Reversible inhibitors of TAFIa can both promote and inhibit fibrinolysis
2003; Elsevier BV; Volume: 1; Issue: 1 Linguagem: Inglês
10.1046/j.1538-7836.2003.00028.x
ISSN1538-7933
AutoresM. Schneider, Michael E. Nesheim,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoSummaryThe plasma carboxypeptidase activated thrombin-activable fibrinolysis inhibitor (TAFIa), is thermally unstable at 37 °C, with a half-life of 8 or 15 min depending on the isoform. The arginine analog, 2-guanidinoethylmercaptosuccinate (GEMSA), not only inhibits TAFIa but also slows the spontaneous inactivation of the enzyme, thereby reducing the activity of TAFIa, while extending its apparent half-life. Because, as shown in previous work, the ability of TAFIa to prolong clot lysis can be more dependent on its half-life than its concentration, in this study we determined whether reversible inhibitors of TAFIa could paradoxically prolong clot lysis. Potato tuber carboxypeptidase inhibitor (PTCI) or GEMSA were titrated into normal pooled human plasma, in the presence of soluble thrombomodulin. Both inhibitors mediate a biphasic antifibrinolytic effect, prolonging clot lysis at lower concentrations and enhancing clot lysis at higher concentrations. The antifibrinolytic effect of GEMSA is maximized at 1 mmol L−1, increasing clot lysis time from 100 min to 350 min. The antifibrinolytic effect of PTCI is maximized at 100 nmol L−1, increasing clot lysis time from 100 min to 240 min. To further characterize the nature of this biphasic effect, TAFI at various concentrations was added to TAFI-immunodepleted human plasma in the presence of PTCI or GEMSA. The magnitude of the effect depends on the concentration of TAFIa, the concentration of inhibitor, and the potency of the inhibitor. We propose that the biphasic antifibrinolytic effect is mediated by the dynamic equilibrium of free TAFIa that inactivates quickly, and TAFIa bound to inhibitor that inactivates slowly. TAFIa inhibitors used as therapeutic agents might not only enhance lysis at higher concentrations, but also stabilize fibrin clots at intermediate concentrations. The plasma carboxypeptidase activated thrombin-activable fibrinolysis inhibitor (TAFIa), is thermally unstable at 37 °C, with a half-life of 8 or 15 min depending on the isoform. The arginine analog, 2-guanidinoethylmercaptosuccinate (GEMSA), not only inhibits TAFIa but also slows the spontaneous inactivation of the enzyme, thereby reducing the activity of TAFIa, while extending its apparent half-life. Because, as shown in previous work, the ability of TAFIa to prolong clot lysis can be more dependent on its half-life than its concentration, in this study we determined whether reversible inhibitors of TAFIa could paradoxically prolong clot lysis. Potato tuber carboxypeptidase inhibitor (PTCI) or GEMSA were titrated into normal pooled human plasma, in the presence of soluble thrombomodulin. Both inhibitors mediate a biphasic antifibrinolytic effect, prolonging clot lysis at lower concentrations and enhancing clot lysis at higher concentrations. The antifibrinolytic effect of GEMSA is maximized at 1 mmol L−1, increasing clot lysis time from 100 min to 350 min. The antifibrinolytic effect of PTCI is maximized at 100 nmol L−1, increasing clot lysis time from 100 min to 240 min. To further characterize the nature of this biphasic effect, TAFI at various concentrations was added to TAFI-immunodepleted human plasma in the presence of PTCI or GEMSA. The magnitude of the effect depends on the concentration of TAFIa, the concentration of inhibitor, and the potency of the inhibitor. We propose that the biphasic antifibrinolytic effect is mediated by the dynamic equilibrium of free TAFIa that inactivates quickly, and TAFIa bound to inhibitor that inactivates slowly. TAFIa inhibitors used as therapeutic agents might not only enhance lysis at higher concentrations, but also stabilize fibrin clots at intermediate concentrations. Thrombin-activable fibrinolysis inhibitor (TAFI) can be activated to form the carboxypeptidase B-like enzyme TAFIa [1Bajzar L. Manuel R. Nesheim M.E. