A high-fat diet delays plasmin generation in a thrombomodulin...

THROMBOSIS AND HEMOSTASIS| MAY 7, 2020

A high-fat diet delays plasmin generation in a thrombomodulin-dependent manner in mice

Adam Miszta, Anna K. Kopec, Asmita Pant, Lori A. Holle, James R. Byrnes, Daniel A. Lawrence, Kirk C. Hansen, Matthew J. Flick, James P. Luyendyk, Bas de Laat, Alisa S. Wolberg

Blood (2020) 135 (19): 1704–1717.

https://doi.org/10.1182/blood.2019004267

Key Points

A novel assay reveals delayed plasmin generation in plasma from mice fed a high-fat diet.

Proteomic and functional analyses suggest delayed plasmin generation results from a thrombomodulin- and TAFI-dependent mechanism.

Abstract

Obesity is a prevalent prothrombotic risk factor marked by enhanced fibrin formation and suppressed fibrinolysis. Fibrin both promotes thrombotic events and drives obesity pathophysiology, but a lack of essential analytical tools has left fibrinolytic mechanisms affected by obesity poorly defined. Using a plasmin-specific fluorogenic substrate, we developed a plasmin generation (PG) assay for mouse plasma that is sensitive to tissue plasminogen activator, α2-antiplasmin, active plasminogen activator inhibitor (PAI-1), and fibrin formation, but not fibrin crosslinking. Compared with plasmas from mice fed a control diet, plasmas from mice fed a high-fat diet (HFD) showed delayed PG and reduced PG velocity. Concurrent to impaired PG, HFD also enhanced thrombin generation (TG). The collective impact of abnormal TG and PG in HFD-fed mice produced normal fibrin formation kinetics but delayed fibrinolysis. Functional and proteomic analyses determined that delayed PG in HFD-fed mice was not due to altered levels of plasminogen, α2-antiplasmin, or fibrinogen. Changes in PG were also not explained by elevated PAI-1 because active PAI-1 concentrations required to inhibit the PG assay were 100-fold higher than circulating concentrations in mice. HFD-fed mice had increased circulating thrombomodulin, and inhibiting thrombomodulin or thrombin-activatable fibrinolysis inhibitor (TAFI) normalized PG, revealing a thrombomodulin- and TAFI-dependent antifibrinolytic mechanism. Integrating kinetic parameters to calculate the metric of TG/PG ratio revealed a quantifiable net shift toward a prothrombotic phenotype in HFD-fed mice. Integrating TG and PG measurements may define a prothrombotic risk factor in diet-induced obesity.

Subjects:

Thrombosis and Hemostasis

Topics:

alteplase, diet, high-fat, fibrin, mice, obesity, plasmin, thrombin, thrombomodulin, plasma, carboxypeptidase u

REFERENCES

1.Blüher M. Obesity: global epidemiology and pathogenesis. Nat Rev Endocrinol. 2019;15(5):288-298.

2.Targher G, Bertolini L, Padovani R, et al. Prevalence of nonalcoholic fatty liver disease and its association with cardiovascular disease among type 2 diabetic patients. Diabetes Care. 2007;30(5):1212-1218.

3.Kernan WN, Inzucchi SE, Sawan C, Macko RF, Furie KL. Obesity: a stubbornly obvious target for stroke prevention. Stroke. 2013;44(1):278-286.

4.Abdollahi M, Cushman M, Rosendaal FR. Obesity: risk of venous thrombosis and the interaction with coagulation factor levels and oral contraceptive use. Thromb Haemost. 2003;89(3):493-498.

5.Mitchell AB, Cole JW, McArdle PF, et al. Obesity increases risk of ischemic stroke in young adults. Stroke. 2015;46(6):1690-1692.

6.Mertens I, Van Gaal LF. Obesity, haemostasis and the fibrinolytic system. Obes Rev. 2002;3(2):85-101.

