Pathway Of Blood Coagulation Research Articles

TFPI (Tissue Factor Pathway Inhibitor) Is Present in Breast Cancer Tumor Cells.

Abnormalities in hemostatic system are common in breast cancer patients. The principal initiator of blood coagulation activation in cancer patients is tissue factor (TF). Tissue factor plays an important role in the processes of angiogenesis, invasion and metastatic dissemination. Tissue factor pathway inhibitor (TFPI) is the main inhibitor of TF-dependent pathway of blood coagulation. The catalytic activity of the TF/VIIa complex triggers activation of factor X, consequently thrombin generation and finally fibrin formation. Thrombin and fibrin facilitate cancer progression. Previous immunohistochemical studies revealed no expression of TFPI in cancer cells of different malignant tumor types (Werling et al, Thromb Haemost 1993; 69: 366–369) The aim of the study was to evaluate the expression of TFPI protein and mRNA in tumor cells of breast cancer.Breast cancer tissues were obtained during surgical treatment of 35 women. The patients undergoing surgery had not received any previous anticancer therapy. Tumor fragments were preserved in 4% paraformaldehyde/phosphate buffered saline and than embedded in paraffin. Immunohistochemical studies (avidin-biotin complex-ABC - technique) were performed using polyclonal antibodies against TFPI. In situ hybridization method employed single-stranded DNA oligonucleotide (probe sequence: 5′Biotin-CATCGCCCAGTGCAGCCTCCGTCAG-Biotin3′) directed against locus NM 006287 of TFPI mRNA.Strong expression of TFPI protein was observed in cancer cell bodies in examined specimens. Weak expression of the above protein was observed in tumor infiltrating macrophages. Normal cells revealed much weaker staining for TFPI than cancer cells. Similarly, mRNA for TFPI was observed in cancer cells of all breast cancer tissues.The above data indicate that TFPI is present in neoplastic cells of breast cancer and may play a role in neutralizing TF procoagulant and signaling functions.

Intermolecular Interactions and Characterization of the Novel Factor Xa Exosite Involved in Macromolecular Recognition and Inhibition: Crystal Structure of Human Gla-domainless Factor Xa Complexed with the Anticoagulant Protein NAPc2 from the Hematophagous Nematode Ancylostoma caninum

NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa ( K i = 8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel β-sheet formed by the segment of the polypeptide chain consisting of residues 74–80 of NAPc2 with the residues 86–93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67–73) and a turn (residues 26–29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233–243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty.

Biological Activity of Asp-hemolysin as a Regulation Factor to Atherogenic Effect by Oxidized Low-Density Lipoprotein

Oxidatively modified low-density lipoprotein (OxLDL) is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis. Asp-hemolysin, a hemolytic toxin from Aspergillus fumigatus, is a binding protein for OxLDL. This study was undertaken to clarify the biological activity of OxLDL and the potentially of Asp-hemolysin as a regulation factor to atherogenic effect by OxLDL. We first analyzed the interaction between OxLDL and blood coagulation factors, which are involved in the blood coagulation pathway. OxLDL caused prolongation of activated partial thromboplastin time (APTT) as a parameter of the intrinsic pathway of blood coagulation in a dose- and oxidation time-dependent manner. In addition, OxLDL significantly inhibited blood coagulation factor VIII, IX, and XI activity. Furthermore, we demonstrated that factor VIII binds to OxLDL. These results indicate that the binding of factor VIII to OxLDL affects the intrinsic pathway of the blood coagulation cascade. Next, to clarify the structure-function relationship of Asp-hemolysin, we expressed Asp-hemolysin in Escherichia coli as a fusion protein with a maltose-binding protein (MBP) and purified it by affinity chromatography. The purified recombinant Asp-hemolysin showed an immunoreactivity with the anti-Asp-hemolysin antibody. In addition, MBP-Asp-hemolysin fusion protein exhibited binding activity to Ox-LDL as did native Asp-hemolysin. Furthermore, to investigate the effect of the Asp-hemolysin-related peptide (P-21), a synthetic peptide derived from a region of Asp-hemolysin that is rich in positive charges, on macrophage proliferation induced by OxLDL. P-21 inhibited OxLDL-induced macrophage proliferation in a dose-dependent manner. In addition, the binding analysis of P-21 to OxLDL indicated that P-21 binds to OxLDL. These results indicate that P-21 inhibits the OxLDL-induced macrophage proliferation through binding of P-21 to OxLDL. In conclusion, we have shown that OxLDL affects the intrinsic pathway of blood coagulation, and its mechanism is dependent on the binding of factor VIII to OxLDL. Furthermore, we indicate the possibility that Asp-hemolysin is a useful tool to investigate the pathophysiological significance of OxLDL. In particular, since the P-21, an Asp-hemolysin-related peptide, inhibits the OxLDL-induced macrophage proliferation through binding of P-21 to OxLDL, further study on the binding mechanism between Asp-hemolysin-related peptide and OxLDL may provide important information on the prevention and treatment of atherosclerosis.

