Identification of human thrombin-activatable fibrinolysis inhibitor in vascular and inflammatory cells

Joellen H. H. Lin; Mathieu Garand; Branislava Zagorac; Steven L. Schadinger; Corey Scipione; Marlys L. Koschinsky; Michael B. Boffa

doi:10.1160/TH10-06-0413

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2011; 105(06): 999-1009
DOI: 10.1160/TH10-06-0413

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Identification of human thrombin-activatable fibrinolysis inhibitor in vascular and inflammatory cells

Authors

Joellen H. H. Lin

¹Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada
Mathieu Garand

²Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada
Branislava Zagorac

²Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada
Steven L. Schadinger

¹Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada
Corey Scipione

²Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada
Marlys L. Koschinsky

²Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada
Michael B. Boffa

²Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada

Abstract

Summary

TAFI (thrombin-activatable fibrinolysis inhibitor) is a carboxypeptidase zymogen originally identified in plasma. The TAFI pathway helps to regulate the balance between the coagulation and fibrinolytic cascades. Activated TAFI (TAFIa) can also inactivate certain pro-inflammatory mediators, suggesting that the TAFI pathway may also regulate communication between coagulation and inflammation. Expression in the liver is considered to be the source of plasma TAFI. TAFI has also been identified in platelets and CPB2 (the gene encoding TAFI) mRNA has been detected in megakaryocytic cell lines as well as in endothelial cells. We have undertaken a quantitative analysis of CPB2 mRNA and TAFI protein in extrahepatic cell types relevant to vascular disease. Using RT-PCR and quantitative RT-PCR, we detected CPB2 mRNA in the human megakaryoblastic cell lines MEG-01 and Dami, the human monocytoid cell line THP-1 as well as THP-1 cells differentiated into a macrophage-like phenotype, and in primary human umbilical vein and coronary artery endothelial cells. CPB2 mRNA abundance in MEG-01, Dami, and THP-1 cells was modulated by the state of differentiation of these cells. Using a recently developed TAFIa assay, we detected TAFI protein in the lysates of the human hepatocellular carcinoma cell line HepG2 as well as in MEG-01 and Dami cells and in the conditioned medium of HepG2 cells, differentiated Dami cells, and THP-1 macrophages. We have obtained clear evidence for extrahepatic expression of TAFI, which has clear implications for the physiological and pathophysiological functions of the TAFI pathway.

Keywords

Thrombin-activatable fibrinolysis inhibitor (TAFI) - gene expression - megakaryocytes - platelets - monocytes/macrophages

