Thrombin Activatable Fibrinolysis Inhibitor, a Potential Regulator of Vascular Inflammation
2003; Elsevier BV; Volume: 278; Issue: 51 Linguagem: Inglês
10.1074/jbc.m306977200
ISSN1083-351X
AutoresTimothy Myles, Toshihiko Nishimura, Thomas H. Yun, Mariko Nagashima, John Morser, Andrew J. Patterson, Ronald G. Pearl, Lawrence Leung,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe latent plasma carboxypeptidase thrombin-activable fibrinolysis inhibitor (TAFI) is activated by thrombin/thrombomodulin on the endothelial cell surface, and functions in dampening fibrinolysis. In this study, we examined the effect of activated TAFI (TAFIa) in modulating the proinflammatory functions of bradykinin, complement C5a, and thrombin-cleaved osteopontin. Hydrolysis of bradykinin and C5a and thrombin-cleaved osteopontin peptides by TAFIa was as efficient as that of plasmin-cleaved fibrin peptides, indicating that these are also good substrates for TAFIa. Plasma carboxypeptidase N, generally regarded as the physiological regulator of kinins, was much less efficient than TAFIa. TAFIa abrogated C5a-induced neutrophil activation in vitro. Jurkat cell adhesion to osteopontin was markedly enhanced by thrombin cleavage of osteopontin. This was abolished by TAFIa treatment due to the removal of the C-terminal Arg168 by TAFIa from the exposed SVVYGLR α4β1 integrin-binding site in thrombin-cleaved osteopontin. Thus, thrombin cleavage of osteopontin followed by TAFIa treatment may sequentially up- and down-modulate the pro-inflammatory properties of osteopontin. An engineered anticoagulant thrombin, E229K, was able to activate endogenous plasma TAFI in mice, and E229K thrombin infusion effectively blocked bradykinin-induced hypotension in wild-type, but not in TAFI-deficient, mice in vivo. Our data suggest that TAFIa may have a broad anti-inflammatory role, and its function is not restricted to fibrinolysis. The latent plasma carboxypeptidase thrombin-activable fibrinolysis inhibitor (TAFI) is activated by thrombin/thrombomodulin on the endothelial cell surface, and functions in dampening fibrinolysis. In this study, we examined the effect of activated TAFI (TAFIa) in modulating the proinflammatory functions of bradykinin, complement C5a, and thrombin-cleaved osteopontin. Hydrolysis of bradykinin and C5a and thrombin-cleaved osteopontin peptides by TAFIa was as efficient as that of plasmin-cleaved fibrin peptides, indicating that these are also good substrates for TAFIa. Plasma carboxypeptidase N, generally regarded as the physiological regulator of kinins, was much less efficient than TAFIa. TAFIa abrogated C5a-induced neutrophil activation in vitro. Jurkat cell adhesion to osteopontin was markedly enhanced by thrombin cleavage of osteopontin. This was abolished by TAFIa treatment due to the removal of the C-terminal Arg168 by TAFIa from the exposed SVVYGLR α4β1 integrin-binding site in thrombin-cleaved osteopontin. Thus, thrombin cleavage of osteopontin followed by TAFIa treatment may sequentially up- and down-modulate the pro-inflammatory properties of osteopontin. An engineered anticoagulant thrombin, E229K, was able to activate endogenous plasma TAFI in mice, and E229K thrombin infusion effectively blocked bradykinin-induced hypotension in wild-type, but not in TAFI-deficient, mice in vivo. Our data suggest that TAFIa may have a broad anti-inflammatory role, and its function is not restricted to fibrinolysis. Thrombin has long been regarded as a multifunctional procoagulant enzyme important in hemostasis. At sites of vascular injury it converts fibrinogen to fibrin, amplifies the clotting cascade by activating factor XI (FXI) and the cofactors FV and FVIII, stabilizes the fibrin clot by activating FXIII, and activating