7.Kotronen A, Joutsi-Korhonen L, Sevastianova K, et al. Increased coagulation factor VIII, IX, XI and XII activities in non-alcoholic fatty liver disease. Liver Int. 2011;31(2):176-183.

8.Kaye SM, Pietiläinen KH, Kotronen A, et al. Obesity-related derangements of coagulation and fibrinolysis: a study of obesity-discordant monozygotic twin pairs. Obesity (Silver Spring). 2012;20(1):88-94.

9.Kopec AK, Abrahams SR, Thornton S, et al. Thrombin promotes diet-induced obesity through fibrin-driven inflammation. J Clin Invest. 2017;127(8):3152-3166.

10.Geyer PE, Wewer Albrechtsen NJ, Tyanova S, et al. Proteomics reveals the effects of sustained weight loss on the human plasma proteome. Mol Syst Biol. 2016;12(12):901-917.

11.Vilahur G, Ben-Aicha S, Badimon L. New insights into the role of adipose tissue in thrombosis. Cardiovasc Res. 2017;113(9):1046-1054.

12.Tripodi A. Thrombin generation assay and its application in the clinical laboratory. Clin Chem. 2016;62(5):699-707.

13.Ay L, Kopp H-P, Brix J-M, et al. Thrombin generation in morbid obesity: significant reduction after weight loss. J Thromb Haemost. 2010;8(4):759-765.

14.Chitongo PB, Roberts LN, Yang L, et al. Visceral adiposity is an independent determinant of hypercoagulability as measured by thrombin generation in morbid obesity. TH Open. 2017;1(2):e146-e154.

15.Targher G, Bertolini L, Scala L, et al. Plasma PAI-1 levels are increased in patients with nonalcoholic steatohepatitis. Diabetes Care. 2007;30(5):e31-e32.

16.Lijnen HR. Role of fibrinolysis in obesity and thrombosis. Thromb Res. 2009;123(Suppl 4):S46-S49.

17.Juhan-Vague I, Alessi MC. PAI-1, obesity, insulin resistance and risk of cardiovascular events. Thromb Haemost. 1997;78(1):656-660.

18.Hori Y, Gabazza EC, Yano Y, et al. Insulin resistance is associated with increased circulating level of thrombin-activatable fibrinolysis inhibitor in type 2 diabetic patients. J Clin Endocrinol Metab. 2002;87(2):660-665.

19.Ma L-J, Mao S-L, Taylor KL, et al. Prevention of obesity and insulin resistance in mice lacking plasminogen activator inhibitor 1. Diabetes. 2004;53(2):336-346.

20.Balagopal P, George D, Sweeten S, et al. Response of fractional synthesis rate (FSR) of fibrinogen, concentration of D-dimer and fibrinolytic balance to physical activity-based intervention in obese children. J Thromb Haemost. 2008;6(8):1296-1303.

21.van Geffen M, Loof A, Lap P, et al. A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis. Hematology. 2011;16(6):327-336.

22.Matsumoto T, Nogami K, Shima M. Simultaneous measurement of thrombin and plasmin generation to assess the interplay between coagulation and fibrinolysis. Thromb Haemost. 2013;110(4):761-768.

23.Simpson ML, Goldenberg NA, Jacobson LJ, Bombardier CG, Hathaway WE, Manco-Johnson MJ. Simultaneous thrombin and plasmin generation capacities in normal and abnormal states of coagulation and fibrinolysis in children and adults. Thromb Res. 2011;127(4):317-323.

24.Tchaikovski SN, VAN Vlijmen BJM, Rosing J, Tans G. Development of a calibrated automated thrombography based thrombin generation test in mouse plasma. J Thromb Haemost. 2007;5(10):2079-2086.

25.Bloemen S, Huskens D, Konings J, et al. Interindividual variability and normal ranges of whole blood and plasma thrombin generation. J Appl Lab Med. 2017;2(2):150-164.