Ceruloplasmin prevents hemostatic disorders during experimental hyperammonemia

Experimental hyperammonemia in rats was accompanied by hemostatic disorders manifesting in coagulopathy (activation of the intrinsic pathway of blood coagulation) and suppression of platelet function. Ceruloplasmin in a total dose of 60 mg/kg effectively normalized coagulation hemostasis and functional activity of platelets by improving secretory processes in platelets and increasing aggregation rate.

Low levels of activated factor VII in systemic sclerosis

Recent investigations show that activated factor VII, the primary enzyme in the extrinsic pathway of blood coagulation, exerts additional extra-coagulant functions, such as apoptosis and angiogenesis. On the basis of these recent acquisitions, the present study was aimed to evaluate activated factor VII in patients with systemic sclerosis and to establish a potential association with pathogenesis and complications of this severe autoimmune disorder. Activated factor VII level was measured in twenty-eight consecutive scleroderma patients (2 men and 26 women, mean age 49.7 +/- 14.8 years). The main clinical correlates of disease, such as disease activity, renal function, skin, vascular and lung involvement, were evaluated by clinical and instrumental investigations. Activated factor VII level was also evaluated in 28 sex and age matched controls. Systemic sclerosis patients exhibited plasma activated factor VII activities significantly lower than those of healthy matched controls (15.2 versus 37.7 U/l, respectively; p < 0.001). No correlation was observed between plasma activated factor VII concentration and age, disease duration, disease subset, disease activity, renal, lung, skin and microvascular involvement. Results of our investigation provide first evidence of low activated factor VII activity in patients with systemic sclerosis. Reduced activated factor VII activity might be involved in the pathogenesis of the ischemic complications, by modulating apoptotic and angiogenetic processes.

Molecular characterization and anticoagulant activity of a novel annexin derived from the Taenia solium

A novel annexin gene was isolated from the Taenia solium cDNA library by degenerate PCR. The purified protein was generated by hydrolysis with thrombin in glutathione S-transferase (GST) affinity chromatography following expression in GST tagged pGEX-5T vector. It shares the common structural features of the annexin family and specially possesses two unique fragments between repeating domains II and III which do not exist in other annexin family members revealed by aligning and homology modeling, hence it was designated as Tso ANXB2 according to the new nomenclature of annexins. According to the parasitic behaviors of the origin, a series of coagulation assays was performed, indicating that Tso ANXB2 inhibits extrinsic blood coagulation pathway and platelet aggregation also has platelet binding activity. Nevertheless, the mutant protein deleting the consensus coagulation-related KGD motif of Tso ANXB2 showed significant decreasing platelet binding and anticoagulation activity.