platelets via the protease-activated receptors (PAR). It can also function as an anticoagulant by binding to thrombomodulin (TM) 1The abbreviations used are: TMthrombomodulinTAFIthrombin-activatable fibrinolysis inhibitorCPBcarboxypeptidase BCPNcarboxypeptidase NOPNosteopontinaPCactivated protein CBKbradykininPPACKd-phenylalanyl-propyl-arginylchlormethylketonePBSphosphate-buffered salineHBSSHanks balanced salt solutionDTTdithiothreitolGSTglutathione S-transferase. on the surface of endothelial cells and activating protein C, which inhibits FVa and FVIIIa, effectively localizing clot formation to the site of vascular injury (1Esmon C.T. J. Exp. Med. 2002; 196: 561-564Crossref PubMed Scopus (107) Google Scholar). TM-bound thrombin can also activate thrombin-activable fibrinolysis inhibitor (TAFI; Refs. 2Bajzar L. Manuel R. Nesheim M.E. J. Biol. Chem. 1995; 270: 14477-14484Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar and 3Bajzar L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2511-2518Crossref PubMed Scopus (183) Google Scholar), a latent plasma procarboxypeptidase also known as carboxypeptidase R, carboxypeptidase U, and procarboxypeptidase B (4Campbell W. Okada H. Biochem. Biophys. Res. Commun. 1989; 162: 933-939Crossref PubMed Scopus (139) Google Scholar, 5Hendriks D. Scharpé S. van S e M. Lommaert M. P. J. Clin. Chem. Clin. Biochem. 1989; 27: 277-285PubMed Google Scholar, 6Eaton D.L. Malloy B.E Tsai S.P. Henzel W. Dratna D. J. Biol. Chem. 1991; 266: 21833-21838Abstract Full Text PDF PubMed Google Scholar). TAFI is now recognized as the second physiological substrate for the thrombin/TM complex. It is important for dampening fibrinolysis by removal of plasmin-exposed lysines on partially digested fibrin clots thereby restricting tissue plasminogen activator binding and further activation of plasminogen (2Bajzar L. Manuel R. Nesheim M.E. J. Biol. Chem. 1995; 270: 14477-14484Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 7Redlitz A Tan A.K. Eaton D.L. Plow E.F. J. Clin. Investig. 1995; 96: 2534-2538Crossref PubMed Scopus (241) Google Scholar). Thus, activated protein C (aPC) and TAFIa may play complementary roles in hemostasis, with aPC dampening the clotting cascade and preventing excessive thrombin generation, while TAFIa serves to protect the clot already formed at the site of injury. thrombomodulin thrombin-activatable fibrinolysis inhibitor carboxypeptidase B carboxypeptidase N osteopontin activated protein C bradykinin d-phenylalanyl-propyl-arginylchlormethylketone phosphate-buffered saline Hanks balanced salt solution dithiothreitol glutathione S-transferase. Thrombin can also act as a proinflammatory molecule by the activation of PAR on monocytes, smooth muscle cells and endothelial cells, thus providing a direct link between coagulation and inflammation (8Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2140) Google Scholar). On the other hand, thrombin/TM activation of PC could also function as a negative regulator of inflammation along with its defined role as an anticoagulant (1). aPC suppressed the expression of proinflammatory cell adhesion molecules and augmented the expression of anti-apoptotic molecules in cultured endothelial cells (9Joyce D.E. Gelbert L. Ciaccia A. DeHoff B. Grinnell B.W. J. Biol. Chem. 2001; 276: 11199-11203Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 10Riewald M. Petrovan R.J. Donner A. Mueller B.M. Ruf W. Science. 