Travers RJ, Morrissey JH. How it all starts: initiation of the clotting cascade. Crit Rev Biochem Mol Biol 2015; 50 (04) 326-336
    CrossrefPubMedGoogle Scholar
  • 24 Renné T, Gailani D. Role of factor XII in hemostasis and thrombosis: clinical implications. Expert Rev Cardiovasc Ther 2007; 5 (04) 733-741
    CrossrefPubMedGoogle Scholar
  • 25 Noubouossie DF, Henderson MW, Mooberry M. et al. Red blood cell microvesicles activate the contact system, leading to factor IX activation via 2 independent pathways. Blood 2020; 135 (10) 755-765
    CrossrefPubMedGoogle Scholar
  • 26 Van Der Meijden PE, Van Schilfgaarde M, Van Oerle R, Renné T, ten Cate H, Spronk HM. Platelet- and erythrocyte-derived microparticles trigger thrombin generation via factor XIIa. J Thromb Haemost 2012; 10 (07) 1355-1362
    CrossrefPubMedGoogle Scholar
  • 27 Rubin O, Delobel J, Prudent M. et al. Red blood cell-derived microparticles isolated from blood units initiate and propagate thrombin generation. Transfusion 2013; 53 (08) 1744-1754
    CrossrefPubMedGoogle Scholar
  • 28 Baas I, Delvasto-Nuñez L, Ligthart P. et al. Complement C3 inhibition by compstatin Cp40 prevents intra- and extravascular hemolysis of red blood cells. Haematologica 2020; 105 (02) e57-e60
    CrossrefPubMedGoogle Scholar
  • 29 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33 (01) 4-15
    CrossrefPubMedGoogle Scholar
  • 30 Dinkelaar J, Molenaar PJ, Ninivaggi M, de Laat B, Brinkman HJ, Leyte A. In vitro assessment, using thrombin generation, of the applicability of prothrombin complex concentrate as an antidote for Rivaroxaban. J Thromb Haemost 2013; 11 (06) 1111-1118
    CrossrefPubMedGoogle Scholar
  • 31 Herczenik E, van Haren SD, Wroblewska A. et al. Uptake of blood coagulation factor VIII by dendritic cells is mediated via its C1 domain. J Allergy Clin Immunol 2012; 129 (02) 501-509
    CrossrefPubMedGoogle Scholar
  • 32 Bakhtiari K, Meijers JCM. In vitro evaluation of factor IX as novel treatment for factor XI deficiency. Blood 2019; 134 (06) 573-575
    CrossrefPubMedGoogle Scholar
  • 33 Josso F, Prou-Wartelle O. Interaction of tissue factor and factor VII at the earliest phase of coagulation. Thromb Diath Haemorrh Suppl 1965; 17: 35-44
    PubMedGoogle Scholar
  • 34 Visser M, van Oerle R, Ten Cate H. et al. Plasma kallikrein contributes to coagulation in the absence of factor XI by activating factor IX. Arterioscler Thromb Vasc Biol 2020; 40 (01) 103-111
    CrossrefPubMedGoogle Scholar
  • 35 Jeney V, Balla J, Yachie A. et al. Pro-oxidant and cytotoxic effects of circulating heme. Blood 2002; 100 (03) 879-887
    CrossrefPubMedGoogle Scholar
  • 36 Omodeo Salè F, Vanzulli E, Caielli S, Taramelli D. Regulation of human erythrocyte glyceraldehyde-3-phosphate dehydrogenase by ferriprotoporphyrin IX. FEBS Lett 2005; 579 (22) 5095-5099
    CrossrefPubMedGoogle Scholar
  • 37 Chu H, McKenna MM, Krump NA. et al. Reversible binding of hemoglobin to band 3 constitutes the molecular switch that mediates O2 regulation of erythrocyte properties. Blood 2016; 128 (23) 2708-2716
    CrossrefPubMedGoogle Scholar
  • 38 Olivieri NF, Brittenham GM. Iron-chelating therapy and the treatment of thalassemia. [published correction appears in Blood 1997 Apr 1;89(7):2621] Blood 1997; 89 (03) 739-761
    CrossrefPubMedGoogle Scholar
  • 39 Dacaranhe CD, Terao J. A unique antioxidant activity of phosphatidylserine on iron-induced lipid peroxidation of phospholipid bilayers. Lipids 2001; 36 (10) 1105-1110
    CrossrefPubMedGoogle Scholar
  • 40 Yang A, Chen F, He C. et al. The procoagulant activity of apoptotic cells is mediated by interaction with factor XII. Front Immunol 2017; 8: 1188
    CrossrefPubMedGoogle Scholar
  • 41 Majumder R, Quinn-Allen MA, Kane WH, Lentz BR. A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex. Blood 2008; 112 (07) 2795-2802
    CrossrefPubMedGoogle Scholar
  • 42 van Dieijen G, Tans G, Rosing J, Hemker HC. The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem 1981; 256 (07) 3433-3442
    CrossrefPubMedGoogle Scholar
  • 43 Pasini EM, Kirkegaard M, Mortensen P, Lutz HU, Thomas AW, Mann M. In-depth analysis of the membrane and cytosolic proteome of red blood cells. Blood 2006; 108 (03) 791-801
    CrossrefPubMedGoogle Scholar
  • 44 Esposito BP, Breuer W, Sirankapracha P, Pootrakul P, Hershko C, Cabantchik ZI. Labile plasma iron in iron overload: redox activity and susceptibility to chelation. Blood 2003; 102 (07) 2670-2677
    CrossrefPubMedGoogle Scholar
  • 45 Lipinski B, Pretorius E, Oberholzer HM, Van Der Spuy WJ. Iron enhances generation of fibrin fibers in human blood: implications for pathogenesis of stroke. Microsc Res Tech 2012; 75 (09) 1185-1190
    CrossrefPubMedGoogle Scholar
  • 46 Barr JD, Chauhan AK, Schaeffer GV, Hansen JK, Motto DG. Red blood cells mediate the onset of thrombosis in the ferric chloride murine model. Blood 2013; 121 (18) 3733-3741
    CrossrefPubMedGoogle Scholar
  • 47 Lauder SN, Allen-Redpath K, Slatter DA. et al. Networks of enzymatically oxidized membrane lipids support calcium-dependent coagulation factor binding to maintain hemostasis. Sci Signal 2017; 10 (507) 2787
    CrossrefPubMedGoogle Scholar
  • 48 Dixon S, Stockwell B. The hallmarks of ferroptosis. Annu Rev Cancer Biol 2019; 3 DOI: 10.1146/annurev-cancerbio-030518-055844.
    CrossrefPubMedGoogle Scholar
  • 49 Carlsen C, Møller J, Skibsted L. Heme-iron in lipid oxidation. Coord Chem Rev 2005; 249: 485-498
    CrossrefPubMedGoogle Scholar
  • 50 Prus E, Fibach E. The labile iron pool in human erythroid cells. Br J Haematol 2008; 142 (02) 301-307
    CrossrefPubMedGoogle Scholar
  • 51 Sparkenbaugh EM, Chantrathammachart P, Wang S. et al. Excess of heme induces tissue factor-dependent activation of coagulation in mice. Haematologica 2015; 100 (03) 308-314
    CrossrefPubMedGoogle Scholar