Relative quantification of glycosaminoglycan-induced upregulation of TFPI-mRNA expression in vitro

Objective Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation and is mainly expressed by endothelial cells. In this study we examined the in vitro effects of heparin and other glycosaminoglycans on TFPI mRNA-expression in cultivated human endothelial ( Ea.hy 926) and in chondrosarcoma ( SW 1353) cells. Methods We used a LightCycler-based method for relative quantification of the TFPI-mRNA expression before and after stimulation. The cells were stimulated with different concentrations of heparin (with and without addition of protamin), heparan sulfate (HS) and chondroitin-6-sulfate (CS). Cells were harvested after incubation times of 4, 8 and 24 h, total RNA was isolated, and cDNA was synthesized and quantified relatively to a constantly expressed housekeeping gene. Results Stimulation of Ea.hy 926 cells with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) caused a time- and dose-dependent upregulation of TFPI-mRNA expression with LMWH showing the stronger effect. In contrast to this, HS led to a strongly and CS to a slightly decreased TFPI-mRNA expression. SW 1353 cells which were stimulated with LMWH/UFH and HS/CS did not show a significant up- or downregulative effect. Conclusion Our results show that we have developed a versatile method for the relative quantification of TFPI-mRNA expression. As a conclusion, the determined heparin-induced upregulation of TFPI-mRNA expression can be considered a major component of the modulation of the anticoagulant properties of the endothelium.

Decreased expressions of hepsin in human hepatocellular carcinomas

Hepsin is a type II transmembrane protein predominantly expressed in the liver and has been implicated in participation in blood coagulation pathway and epithelial carcinogenesis. The aim of this current study is to investigate the role of hepsin in hepatocarcinogenesis. Quantitative real-time RT-PCR was used to investigate the expression levels of hepsin in a total of 50 paired hepatocellular carcinomas (HCCs) and the corresponding non-tumor liver tissues. Hepsin was transfected to the hepsin-non-expressing SK-HEP-1 cells to study the change of cell proliferation. In 62% (31/50) patients, the expression levels of hepsin in non-tumor liver tissues are at least twofold higher than those in the corresponding HCC tissues. Positive hepatitis B surface antigen was more often detected in patients with hepsin underexpressed in the HCC tissues (74.2% vs. 31.6%, P=0.007). Patients with hepsin underexpressed in the HCC tissues survived shorter time than those without hepsin underexpression in the HCC tissues. The cell proliferation and for a colony formation of SK-HEP-1 HCC cells were inhibited by hepsin. Most HCC patients had hepsin underexpressed in the HCC tissues. These patients survived for a shorter time compared with those without hepsin underexpression in the HCC tissues. Hepsin expression could inhibit cell proliferation and colony formation of HCC cells.

Anticoagulant effect of Naja naja venom 5′nucleotidase: Demonstration through the use of novel specific inhibitor, vanillic acid

The snake venom proteins affect hemostasis by either advancing/delaying blood coagulation. Apart from proteases and phospholipase A(2)s (PLA(2)s), 5'nucleotidase is known to affect hemostasis by inhibiting platelet aggregation. In this study, the possible involvement of Naja naja venom 5'nucleotidase in mediating anticoagulant affect is evaluated. Vanillic acid selectively and specifically inhibited 5'nucleotidase activity among other enzymes present in N. naja venom. It is a competitive inhibitor as evident of inhibition relieving upon increased substrate concentration. Vanillic acid dose dependently inhibited the anticoagulant effect of N. naja venom up to 40%. This partial involvement of 5'nucleotidase in mediating anticoagulant effect is substantiated by concanavalin-A (Con-A) inhibition studies. Con-A, competitively inhibited in vitro protease and 5'nucleotidase activity up to 100%. However, it did not exhibit inhibitory activity on PLA(2). The complete inhibition of anticoagulant effect by Con-A upon recalcification time suggests the participation of both 5'nucleotidase and protease in mediating anticoagulant effect of N. naja venom. Vanillic acid and Con-A inhibition studies together suggest that probably 5'nucleotidase interacts with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Thus, this study for the first time demonstrates the involvement of 5'nucleotidase in mediating N. naja venom anticoagulant effect.

