Role of lone-pair electron localization in temperature-induced phase transitions in mimetite.

The crystal structure of mimetite Pb5(AsO4)3Cl, a phosphate with apatite structure-type has been investigated in situ at 123, 173, 273, 288, 353 and 393 K. A careful inspection of the diffraction pattern and subsequent structure refinements indicated that mimetite transforms from the monoclinic to the hexagonal polymorph with increasing temperature. At 123 K, a monoclinic superstructure, mimetite-2M, with cell parameters a = 20.4487 (9),  b = 7.4362 (2), c = 20.4513 (9) Å, ß = 119.953 (6)°, V = 2694.5 (2) Å3 and space group P21 was observed. From 173 to 353 K, the reflections of the supercell were evident only along one direction of the corresponding hexagonal apatite-cell and the structure transforms to the polymorph mimetite-M with space group P21/b and unit-cell parameters a = 10.2378 (3), b = 20.4573 (7), c = 7.4457 (2) Å, ß = 120.039 (5)°, V = 1349.96 (9) Å3. Only at higher temperature, i.e. 393 K, does mimetite adopt the hexagonal space group P63/m characteristic of apatite structure-types. The role of the electron lone pairs of Pb atoms in the phase transition was investigated through the analysis of the electron localization function (ELF) calculated based on the DFT-geometry optimized structures of the three polymorphs. The changes in spatial distribution of the 6s2 electron density during the phase transitions were explored by means of the Wannier Function Centres (WFCs) derived from ab initio molecular dynamics trajectories. In the high-temperature hexagonal structure the 6s2 electrons are spherically symmetric relative to the position of Pb atoms. At low temperature the maximum of 6s2 electron density is displaced relative to the position of Pb atom contributing to the polar interaction in the monoclinic polymorphs.

Variants of Lipid-Related Genes in Adult Japanese Patients with Severe Hypertriglyceridemia.

AIM: Hypertriglyceridemia is a type of dyslipidemia that contributes to atherosclerosis and coronary heart disease. Variants in lipoprotein lipase (LPL), apolipoprotein CII (APOC2), apolipoprotein AV (APOA5), glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), lipase maturation factor 1 (LMF1), and glucokinase regulator (GCKR) are responsible for hypertriglyceridemia. We investigated the molecular basis of severe hypertriglyceridemia in adult patients referred to the Clinical Laboratory at Fukuoka University Hospital. METHODS: Twenty-three adult patients with severe hypertriglyceridemia (＞1,000 mg/dL, 11.29 mmol/L) were selected. The coding regions of candidate genes were sequenced by next-generation sequencing. Forty-nine genes reportedly associated with hypertriglyceridemia were analyzed. RESULTS: In the 23 patients, we detected 70 variants: 28 rare and 42 common ones. Among the 28 rare variants with ＜1% allele frequency, p.I4533L in APOB, p.M490I in MLXIPL, p.L152M in NCAN, and p.S264T in TIMD4 were novel. We did not observe single gene homozygous or compound heterozygous disease-causing rare variants in any of the 23 hypertriglyceridemia cases. However, in silico algorithms and previous reports indicated that five rare variants, APOA5 (p.T184S), GCKR (c.354＋1G＞A), LMF1 (p.G410R), and LRP1 (p.G813R; p.R2173Q), and seven common variants, APOA5 (pG185C), APOE (p.C130R; p.E262K/p.E263K), GCKR (p.V103M), GPIHBP1 (p.C14F), LRP1 (p.Y4054F), and MLXIPL (p.Q241H), can cause hypertriglyceridemia. However, all five disease-causing rare variants detected in this study were heterozygous. CONCLUSIONS: The prevalence of disease-causing rare variants in candidate genes in severe hypertriglyceridemia patients was low. The major causes of severe hypertriglyceridemia were not single gene abnormalities, but involved multiple gene variations and environmental factors.

Treatment Technology of Hazardous Water Contaminated with Radioisotopes with Paper Sludge Ash-Based Geopolymer-Stabilization of Immobilization of Strontium and Cesium by Mixing Seawater.

