Human megakaryocytes confer tissue factor to a subset of shed platelets to stimulate thrombin generation

Summary

Tissue factor (TF), the main activator of the blood coagulation cascade, has been shown to be expressed by platelets. Despite the evidence that both megakaryocytes and platelets express TF mRNA, and that platelets can make de novo protein synthesis, the main mechanism thought to be responsible for the presence of TF within platelets is through the uptake of TF positive microparticles. In this study we assessed 1) whether human megakaryocytes synthesise TF and transfer it to platelets and 2) the contribution of platelet-TF to the platelet hemostatic capacity. In order to avoid the cross-talk with circulating microparticles, we took advantage from an in vitro cultured megakaryoblastic cell line (Meg-01) able to differentiate into megakaryocytes releasing platelet-like particles. We show that functionally active TF is expressed in human megakaryoblasts, increased in megakaryocytes, and is transferred to a subset of shed platelets where it contributes to clot formation. These data were all confirmed in human CD34^pos- derived megakaryocytes and in their released platelets. The effect of TF silencing in Meg-megakaryoblasts resulted in a significant reduction of TF expression in these cells and also in Meg-megakaryocytes and Meg-platelets. Moreover, the contribution of platelet-TF to the platelet hemostatic capacity was highlighted by the significant delay in the kinetic of thrombin formation observed in platelets released by TF-silenced megakaryocytes. These findings provide evidences that TF is an endogenously synthesised protein that characterises megakaryocyte maturation and that it is transferred to a subset of newly-released platelets where it is functionally active and able to trigger thrombin generation.

Keywords

Megakaryocytes - platelets - tissue factor - thrombin generation

^* These authors contributed equally to this article.