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor.J Biol Chem. 1995; 270: 14477-84Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar], which can remove carboxy-terminal lysine residues from a fibrin clot, thereby diminishing the cofactor activity for plasminogen activation [2Wang W. Boffa M.B. Bajzar L. Walker J.B. Nesheim M.E. A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 1998; 273: 27176-81Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar]. TAFI is also known as plasma procarboxypeptidase B [3Tan A.K. Eaton D.L. Biochemistry. Activation and characterization of procarboxypeptidase B from human plasma.Biochemistry. 1995; 34: 5811-6Crossref PubMed Scopus (137) Google Scholar] and procarboxypeptidase U [4Wang W. Hendriks D.F. Scharpe S.S. Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen.J Biol Chem. 1994; 269: 15937-44Abstract Full Text PDF PubMed Google Scholar]. An interesting property of TAFIa is its profound thermal instability, which, depending on the isoform, has been found to be 8 or 15 min at 37 °C [5Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme.J Biol Chem. 2002; 277: 1021-30Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar]. As there are no known inhibitors of TAFIa in plasma, this instability is probably a mechanism for the regulation of TAFIa activity in vivo. In support of this proposed mechanism, we found previously that TAFIa variants with altered thermal stabilities have correspondingly altered antifibrinolytic potentials [5Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme.J Biol Chem. 2002; 277: 1021-30Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. For example, compared with human plasma clots lyzed in the absence of TAFIa, the less thermally stable naturally occurring Thr325 variant of TAFIa attenuates clot lysis by 2.7-fold whereas the more thermally stable Ile325 variant of TAFIa attenuates clot lysis by 3.9-fold [5Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme.J Biol Chem. 2002; 277: 1021-30Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar]. Alternatively, a recombinant variant of TAFI (Arg302Gln) with a half-life of only 1.4 min attenuates clot lysis by only 1.6-fold [6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. We might expect that the variation in the antifibrinolytic potential of TAFIa mediated by differing thermal stabilities could be overcome by increasingly large concentrations of the enzyme; however, this does not appear to be the case. Rather, we find that isoforms of TAFIa with altered thermal stabilities each exhibit a maximum ability to attenuate clot lysis, and the magnitude of this maximum is dictated by the thermal stability of the TAFIa variant [5Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme.J Biol Chem. 2002; 277: 1021-30Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. No matter how much of a less stable TAFIa variant is added to a clot lysis assay, it cannot stabilize a clot as well as a more stable TAFIa variant. Equally interesting is that the half-maximal antifibrinolytic effect is observed at 1 nmol L−1 TAFIa, and the maximum antifibrinolytic effect is reached by 10 nmol L−1 TAFIa. At concentrations above 10 nmol L−1 there is little change in the antifibrinolytic potential of TAFIa, and the system is saturated with respect to the antifibrinolytic potential. Therefore, we have the curious situation where at higher concentrations of TAFIa (close to or above 10 nmol L−1), the half-life of the enzyme dictates the maximum antifibrinolytic potential of TAFIa, whereas changes in the concentration of TAFIa are not important with respect to the antifibrinolytic potential of the enzyme. 2-Guanidinoethylmercaptosuccinate (GEMSA) is an arginine analog that can reversibly inhibit TAFIa [6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. While studying the kinetics of TAFIa thermal decay, we found that GEMSA slows the rate of spontaneous TAFIa inactivation by 64-fold [6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. A TAFIa pool incubated in the presence of GEMSA has less activity corresponding to the amount of inhibitor present; however, this less active pool also exhibits an increased apparent half-life. When GEMSA resides in the active site of TAFIa, it makes the thermal inactivation process much less favorable, and we thought that this stabilizing effect could be common to other reversible inhibitors that occupy the TAFIa active site, such as potato tuber carboxypeptidase inhibitor (PTCI). Because thermal stability is more important than concentration with respect to the antifibrinolytic activity of TAFIa, and