Alterations in the expression of tissue factor and tissue factor pathway inhibitor induced by the onset of cardiac hypertrophy

Tissue factor (TF), a transmembrane glycoprotein and its regulator, tissue factor pathway inhibitor (TFPI), are the major regulatory of the extrinsic pathway of blood coagulation. TF is a multifunctional protein that can also induce changes in the patterns of gene expression. TF expression is thought to be up-regulated during the onset of cardiac hypertrophy. We examined the expression of TF and TFPI at the onset of cardiac hypertrophy. Hypertrophy was induced surgically via abdominal aortic constriction with a 0.5 mm (OD) needle. TF and TFPI expression was examined at 1, 3, 7 and 14 days post-surgery, in isolated cardiomyocytes and left ventricular tissue using semi-quantitative RT-PCR and western blot analysis. Progressive cardiac hypertrophy was confirmed by the expression of atrial natriuretic factor (ANF). TFPI mRNA expression in isolated cardiomyocytes peaked on day 7 in the constricted animals, but normalised by day 14. TF expression in the isolated cardiomyocytes was undetectable throughout the 14 days. In contrast, in left ventricular tissue TFPI expression transiently increased within 24 h with a second, ore persistent wave at day 7, while TF expression remained constant throughout. TFPI protein expression in the isolated cardiomyocytes decreased over the period of investigation, whereas in left ventricular tissue, expression peaked at day 7 returning to the original level by day 14. TF protein expression remained at baseline level for the first 7 days in the experimental group and then doubled by the 14th day. Thus, the altered expressions of TF and TFPI in the hypertrophied heart reflect an imbalance in haemostatic and tissue homeostatic mechanisms, arising during the onset of cardiac hypertrophy.

Development of an immunoassay for oxidized or unoxidized forms of thrombomodulin

Cardiovascular disease is the leading cause of premature death in smokers, and this elevated risk of death is thought to be linked to the hypercoagulability of smokers. Although it is well known that smokers are in a prothrombotic state the underlying molecular mechanisms for this state remain obscure. Thrombomodulin is a key component in the blood coagulation pathway. The thrombomodulin-thrombin complex activates protein C, which in turn inhibits clot formation. Oxidation of methionine 388 in thrombomodulin is known to greatly decrease the activation of protein C. Smoking imposes an oxidative stress on the body and is known to oxidize methionine in other proteins. We hypothesized that the oxidation of methionine 388 is elevated in smokers relative to non-smokers, and this may be a root molecular cause of elevated cardiovascular disease in smokers. The aim of this study is to develop an immunoassay specific for either the oxidized or unoxidised form of thrombomodulin. The accuracy of the immunoassay can be assessed by chemical analysis of the oxidized and unoxidised forms of the protein. These tools will be utilized to conduct a small scale study of the difference in oxidation state of thrombomodulin in smokers and non-smokers. In addition, these tools will be used to compare the oxidation status of thrombomodulin in plasma and urine.

Smoking: Bans, Bans, Good for the Heart!

Could moving to Pueblo, Colorado, be a new way to reduce the risk of suffering a heart attack? A study reported at the American Heart Association’s Scientific Sessions 2005 Conference in Dallas last November suggests that the city’s ban on smoking in all public buildings helped reduce the local heart attack rate by 27% over a year and a half. The finding appears to bolster recent decisions to ban smoking in workplaces and other public buildings across the United States, Spain, Ireland, and parts of the United Kingdom. Tobacco smoke—including secondhand smoke—activates blood coagulation pathways that could lead to coronary thrombosis, or heart attack. Smoke is particularly dangerous for people whose arteries are already hardened by age, cholesterol deposits, or smoking itself. Indeed, some 30% of heart attacks are thought directly related to smoke-induced thrombosis. In the United States, the annual direct medical cost of coronary heart disease caused by secondhand smoke alone is some $2.45 billion, according to the U.S. Society of Actuaries. In an attempt to reduce the incidence of heart attacks and other smoking-related illnesses, Pueblo introduced a ban 1 July 2003 on smoking in all indoor public areas within the city limits. Eighteen months later the Pueblo Public Health Department instigated an observational study to determine whether the ban had had any effect on the incidence of coronary events. “We recorded the number of heart attacks that occurred in the city for the eighteen months before and after the introduction of the ban,” explains Mori Krantz, director of the Prevention Department of the Colorado Prevention Center in Denver, “and found a post-introduction fall of around twenty-seven percent. This was significantly greater than the small reduction we saw among the population living outside the city limits, and much greater than the virtually unchanged rate among residents of [adjacent] El Paso County, which does not have such an ordinance.” Similar findings were reported in an earlier study published 24 April 2004 in BMJ after a smoking ban was imposed in Helena, Montana. “Our study builds on this work by involving a sample three times as large, and suggests that these ordinances may be having a positive effect on cardiac health,” says Krantz. “This observational study is limited in that it does not distinguish between smokers and nonsmokers, and did not check the medical records of those involved,” remarks Jose Ramon Banegas, a professor of preventive medicine and public health at the Universidad Autonoma in Madrid, Spain. He adds, however, that “even if the effect were half that reported, it would mean this type of ban could save many lives while reducing health spending.” Banegas points out that the results are also relevant given the new Spanish ban, enacted 1 January 2006, which still allows restaurateurs to declare their locales either smoking or nonsmoking. “Unfortunately,” he says, “ninety-eight percent are scared of losing income by going smoke-free.”

