Carboxy-terminal Lysine Residues Research Articles

Surface Protein Dispersin of Enteroaggregative Escherichia coli Binds Plasminogen That Is Converted Into Active Plasmin.

Dispersin is a 10.2 kDa-immunogenic protein secreted by enteroaggregative Escherichia coli (EAEC). In the prototypical EAEC strain 042, dispersin is non-covalently bound to the outer membrane, assisting dispersion across the intestinal mucosa by overcoming electrostatic attraction between the AAF/II fimbriae and the bacterial surface. Also, dispersin facilitates penetration of the intestinal mucus layer. Initially characterized in EAEC, dispersin has been detected in other E. coli pathotypes, including those isolated from extraintestinal sites. In this study we investigated the binding capacity of purified dispersin to extracellular matrix (ECM), since dispersin is exposed on the bacterial surface and is involved in intestinal colonization. Binding to plasminogen was also investigated due to the presence of conserved carboxy-terminal lysine residues in dispersin sequences, which are involved in plasminogen binding in several bacterial proteins. Moreover, some E. coli components can interact with this host protease, as well as with tissue plasminogen activator, leading to plasmin production. Recombinant dispersin was produced and used in binding assays with ECM molecules and coagulation cascade compounds. Purified dispersin bound specifically to laminin and plasminogen. Interaction with plasminogen occurred in a dose-dependent and saturable manner. In the presence of plasminogen activator, bound plasminogen was converted into plasmin, its active form, leading to fibrinogen and vitronectin cleavage. A collection of E. coli strains isolated from human bacteremia was screened for the presence of aap, the dispersin-encoding gene. Eight aap-positive strains were detected and dispersin production could be observed in four of them. Our data describe new attributes for dispersin and points out to possible roles in mechanisms of tissue adhesion and dissemination, considering the binding capacity to laminin, and the generation of dispersin-bound plasmin(ogen), which may facilitate E. coli spread from the colonization site to other tissues and organs. The cleavage of fibrinogen in the bloodstream, may also contribute to the pathogenesis of sepsis caused by dispersin-producing E. coli.

Small size apolipoprotein(a) isoforms enhance inflammatory and proteolytic potential of collagen-primed monocytes

BackgroundAtherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease.Design and methodsThe aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen.ResultsApo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites.ConclusionsThese results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis.