2002; 296: 1880-1882Crossref PubMed Scopus (748) Google Scholar). This anti-inflammatory role of aPC may contribute to its clinical efficacy in the treatment of severe sepsis (11Bernard G.R. Vincent J.L. Laterre P.F. LaRosa S.P. Dhainaut J.F. Lopez-Rodriguez A. Steingrub J.S. Garber G.E. Helterbrand J.D. Ely E.W. Fisher Jr., C.J. N. Engl. J. Med. 2001; 344: 699-709Crossref PubMed Scopus (5070) Google Scholar). It has been proposed that TAFIa could also play a role in regulating inflammation by inactivation of kinins and anaphylatoxins through its arginine/lysine specific carboxypeptidase activity (12Shinohara T. Sakurada C. Suzuki T. Takeuchi O. Campbell W.D. Ikeda S. Okada N. Okada H. Int. Arch. Allergy Immunol. 1994; 103: 400-404Crossref PubMed Scopus (64) Google Scholar, 13Campbell W.D Lazoura E. Okada N. Okada H. Microbiol. Immunol. 2002; 46: 131-134Crossref PubMed Scopus (188) Google Scholar). However, the efficiency of TAFIa in inactivating these biologically active mediators in comparison with carboxypeptidase N, the constitutively active plasma anaphylatoxin inhibitor, has not been defined. Recently, a new mechanism by which thrombin can regulate inflammation and tissue repair is revealed by its interaction with osteopontin (OPN, 14Senger D.R. Perruzzi C.A. Papadopoulos A. Tenen D.G. Biochim. Biophys. Acta. 1989; 996: 43-48Crossref PubMed Scopus (177) Google Scholar, 15Senger D.R. Perruzzi C.A. Papadopoulos-Sergiou A. Van de Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar, 16Xuan J.W. Hota C Chambers A.F. J. Cell. Biochem. 1994; 54: 247-255Crossref PubMed Scopus (74) Google Scholar). OPN is a multifunctional RGD-containing phosphoprotein with adhesive and cell-signaling functions involved in cell-cell and cell-matrix interactions important in inflammatory responses (17Denhardt D.T. Noda M. O'Regan A.W. Pavlin D. Berman J.S. J. Clin. Investig. 2001; 107: 1055-1061Crossref PubMed Scopus (917) Google Scholar). It is present as an extracellular matrix component in mineralized tissues and in the subendothelial matrix of blood vessels involved by atherosclerosis. OPN also circulates as a soluble proinflammatory cytokine and is widely expressed by many inflammatory cells in culture, including T cells, macrophages, and NK cells. Its expression is enhanced in response to inflammation, tissue injury and stress. It interacts with many cells via RGD-dependent (αvβ1, αvβ3, or αvβ5) and RGD-independent integrins (α4β1, α5β1, α8β1, or α9β1) and also CD44. It is chemotactic for various cell types, notably monocytes and macrophages and stimulates cell motility and cell survival (17Denhardt D.T. Noda M. O'Regan A.W. Pavlin D. Berman J.S. J. Clin. Investig. 2001; 107: 1055-1061Crossref PubMed Scopus (917) Google Scholar). Of interest, thrombin cleavage of OPN increases the adhesion, spreading, and migration of a variety of cells significantly in vitro, and this can occur in vivo suggesting an important functional role of OPN cleavage at sites of thrombin generation (15Senger D.R. Perruzzi C.A. Papadopoulos-Sergiou A. Van de Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar, 18Senger D.R. Peruzzi C.A. Gracey C.F. Papadopoulos A. Tenen D.G. Cancer Res. 1988; 48: 5770-5774PubMed Google Scholar). Thrombin cleavage of OPN generates an N-terminal fragment that exposes a cryptic integrin-binding motif 161SVVYGLR168 on its C terminus, allowing the specific interaction to cells bearing the integrins α4β1 (19Bayless K.J. Meininger G.A. Scholtz J.M. Davis G.E. J. Cell Sci. 1998; 111: 1165-1174Crossref PubMed Google Scholar, 20Barry S.T. Ludbrook S.B. Murrison E. Horgan C.M.T. Exp. Cell Res. 2000; 258: 342-351Crossref PubMed Scopus (96) Google Scholar, 21Green P.M. Ludbrook S.B. Miller D.D. Horgan C.M.T. Barry S.T. FEBS Lett. 2001; 503: 75-79Crossref PubMed Scopus (90) Google Scholar, 22Bayless K.J. Davis G.E. J. Biol. Chem. 2001; 276: 13483-13489Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) or α9β1 (23Smith L.L. Cheung H-K. ling L.E. Chen J. Sheppard D. Pytela R. Giachelli C.M. J. Biol. Chem. 1996; 271: 