Low‐molecular‐weight heparins and angiogenesis

The involvement of the vascular system in malignancy encompasses not only angiogenesis, but also systemic hypercoagulability and a pro-thrombotic state, and there is increasing evidence that pathways of blood coagulation and angiogenesis are reciprocally linked. In fact, cancer atients often display hypercoagulability resulting in markedly increased thromboembolism, which requires anti-coagulant treatment using heparins, for example. Clinical trials reveal that treatment with various low-molecular-weight heparins (LMWHs) improves the survival time in cancer patients receiving chemotherapy compared with those receiving unfractionated standard heparin (UFH) or no heparin treatment, as well as in cancer patients receiving LMWH as thrombosis prophylaxis during primary surgery. This anti-tumor effect of the heparins appears to be unrelated to their anti-coagulant activity, but the mechanisms involved are not fully understood. Tumor growth and spread are dependent on angiogenesis and it is noteworthy that the most potent endogenous pro- and anti-angiogenic factors are heparin-binding proteins that may be affected by systemic treatment with heparins. Heparin and other glycosaminoglycans play a role in vascular endothelial cell function, as they are able to modulate the activities of angiogenic growth factors by facilitating the interaction with their receptor and promoting receptor activation. To date, preclinical studies have demonstrated that only LMWH fragments produced by the heparinase digestion of UFH, i.e. tinzaparin, exert anti-angiogenic effects in any type of tissue in vivo. These effects are fragment-mass-specific and angiogenesis-type-specific. Data on the effect of various LMWHs and UFH on endothelial cell capillary tube formation and proliferation in vitro are also presented. We hope that this paper will stimulate and facilitate future research designed to elucidate whether the anti-angiogenic or anti-tumor effects of commercial LMWHs in their own right are agent specific and whether anti-angiogenic properties increase the anti-tumor properties of the LMWHs in the clinic.

Expression and characterization of wild-type TFPI and the [P151L]TFPI mutant in insect cells

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation. In earlier studies we could identify the [P151L]TFPI mutant, and we could also demonstrate that heterozygous carriers of this mutant show a nine-fold increased risk for deep venous thrombosis (DVT). To express greater amounts of both proteins and to enable their characterization, we expressed wild-type TFPI as well as [P151L]TFPI in High Five insect cells with expression rates of up to 215 ng/ml for wild-type TFPI and 214 ng/ml for [P151L]TFPI. The specific inhibitory activities for the recombinant proteins were determined as 11.3 and 11.5 mU/ng, respectively. Both proteins were detected via Western blot analysis and ELISA. The recombinant proteins' inhibitory activities were characterized by a chromogenic assay and by the determination of a modified activated thromboplastin time (aPTT) in which both of them proved to be inhibitorily active. We also examined both recombinant proteins' binding properties to glycoproteins, glycosaminoglycans, lipoproteins and tissue factor. Our results show that we have developed an efficient model system for the recombinant expression of inhibitorily active wild-type TFPI as well as [P151L]TFPI in insect cells, and we were able to characterize both proteins' inhibitory properties by determination of their influence on the aPTT and also their binding properties. Although both recombinant proteins did not show a significant difference in their effect on the aPTT, their binding properties differed significantly between the wild type and mutant protein.