The preparation of a t-PA/nanoparticle conjugate via bio-affinity ligation

Event Abstract Back to Event The preparation of a t-PA/nanoparticle conjugate via bio-affinity ligation Xiaoli Liu1, Zengchao Tang1, Dan Li1* and Hong Chen1* 1 Soochow University, China Introduction: Tissue plasminogen activator (t-PA) is commonly used as a thrombolytic agent in the treatment of thrombotic diseases such as acute myocardial infarction and ischemic stroke. A major issue in the therapeutic use of t-PA is its very short half-life in the circulation (2–6 min) due to the effects of inhibitors, enzymes and antibodies in blood. Efforts have been made to overcome this deficiency by conjugating t-PA to synthetic materials to limit access of inhibitors or to materials with thrombus-targeted ability.[1] However, the linkages between t-PA and synthetic materials are commonly based on covalent bonding or electrostatic interactions, which are “invasive” and generally accompanied by loss of protein activity and immunogenic responses. Bio-affinity ligation based on the specific interactions between ligands and receptors has many advantages over covalent bonding and other nonspecific interactions for the conjugation of proteins to synthetic materials since it is benign to protein, highly specific and efficient, proceeds under physiological conditions and has no unwanted side reactions. Most proteins possess specific domains that can be recognized by some affinity ligand, so the interaction between such a domain and its affinity ligand can be utilized to link protein to materials. For t-PA, there is a high affinity lysine binding site in one of its two kringle domains that mediates its binding to carboxy-terminal lysine residues (ε-lysines) exposed on the surface of fibrin. Therefore ε-lysine is a natural affinity ligand that can be utilized to link t-PA to a synthetic material. Moreover, t-PA conjugated through ε-lysine mimics the one bound on the surface of fibrin in physiological fibrinolytic process, which is advantageous for limiting access of inhibitors. In this study, a t-PA/gold nanoparticle (t-PA/AuNP) conjugate was prepared via bio-affinity ligation. Materials and Methods: Here AuNPs were used as the model nanoparticles and modified with ε-lysine-terminated polyvinyl pyrrolidone (PVP), referred to as AuNPs-PVP-Lys. PVP functions as a bio-inert moiety to prevent the conjugate from interacting nonspecifically with other components in blood. t-PA was conjugated to AuNPs-PVP-Lys under physiological conditions through affinity interactions between ε-lysine exposed on AuNP surface and the specific domain in t-PA. Results and Discussion: Activity of the conjugated t-PA was compared with free t-PA both in vitro and in vivo. It is found that the t-PA/AuNP conjugate can not only maintain almost all enzyme activity of t-PA but also reduce the inhibition effect, leading to a prolonged circulation time (20.5 min). Conclusions: In conclusion, a t-PA/AuNPs-PVP-Lys conjugate was prepared under physiological conditions via bio-affinity ligation based on the bio-affinity interactions under physiological conditions. Compared with free t-PA, the conjugate retained almost full enzyme activity and clot-dissolving efficiency, and the t-PA was protected from inhibition by PAI-1 to some extent. Moreover, the conjugate showed prolonged circulation time in vivo. It will be of interest to apply this bio-affinity ligation more generally to conjugate synthetic materials with other proteins, especially those with important biomedical functions. National Natural Science Foundation of China (21204055, 21304062, 21334004); National Natural Science Foundation Fund for Distinguished Young Scholars (21125418); The Natural Science Foundation of the Jiangsu Higher Education Institutions of China (13KJB430020); China Postdoctoral Science Foundation (2013M541714)

Plg‐RKT Deficient Mice Exhibit Defective Macrophage Migration and Plasminogen Binding

Plg‐RKT is a novel transmembrane plasminogen receptor that binds plasminogen via a carboxy‐terminal lysine residue. In order to evaluate the role of Plg‐RKT in vivo we generated Plg‐RKT‐/‐ mice using homologous recombination and backcrossed the mice ten generations into the C57BL/6J background. The mice were viable and fertile. Among 2,340 littermates from heterozygous matings 611 were Plg‐RKT+/+ (26%), 1,136 were Plg‐RKT +/‐ (49%) and 593 were Plg‐RKT ‐/‐ (25%). This distribution was not significantly different (P>0.05, Chi‐square test) from the expected Mendelian 1:2:1 ratios and indicate equal viability at 3 weeks of age of Plg‐RKT +/+, Plg‐RKT +/‐, and Plg‐RKT ‐/‐ mice. Hematological parameters were determined. There was no effect of genotype on activated partial thromboplastin time, prothrombin time, plasma plasminogen and fibrinogen levels or hematocrit. Plasminogen binding to mouse macrophages was evaluated using quantitative fluorescence activated cell sorting. Plasminogen binding to Plg‐RKT ‐/‐ macrophages was only 20% of that of Plg‐RKT +/+ macrophages (Bmax = 16,386 ± 1,315 molecules/cell and 81,399 ±9,585 molecules/cell, respectively). Furthermore, macrophage migration to the peritoneum 72 hr following thioglycollate injection was 60% less in Plg‐RKT ‐/‐ mice compared to Plg‐RKT +/+ littermates. In conclusion, Plg‐RKTis required for plasminogen binding and macrophage migration in vivo, key features of the inflammatory response.

Carboxypeptidase N-deficient mice present with polymorphic disease phenotypes on induction of experimental autoimmune encephalomyelitis