28485-28491Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 24Smith L.L. Giachelli C.M. Exp. Cell Res. 1998; 242: 351-360Crossref PubMed Scopus (91) Google Scholar). In certain cell types, such as melanoma cells, cell binding only occurs with the thrombin-cleaved, but not the intact OPN, suggesting that thrombin cleavage is critical for certain OPN-cell interactions (15Senger D.R. Perruzzi C.A. Papadopoulos-Sergiou A. Van de Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar, 24Smith L.L. Giachelli C.M. Exp. Cell Res. 1998; 242: 351-360Crossref PubMed Scopus (91) Google Scholar). Here we address the potential role of TM-dependent thrombin activation of TAFI, and the subsequent TAFIa inactivation of bradykinin, C3a, C5a, and thrombin-cleaved OPN. Our data suggest that along with aPC, TAFIa serves to counterbalance the prothrombotic and proinflammatory effects of thrombin generation and thus its biological role is not limited to fibrin clot stabilization. Materials—Synthetic peptides were synthesized and purified by the peptide synthesis core facility at the Stanford University Beckman Center (Stanford, CA). The peptides were based on residues 160-168 of the C terminus of thrombin-cleaved osteopontin (OPN160-168), residues 69-77 of activated complement C3a (C3a69-77), residues 66-74 of C5a (C5a66-74) and peptides at the fibrin plasmin cleavage sites on α-chain α-Arg104 (FBα-Arg96-104), β-chain β-Lys133 (FBβ-Lys125-133), and γ-chain γ-Lys62 (FBγ-Lys54-62) and γ-Lys85 (FBγ-Lys77-85). The same peptides were synthesized in their des-Arg or des-Lys forms for calibration curves. The anti-integrin antibodies HP2/1 (α4, CD49d), AMF7 (αV, CD51), SAM1 (α5, CD49e), 7E4 (β2, CD18), 25.3.1 (αL, CD11a), BEAR 1 (αM, CD11b), and 4B4 (β1, CD29) were from Beckman Coulter (Brea, CA). The sheep anti-human TAFI antibody SATAFI-AP was from Affinity Biologicals (Ontario, Canada) and horseradish peroxidase-conjugated rabbit anti-sheep antibody was from Pierce. Recombinant soluble human TM and human carboxypeptidase N (CPN) are from Berlex Biosciences (Richmond, CA). Human TAFI was purchased from Hematological Technologies (Essex Junction, VT). Bradykinin (BK), benzamidine, d-phenylalanyl-propyl-arginylchlormethylketone (PPACK), C5a, cytochalasin B, o-dianisidine, potato carboxypeptidase inhibitor, glutathione, and Thrombomax-HS reagent were purchased from Sigma Chemicals. The protease inhibitor mixture set III and Bugbuster™ reagent were purchased from Novagen (Madison, WI). Recombinant human wild-type and E229K thrombins were expressed and purified as described previously (25Tsiang M. Paborsky L.R. Li W.X. Jain A.K. Mao C.T. Dunn K.E. Lee D.W. Matsumura S.Y. Matteucci M.D. Coutre S.E. Leung L.L. Gibbs C.S. Biochemistry. 1996; 35: 16449-16457Crossref PubMed Scopus (76) Google Scholar). TAFIa-mediated Hydrolysis of C-terminal Peptides—Soluble recombinant TM (40 nm) was complexed with 1 nm thrombin. TAFI (100 nm) was added and incubated at 25 °C for 20 min. The reaction was terminated by the addition of PPACK (5 μm). The concentration of TAFIa was determined using an Actichrome TAFI activity kit (American Diagnostica). The peptides based on BK, OPN160-168, C3a69-77, C5a66-74, FBα-Arg96-104, FBβ-Lys125-133, FBγ-Lys54-62, and FBγ-Lys77-85 with concentrations ranging from 15 μm to 2 mm were digested with either 5 nm TAFIa or CPN for 2-5 min at 37 °C in assay buffer. Reactions were stopped by boiling for 5 min. Cleaved peptides were resolved by HPLC and the area of the cleaved peptide peak was calculated using the Waters HPLC software then converted to nmoles of peptide using a calibration curve determined with the purified des-Arg or des-Lys form of each peptide. The values for Km and kcat were determined by plotting the