26.Honjo K, Munakata S, Tashiro Y, et al. Plasminogen activator inhibitor-1 regulates macrophage-dependent postoperative adhesion by enhancing EGF-HER1 signaling in mice. FASEB J. 2017;31(6):2625-2637.

27.Zhou H, Wu X, Lu X, Chen G, Ye X, Huang J. Evaluation of plasma urokinase-type plasminogen activator and urokinase-type plasminogen-activator receptor in patients with acute and chronic hepatitis B. Thromb Res. 2009;123(3):537-542.

28.Pieters M, Wolberg AS. Fibrinogen and fibrin: an illustrated review. Res Pract Thromb Haemost. 2019;3(2):161-172.

29.Medved L, Nieuwenhuizen W. Molecular mechanisms of initiation of fibrinolysis by fibrin. Thromb Haemost. 2003;89(3):409-419.

30.Prasad JM, Gorkun OV, Raghu H, et al. Mice expressing a mutant form of fibrinogen that cannot support fibrin formation exhibit compromised antimicrobial host defense. Blood. 2015;126(17):2047-2058.

31.Wolberg AS. Thrombin generation and fibrin clot structure. Blood Rev. 2007;21(3):131-142.

32.Bannish BE, Chernysh IN, Keener JP, Fogelson AL, Weisel JW. Molecular and physical mechanisms of fibrinolysis and thrombolysis from mathematical modeling and experiments. Sci Rep. 2017;7(1):6914-6925.

33.Wolberg AS, Aleman MM, Leiderman K, Machlus KR. Procoagulant activity in hemostasis and thrombosis: Virchow’s triad revisited. Anesth Analg. 2012;114(2):275-285.

34.Nagai N, Hoylaerts MF, Cleuren ACA, Van Vlijmen BJM, Lijnen HR. Obesity promotes injury induced femoral artery thrombosis in mice. Thromb Res. 2008;122(4):549-555.

35.Hekman CM, Loskutoff DJ. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem. 1985;260(21):11581-11587.

36.Brown EW, Ravindran S, Patston PA. The reaction between plasmin and C1-inhibitor results in plasmin inhibition by the serpin mechanism. Blood Coagul Fibrinolysis. 2002;13(8):711-714.

37.Wang W, Boffa MB, Bajzar L, Walker JB, Nesheim ME. A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor. J Biol Chem. 1998;273(42):27176-27181.

38.Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem. 1996;271(28):16603-16608.

39.Boffa MB, Wang W, Bajzar L, Nesheim ME. Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties. J Biol Chem. 1998;273(4):2127-2135.

40.Wang W, Nagashima M, Schneider M, Morser J, Nesheim M. Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation. J Biol Chem. 2000;275(30):22942-22947.

41.Dharmasaroja P, Dharmasaroja PA, Sobhon P. Increased plasma soluble thrombomodulin levels in cardioembolic stroke. Clin Appl Thromb Hemost. 2012;18(3):289-293.

42.Lin S-M, Wang Y-M, Lin H-C, et al. Serum thrombomodulin level relates to the clinical course of disseminated intravascular coagulation, multiorgan dysfunction syndrome, and mortality in patients with sepsis. Crit Care Med. 2008;36(3):683-689.

43.Porreca E, Di Febbo C, Fusco L, Moretta V, Di Nisio M, Cuccurullo F. Soluble thrombomodulin and vascular adhesion molecule-1 are associated to leptin plasma levels in obese women. Atherosclerosis. 2004;172(1):175-180.

44.Rega-Kaun G, Kaun C, Ebenbauer B, et al. Bariatric surgery in morbidly obese individuals affects plasma levels of protein C and thrombomodulin. J Thromb Thrombolysis. 2019;47(1):51-56.

45.Pawlak K, Myśliwiec M, Pawlak D. Kynurenine pathway - a new link between endothelial dysfunction and carotid atherosclerosis in chronic kidney disease patients. Adv Med Sci. 2010;55(2):196-203.