Carboxypeptidase N (CPN) is a member of the carboxypeptidase family of enzymes that cleave carboxy-terminal lysine and arginine residues from a large number of biologically active peptides and proteins. These enzymes are best known for their roles in modulating the activity of kinins, complement anaphylatoxins and coagulation proteins. Although CPN makes important contributions to acute inflammatory events, little is known about its role in autoimmune disease. In this study we used CPN−/− mice in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. Unexpectedly, we observed several EAE disease phenotypes in CPN−/− mice compared to wild type mice. The majority of CPN−/− mice died within five to seven days after disease induction, before displaying clinical signs of disease. The remaining mice presented with either mild EAE or did not develop EAE. In addition, CPN−/− mice injected with complete or incomplete Freund's adjuvant died within the same time frame and in similar numbers as those induced for EAE. Overall, the course of EAE in CPN−/− mice was significantly delayed and attenuated compared to wild type mice. Spinal cord histopathology in CPN−/− mice revealed meningeal, but not parenchymal leukocyte infiltration, and minimal demyelination. Our results indicate that CPN plays an important role in EAE development and progression and suggests that multiple CPN ligands contribute to the disease phenotypes we observed.

Levels of thrombin activatable fibrinolysis inhibitor in gestational diabetes mellitus

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase, which is synthesised in liver and activated by thrombin and the thrombin–thrombomodulin complex. TAFI suppresses fibrinolysis by removing carboxy–terminal lysine residues from partially degraded fibrin. In this study we aimed to assess the circulating levels of TAFI antigen, ‘a fibrinolytic parameter’ in women with gestational diabetes (GDM). Thirty-four pregnant women with GDM and 50 pregnant women with normal glucose tolerance were included in the study. Plasma TAFI antigen levels were significantly higher in pregnant women with GDM when compared with controls. Increased TAFI levels may contribute to the decreased fibrinolytic potency, causing a thrombophilic state. GDM is regarded as a specific form of diabetes, and it could in addition be a predictor of type 2 diabetes mellitus in the future and the risk of complications due to hypercoagulability increases in this disease. Increased TAFI levels may also have a role in increased risk of hypercoagulability.

Role of Thrombin Activatable Fibrinolysis Inhibitor in Endocrine and Cardiovascular Disorders: An Update

Thrombin activatable fibrinolysis inhibitor (TAFI) is a zymogene that potently inhibits fibrinolysis through the removal of the carboxy-terminal lysine and arginine residues from partially degraded fibrin polymers. In addition, TAFI has a suppressor effect on conversion of inactive plasminogen to plasmin. Since impaired fibrinolysis is a very well established risk factor for cardiovascular morbidity and mortality, understanding the role of TAFI in cardiovascular disorders, insulin resistance, diabetes and other endocrine problems may hold promise for improving management of these diseases. This paper includes a review of current evidence on TAFI pathway and its alteration in endocrine and cardiovascular disorders and relevant patents.

Intravenous and oral administrations of DD2 [7-Amino-2-(sulfanylmethyl)heptanoic acid] produce thrombolysis through inhibition of plasma TAFIa in rats with tissue factor-induced microthrombosis

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen that is activated by thrombin in plasma. In fibrinolytic processes, carboxy-terminal lysine (Lys) residues in partially degraded fibrin are important sites for plasminogen binding and activation, and an active form of TAFI (TAFIa) inhibits fibrinolysis by eliminating these residues proteolytically. We synthesized DD2 [7-Amino-2-(sulfanylmethyl)heptanoic acid], a Lys analogue containing sulfur, as an inhibitor of TAFIa and investigated its pharmacological profile and pathophysiological role in thrombolysis via in vitro and in vivo studies. DD2 specifically inhibited plasma TAFIa activity with an apparent IC50 (50% inhibitory concentration) value of 3.4×10-8M under the present experimental condition and enhanced tissue plasminogen activator-mediated clot lysis in a concentration-dependent manner. In order to study tissue factor (TF)-induced microthrombosis in an animal model, rats were given intravenous injection (2.5mg/kg and higher) or oral administration (10mg/kg and higher) of DD2. This attenuated TF-induced glomerular fibrin deposition and increased the plasma levels of fibrin degradation products and D-dimer in a dose-dependent manner. A DD2 dose approximately 4X higher than the dose used in intravenous injections was required to achieve an equivalent thrombolytic effect to that seen following oral administration. Moreover, the oral absorption efficiency of DD2 into the vasculature was 29.8%. These results indicate that both intravenous and oral administration of DD2 enhanced endogenous fibrinolysis and reduced thrombi in a TF-induced microthrombosis model.