Long-term immobilization ratios of strontium (Sr2+) and cesium (Cs⁺) in paper sludge ash-based geopolymer (PS-GP) were investigated in one year. PS-GP paste specimens were prepared in the conditions of 20 °C and 100% R.H., using two kinds of paper sludge ash (PS-ash). Two kinds of alkaline solution were used in the PS-GP as activator. One was prepared by diluting aqueous Na-disilicate (water glass) with seawater. Another was a mixture of this solution and caustic soda of 10 M concentration. When seawater was mixed into the alkaline solution, unstable fixations of Sr2+ and Cs⁺ were greatly improved, resulting stable and high immobilization ratios at any age up to one year, no matter what kind of PS-ash and alkaline solution were used. Element maps obtained by EPMA exhibited nearly even distribution of Cs⁺. However Sr2+ was biased, making domains so firmly related to Ca2+ presence. The mechanism that seawater stabilizes immobilization of Sr2+ and Cs⁺ was discussed in this study, but still needs to further investigation. Chemical composition analyses of PS-GP were also conducted by SEM-EDS. Two categories of GP matrix were clearly observed, so called N-A-S-H and C-A-S-H gels, respectively. By plotting in ternary diagrams of SiO2-(CaO + Na2O)-Al2O3 and Al2O3-CaO-Na2O, compositional trends were discussed in view of 'plagioclase gels' newly found in this study. As a result, it is suggested that the N-A-S-H and C-A-S-H gels should be strictly called Na-rich N-C-A-S-H and Ca-rich N-C-A-S-H gels, respectively.

Lipocalin-type prostaglandin D synthase scavenges biliverdin in the cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the second major protein in human cerebrospinal fluid (CSF) and belongs to the lipocalin superfamily composed of various secretory lipophilic ligand transporter proteins. However, the endogenous ligand of L-PGDS has not yet been elucidated. In this study, we purified L-PGDS from the CSF of aneurysmal subarachnoid hemorrhage (SAH) patients. Lipocalin-type PG D synthase showed absorbance spectra with major peaks at 280 and 392 nm and a minor peak at around 660 nm. The absorbance at 392 nm of L-PGDS increased from 1 to 9 days and almost disappeared at 2 months after SAH, whereas the L-PGDS activity decreased from 1 to 7 days and recovered to normal at 2 months after SAH. These results indicate that some chromophore had accumulated in the CSF after SAH and bound to L-PGDS, thus inactivating it. Matrix assisted laser desorption ionization time-of-flight mass spectrometry of L-PGDS after digestion of it with endoproteinase Lys-C revealed that L-PGDS had covalently bound biliverdin, a by-product of heme breakdown. These results suggest that L-PGDS acted as a scavenger of biliverdin, which is a molecule not found in normal CSF. This is the first report of identification of a pathophysiologically important endogenous ligand for this lipocalin superfamily protein in humans.

Thrombin-activatable carboxypeptidase B cleavage of osteopontin regulates neutrophil survival and synoviocyte binding in rheumatoid arthritis.

OBJECTIVE: Osteopontin (OPN) is a proinflammatory cytokine that plays an important role in the pathogenesis of rheumatoid arthritis (RA). OPN can be cleaved by thrombin, resulting in OPN-R and exposing the cryptic C-terminal alpha4beta1 and alpha9beta1 integrin-binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also called thrombin-activatable fibrinolysis inhibitor, removes the C-terminal arginine from OPN-R, generating OPN-L and abrogating its enhanced cell binding. We undertook this study to investigate the roles of OPN-R and OPN-L in synoviocyte adhesion, which contributes to the formation of invasive pannus, and in neutrophil survival, which affects inflammatory infiltrates in RA. METHODS: Using specifically developed enzyme-linked immunosorbent assays, we tested the synovial fluid of patients with RA, osteoarthritis (OA), and psoriatic arthritis (PsA) to determine OPN-R, OPN-L, and full-length OPN (OPN-FL) levels. RESULTS: Elevated levels of OPN-R and OPN-L were found in synovial fluid samples from RA patients, but not in samples from OA or PsA patients. Increased levels of OPN-R and OPN-L correlated with increased levels of multiple inflammatory cytokines, including tumor necrosis factor alpha and interleukin-6. Immunohistochemical analyses revealed robust expression of OPN-FL, but only minimal expression of OPN-R, in RA synovium, suggesting that cleaved OPN is released into synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an antiapoptotic effect on neutrophils. OPN-R augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to alpha4beta1, whereas OPN-L did not. CONCLUSION: Thrombin activation of OPN (resulting in OPN-R) and its subsequent inactivation by thrombin-activatable CPB (generating OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA by regulating neutrophil viability and reducing synoviocyte adhesion.