Fibrinogen Substrate Recognition by Staphylocoagulase·(Pro)thrombin Complexes

Thrombin generation and fibrinogen (Fbg) clotting are the ultimate proteolytic reactions in the blood coagulation pathway. Staphylocoagulase (SC), a protein secreted by the human pathogen Staphylococcus aureus, activates prothrombin (ProT) without proteolysis. The SC.(pro)thrombin complex recognizes Fbg as a specific substrate, converting it directly into fibrin. The crystal structure of a fully active SC fragment containing residues 1-325 (SC-(1-325)) bound to human prethrombin 2 showed previously that SC inserts its Ile(1)-Val(2) N terminus into the Ile(16) pocket of prethrombin 2, inducing a functional active site in the cognate zymogen conformationally. Exosite I of alpha-thrombin, the Fbg recognition site, and proexosite I on ProT are blocked by domain 2 of SC-(1-325). In the present studies, active site-labeled fluorescent ProT analogs were used to quantitate Fbg binding to the SC-(1-325).ProT complex. Fbg binding and cleavage are mediated by expression of a new Fbg-binding exosite on the SC-(1-325).ProT complex, resulting in formation of an (SC-(1-325).ProT)(2).Fbg pentameric complex with a dissociation constant of 8-34 nm. In both crystal structures, the SC-(1-325).(pre)thrombin complexes form dimers, with both proteinases/zymogens facing each other over a large U-shaped cleft, through which the Fbg substrate could thread. On this basis, a molecular model of the pentameric (SC-(1-325).thrombin)(2).Fbg encounter complex was generated, which explains the coagulant properties and efficient Fbg conversion. The results provide new insight into the mechanism that mediates high affinity Fbg binding and cleavage as a substrate of SC.(pro)thrombin complexes, a process that is central to the molecular pathology of S. aureus endocarditis.

Structural Basis for Reduced Staphylocoagulase-mediated Bovine Prothrombin Activation

Staphylocoagulase (SC) is a protein secreted by the human pathogen, Staphylococcus aureus, that activates human prothrombin (ProT) by inducing a conformational change. SC-bound ProT efficiently clots fibrinogen, thus bypassing the physiological blood coagulation pathway. The crystal structure of a fully active SC fragment, SC-(1-325), bound to human prethrombin 2 showed that the SC-(1-325) N terminus inserts into the Ile(16) pocket of prethrombin 2, thereby inducing expression of a functional catalytic site in the cognate zymogen without peptide bond cleavage. As shown here, SC-(1-325) binds to bovine and human ProT with similar affinity but activates the bovine zymogen only very poorly. By contrast to the approximately 2-fold difference in chromogenic substrate kinetic constants between human thrombin and the SC-(1-325).human (pro)thrombin complexes, SC-(1-325).bovine ProT shows a 3,500-fold lower k(cat)/K(m) compared with free bovine thrombin, because of a 47-fold increase in K(m) and a 67-fold decrease in k(cat). The SC-(1-325).bovine ProT complex is approximately 5,800-fold less active compared with its human counterpart. Comparison of human and bovine fibrinogen as substrates of human and bovine thrombin and the SC-(1-325).(pro)thrombin complexes indicates that the species specificity of SC-(1-325) cofactor activity is determined primarily by differences in conformational activation of bound ProT. These results suggest that the catalytic site in the SC-(1-325).bovine ProT complex is incompletely formed. The current crystal structure of SC-(1-325).bovine thrombin reveals that SC would dock similarly to the bovine proenzyme, whereas the bovine (pro)thrombin-characteristic residues Arg(144) and Arg(145) would likely interfere with insertion of the SC N terminus, thus explaining the greatly reduced activation of bovine ProT.