initial velocities of cleavage against the different substrate concentrations, then fitting to the Michaelis-Menten equation by non-linear regression analysis. Experiments were performed in duplicate and the data pooled for analysis. Escherichia coli Expression of Recombinant Osteopontin—The cDNA sequences for mature full-length human OPN (OPN-FL: amino acids 17-314), N-terminal OPN mimicking thrombin-cleaved OPN (OPN-Arg168: amino acids 17-168) and the N-terminal OPN mimicking thrombin-cleaved and TAFIa-treated OPN (OPN-LeuΔArg: amino acids 17-167) were inserted as C-terminal in-frame fusions to GST using the E. coli expression vector pGEX-6P-3 (Amersham Biosciences). The constructs pOPN-FL, pOPN-Arg168, and pOPN-LeuΔArg also incorporated the 159RGD161 to 159RAA161 substitutions by site-directed mutagenesis using the Quick-change site directed mutagenesis kit (Stratagene, La Jolla, CA) to form the constructs pRAA/OPN-FL, pRAA/OPN-Arg168, and pRAA/OPN-LeuΔArg. Constructs were sequenced and then transformed into E. coli BL21 Gold (Stratagene, La Jolla, CA). For large scale expression of the constructs, transformed cells were grown in 1 liter of LB broth supplemented with ampicillin (100 μg/ml) at 37 °C until an OD600 of 0.6-0.7. Isopropyl-1-thio-β-d-galactopyranoside (0.2 mm) was added and grown for a further 2 h at 37 °C. Cells were pelleted and washed twice with PBS, pH 7.2. Cells were lysed with Bugbuster™ reagent supplemented with the protease inhibitor III mixture set and 2 mm DTT. The cell lysate was clarified, diluted 5-fold in low salt buffer (50 mm Tris, 50 mm NaCl, 2 mm DTT, pH 7.5, and the protease inhibitors) and loaded onto a hiPrep QXL column utilizing an FPLC system (Amersham Biosciences) at 4 °C. The column was washed extensively with wash buffer (50 mm Tris, 200 mm NaCl, 2 mm DTT, pH 7.5, the protease inhibitors), then eluted with high salt buffer (50 mm Tris, 600 mm NaCl, 2 mm DTT, pH 7.5) containing 1 mm benzamidine. Fractions containing GST-OPN fusion proteins were pooled and loaded onto a 10-ml glutathione-Sepharose column equilibrated with low salt buffer with 2 mm DTT and 1 mm benzamidine. The column was washed with low salt buffer and OPN was eluted with low salt buffer containing 10 mm glutathione. Fractions containing GST-OPN fusion proteins were pooled and dialyzed against 50 mm Tris, 100 mm NaCl, pH 7.5. The recombinant proteins were greater than 95% pure as judged by SDS-PAGE and Coomassie Blue staining. Adhesion Assays—Jurkat cells were grown in RPMI 1640 media supplemented with 10% fetal calf serum. Cells with greater than 95% viability as demonstrated by Trypan Blue exclusion assay were washed with Hanks balanced salt solution (HBSS) with 50 mm Hepes, pH 7.5 and adjusted to a concentration of 2 × 106 cells/ml in buffer containing 0.2 mm MnCl2 (21Green P.M. Ludbrook S.B. Miller D.D. Horgan C.M.T. Barry S.T. FEBS Lett. 2001; 503: 75-79Crossref PubMed Scopus (90) Google Scholar). Recombinant wild-type and mutant OPN fusion proteins were diluted in PBS (concentrations ranging from 0, 0.1, 1, 10, 100 μg/ml) and coated onto high protein binding 96-well microtiter plates (Greiner Labortechnik, Ocala, FL) with a final volume of 100 μl per well, then incubated overnight at 4 °C. For experiments studying the effect of thrombin and TAFIa on OPN-FL mediated Jurkat cell adhesion, the plates were washed three times with thrombin assay buffer (25 mm Hepes, 150 mm, 5 mm CaCl2, 0.1% PEG6000, pH 7.5). To some of the wells, 100 μl of thrombin (100 nm final concentration) was added, and incubated for 1 h at 37 °C. Wells were washed three times with thrombin assay buffer. To some of the wells that had been treated with thrombin, 100 μl of TAFIa (1.7 nm final concentration) was added and