The roles of selected arginine and lysine residues of TAFI (Pro-CPU) in its activation to TAFIa by the thrombin-thrombomodulin complex.

Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

A novel inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) - part I: pharmacological characterization.

We have discovered a novel small-molecule (3-phosphinoylpropionic acid) inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa), BX 528, which had an IC (50) of 2 nM in an enzymatic assay and 50 nM in an in-vitro clot lysis assay, with 3,500- to 35,000-fold selectivity against other carboxypeptidases, such as CPN, CPZ and CPD, and 5- and 12-fold selectivity against CPE (CPH) and CPB, respectively. At 10 micro M, BX 528 had no significant activity (<50% inhibition or antagonism) in a panel of 137 enzymes and receptors. It had no effects on blood coagulation and platelet aggregation up to 300 and 10 micro M, respectively. The plasma half-life following intravenous administration was 0.85 hours in rats and 4.5 hours in dogs. No significant metabolism was detected in human, dog or rabbit hepatic microsomes, and no significant inhibition of cytochrome P450 3A4 and 2D6 up to 30 micro M. No cytotoxic or cell proliferative effects were found in three hepatic and renal cell lines up to 300 micro M and no mutagenic activity was seen in the Ames II screen. There were no significant hemodynamic effects in rats and dogs up to 100 and 30 mg/kg with peak plasma drug concentrations of approximately 1,000 and 300 micro M, respectively. In an in-vivo complement activation model in guinea pigs, BX 528 showed minimal inhibition of plasma CPN activity up to 60 mg/kg with peak plasma concentrations up to 250 micro M. Thus, these data demonstrate that BX 528 is a novel, potent, selective and safe TAFIa inhibitor.

A novel inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa) - part II: enhancement of both exogenous and endogenous fibrinolysis in animal models of thrombosis.

We have discovered a novel small-molecule TAFIa inhibitor, BX 528, which is potent, highly selective against other carboxypeptidases and safe. The present study was to determine if BX 528 can enhance exogenous and endogenous thrombolysis in four different animal models. In the first three models, a thrombus was induced by FeCl (2) (dogs) or laser (rats) injury of the femoral artery, or formed ex vivo and implanted in the jugular vein in rabbits. A low dose of exogenous t-PA was given to induce a low-level thrombolysis on an established thrombus. Co-treatment with BX 528 further enhanced the thrombolytic effects induced by the exogenous t-PA and, thus, reduced thrombosis in all three animal models. In a second rat model, fibrin deposition in the lungs was induced by batroxobin, which was spontaneously resolved in 30 minutes due to the activation of endogenous fibrinolysis. Pre-treatment with lipopolysaccharide (LPS) attenuated this spontaneous fibrinolysis. Co-treatment with 10 mg/kg BX 528 prevented the LPS-induced attenuation of endogenous fibrinolysis. Thus, these studies demonstrated that inhibition of TAFIa by BX 528, our newly discovered small-molecule TAFIa inhibitor, enhanced both the exogenous (induced by a low dose of t-PA) and endogenous (LPS-induced resistance) thrombolysis without increasing the bleeding risk in four different animal models of thrombosis in different species (rat, dog and rabbit) employing different thrombogenic stimuli (FeCl (2) , laser, ex vivo and batroxobin) to induce thrombus formation in different tissues (artery, vein and lung microcirculation).

Thrombin-activatable fibrinolysis inhibitor deficiency attenuates bleomycin-induced lung fibrosis.