• References

• 1 Mallat Z, Benamer H, Hugel B. et al. Elevated levels of shed membrane micro-particles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation 2000; 101: 841-843.
• 2 Nieuwland R, Berckmans RJ, McGregor S. et al. Cellular origin and procoagulant properties of microparticles in meningococcal sepsis. Blood 2000; 95: 930-935.
• 3 Hughes M, Hayward CP, Warkentin TE. et al. Morphological analysis of micro-particle generation in heparin-induced thrombocytopenia. Blood 2000; 96: 188-194.
• 4 Giesen PL, Rauch U, Bohrmann B. et al. Blood-borne tissue factor: another view of thrombosis. Proc Natl Acad Sci USA 1999; 96: 2311-2315.
• 5 Muller I, Klocke A, Alex M. et al. Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets. FASEB J 2003; 17: 476-478.
• 6 Del Conde I, Shrimpton CN, Thiagarajan P. et al. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005; 106: 1604-1611.
• 7 Zillmann A, Luther T, Muller I. et al. Platelet-associated tissue factor contributes to the collagen-triggered activation of blood coagulation. Biochem Biophys Res Commun 2001; 281: 603-609.
• 8 Siddiqui FA, Desai H, Amirkhosravi A. et al. The presence and release of tissue factor from human platelets. Platelets 2002; 13: 247-253.
• 9 Camera M, Frigerio M, Toschi V. et al. Platelet activation induces cell-surface immunoreactive tissue factor expression, which is modulated differently by antiplatelet drugs. Arterioscl Thromb Vasc Biol 2003; 23: 1690-1696.
• 10 Schwertz H, Tolley ND, Foulks JM. et al. Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenicity of human platelets. J Exp Med 2006; 203: 2433-2440.
• 11 Panes O, Matus V, Saez CG. et al. Human platelets synthesize and express functional tissue factor. Blood 2007; 109: 5242-5250.
• 12 Brambilla M, Camera M, Colnago D. et al. Tissue factor in patients with acute coronary syndromes: expression in platelets, leukocytes, and platelet-leukocyte aggregates. Arterioscl Thromb Vasc Biol 2008; 28: 947-953.
• 13 Gerrits AJ, Koekman CA, van Haeften TW. et al. Platelet tissue factor synthesis in type 2 diabetic patients is resistant to inhibition by insulin. Diabetes 2010; 59: 1487-1495.
• 14 Falanga A, Marchetti M, Vignoli A. et al. V617F JAK-2 mutation in patients with essential thrombocythemia: relation to platelet, granulocyte, and plasma hemostatic and inflammatory molecules. Exp Hematol 2007; 35: 702-711.
• 15 Tilley RE, Holscher T, Belani R. et al. Tissue factor activity is increased in a combined platelet and microparticle sample from cancer patients. Thromb Res 2008; 122: 604-609.
• 16 Ogura M, Morishima Y, Ohno R. et al. Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. Blood 1985; 66: 1384-1392.
• 17 Balduini A, Pallotta I, Malara A. et al. Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes. J Thromb Haemost 2008; 06: 1900-1907.
• 18 Schweinfurth N, Hohmann S, Deuschle M. et al. Valproic acid and all trans retinoic acid differentially induce megakaryopoiesis and platelet-like particle formation from the megakaryoblastic cell line MEG-01. Platelets 2010; 21: 648-657.
• 19 Ollivier V, Wang J, Manly D. et al. Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay. Thromb Res 2010; 125: 90-96.
• 20 Mallat Z, Hugel B, Ohan J. et al. Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: a role for apoptosis in plaque thrombogenicity. Circulation 1999; 99: 348-353.
• 21 Soejima H, Ogawa H, Yasue H. et al. Heightened tissue factor associated with tissue factor pathway inhibitor and prognosis in patients with unstable angina. Circulation 1999; 99: 2908-2913.
• 22 Flaumenhaft R, Dilks JR, Richardson J. et al. Megakaryocyte-derived microparticles: direct visualisation and distinction from platelet-derived microparticles. Blood 2009; 113: 1112-1121.
• 23 Camera M, Brambilla M, Toschi V. et al. Tissue factor expression on platelets is a dynamic event. Blood 2010; 116: 5076-5077.
• 24 Camera M, Brambilla M, Boselli D. et al. Functionally active platelets do express tissue factor. Blood 2012; 119: 4339-4341.
• 25 Mackman N, Luther T. Platelet tissue factor: To be or not to be. Thromb Res 2013; 132: 3-5.
• 26 Bouchard BA, Mann KG, Butenas S. No evidence for tissue factor on platelets. Blood 2010; 116: 854-855.
• 27 Bouchard BA, Krudysz-Amblo J, Butenas S. Platelet tissue factor is not expressed transiently after platelet activation. Blood 2012; 119: 4338-4339 author reply 4339–4341.
• 28 Bouchard BA, Gissel MT, Whelihan MF. et al. Platelets do not express the oxidized or reduced forms of tissue factor. Biochim Biophys Acta 2014; 1840: 1188-1193.
• 29 Osterud B, Olsen JO. Human platelets do not express tissue factor. Thromb Res 2013; 132: 112-115.
• 30 Gnatenko DV, Dunn JJ, McCorkle SR. et al. Transcript profiling of human platelets using microarray and serial analysis of gene expression. Blood 2003; 101: 2285-2293.
• 31 Weyrich AS, Dixon DA, Pabla R. et al. Signal-dependent translation of a regulatory protein, Bcl-3, in activated human platelets. Proc Natl Acad Sci USA 1998; 95: 5556-5561.
• 32 Lindemann S, Tolley ND, Dixon DA. et al. Activated platelets mediate inflammatory signaling by regulated interleukin 1beta synthesis. J Cell Biol 2001; 154: 485-490.
• 33 Evangelista V, Manarini S, Di Santo A. et al. De novo synthesis of cyclooxygenase-1 counteracts the suppression of platelet thromboxane biosynthesis by aspirin. Circulation Res 2006; 98: 593-595.
• 34 Egorina EM, Sovershaev MA, Bjorkoy G. et al. Intracellular and surface distribution of monocyte tissue factor: application to intersubject variability. Arterioscler Thromb Vasc Biol 2005; 25: 1493-1498.
• 35 Rao LV, Pendurthi UR. Regulation of tissue factor coagulant activity on cell surfaces. J Thromb Haemost 2012; 10: 2242-2253.
• 36 Chen VM, Hogg PJ. Encryption and decryption of tissue factor. J Thromb Hae-most 2013; 11 (Suppl. 01) 277-284.
• 37 Stalker TJ, Traxler EA, Wu J. et al. Hierarchical organisation in the hemostatic response and its relationship to the platelet-signaling network. Blood 2013; 121: 1875-1885.
• 38 Palmerini T, Tomasi L, Barozzi C. et al. Detection of tissue factor antigen and coagulation activity in coronary artery thrombi isolated from patients with ST-segment elevation acute myocardial infarction. PLoS One 2013; 08: e81501.
• 39 Pawlinski R, Wang JG, Owens AP. 3rd, et al. Hematopoietic and nonhematopoietic cell tissue factor activates the coagulation cascade in endotoxemic mice. Blood 2010; 116: 806-814.
• 40 Tyagi T, Ahmad S, Gupta N. et al. Altered expression of platelet proteins and cal-pain activity mediate hypoxia-induced prothrombotic phenotype. Blood 2014; 123: 1250-1260.
• 41 Bugert P, Dugrillon A, Gunaydin A. et al. Messenger RNA profiling of human platelets by microarray hybridisation. Thromb Haemost 2003; 90: 738-748.
• 42 Podmore A, Smith M, Savidge G. et al. Real-time quantitative PCR analysis of factor XI mRNA variants in human platelets. J Thromb Haemost 2004; 02: 1713-1719.
• 43 Rox JM, Bugert P, Muller J. et al. Gene expression analysis in platelets from a single donor: evaluation of a PCR-based amplification technique. Clin Chem 2004; 50: 2271-2278.