Green tea polyphenols increase p53 transcriptional activity and acetylation by suppressing class�I histone deacetylases

Acetylation of the tumor suppressor gene p53 at the carboxy-terminal lysine (Lys) residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Histone deacetylases (HDACs), a family of evolutionarily conserved enzymes, counterbalance the acetylation of lysine residues on histone and non-histone proteins. In this study, we demonstrate that green tea polyphenols (GTPs) and their major constituent, (-) epigallocatechin-3-gallate (EGCG), activate p53 through acetylation at the Lys373 and Lys382 residues by inhibiting class I HDACs in LNCaP human prostate cancer cells. Treatment of cells with GTPs (2.5-10 µg/ml) and EGCG (5-20 µM) resulted in dose- and time-dependent inhibition of class I HDACs (HDAC1, 2, 3 and 8), albeit at varying levels. Discontinuation of treatment with GTP/EGCG resulted in the loss of p53 acetylation at both the sites in these cells. GTP/EGCG treatment also resulted in increased expression of p21/waf1 and Bax at the protein and message levels in these cells. The increased GTP/EGCG-mediated p53 acetylation enhanced its binding on the promoters of p21/waf1 and Bax, which was associated with increased accumulation of cells in the G0/G1 phase of the cell cycle and induction of apoptosis. Our findings indicate that GTP/EGCG causes acetylation of p53 by inhibiting class I HDACs, a function that is likely to be part of the mechanisms that control the physiological activity of p53.

Fibrinolytic cross-talk: a new mechanism for plasmin formation

AbstractFibrinolysis and pericellular proteolysis depend on molecular coassembly of plasminogen and its activator on cell, fibrin, or matrix surfaces. We report here the existence of a fibrinolytic cross-talk mechanism bypassing the requirement for their molecular coassembly on the same surface. First, we demonstrate that, despite impaired binding of Glu-plasminogen to the cell membrane by ϵ-aminocaproic acid (ϵ-ACA) or by a lysine-binding site–specific mAb, plasmin is unexpectedly formed by cell-associated urokinase (uPA). Second, we show that Glu-plasminogen bound to carboxy-terminal lysine residues in platelets, fibrin, or extracellular matrix components (fibronectin, laminin) is transformed into plasmin by uPA expressed on monocytes or endothelial cell–derived microparticles but not by tissue-type plasminogen activator (tPA) expressed on neurons. A 2-fold increase in plasmin formation was observed over activation on the same surface. Altogether, these data indicate that cellular uPA but not tPA expressed by distinct cells is specifically involved in the recognition of conformational changes and activation of Glu-plasminogen bound to other biologic surfaces via a lysine-dependent mechanism. This uPA-driven cross-talk mechanism generates plasmin in situ with a high efficiency, thus highlighting its potential physiologic relevance in fibrinolysis and matrix proteolysis induced by inflammatory cells or cell-derived microparticles.

Role of plasminogen activation in neuronal organization and survival

We characterized the interactions between plasminogen and neurons and investigated the associated effects on extracellular matrix proteolysis, cell morphology, adhesion, signaling and survival. Upon binding of plasminogen to neurons, the plasmin formed by constitutively expressed tissue plasminogen activator (tPA) degrades extracellular matrix proteins, leading to retraction of the neuron monolayer that detaches from the matrix. This sequence of events required both interaction of plasminogen with carboxy-terminal lysine residues and the proteolytic activity of plasmin. Surprisingly, 24 h after plasminogen addition, plasmin-detached neurons survived and remained associated in clusters maintaining focal adhesion kinase phosphorylation contrasting with other adherent cell types fully dissociated by plasmin. However, long-term incubation (72 h) with plasminogen was associated with an increased rate of apoptosis, suggesting that prolonged exposure to plasmin may cause neurotoxicity. Regulation of neuronal organization and survival by plasminogen may be of pathophysiological relevance, as plasminogen is expressed in the brain and/or extravasate during vascular accidents or inflammatory processes.

High Thrombin Activatable Fibrinolysis Inhibitor (TAFI) Levels May Protect against Recurrent Fetal Loss.

Trombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase which suppresses fibrinolysis by removing carboxy-terminal lysine residues from partially degraded fibrin. A recent study in pregnant mice suggested that fibrin degradation products induced by the subsequent generation of thrombin and fibrin, cause apoptosis of throphoblasts and consequently fetal loss. This effect was reversed by anticoagulant and antifibrinolytic drugs, and by depletion of fibrinogen. We hypothesized that increased levels of TAFI during normal pregnancy in humans may similarly protect against fetal loss. This effect might be more pronounced in women with increased thrombin generation due to thrombophilic defects. To test this hypothesis, we analysed data from four pooled family cohort studies, which originally were designed to estimate the absolute risk of venous thromboembolism, associated with either hereditary deficiencies of antithrombin, protein C or protein S, factor V Leiden, the prothrombin 20210A mutation, elevated plasma levels of factor VIII:C, or hyperhomocysteinemia. The present study addressed fetal loss in female probands and relatives. In addition to above mentioned thrombophilic defects, TAFI activity was measured. Fetal loss rates were compared in women with high TAFI levels (³ 126 U/dl) and women with normal TAFI levels.Of 1557 women, 175 were excluded because they were younger than 15 years of age, had deceased or did not consent. Another 444 women were not evaluable because they had never been pregnant or had had only terminated pregnancies, and 95 women because of missing TAFI measurements. The remaining 843 women (including probands) were analysed, of whom 95 women had high TAFI levels and 748 women had normal TAFI levels. Age at time of first pregnancy was comparable in both groups, as was the distribution of thrombophilic defects. Results are summarized in the table.18 women (18.9 %) with high TAFI levels experienced any, i.e. early or late fetal loss, compared to 205 women (27.5 %) with normal TAFI levels (p= 0.074). Overall 17 women (17.9 %) with high TAFI levels experienced one or recurrent early fetal loss compared to 180 women (24.2 %) with normal TAFI levels (p= 0.173). 1 woman (1.1 %) with high TAFI level had experienced recurrent early fetal loss, compared to 53 women (7.1 %) with normal TAFI levels (p=0.023). The number of fetal losses per woman ranged from 2 to 8 in women with normal TAFI levels (median was 2 fetal losses), while it was 2 in the woman with high TAFI level. In conclusion, women with high TAFI levels had less recurrent fetal loss in comparison with women with normal TAFI levels. Our results support the assumption that high TAFI levels protects against recurrent fetal loss.Normal TAFI levelsHigh TAFI levelsp-valueAge at 1st pregnancy, median (range), yr24.8 (11-42)24.1 (17-40 )TAFI levels, U/dL, mean (SD)99.7 (13.6)137.1 (10.6)Women, n(n=748)(n=95)Total fetal loss, n (%)206 (27.5)18 (18.9) 0.074early180 (24.2)17 (17.9)0.173late29 (3.9)2 (2.1)0.383Total recurrent fetal loss, n (%)56 (7.5)2 (2.1)0.050early53 (7.1)1 (1.1)0.023late2 (0.3)0 (0)0.613

TAFI antigen level variability in young healthy Asian Indians; First report from Asia

Thrombin Activatable Fibrinolytic Inhibitor (TAFI) is a plasma protein, which inhibits fibrinolysis by removing carboxyterminal lysine residues from partially degraded fibrin thereby decreasing plasminogen binding on its surface. In this study we have investigated the antigenic level variability (Inter and Intraindividual) of Thrombin Activatable Fibrinolysis Inhibitor in 120 healthy Asian Indians since no data on this is available regarding this population. TAFI antigen levels did not show a normal distribution in our population ( p < 0.001). Median TAFI antigen levels were found to be 11.683 µg/ml. It ranged from 33.9–202.5%of normal pool plasma (3.9–23.5 µg/ml). TAFI antigenic level showed high level of variability in the Indian population (coefficient of variation: 37.4%). TAFI antigenic levels were stable intraindividually ( p = 0.218).