as the reversible inhibitor GEMSA can increase the apparent half-life of TAFIa, in this study we have characterized the antifibrinolytic effect of TAFIa in the presence of reversible inhibitors. We studied the effect of PTCI and GEMSA on the time it takes to lyze clots formed from normal human plasma and found that both reversible carboxypeptidase inhibitors can mediate a biphasic antifibrinolytic effect; both enhancing and inhibiting the antifibrinolytic potential of TAFIa, depending on the concentration of inhibitor used. By supplementing TAFI-immunodepleted human plasma with various concentrations of TAFIa and either PTCI or GEMSA we have found that this biphasic antifibrinolytic effect depends on the concentration of TAFIa, the concentration of inhibitor and its potency. We propose that the paradoxical antifibrinolytic effect of reversible TAFIa inhibitors is a result of their ability to maintain a pool of TAFIa bound to inhibitor that inactivates slowly, which can serve to buffer the free TAFIa pool that inactivates quickly. The synthetic carboxypeptidase substrate anisylazoformyllysine (AAFK) [7] was a generous gift from Dr William L. Mock (Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA). CNBr-activated Sepharose 4B was purchased from Sigma-Aldrich Canada LTD (Oakville, ON, Canada). Ultrafree-4 centrifugal filter devices were purchased from Millipore Corp (Bedford, MA, USA). The thrombin inhibitors PPAck and dEGRck were purchased from Calbiochem (San Diego, CA, USA). Dulbecco's modified Eagle's medium/nutrient mixture F-12, newborn calf serum, Opti-MEM, penicillin/streptomycin/fungizone (PSF) mixture and reduced glutathione were purchased from Life Technologies Inc. Methotrexate was obtained from Kingston General Hospital pharmacy (Wyeth-Ayerst, Canada, Ltd., Montreal, QU, Canada). Baby hamster kidney (BHK) cells and the pNUT mammalian expression vector were a gift from Dr Ross MacGillivray (University of British Columbia, Vancouver, BC, Canada). For immunoadsorption chromatography, a monoclonal antibody raised against purified TAFI (mAb16) was coupled to CNBr-activated Sepharose 4B [8Bajzar L. Nesheim M.E. Tracy P. The profibrinolytic effect of activated protein c in clots formed from plasma is TAFI-dependent.Blood. 1996; 88: 2093-100Crossref PubMed Google Scholar]. Thrombin was prepared from human plasma-derived prothrombin as described previously [9Walker J.B. Nesheim M.E. The molecular weights, mass distribution, chain composition, and structure of soluble fibrin degradation products released from a fibrin clot perfused with plasmin.J Biol Chem. 1999; 274: 5201-12Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. Human fibrinogen, human plasminogen, and antiplasmin were purified as described previously [9Walker J.B. Nesheim M.E. The molecular weights, mass distribution, chain composition, and structure of soluble fibrin degradation products released from a fibrin clot perfused with plasmin.J Biol Chem. 1999; 274: 5201-12Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. Recombinant tPA (Activase) was generously provided by Dr Gordon Vehar (Genentech, Inc., South San Francisco, CA, USA). Recombinant soluble thrombomodulin (Solulin) was a generous gift of Dr John Morser (Berlex Biosciences, Inc., Richmond, CA, USA). Synthetic 75% phosphatidyl choline: 25% phosphatidyl serine (PCPS) vesicles were prepared as described previously [10Bloom J.W. Nesheim M.E. Mann K.G. Phospholipid-binding properties of bovine factor V and factor Va.Biochemistry. 1979; 18: 4419-25Crossref PubMed Scopus (115) Google Scholar]. The cDNA for the TAFI isoform corresponding to the amino acid sequence determined by Eaton and coworkers [11Eaton D.L. Malloy B.E. Tsai S.P. Henzel W. Drayna D. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma.J Biol Chem. 1991; 266: 21833-8Abstract Full Text PDF PubMed Google Scholar] was inserted into the pNUT expression vector, and transfected into BHK cells as described previously [5Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme.J Biol Chem. 2002; 277: 1021-30Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 12Boffa M.B. Wang W. Bajzar L. Nesheim M.E. 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: 2127-35Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 13Chen C. Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA.Mol Cell Biol. 1987; 7: 2745-52Crossref PubMed Scopus (4817) Google Scholar]. For recombinant TAFI expression, stably expressing lines were cultured in triple flasks (500 cm2, Nunc, Roskilde, Denmark) in Opti-MEM with 1% PSF (v/v), and 40 µmol L−1 ZnCl2. Conditioned medium was harvested at 48-h intervals and replaced with fresh medium. Harvested medium was centrifuged at 1000 g for 10 min, supplemented with Tris-HCl, pH 8.0 (5 mmol L−1 final concentration), reduced glutathione (0.5 mmol L−1 final concentration), and dEGRck (2 µmol L−1 final concentration) and stored at − 20 °C. To isolate the TAFI variants, typically 1 L of frozen, conditioned medium was thawed at room temperature, passed through a 0.22-µmol L−1 filter, and then over a 4.0-mL mAb16-Sepharose 4B column (6 mg antibody mL−1) at 4 °C. The column was washed extensively with 20 mmol L−1 HEPES, 150 mmol L−1 NaCl, 0.01% Tween 80, at pH 7.4 (HBS/Tween 0.01%). TAFI was eluted with 1.0 mL fractions of glycine (0.2 mol L−1), pH 3, into 1.0 mL aliquots of Tris-HCl (1 mol L−1), pH 8. Protein-containing fractions were pooled and the buffer was changed to HBS/Tween 0.01% using the Ultrafree-4 centrifugal filter device. Purified TAFI was quantified by measurement of absorbance at 280 nm (ε1%, 280nm = 26.4, Mr = 60 000 Da), aliquoted, and stored at − 20 °C. We have observed some variability in the potency of commercial PTCI preparations with respect to their ability to inhibit TAFIa; therefore we have determined the potency of all inhibitor preparations used in this study, including two different lots of commercially available PTCI (Sigma-Aldrich), and GEMSA (Calbiochem). To estimate the potencies of the two PTCI preparations, various concentrations of each PTCI (0–80 nmol L−1) preparation were incubated with various concentrations of AAFK (100–300 µmol L−1) in the presence of 5 nmol L−1 TAFIa. The reactions were monitored at 350 nm and the rate of AAFK hydrolysis was determined by the initial rate of the change in absorbance vs. time relationship, as described previously [5Schneider M. Boffa M. Stewart R. Rahman M. Koschinsky M. Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme.J Biol Chem. 2002; 277: 1021-30Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar]. At each AAFK concentration, the rates of hydrolysis in the presence of PTCI were made relative to the rate in the absence of PTCI. PTCI is a potent inhibitor of TAFIa, and the concentration of TAFIa cannot be made low enough to be insignificant with respect to the PTCI concentration. Therefore the following model was used to estimate the potency of PTCI to inhibit TAFIa: 1 where r/ro represents the rate of AAFK hydrolysis in the presence of inhibitor relative the rate of AAFK hydrolysis in the absence of inhibitor, Io represents the concentration of PTCI, Km represents the binding of TAFIa to AAFK, To represents the total concentration of TAFIa, S represents the concentration of AAFK, and KI is the dissociation constant for the binding of PTCI to TAFIa. The KI was estimated by non-linear regression of the data using the Nonlin function of Systat 9 (SPSS, Inc., Chicago, IL, USA), with the standard error calculated by the regression algorithm. To estimate the potency of the GEMSA preparation, various concentrations of GEMSA (0–150 µmol L−1) were incubated with various concentrations of AAFK (100–300 µmol L−1) in the presence of 5 nmol L−1 TAFIa. The reactions were monitored at 350 nm and the rate of AAFK hydrolysis was determined by the initial rate of the change in absorbance vs. time relationship. At each AAFK concentration, the rates of hydrolysis in the presence of GEMSA were made relative to the rate in the absence of GEMSA. The KI was estimated using the equation for competitive inhibition. To minimize adsorption of proteins to plastic, all microtiter plates were presoaked overnight with HBS/Tween 1%, rinsed extensively with deionized distilled water and air-dried. TAFI was activated to TAFIa by incubating the zymogen with thrombin (25 nmol L−1 final concentration) and Solulin (100 nmol L−1 final concentration) for 10 min at 25 °C. The thrombin was then quenched by the addition of PPAck (1 µmol L−1 final concentration). As activated TAFIa is thermally unstable at 25 °C, the TAFIa was placed on ice until use. To measure the magnitude of the stabilizing effect of GEMSA, TAFIa was diluted to 500 nmol L−1 