incubated for 60 min at room temperature (RT). The plates were washed three times with PBS, then blocked for 1 h with 3% BSA in PBS. The wells were then washed three times with HBSS-Hepes buffer with 0.2 mm MnCl2. To each well, 100 μl of 2 × 106 cells/ml were added in the same HBSS-Hepes buffer with 0.2 mm MnCl2, and cells were allowed to adhere for 60 min at 37 °C. Cells were washed twice with PBS, once with absolute ethanol and fixed for 20 min with absolute ethanol. Wells were then washed three times with PBS, stained with 0.1% crystal violet, and again washed three times with PBS. Cells were then lysed with 0.5% Triton X-100. Lysates were read at A570 nm using a SpectroMAX plate reader (Molecular Dynamics, Sunnyvale, CA). For studies using recombinant GST-OPN fusion proteins (OPN-Arg168 and OPN-LeuΔArg with or without the RGD → RAA substitution), protein adsorption to the microtiter wells was carried out and Jurkat cell binding determined as described above. For antibody inhibition of Jurkat cell binding to OPN-Arg168 and OPN-LeuΔArg, cells were preincubated with 10 μg/ml of the following anti-integrin antibodies: HP2/1, AMF7, SAM1, 7E4, 25.3.1, 4B4, and BEAR1 for 10 min on ice. Cells were then allowed to attach for 60 min and the amount of cell binding determined. Neutrophil Myeloperoxidase Release Assay—Neutrophils were prepared from buffy coat concentrates obtained from Stanford Blood Bank following a published protocol (26Henson P.M. Zanolari B. Schwartzman N.A. Hong S.R. J. Immunol. 1978; 121: 851-855PubMed Google Scholar). Neutrophils (4 × 106 cells/ml) were resuspended in Hanks solution with 0.25% bovine serum albumin. To test the effect of TAFIa on C5a-mediated myeloperoxidase release, 60 nm recombinant soluble TM was complexed with 3 nm thrombin for 10 min. TM-complexed thrombin was used to activate TAFI (200 nm). Human C5a (10 μm) was hydrolyzed by 0, 1, 10, 100 nm TAFIa at RT. 10-μl aliquots were removed at 1, 5, 10, 30, and 60 min and the TAFIa inhibited with potato carboxypeptidase inhibitor. The samples were then diluted 10-fold such that the concentration of C5a/C5adesArg was 1.0 μm. 1 ml of neutrophils (4 × 106 cell/ml) was treated with 5 μg/ml cytochalasin B for 5 min at 37 °C. 1 μl of each diluted C5a/TAFIa reaction was added to the cell suspension and incubated for 15 min at 37 °C, then centrifuged. Myeloperoxidase released to the supernatant was measured by adding 20 μl of supernatant to 130 μl of 33 mm phosphate buffer pH 6.2, 0.002% H2O2, 6.7% o-dianisidine-HCl. Reaction velocities were followed at A460 for 30 min. Measurement of Mouse aPTT—Mice were anesthetized with ketamine followed by isoflurane. A midline incision was made and the internal jugular vein cannulated. Saline, wild-type, or E229K thrombin solutions were infused through the cannulated jugular vein at rates ranging from 0 to 50 μg thrombin/kg/min. Heart and respiratory rates were monitored and blood samples drawn after a 10-min infusion time. Blood was taken through a syringe inserted into the left ventricle and collected in 0.32% citrate. Blood samples were spun down for 1 min, 100 μl of plasma was warmed to 37 °C then mixed with 200 μl of prewarmed Thrombomax-HS reagent, and the aPTT determined using a BBL fibrometer. Fibrinogen levels were taken from similarly processed plasma while platelets counts were performed on whole blood collected into citrate anticoagulant tubes by the Stanford Animal Laboratory Facility (Stanford, CA). At each E229K thrombin dose, at least three mice were used. Results are presented as the mean a PTT ± S.E. (n = 5 for each data point in E229K, n = 3 for each data point in wild type thrombin). The study was approved by the Stanford Panel on Laboratory Animal Care and was conducted