46.Weiler-Guettler H, Christie PD, Beeler DL, et al. A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state. J Clin Invest. 1998;101(9):1983-1991.

47.Lopez-Ramirez MA, Pham A, Girard R, et al. Cerebral cavernous malformations form an anticoagulant vascular domain in humans and mice. Blood. 2019;133(3):193-204.

48.Mosnier LO, Meijers JCM, Bouma BN. Regulation of fibrinolysis in plasma by TAFI and protein C is dependent on the concentration of thrombomodulin. Thromb Haemost. 2001;85(1):5-11.

49.Leung LLK, Morser J. Carboxypeptidase B2 and carboxypeptidase N in the crosstalk between coagulation, thrombosis, inflammation, and innate immunity. J Thromb Haemost. 2018;16(8):1474-1486.

50.Semeraro F, Giordano P, Faienza MF, Cavallo L, Semeraro N, Colucci M. Evidence that fibrinolytic changes in paediatric obesity translate into a hypofibrinolytic state: relative contribution of TAFI and PAI-1. Thromb Haemost. 2012;108(2):311-317.

51.Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest. 1985;76(6):2178-2181.

52.Suzuki K, Nishioka J, Hayashi T, Kosaka Y. Functionally active thrombomodulin is present in human platelets. J Biochem. 1988;104(4):628-632.

53.Dittman WA, Majerus PW. Structure and function of thrombomodulin: a natural anticoagulant. Blood. 1990;75(2):329-336.

54.Conway EM, Nowakowski B, Steiner-Mosonyi M. Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation. Blood. 1992;80(5):1254-1263.

55.Nawroth PP, Handley DA, Esmon CT, Stern DM. Interleukin 1 induces endothelial cell procoagulant while suppressing cell-surface anticoagulant activity. Proc Natl Acad Sci USA. 1986;83(10):3460-3464.

56.Nawroth PP, Stern DM. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med. 1986;163(3):740-745.

57.Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science. 1993;259(5091):87-91.

58.Boehme MW, Deng Y, Raeth U, et al. Release of thrombomodulin from endothelial cells by concerted action of TNF-alpha and neutrophils: in vivo and in vitro studies. Immunology. 1996;87(1):134-140.

59.Maruyama I, Nakata M, Yamaji K. Effect of leptin in platelet and endothelial cells. Obesity and arterial thrombosis. Ann N Y Acad Sci. 2000;902(1):315-319.

60.Sohn RH, Deming CB, Johns DC, et al. Regulation of endothelial thrombomodulin expression by inflammatory cytokines is mediated by activation of nuclear factor-kappa B. Blood. 2005;105(10):3910-3917.

61.Boehme MWJ, Galle P, Stremmel W. Kinetics of thrombomodulin release and endothelial cell injury by neutrophil-derived proteases and oxygen radicals. Immunology. 2002;107(3):340-349.

62.Luyendyk JP, Sullivan BP, Guo GL, Wang R. Tissue factor-deficiency and protease activated receptor-1-deficiency reduce inflammation elicited by diet-induced steatohepatitis in mice. Am J Pathol. 2010;176(1):177-186.

63.Lijnen HR, Maquoi E, Morange P, et al. Nutritionally induced obesity is attenuated in transgenic mice overexpressing plasminogen activator inhibitor-1. Arterioscler Thromb Vasc Biol. 2003;23(1):78-84.

64.Mosnier LO, von dem Borne PAK, Meijers JCM, Bouma BN. Plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation. Thromb Haemost. 1998;80(5):829-835.

65.van Tilburg NH, Rosendaal FR, Bertina RM. Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis. Blood. 2000;95(9):2855-2859.

66.Juhan-Vague I, Morange PE, Aubert H, et al; HIFMECH Study Group. Plasma thrombin-activatable fibrinolysis inhibitor antigen concentration and genotype in relation to myocardial infarction in the north and south of Europe. Arterioscler Thromb Vasc Biol. 2002;22(5):867-873.