and incubated with various concentrations of GEMSA (0–400 µmol L−1) at 37 °C. At various times during the incubation, 10 µL aliquots were removed and quenched with 190 µL AAFK (120 µmol L−1 final concentration). The reactions were monitored at 350 nm and the initial rate of hydrolysis was determined from the initial slope of the absorbance vs. time relationship. The measured slopes were made relative to the initial TAFIa activity, and the data was fitted with the exponential decay function using Sigmaplot (SPSS Inc). To determine whether PTCI could also slow the spontaneous TAFIa inactivation, TAFIa was diluted to 100 nmol L−1 in the presence of various concentrations PTCI (0–200 nmol L−1) and placed in a 37 °C waterbath. At various times during the incubation, 100 µL aliquots of the reaction mixture were removed and quenched with 100 µL of AAFK (120 µmol L−1 final concentration), in the well of a microtiter plate. The reactions were monitored at 350 nm and the initial rate of hydrolysis was determined from the initial slope of the absorbance vs. time relationship. The measured slopes were made relative to the initial TAFIa activity, and the data was fitted with the double exponential decay function using Sigmaplot (SPSS Inc). All microtiter plates were pretreated with HBS/Tween 1% as described. Fresh frozen citrated human plasma was thawed at 37 °C and passed over a 1-mL mAb16-Sepharose 4B column at room temperature. The column was then washed extensively with HBS/Tween 0.01%, the bound protein was eluted with 0.2 mol L−1 glycine, pH 3, and the column was re-equilibrated in HBS/Tween 0.01%. In total, this process was repeated three times with the same plasma sample. To be certain that the immunodepleted plasma was TAFI deficient, clot lysis assays were performed with the plasma either in the presence or in the absence of 10 nmol L−1 Solulin. As the time to 50% clot lysis was the same in both cases, we concluded that the plasma was TAFI-deficient. In every case, either normal pooled human plasma (Precision Biologic, Dartmouth, Nova Scotia, Canada) or TAFI-Immunodepleted human plasma was diluted 1 : 3 with HBS/Tween 0.01% in a well of a 96-well microtiter plate that had been pretreated with HBS/Tween 1% as described. Clotting was initiated by the addition of human thrombin (5 nmol L−1 final concentration), CaCl2 (10 mmol L−1 final concentration) and PCPS (20 µmol L−1 final concentration) in the presence of tPA (0.5 nmol L−1 final concentration). All reactions were covered to eliminate evaporation and incubated at 37 °C while the turbidity was monitored at 400 nm in a Spectramax Plus plate reader (Molecular Devices, La Jolla, CA, USA). The time to 50% clot lysis was determined graphically as the midpoint between the maximum absorbance after clot formation and the minimum absorbance after complete clot lysis. Clot lysis assays performed using TAFI-Immunodepleted plasma were supplemented with TAFI (0–80 nmol L−1), and PTCI (0–2 µmol L−1) or GEMSA (0–6 mmol L−1), in the presence of Solulin (10 nmol L−1 final concentration). Clot lysis assays performed using pooled fresh frozen human plasma (Precision Biologic) were supplemented with either PTCI (0–2 µmol L−1) or GEMSA (0–6 mmol L−1), in the presence of Solulin (10 nmol L−1 final concentration). Clots were formed in the wells of a microtiter plate from fibrinogen (3 µmol L−1), plasminogen (600 nmol L−1), antiplasmin (300 nmol L−1), thrombin (5 nmol L−1), tPA (0.5 nmol L−1), Solulin (10 nmol L−1), CaCl2 (5 mmol L−1), and PTCI (0–2 µmol L−1) in the presence or absence of TAFI (40 nmol L−1). All reactions were covered to eliminate evaporation and incubated at 37 °C while the turbidity was monitored at 400 nm in a Spectramax Plus plate reader (Molecular Devices, La Jolla, CA, USA). The times to 50% clot lysis were determined as described. All microtiter plates were pretreated with HBS/Tween 1% as described. We have observed variation in the ability of commercially obtained preparations of PTCI to inhibit TAFIa. Therefore, we believe that it is critical to assess the potency of all PTCI preparations against TAFIa before relying on their inhibitory potential in experimental models. To illustrate this point we have determined the inhibition kinetics of two separate lots of PTCI against TAFIa, and have found a ninefold difference in the KI. The more potent PTCI