in compliance with university regulations. The Effect of E229K on Bradykinin-induced Hypotension in Vivo—Male C57BL/6 mice (8-12 weeks old, 25-32 g) or TAFI-deficient mice and their wild-type littermates (in a mixed background of C57BL/6 and 129/Sv) were anesthetized with isoflurane 2%. A midline incision was made in the upper thorax to expose the carotid artery and jugular vein. The carotid artery was cannulated with a pressure transduction catheter connected to a computerized pressure monitor (Powerlab, Colorado Springs, CO) to record blood pressure. The transduction system was calibrated using a sphygmomanometer. Another catheter (PE-10) was inserted into the left jugular vein serving as a route of delivery for experimental treatments. After the level of isoflurane was reduced to 1%, the blood pressure and respiratory rate of the mouse was allowed to stabilize for several minutes. Then 100 μl of either E229K thrombin (40 μg/kg) or a saline control was administered to the jugular vein. Immediately afterward, 50 μl of saline was injected to flush out the catheter, then 100 μl of BK in saline (10 nmol/kg) or the equivalent molar dosage of des-ArgBK was given to the mouse through the same jugular vein catheter. Blood pressure tracings were monitored and recorded. Data are presented as maximum drops in mean arterial pressure after administration of BK or des-Arg BK. Bradykinin, C5a and Thrombin-cleaved Osteopontin Were Good Substrates for TAFIa—The established view on TAFIa function is that it modulates clot fibrinolysis by removing exposed C-terminal lysines from partially plasmin-degraded clots. Removal of the lysines reduces t-PA and plasminogen binding thereby reducing t-PA enhanced activation of plasmin (2Bajzar L. Manuel R. Nesheim M.E. J. Biol. Chem. 1995; 270: 14477-14484Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 3Bajzar L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2511-2518Crossref PubMed Scopus (183) Google Scholar, 5Hendriks D. Scharpé S. van S e M. Lommaert M. P. J. Clin. Chem. Clin. Biochem. 1989; 27: 277-285PubMed Google Scholar, 6Eaton D.L. Malloy B.E Tsai S.P. Henzel W. Dratna D. J. Biol. Chem. 1991; 266: 21833-21838Abstract Full Text PDF PubMed Google Scholar, 7Redlitz A Tan A.K. Eaton D.L. Plow E.F. J. Clin. Investig. 1995; 96: 2534-2538Crossref PubMed Scopus (241) Google Scholar). Recent studies also imply the possible role of TAFIa inactivation of kinins and anaphylatoxins (4Campbell W. Okada H. Biochem. Biophys. Res. Commun. 1989; 162: 933-939Crossref PubMed Scopus (139) Google Scholar, 12Shinohara T. Sakurada C. Suzuki T. Takeuchi O. Campbell W.D. Ikeda S. Okada N. Okada H. Int. Arch. Allergy Immunol. 1994; 103: 400-404Crossref PubMed Scopus (64) Google Scholar, 13Campbell W.D Lazoura E. Okada N. Okada H. Microbiol. Immunol. 2002; 46: 131-134Crossref PubMed Scopus (188) Google Scholar). To show that kinins such as BK, anaphylatoxins such as complement C3a and C5a, and the thrombin-cleaved cytokine, OPN, are potential substrates of TAFIa, we determined their Michaelis-Menten constants for hydrolysis by TAFIa. These were compared with the hydrolysis of peptides based on four major plasmin cleavage sites on fibrin clots (27Collen D. Kudryk B.J. Hessel B. Blömback B. J. Biol. Chem. 1975; 250: 5808-5817Abstract Full Text PDF PubMed Google Scholar), the fibrin α-chain Arg104-Asp105, the β-chain Lys133-Asp134, and the γ-chain Lys62-Ala63 and Lys85-Ser86 sites (Table I). TAFIa is thermolabile (5Hendriks D. Scharpé S. van S e M. Lommaert M. P. J. Clin. Chem. Clin. Biochem. 