Decreased fibrinolytic function favors the development of pulmonary fibrosis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a strong suppressor of fibrinolysis, but its role in lung fibrosis is unknown. Therefore, we compared bleomycin-induced lung fibrosis in TAFI-deficient, heterozygous, and wild-type mice. The animals were sacrificed 21 days after bleomycin administration, and markers of lung fibrosis and inflammation were measured. The bronchoalveolar lavage fluid levels of total protein, neutrophil proteases (elastase, myeloperoxidase), cytokines (tumor necrosis factor-alpha, interleukin-13), chemokine (monocyte chemoattractant protein-1), coagulation activation marker (thrombin-antithrombin complex), total soluble collagen, and growth factors (platelet-derived growth factor, transforming growth factor-beta1, granulocytic-macrophage growth factor) were significantly decreased in knockout mice compared to wild-type mice. Further, histological findings of fibrosis, fibrin deposition, and hydroxyproline and collagen content in the lung were significantly decreased in knockout mice compared to wild-type mice. Depletion of fibrinogen by ancrod treatment led to equalization in the amount of fibrosis and collagen deposition in the lungs of knockout and wild-type mice. No difference was detected in body temperature or arterial pressure between the different mouse phenotypes. These results suggest that the anti-fibrinolytic activity of TAFI promotes lung fibrosis by hindering the rate at which fibrin is degraded.

Genotypes of the pancreatic beta-cell K-ATP channel and clinical phenotypes of Japanese patients with persistent hyperinsulinaemic hypoglycaemia of infancy.

OBJECTIVE: Persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI) is a disorder of glucose metabolism that is characterized by dysregulated secretion of insulin from pancreatic beta-cells. This disease has been reported to be associated with mutations of the sulfonylurea receptor SUR1 (ABCC8) or the inward-rectifying potassium channel Kir6.2 (KCNJ11), which are two subunits of the pancreatic beta-cell ATP-sensitive potassium channel. PATIENTS AND METHODS: In 14 Japanese PHHI patients, all exons of SUR1 and Kir6.2 genes were analysed by polymerase chain reaction (PCR) and direct sequencing. Four patients responded to diazoxide, and nine patients underwent a subtotal pancreatectomy. Histologically, seven patients were diagnosed to have a focal form and two a diffuse form of the disease. RESULTS: We found nine novel mutations in the SUR1 gene and two in the Kir6.2 gene. In the SUR1 gene mutations, three were nonsense mutations (Y512X, Y1354X and G1469X), one was a one-base deletion in exon 7, and two were missense mutations in the nucleotide-binding domain 2 (K1385Q, R1487K). The other three mutations occurred in introns 14, 29 and 36, which might cause aberrant splicing of RNA. Two siblings in one family were heterozygotes for a missense mutation, K1385Q, which was maternally inherited. In Kir6.2 gene screening, one patient was found to be a compound heterozygote of a missense mutation (R34H) and a one-base deletion (C344fs/ter). CONCLUSION: The novel mutations reported here could be pathological candidates for PHHI in Japan. They also reveal that SUR1 and Kir6.2 mutations in the Japanese population exhibit heterogeneity and that they occurred at a frequency similar to other genetic populations.

The pattern recognition receptor (RAGE) is a counterreceptor for leukocyte integrins: a novel pathway for inflammatory cell recruitment.

The pattern recognition receptor, RAGE (receptor for advanced glycation endproducts), propagates cellular dysfunction in several inflammatory disorders and diabetes. Here we show that RAGE functions as an endothelial adhesion receptor promoting leukocyte recruitment. In an animal model of thioglycollate-induced acute peritonitis, leukocyte recruitment was significantly impaired in RAGE-deficient mice as opposed to wild-type mice. In diabetic wild-type mice we observed enhanced leukocyte recruitment to the inflamed peritoneum as compared with nondiabetic wild-type mice; this phenomenon was attributed to RAGE as it was abrogated in the presence of soluble RAGE and was absent in diabetic RAGE-deficient mice. In vitro, RAGE-dependent leukocyte adhesion to endothelial cells was mediated by a direct interaction of RAGE with the beta2-integrin Mac-1 and, to a lower extent, with p150,95 but not with LFA-1 or with beta1-integrins. The RAGE-Mac-1 interaction was augmented by the proinflammatory RAGE-ligand, S100-protein. These results were corroborated by analysis of cells transfected with different heterodimeric beta2-integrins, by using RAGE-transfected cells, and by using purified proteins. The RAGE-Mac-1 interaction defines a novel pathway of leukocyte recruitment relevant in inflammatory disorders associated with increased RAGE expression, such as in diabetes, and could provide the basis for the development of novel therapeutic applications.

Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation.

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 alpha 4 beta 1 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.

Activated thrombin-activatable fibrinolysis inhibitor attenuates spontaneous fibrinolysis of batroxobin-induced fibrin deposition in rat lungs.

Studies have shown that inhibition of TAFI by small peptides enhances pharmacological effects of tPA in animal models of thrombosis, suggesting that TAFI modulates the fibrinolytic system. In this study, we investigated the effect of activated human TAFI (TAFIa) on endogenous fibrinolysis in a rat model of intravascular fibrin deposition. (125)I-labeled fibrinogen was injected intravenously followed by a bolus injection of batroxobin, a thrombin-like enzyme. Batroxobin cleaved fibrinogen to form insoluble fibrin that was deposited in tissues, including the lungs. This was shown by a decrease of radioactivity in the blood as a result of consumption of (125)I-labeled fibrinogen and an elevation of radioactivity in the lungs 5 min following batrox-obin administration. Endogenous fibrinolysis was detected by a gradual increase in radioactivity in the blood and a decrease in radioactivity in the lungs at 30 min, an indication of radiolabeled fibrin degradation products (FDPs) being released into the circulation from the tissues. Intravenous administration of human TAFIa dose-dependently attenuated the later phase reduction of radioactivity in the lungs. When the dose of TAFIa was 218 micro g/kg, giving a peak plasma level of TAFIa 0.9 +/- 0.05 micro g/ml, the spontaneous fibrinolysis was completely prevented. These results provide direct evidence that an increase in circulating TAFIa impairs endogenous clot lysis in a rat model of fibrin deposition.

Mutations in the substrate binding site of thrombin-activatable fibrinolysis inhibitor (TAFI) alter its substrate specificity.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a zymogen that inhibits the amplification of plasmin production when converted to its active form (TAFIa). TAFI is structurally very similar to pancreatic procarboxypeptidase B. TAFI also shares high homology in zinc binding and catalytic sites with the second basic carboxypeptidase present in plasma, carboxypeptidase N. We investigated the effects of altering residues involved in substrate specificity to understand how they contribute to the enzymatic differences between TAFI and carboxypeptidase N. We expressed wild type TAFI and binding site mutants in 293 cells. Recombinant proteins were purified and characterized for their activation and enzymatic activity as well as functional activity. Although the thrombin/thrombomodulin complex activated all the mutants, carboxypeptidase B activity of the activated mutants against hippuryl-arginine was reduced. Potato carboxypeptidase inhibitor inhibited the residual activity of the mutants. The functional activity of the mutants in a plasma clot lysis assay correlated with their chromogenic activity. The effect of the mutations on other substrates depended on the particular mutation, with some of the mutants possessing more activity against hippuryl-His-leucine than wild type TAFIa. Thus mutations in residues around the substrate binding site of TAFI resulted in altered C-terminal substrate specificity.

Amino acid residues in the P6-P'3 region of thrombin-activable fibrinolysis inhibitor (TAFI) do not determine the thrombomodulin dependence of TAFI activation.