Role of Thrombin Activatable Fibrinolysis Inhibitor in Endocrine and Cardiovascular Disorders

Thrombin activatable fibrinolysis inhibitor (TAFI) is a zymogene that potently inhibits fibrinolysis. It is synthesized by the liver and has an inhibitor role in fibrinolysis through the removal of the carboxy-terminal lysine and arginine residues from partially degraded fibrin polymers. Besides, TAFI has a suppressor effect on conversion of inactive plasminogen to plasmin. Alterations in TAFI pathway have been reported in many conditions. Few data are present in the literature regarding the relationship between TAFI and endocrine disorders. The results from few studies in patients with cardiovascular disease are conflicting. Understanding the role of TAFI in the pathogenesis of endocrine and cardiovascular disorders may hold promise for improving management of these diseases. This paper includes a review of the studies and patents concerned with TAFI pathway and endocrine and cardiovascular problems.

TAFI and Pancreatic Carboxypeptidase B Modulate In Vitro Capillary Tube Formation by Human Microvascular Endothelial Cells

Besides having a key role in fibrinolysis, the plasminogen system has been implicated in cell migration and angiogenesis. A common mechanism is the binding of plasminogen to carboxy-terminal lysine residues in partially degraded fibrin or on cellular surfaces. Here we examined the involvement of thrombin activatable fibrinolysis inhibitor (TAFI) and pancreatic carboxypeptidase B (CPB) in an in vitro capillary tube formation system, which is largely plasminogen-dependent. Human microvascular endothelial cells (hMVECs) were seeded on a 3D plasma clot matrix and subsequently stimulated with bFGF/tumor necrosis factor (TNF)-alpha. Tube formation was analyzed and fibrin degradation products (FbDP) were determined in the medium. Supplementation of the matrix with additional TAFI or CPB produced a reduction in tube formation. Pretreatment of hMVECs with CPB before seeding resulted in a similar effect. FbDP-levels indicated a concomitant reduction in matrix proteolysis. A TAFIa inhibitor increased tube formation and FbDP release into the medium. In separate assays, CPB impaired the migration of hMVECs in a dose-dependent manner, whereas proliferation and adhesion remained unaffected. Overall, these results demonstrate that TAFI and CPB in these systems modulate the plasminogen system both in the matrix and on the cell surface, thus leading to the inhibition of endothelial cell movement and tube formation.

Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function

During the analytical characterization of the humanized Lewis-Y specific monoclonal antibody IGN311 (IgG1/κ) used for passive anti-cancer therapy in humans, isoelectric focusing (IEF) experiments revealed that IGN311 batches produced in serum-containing and serum-free medium, respectively, displayed different banding patterns. The additional bands in the IEF pattern correlated with additional peaks observed by subsequent cation exchange (CEX)-HPLC analysis. Since the IEF pattern is one of the specification criteria in the quality control of monoclonal antibodies and a non-matching pattern may be indicative for lot-to-lot inconsistency, this phenomenon was investigated in detail. First, we investigated whether a difference in antibody glycosylation was the cause for the observed charge heterogeneity. De- N-glycosylation experiments demonstrated that charge heterogeneity observed in the IEF pattern is not a consequence of glycosylation. In contrast, sample treatment by carboxypeptidase B, removing the carboxy-terminal lysine residues from the two heavy chains of the antibody, resulted in reduced charge heterogeneity eliminating the two most basic bands observed in IEF. These data were supported by reversed phase HPLC–MALDI-TOF-MS analysis of enzymatically cleaved peptides of the antibody as well as by carboxy-terminal sequencing of the heavy chains. It was demonstrated that the differences in the IEF banding pattern were due to lysine clipping occurring during the production of the antibody. The antibody batch produced under serum-free conditions was less affected by lysine clipping. Both antibody variants – clipped and unclipped – elicited the same potency in a complement dependent cytotoxicity (CDC) assay demonstrating that lysine clipping of IGN311 does not impair Fc-mediated effector functions.