Full Text

References

References
1 Nesheim M. Thrombin and fibrinolysis. Chest 2003; 124: 33S-39S.
2 Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem 1995; 270: 14477-14484.
3 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: 16603-16608.
4 Mao SS, Cooper CM, Wood T. et al. Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans. J Biol Chem 1999; 274: 35046-35052.
5 Wang W, Boffa MB, Bajzar L. et al. A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 1998; 273: 27176-27181.
6 Eaton DL, Malloy BE, Tsai SP. et al. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. J Biol Chem 1991; 266: 21833-21838.
7 Campbell W, Okada H. An arginine specific carboxypeptidase generated in blood during coagulation or inflammation which is unrelated to carboxypeptidase N or its subunits. Biochem Biophys Res Commun 1989; 162: 933-939.
8 Hendriks D, Scharpé S, van Sande M. et al. Characterisation of a carboxypeptidase in human serum distinct from carboxypeptidase N. J Clin Chem Clin Biochem 1989; 27: 277-285.
9 Schneider M, Nesheim M. A study of the protection of plasmin from antiplasmin inhibition within an intact fibrin clot during the course of clot lysis. J Biol Chem 2004; 279: 13333-13339.
10 Schneider M, Brufatto N, Neill E. et al. Activated thrombin-activatable fibrinolysis inhibitor reduces the ability of high molecular weight fibrin degradation products to protect plasmin from antiplasmin. J Biol Chem 2004; 279: 13340-13345.
11 Frère C, Morange PE, Saut N. et al. Quantification of thrombin activatable fibrinolysis inhibitor (TAFI) gene polymorphism effects on plasma levels of TAFI measured with assays insensitive to isoform-dependent artefact. Thromb Haemost 2005; 94: 373-379.
12 van Tilburg NH, Rosendaal FR, Bertina RM. Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis. Blood 2000; 95: 2855-2859.
13 Meltzer ME, Lisman T, de Groot PG. et al. Venous thrombosis risk associated with plasma hypofibirnolysis is explained by elevated plasma levels of TAFI and PAI-1. Blood 2010; 116: 113-121.
14 Eichinger S, Schönauer V, Weltermann A. et al. Thrombin-activatable fibrinolysis inhibitor and the risk for recurrent venous thromboembolism. Blood 2004; 103: 3773-3776.
15 Leebeek FW, Goor MP, Guimaraes AH. et al. High functional levels of thrombin-activatable fibrinolysis inhibitor are associated with an increased risk of first ischemic stroke. J Thromb Haemost 2005; 3: 2211-2218.
16 Santamaría A, Oliver A, Borrell M. et al. Risk of ischemic stroke associated with functional thrombin-activatable fibrinolysis inhibitor plasma levels. Stroke 2003; 34: 2387-2391.
17 Rooth E, Wallen H, Antovic A. et al. Thrombin activatable fibrinolysis inhibitor and its relationship to fibrinolysis and inflammation during the acute and convalescent phase of ischemic stroke. Blood Coag Fibrinolysis 2007; 18: 365-370.
18 Ladenvall C, Gils A, Jood K. et al. Thrombin activatable fibrinolysis inhibitor activation peptide shows association with all major subtypes of ischemic stroke and with TAFI gene variation. Arterioscler Thromb Vasc Biol 2007; 27: 955-962.
19 de Bruijen EL, Gils A, Guimarães AH. et al. The role of thrombin activatable fibrinolysis inhibitor in arterial thrombosis at a young age: the ATTAC study. J Thromb Haemost 2009; 7: 919-927.
20 Trégouet DA, Schnabel R, Alessi MC. et al. Activated thrombin activatable fibrnolysis inhibitor levels are associated with the risk of cardiovascular death in patients with coronary artery disease: the Athero Gene study. J Thromb Haemost 2009; 7: 49-57.
21 Campbell WD, Lazoura E, Okada N. et al. Inactivation of C3a and C5a octapep-tides by carboxypeptidase R and carboxypeptidase N. Microbiol Immunol 2002; 46: 131-134.
22 Hendriks D, Wang W, van Sande M. et al. Human serum carboxypeptidase U: a new kininase?. Agents Actions Suppl 1992; 38: 407-413.
23 Myles T, Nishimura T, Yun TH. et al. Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation. J Biol Chem 2003; 278: 51059-51067.
24 Leung LL, Myles T, Nishimura T. et al. Regulation of tissue inflammation by thrombin-activatable carboxypeptidase B (or TAFI). Mol Immunol 2008; 45: 4080-4083.
25 Du XY, Zabel BA, Myles T. et al. Regulation of chemerin bioactivity by plasma carboxypeptidase N, carboxypeptidase B (activated thrombin-activable fibrinolysis inhibitor), and platelets. J Biol Chem 2009; 284: 751-758.