Journal of Internal Medicine: a journal for the future

<i>Journal of Internal Medicine</i>: a journal for the future

Expression, Crystallization, and Three-dimensional Structure of the Catalytic Domain of Human Plasma Kallikrein

Plasma kallikrein is a serine protease that has many important functions, including modulation of blood pressure, complement activation, and mediation and maintenance of inflammatory responses. Although plasma kallikrein has been purified for 40 years, its structure has not been elucidated. In this report, we described two systems (Pichia pastoris and baculovirus/Sf9 cells) for expression of the protease domain of plasma kallikrein, along with the purification and high resolution crystal structures of the two recombinant forms. In the Pichia pastoris system, the protease domain was expressed as a heterogeneously glycosylated zymogen that was activated by limited trypsin digestion and treated with endoglycosidase H deglycosidase to reduce heterogeneity from the glycosylation. The resulting protein was chromatographically resolved into four components, one of which was crystallized. In the baculovirus/Sf9 system, homogeneous, crystallizable, and nonglycosylated protein was expressed after mutagenizing three asparagines (the glycosylation sites) to glutamates. When assayed against the peptide substrates, pefachrome-PK and oxidized insulin B chain, both forms of the protease domain were found to have catalytic activity similar to that of the full-length protein. Crystallization and x-ray crystal structure determination of both forms have yielded the first three-dimensional views of the catalytic domain of plasma kallikrein. The structures, determined at 1.85 A for the endoglycosidase H-deglycosylated protease domain produced from P. pastoris and at 1.40 A for the mutagenically deglycosylated form produced from Sf9 cells, show that the protease domain adopts a typical chymotrypsin-like serine protease conformation. The structural information provides insights into the biochemical and enzymatic properties of plasma kallikrein and paves the way for structure-based design of protease inhibitors that are selective either for or against plasma kallikrein.

Analysis of sequence variations in the promoter region of the human tissue factor pathway inhibitor 2 gene in apoplectic patients and blood donors

Tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine protease inhibitor with homology to TFPI-1, an important regulator of the extrinsic pathway of blood coagulation. Recent studies have focused on TFPI-2 and its implications for atherosclerosis. The promoter region and the exons of the human TFPI-2 gene were screened for sequence variations in 41 apoplectic patients and 140 blood donors with no history of ischemic stroke. The sequence variations -567T>C, -546T>C, -353A>G, -161G>C, -167G>A, -47C>A, and -18C>A, which are located in the TFPI-2 promoter, were discovered in both cohorts with allelic frequencies ranging from 0.3 to 2.4%. The influence of these sequence variations on the transcriptional activity of the TFPI-2 gene was investigated in HEK-293 cells using a promoter test system. A wild-type TFPI-2 promoter fragment 716 bp upstream of the translation start site was cloned into a secreted alkaline phosphatase expression vector, and the sequence variations were introduced by site-directed mutagenesis. Interestingly, the promoter activity of the tested mutants was reduced by 1.3- to 2.8-fold compared to that of wild-type control. The variation -18C>A, where a putative binding site of the transcription factor Sp-1 is located, had the strongest effect on transcriptional activity. In conclusion, our present study shows that the transcription of TFPI-2 is changed by single nucleotide polymorphisms and that the sequence variations in transcription factor binding sites of the TFPI-2 promoter may influence the regulation of this gene.

Structural and Mutational Analyses of the Molecular Interactions between the Catalytic Domain of Factor XIa and the Kunitz Protease Inhibitor Domain of Protease Nexin 2

Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 angstroms (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI loop 1 11TGPCRAMISR20 and loop 2 34FYGGC38 were tested for their ability to inhibit FXIa. The P1 site mutation R15A completely abolished its ability to inhibit FXIa. IC50 values for the wild type protein and the remaining mutants were as follows: PN2KPI WT, 1.28 nM; P13A, 5.92 nM; M17A, 1.62 nM; S19A, 1.86 nM; R20A, 5.67 nM; F34A, 9.85 nM. The IC50 values for the M17A and S19A mutants were not significantly different from those obtained with wild type PN2KPI. These functional studies and activated partial thromboplastin time analysis validate predictions made from the PN2KPI-FXIac co-crystal structure and implicate PN2KPI residues, in descending order of importance, Arg15, Phe34, Pro13, and Arg20 in FXIa inhibition by PN2KPI.