Thrombin bound to thrombomodulin activates thrombin-activable fibrinolysis inhibitor (TAFI) and protein C much more efficiently than thrombin alone. Although thrombomodulin has been proposed to alter the thrombin active site, the recently determined structure of the thrombin-thrombomodulin complex does not support this proposal. In this study, the contribution of amino acids near the activation site of TAFI toward thrombomodulin dependence was determined, utilizing four variants of TAFI with specific substitutions in the P6-P'3 region surrounding the Arg-92 cleavage site. Two point mutants had either the Ser-90 or Asp-87 of TAFI replaced with Ala, a third mutant had the thrombin activation site of the fibrinogen Bbeta-chain substituted into positions 91-95 of TAFI, and a fourth mutant had the thrombin activation site of protein C substituted into positions 90-95 of TAFI. Each of these mutants was expressed, purified, and characterized with respect to activation kinetics and functional properties of the enzyme. Even though fibrinogen is poorly cleaved by thrombin-thrombomodulin, the fibrinogen activation site does not significantly alter the thrombomodulin dependence of TAFI activation. The TAFI variant with the protein C activation sequence is only slowly activated by thrombin-thrombomodulin, and not at all by free thrombin. Mutating Asp-87 to Ala increases the catalytic efficiency of activation 3-fold both in the presence and absence of thrombomodulin, whereas mutating Ser-90 to Ala effects only minor kinetic differences compared with wild type TAFI. The thermal stabilities and antifibrinolytic properties of the enzymes were not substantially altered by any of the mutations that allowed for efficient activation of the enzyme. We conclude that residues in the P6-P'3 region of TAFI do not determine the thrombomodulin dependence of activation, which lends support to the argument that the role of thrombomodulin is to optimally orient thrombin and its substrate, rather than to allosterically alter the specificity of the thrombin active site.

Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible with murine life.

To investigate the consequence of deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI), we generated homozygous TAFI-deficient mice by targeted gene disruption. Intercrossing of heterozygous TAFI mice produced offspring in the expected Mendelian ratio, indicating that transmission of the mutant TAFI allele did not lead to embryonic lethality. TAFI-deficient mice developed normally, reached adulthood, and were fertile. No gross physical abnormalities were observed up to 24 months of age. Hematological analysis of TAFI-deficient mice did not show any major differences including plasma fibrinogen level, prothrombin time, and activated partial thromboplastin time. TAFI-deficient mice did not suffer from excess bleeding as determined by blood loss following tail transection, although their plasma failed to prolong clot lysis time in vitro. In vivo, TAFI deficiency did not influence occlusion time in either an arterial or a venous injury model. TAFI deficiency did not improve survival rate compared with the wild-type in thrombin-induced thromboembolism, factor X coagulant protein-induced thrombosis, and endotoxin-induced disseminated intravascular coagulation. Furthermore, TAFI deficiency did not alter kaolin-induced writhing response, implying that TAFI does not play a major role in bradykinin catabolism. The current study demonstrates that TAFI deficiency does not change normal responses to acute challenges.

Thrombin-activatable fibrinolysis inhibitor (TAFI) deficient mice.

In order to examine the physiological role of thrombin-activatable fibrinolysis inhibitor (TAFI), we generated homozygous TAFI deficient mice by targeted gene disruption. Intercrossing of heterozygous TAFI mice showed that TAFI mice were born in the expected Mendelian ratio, indicating that transmission of the mutant TAFI allele did not lead to embryonic lethality. TAFI deficient mice developed normally and reached adulthood. No physical abnormalities were observed. They were fertile and pregnancies were carried to full term. Hematological analysis of TAFI deficient mice did not show any major differences compared with their wild type littermates, including plasma fibrinogen level, PT and aPTT. Prolongation of lysis time upon activation of TAFI was observed only with plasma from wild type and heterozygous mice in an in vitro clot lysis assay. TAFI deficiency did not lead to increased bleeding as determined by blood loss following tail transection. In vivo, TAFI deficiency did not influence occlusion time in either an arterial or a venous thrombosis model. The effects of TAFI deficiency were also investigated in thrombin-induced pulmonary thromboembolism, Factor X coagulant protein-induced thrombosis and endotoxin-induced disseminated intravascular coagulation models. In these models, TAFI deficiency did not improve the morbidity or mortality. Based on the kaolin-induced writhing test, TAFI did not play a major role in bradykinin degradation under normal conditions. These studies demonstrate that TAFI deficiency is compatible with murine life