1989; 27: 277-285PubMed Google Scholar); however, using reaction conditions in the presence of excess substrate with short incubation times at 37 °C, no significant TAFIa thermal instability and inactivation was observed (data not shown). The Km and kcat values for hydrolysis of the fibrin peptides ranged from 14.3 μm and 13.6 s-1 for FBβLys125-133 to 361.4 μm and 1.5 s-1 for FBαArg96-104. Differences were both in Km and kcat for the fibrin peptides with FBβLys125-133 and FBγLys54-65 being the best substrates for TAFIa while FBαArg96-104 and FBγLys77-85 were the worst. By comparison, the overall specificity constants for BK (2.8 × 105m-1·s -1), OPN159-168 (4.1 × 105m-1·s-1), C5a66-74 (1.3 × 105m-1·s-1) and C3a69-77 (2.3 × 105m-1·s-1) were similar to the two best fibrin substrates FBβLys125-133 (9.5 × 105m-1·s-1) and FBγLys54-62 (7.6 × 104m-1·s-1). The in vitro data suggests that BK, C5a, C3a, thrombin-cleaved OPN and the surface exposed basic residues on partially degraded clots are all good substrates for TAFIa in vivo. It is notable that CPN, commonly regarded as the physiological inactivator of kinins and anaphylatoxins, was less efficient for hydrolysis of BK (3.0 × 104m-1·s-1), OPN159-168 (1.6 × 104m-1·s-1) and C5a66-74 (1.5 × 104m-1·s-1) with kcat/Km values ∼9- and 26-fold lower. The peptides C3a69-77, FBαArg96-104, and FBβLys125-133 appeared to be slightly better substrates for CPN (kcat/Km = 7.5 × 105m-1·s-1, 4.2 × 103m-1·s-1 and 2.1 × 106m-1·s-1, respectively).Table IInactivation of BK and OPN Peptides by TAFIa and CPNSubstrateEnzymeKmkcatkcat/Kmμms−1M−1 S−1BKTAFIa70.6 ± 4.819.7 ± 4.82.8 × 105OPN159-168TAFIa57.7 ± 5.323.6 ± 0.64.1 × 105C5a66-74TAFIa219.0 ± 16.229.5 ± 0.71.3 × 105C3a69-77TAFIa35.9 ± 6.68.4 ± 0.62.3 × 105FBα-Arg96-104TAFIa361.4 ± 20.31.5 ± 0.14.2 × 103FBβ-Lys125-133TAFIa14.3 ± 0.713.6 ± 0.29.5 × 105FBγ-Lys54-62TAFIa34.0 ± 4.12.6 ± 0.17.6 × 104FBγ-Lys77-85TAFIa238.9 ± 24.25.9 ± 0.32.5 × 104BKCPN302.7 ± 29.19.1 ± 0.23.0 × 104OPN159-168CPN141.6 ± 11.62.3 ± 0.11.6 × 104C5a66-74CPN602.2 ± 74.39.3 ± 0.41.5 × 104C3a69-77CPN77.1 ± 11.257.9 ± 2.17.5 × 105FBα-Arg96-104CPN448.9 ± 43.82.9 ± 0.16.5 × 103FBβ-Lys125-133CPN53.2 ± 4.9109.1 ± 3.62.1 × 106FBγ-Lys54-62CPN657.6 ± 20.53.5 ± 0.15.3 × 104FBγ-Lys77-85CPN3727.0 ± 408.611.8 ± 0.83.2 × 104 Open table in a new tab TAFIa Reduced Jurkat Cell Adhesion to Thrombin-cleaved Osteopontin—Recombinant full-length OPN (OPN-FL) was adsorbed to 96-well microtiter plates at various concentrations and then treated with either thrombin or thrombin followed by TAFIa and compared with untreated OPN-FL for Jurkat cell binding (Fig. 1A). Thrombin-treated OPN-FL at a concentration of 1.0 μg/ml supported a 4.7-fold increase in Jurkat cell adhesion (A570 = 0.47 ± 0.12, n = 6) compared with untreated OPN-FL (A570 = 0.10 ± 0.03, n = 13) (Fig. 1, A and B). These results are consistent with previous studies showing that the increased cell adhesion is due to exposure of a cryptic integrin-binding motif SVVYGLR at the C terminus of the thrombin-cleaved OPN fragment (15Senger D.R. Perruzzi C.A. Papadopoulos-Sergiou A. Van de Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar, 16Xuan J.W. Hota C Chambers A.F. J. Cell. Biochem. 1994; 54: 247-255Crossref PubMed Scopus (74) Google Scholar, 17Denhardt D.T. Noda M. O'Regan A.W. Pavlin D. Berman J.S. J. Clin. Investig. 2001; 107: 1055-1061Crossref PubMed Scopus (917) Google Scholar, 18Senger D.R. Peruzzi C.A. Gracey C.F. Papadopoulos A. Tenen D.G. Cancer Res. 1988; 48: 5770-5774PubMed Google Scholar). TAFIa treatment of thrombin-cleaved OPN-FL reduced Jurkat cell adhesion to levels seen with OPN-FL (A570 = 0.12 ± 0.03, n = 6), suggesting a potential role for TAFIa regulating integrin-mediated cell adhesion to thrombin-cleaved OPN. At higher concentrations of OPN (>10.0 μg/ml), cell adhesion between the differently treated OPN were similar to OPN-FL, suggesting saturation of secondary lower affinity b
Referência(s)