26 Van Thiel DH, George M, Fareed J. Low levels of thrombin activatable fibrinolysis inhibitor (TAFI) in patients with chronic liver disease. Thromb Haemost 2001; 85: 667-670.
27 Boffa MB, Reid TS, Joo E. et al. Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B). Biochemistry 1999; 38: 6547-6558.
28 Mosnier LO, Buijtenhuijs P, Marx PF. et al. Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets. Blood 2003; 101: 4844-4846.
29 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: 660-665.
30 Aubert H, Frère C, Aillaud MF. et al. Weak and non-independent association between plasma TAFI antigen levels and the insulin resistance syndrome. J Thromb Haemost 2003; 1: 791-797.
31 Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 1980; 209: 497-499.
32 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.
33 Greenberg SM, Rosenthal DS, Greeley TA. et al. Characterization of a new megakaryocytic cell line: the Dami cell. Blood 1988; 72: 1968-1977.
34 Tsuchiya S, Yamabe M, Yamaguchi Y. et al. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 1980; 26: 171-176.
35 Zhao SP, Deng P, Huang H. et al. Expression of COX-2 mRNA in peripheral blood monocytes from patients with acute myocardial infarction and its significance. 2005; Clinical Chemistry 51: 2170-2173.
36 Maret D, Boffa MB, Brien DF. et al. Role of mRNA transcript stability in modulation of expression of the gene encoding thrombin activable fibrinolysis inhibitor. J Thromb Haemost 2004; 2: 1969-1979.
37 Boffa MB, Nesheim ME, Koschinsky ML. Thrombin activable fibrinolysis inhibitor (TAFI): molecular genetics of an emerging potential risk factor for thrombotic disorders. Curr Drug Targets Cardiovasc Haematol Disord 2001; 1: 59-74.
38 Boffa MB, Wang W, Bajzar L. et al. 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: 2127-2135.
39 Gils A, Alessi MC, Brouwers E. et al. Development of a genotype 325-specific proCPU/TAFI ELISA. Arterioscler Thromb Vasc Biol 2003; 23: 1122-1127.
40 Kim PY, Foley J, Hsu G. et al. An assay for measuring functional activated thrombin-activatable fibrinolysis inhibitor in plasma. Anal Biochem 2008; 372: 32-40.
41 Isakari Y, Sogo S, Ishida T. et al. Gene expression analysis during platelet-like particle production in phorbol myristate acetate-treated MEG-01 cells. Biol Pharm Bull 2009; 32: 354-358.
42 Perez A, Thuillard JL, Bentzen CL. et al. Expression of nuclear receptors and apo E secretion during the differentiation of monocytic THP-1 cells into macrophages. Cell Biol Toxicol 2003; 19: 95-105.
43 Fugman DA, Witte DP, Jones CL. et al. In vitro establishment and characterization of a human megakaryoblastic cell line. Blood 1990; 75: 1252-1261.
44 Briquet-Laugier V, El Golli N, Nurden P. et al. Thrombopoietin-induced Dami cells as a model for alpha-granule biogenesis. Platelets 2004; 15: 341-344.
45 Schadinger SL, Lin JHH, Garand M. et al. Secretion and antifibrinolytic function of thrombin-activatable fibrinolysis inhibitor from human platelets. J Thromb Haemost 2010; 8: 2523-2529.
46 Auwerx J. The human leukemia cell line, THP-1: a multifecetted model for the study of monocyte-macrophage differentiation. Experentia 1991; 47: 22-31.
47 te Velde EA, Wagenaar GT, Reijerkerk A. et al. Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor. J Thromb Haemost 2003; 1: 2087-2096.
48 Swaisgood CM, Schmitt D, Eaton D. et al. In vivo regulation of plasminogen function by plasma carboxypeptidase B. J Clin Invest 2002; 110: 1275-1282.
49 Verbon A, Meijers JC, Spek CA. et al. Effects of IC14, an anti-CD14 antibody, on coagulation and fibrinolysis during low-grade endotoxemia in humans. J Infect Dis 2003; 187: 55-61.
50 Emonts M, de Bruijne EL, Guimarães AH. et al. Thrombin-activatable fibrinolysis inhibitor is associated with severity and outcome of severe meningococcal infection in children. J Thromb Haemost 2008; 6: 268-276.
51 Bengtson SH, Sandén C, Mörgelin M. et al. Activation of TAFI on the surface of Streptococcus pyogenes evokes inflammatory reactions by modulating the kallikrein/kinin system. J Innate Immun 2008; 1: 18-28.
52 Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004; 84: 767-801.