67.Meltzer ME, Lisman T, de Groot PG, et al. Venous thrombosis risk associated with plasma hypofibrinolysis is explained by elevated plasma levels of TAFI and PAI-1. Blood. 2010;116(1):113-121.

68.Marx PF, Wagenaar GTM, Reijerkerk A, et al. Characterization of mouse thrombin-activatable fibrinolysis inhibitor. Thromb Haemost. 2000;83(2):297-303.

69.Gils A, Ceresa E, Macovei AM, et al. Modulation of TAFI function through different pathways--implications for the development of TAFI inhibitors. J Thromb Haemost. 2005;3(12):2745-2753.

70.Hillmayer K, Macovei A, Pauwels D, Compernolle G, Declerck PJ, Gils A. Characterization of rat thrombin-activatable fibrinolysis inhibitor (TAFI)--a comparative study assessing the biological equivalence of rat, murine and human TAFI. J Thromb Haemost. 2006;4(11):2470-2477.

71.Wang X, Smith PL, Hsu M-Y, Ogletree ML, Schumacher WA. Murine model of ferric chloride-induced vena cava thrombosis: evidence for effect of potato carboxypeptidase inhibitor. J Thromb Haemost. 2006;4(2):403-410.

72.Wang X, Smith PL, Hsu M-Y, Tamasi JA, Bird E, Schumacher WA. Deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI) protected mice from ferric chloride-induced vena cava thrombosis. J Thromb Thrombolysis. 2007;23(1):41-49.

73.Nagashima M, Werner M, Wang M, et al. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor potentiates tissue-type plasminogen activator-induced thrombolysis in a rabbit jugular vein thrombolysis model. Thromb Res. 2000;98(4):333-342.

74.Nagashima M, Yin ZF, Zhao L, et al. Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible with murine life. J Clin Invest. 2002;109(1):101-110.

75.Binette TM, Taylor FB Jr., Peer G, Bajzar L. Thrombin-thrombomodulin connects coagulation and fibrinolysis: more than an in vitro phenomenon. Blood. 2007;110(9):3168-3175.

76.Hemker HC, Kremers R. Data management in thrombin generation. Thromb Res. 2013;131(1):3-11.

77.De Smedt E, Wagenvoord R, Coen Hemker H. The technique of measuring thrombin generation with fluorogenic substrates: 3. The effects of sample dilution. Thromb Haemost. 2009;101(1):165-170.

78.Lijnen HR, van Hoef B, Beelen V, Collen D. Characterization of the murine plasma fibrinolytic system. Eur J Biochem. 1994;224(3):863-871.

79.Svensson J, Bergman A-C, Adamson U, Blombäck M, Wallén H, Jörneskog G. Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: a mass spectrometric and isotope labeling study. Biochem Biophys Res Commun. 2012;421(2):335-342.

80.Ajjan RA, Gamlen T, Standeven KF, et al. Diabetes is associated with posttranslational modifications in plasminogen resulting in reduced plasmin generation and enzyme-specific activity. Blood. 2013;122(1):134-142.

81.Gugliucci A, Menini T, Stahl AJC. Glycation of fibrinogen in diabetic patients: a practical colorimetric assay. Glycosyl Dis. 1994;1(3):177-183.

82.Pieters M, van Zyl DG, Rheeder P, et al. Glycation of fibrinogen in uncontrolled diabetic patients and the effects of glycaemic control on fibrinogen glycation. Thromb Res. 2007;120(3):439-446.

83.Lisman T, Stravitz RT. Rebalanced hemostasis in patients with acute liver failure. Semin Thromb Hemost. 2015;41(5):468-473.

© 2020 by The American Society of Hematology

This program is developed by Focus Insight with the permission of American Society of Hematology, Inc. The content are excerpted from the journal Blood. Copyright © 2019 The American Society of Hematology. All rights reserved. 「American Society of Hematology」, 「ASH」 and the ASH Logo are registered trademarks of the American Society of Hematology.