A functional assay for measuring activated thrombin-activatable fibrinolysis inhibitor in plasma

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase found in plasma that is activated by thrombin, the thrombin–thrombomodulin complex, or plasmin. The active carboxypeptidase, TAFIa, attenuates fibrinolysis by removing newly exposed carboxy-terminal lysine residues on fibrin. The half-maximal effect of TAFIa on clot lysis occurs at 1 nM and the maximal effect occurs at 20 nM. Since the circulating concentration of the procarboxypeptidase is approximately 75 nM, only a small portion needs to be activated to have a significant effect on clot lysis. Several assays to measure total plasma TAFI levels and plasma TAFIa levels after it is fully activated exist. However, no currently available assay is sufficiently sensitive and specific to measure endogenous TAFIa in plasma. We have devised a new sensitive and specific assay for TAFIa in plasma that is based on physiologic function. This assay is based on the fact that TAFIa decreases the cofactor activity of high-molecular-weight fibrin degradation products in the stimulation of plasminogen cleavage in a concentration-dependent fashion. With this assay, we can measure TAFIa concentrations as low as 10 pM in plasma and it is not affected by variability in other hemostatic factors. This assay is reliable and repeatable with intra- and interassay variabilities of 6.5 and 6.1%, respectively.

Tumor growth and metastasis are not affected in thrombin‐activatable fibrinolysis inhibitor‐deficient mice

Many studies have indicated that the plasminogen activation system may have a prominent role in cancer. Activation of the zymogen plasminogen into the serine protease plasmin by plasminogen activator is mediated by carboxyterminal basic amino acids in fibrin, including lysines and arginines. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating carboxypeptidase B-type proenzyme that, after activation, removes carboxyterminal lysine or arginine residues in fibrin, resulting in decreased plasminogen activation and attenuated fibrinolysis. To determine directly whether TAFI is involved in primary tumor growth and metastasis formation, we examined the effects of TAFI deficiency on subcutaneous growth and experimentally or spontaneously induced pulmonary metastasis formation of different tumor cell types in mice. In all tumor models TAFI deficiency did not affect the formation and growth of primary and metastasized tumors.

Carboxypeptidase B inhibitors reduce tissue factor-induced renal microthrombi in rats

Procarboxypeptidase B (also known as thrombin-activatable fibrinolysis inhibitor) is a recently described plasma zymogen known to be activated by thrombin in plasma. Carboxy-terminal lysine residues from partially degraded fibrin are important for the binding and activation of plasminogen, and carboxypeptidase B, an active form of procarboxypeptidase B, has been shown to inhibit fibrinolysis by eliminating these residues. The present paper investigates the effects of carboxypeptidase B inhibitors, dl-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGPA) and potato-derived carboxypeptidase inhibitor (CPI), on tissue factor (TF)-induced microthrombosis in rats. Intravenous injection of MGPA (3 mg/kg and higher) or CPI (0.3 mg/kg and higher) after microthrombi formation dramatically attenuated TF-induced glomerular fibrin deposition with an increase in plasma levels of d-dimer. These results indicate that carboxypeptidase B inhibitors can enhance endogenous fibrinolysis and reduce thrombi in the TF-induced microthrombosis model after systemic administration even after thrombi formation.

Le système fibrinolytique : activation et inhibition. Ses modifications en cas de CIVD

The blood fibrinolytic system comprises an inactive proenzyme, plasminogen, that can be converted to the active enzyme, plasmin. Plasmin degrades fibrin and participates in the extracellular matrix remodelling. This conversion implies two physiological plasminogen activators (PA), the tissue type (t-PA) and the urinary type (u-PA). t-PA mediated plasminogen activation is mainly involved in the dissolution of fibrin in the circulation. u-PA binds to a specific cellular receptor (u-PAR), resulting in enhanced activation of cell bound plasminogen. Inhibition of the fibrinolytic system may occur by specific plasminogen activator inhibitors (PAI), or at the level of plasmin, mainly by α 2-antiplasmin. Recently a thrombin activatable fibrinolysis inhibitor (TAFI) has been described. By eliminating carboxyterminal lysine residues from partially degraded fibrin, it could participate to the control of in vivo fibrinolysis. During DIC, fibrinolysis is induced and septic DIC is characterized by a sustained increase in PAI-1 synthesis due to the presence of proinflammatory cytokines, mainly TNFα.