preparation had a KI of 1.2 ± 0.3 nmol L−1 while the less potent PTCI preparation had a KI of 11 ± 1.3 nmol L−1. Both of these PTCI preparations are high affinity TAFIa inhibitors; however, they are still much less potent than reported previously for PTCI (KI = 0.4 nmol L−1) [6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. We have also determined the potency of GEMSA against TAFIa, and estimated a KI of 11.3 ± 0.4 µmol L−1, which is similar to what has been reported previously for this compound [6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. All subsequent work in this study used the more potent PTCI preparation (KI = 1.2 ± 0.3 nmol L−1) or GEMSA. Previous work has shown that GEMSA can slow the spontaneous inactivation of TAFIa [6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. To determine whether this stabilizing effect is a property peculiar to GEMSA, or whether it is a more general property of reversible TAFIa inhibitors that occupy the active site we have measured the apparent half-life TAFIa in the presence of various concentrations of either GEMSA in Fig. 1 or PTCI in Fig. 2. The activity remaining is reported relative to the initial activity.Figure 2Stabilization of TAFIa Activity with PTCI. TAFI was activated with thrombin/thrombomodulin, diluted to 100 nmol L−1, and incubated at 37 °C with 0 nmol L−1 (●), 50 nmol L−1 (▵), 100 nmol L−1 (▴), 150 nmol L−1 (□), or 200 nmol L−1 (▪) PTCI. Activity was reported relative to the initial and the data are fitted with the double exponential decay equation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the presence of GEMSA, TAFIa is stabilized, with the apparent half-life increasing in proportion to the concentration of inhibitor present. As GEMSA is a weak inhibitor of TAFIa, changes in the concentration of TAFIa that occur as it spontaneously inactivates are insignificant with respect to the concentrations of GEMSA used, and we were able to estimate the apparent half-lives using a single exponential decay function. In the presence of GEMSA (0, 5, 10, 25, 50 µmol L−1) we estimated the half-lives to be 8.2 ± 0.2 min, 29.6 ± 1.5 min, 39.7 ± 1.3 min, 64 ± 5.2 min, 106 ± 7.2 min, respectively. By 400 µmol L−1 GEMSA, no loss of TAFIa activity could be measured over the 90-min incubation. Thus, as shown previously [6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar], GEMSA can prolong the apparent half-life of a TAFIa pool. Similar to GEMSA, in the presence of PTCI, TAFIa exhibits a prolongation of its apparent half-life in proportion to the concentration of inhibitor; however, since PTCI is a much more potent inhibitor of TAFIa, we were limited in the range of PTCI concentrations that we could use and still reliably measure TAFIa activity. This limitation also means that the concentration of TAFIa is not insignificant with respect to the PTCI concentrations used, and as the initial TAFIa pool inactivates, the change in the concentration of TAFIa alters the fraction of enzyme bound to PTCI during the course of the incubation. To take this process into account, the apparent decay of TAFIa is fitted with the double exponential decay function. We have estimated the half-life of TAFIa by calculating how long it takes for the residual TAFIa activity to decrease to half of its original value. In the presence of PTCI (0, 50, 100, 150, 200 µmol L−1) we estimated the half-lives to be 8 min, 10 min, 15 min, 35 min, and 85 min, respectively. Although GEMSA and PTCI can both reversibly inhibit TAFIa, they are quite different substances. GEMSA is a small molecule while PTCI is a 4-kDa protein that inserts a pentapeptide tail into the active site of carboxypeptidase-B like enzymes [14Rees D.C. Lipscomb W.N. Structure of the potato inhibitor complex of carboxypeptidase A at 2.5-Å resolution.Proc Natl Acad Sci USA. 1980; 77: 4633-7Crossref PubMed Scopus (48) Google Scholar]. As two fundamentally different types of inhibitor can stabilize TAFIa, this effect is probably a common property of compounds that occupy the active site, perhaps even including physiologically relevant substrates such as partially plasmin-degraded fibrin. PTCI and GEMSA can increase the apparent half-life of TAFIa while simultaneously decreasing the apparent total concentration of TAFIa. Therefore, we were interes
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