Metformin Is Associated with Reduced Tissue Factor Procoagulant Activity in Patients with Poorly Controlled Diabetes.

PURPOSE: Metformin is the first-line antidiabetic drug and shown to reduce cardiovascular risk independent from its glucose lowering action. Particularly in poorly controlled diabetes, tissue factor (TF) is expressed in the vasculature and accounts for thromboembolic complications. Here, we aimed to assess the effect of metformin on TF activity and markers of vascular inflammation in poorly controlled type 2 diabetes. METHODS: In a cohort of patients with uncontrolled type 2 diabetes (glycosylated hemoglobin 8.39 ± 0.24%, 68.1 ± 2.6 mmol/mol, n = 46) of whom half of the individuals were treated with metformin and the other half did not receive metformin as part of an anti-diabetic combination therapy, we assessed TF activity and markers of vascular inflammation. In vitro, human monocytic cells (THP-1) were exposed to metformin and TF expression measured in the presence and absence of the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide riboside (AICAR) or the AMPK inhibitor compound C. RESULTS: In the patients, metformin treatment was associated with lower levels of TF protein (241.5 ± 19 vs. 315.4 ± 25 pg/mL, p = 0.03) and reduced TF activity (408.9 ± 49 vs. 643.8 ± 47 U/mL, p = 0.001) compared with controls. Moreover, the patients on metformin showed lower levels of vascular cell adhesion molecule (VCAM)1 (26.6 ± 1.4 vs. 35.03 ± 3.1 ng/mL, p = 0.014) and higher expression of miR-126-3p/U6sno (11.39 ± 2.8 vs. 4.26 ± 0.9, p = 0.006), a known post-transcriptional down regulator of TF and VCAM1. In vitro, metformin dose-dependently reduced lipopolysaccharide (LPS)-induced TF expression in THP-1 cells. The AMPK activator AICAR alone lowered TF expression in THP-1, while the AMPK inhibitor compound C abrogated the metformin-dependent reduction in TF expression. CONCLUSIONS: Our data are the first to report that metformin is associated with reduced plasma TF procoagulant activity possibly explaining-at least in part-the vasculoprotective properties of metformin.

Thioredoxin and thioredoxin reductase control tissue factor activity by thiol redox-dependent mechanism.

Abnormally enhanced tissue factor (TF) activity is related to increased thrombosis risk in which oxidative stress plays a critical role. Human cytosolic thioredoxin (hTrx1) and thioredoxin reductase (TrxR), also secreted into circulation, have the power to protect against oxidative stress. However, the relationship between hTrx1/TrxR and TF remains unknown. Here we show reversible association of hTrx1 with TF in human serum and plasma samples. The association is dependent on hTrx1-Cys-73 that bridges TF-Cys-209 via a disulfide bond. hTrx1-Cys-73 is absolutely required for hTrx1 to interfere with FVIIa binding to purified and cell-surface TF, consequently suppressing TF-dependent procoagulant activity and proteinase-activated receptor-2 activation. Moreover, hTrx1/TrxR plays an important role in sensing the alterations of NADPH/NADP(+) states and transducing this redox-sensitive signal into changes in TF activity. With NADPH, hTrx1/TrxR readily facilitates the reduction of TF, causing a decrease in TF activity, whereas with NADP(+), hTrx1/TrxR promotes the oxidation of TF, leading to an increase in TF activity. By comparison, TF is more likely to favor the reduction by hTrx1-TrxR-NADPH. This reversible reduction-oxidation reaction occurs in the TF extracellular domain that contains partially opened Cys-49/-57 and Cys-186/-209 disulfide bonds. The cell-surface TF procoagulant activity is significantly increased after hTrx1-knockdown. The response of cell-surface TF procoagulant activity to H(2)O(2) is efficiently suppressed through elevating cellular TrxR activity via selenium supplementation. Our data provide a novel mechanism for redox regulation of TF activity. By modifying Cys residues or regulating Cys redox states in TF extracellular domain, hTrx1/TrxR function as a safeguard against inappropriate TF activity.

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta.

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His(6)-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni-nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P.angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.

A fusion protein containing murine vascular endothelial growth factor and tissue factor induces thrombogenesis and suppression of tumor growth in a colon carcinoma model.

Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.

Diagnosis of factor VIII deficiency.

The correct diagnosis of factor VIII deficiency and the assessment of severity of the disease are essential for a patient-tailored treatment strategy. An optimal diagnostic procedure comprises sensitive and specific screening methods and factor VIII activity assays. Different screening reagents show variable characteristics and receiver operator characteristic curves are presented showing the relation between sensitivity and specificity of eleven activated partial thromboplastin time reagents. The details of the three methods for factor VIII activity assay, one-stage and two-stage assay and chromogenic assays, are discussed. The chromogenic assay seems to be more sensitive than the one-stage assay with regard to the detection of severe haemophilia. Discrepant results obtained with one-stage and two-stage assays are reviewed and discussed.

Prothrombotic effects of prostasomes isolated from prostatic cancer cell lines and seminal plasma.

Thromboembolism is well recognized as a major complication of cancer. Many tumor cells overexpress tissue factor (TF), which activates blood coagulation in cancer patients. Inflammatory cells expressing TF are also contributors to this activation. In prostate cancer, we believe that prostasomes may also be involved in the initiation of blood coagulation. Prostasomes are submicron secretory granules derived from the prostate gland. They are surrounded by membrane and their extracellular appearance and membrane architecture are complex. Seminal prostasomes are believed to be necessary for successful fertilization and act as protectors of the spermatozoa in the lower and upper female genital tract. Cells from prostate cancer and its metastases are able to produce and export prostasomes to the extracellular environment. These prostasomes may differ quantitatively rather than qualitatively from their normal counterparts with regard to protein composition and function. A majority of human prostate cancers have been found to overexpress TF, and we have demonstrated by various methods that prostasomes derived from prostate cancer cells express considerably higher levels of TF compared with prostasomes of nonmalignant cell origin. The mechanism related to thromboembolic disease generated by prostasomes in prostatic cancer patients may be the early release of prostasomes from prostate cancer cells into the blood circulation, where they will evoke their blood-clotting effects.

International collaborative study for the calibration of a proposed international standard for thromboplastin, rabbit, plain.

BACKGROUND: A preparation of rabbit brain thromboplastin, provisionally coded 04/162, is proposed as a candidate for the World Health Organization (WHO) International Standard (IS) for thromboplastin (rabbit, plain), meant to replace the IS coded RBT/90 (rabbit, plain), stocks of which are now exhausted. RESULTS: The preparation was calibrated in an international collaborative study involving 21 laboratories from 13 countries and the calibration was performed against the existing WHO-IS (i.e. rTF/95 and OBT/79) and other Certified Reference Materials from the Institute for Reference Materials and Measurements of the European Commission (i.e. CRM149 S) and from the European Action on Anticoagulation (i.e. EUTHR-01). An additional candidate rabbit brain thromboplastin coded as 04/106 was also included in the study. On the basis of predefined criteria (the within- and between-laboratory precision of the calibration and the conformity to the calibration model), 04/162 was the preferred candidate. CONCLUSIONS: The assigned International Sensitivity Index value was 1.15 and the inter-laboratory SD and coefficient of variation were 0.057% and 4.9%, respectively.

Detached endothelial cells and microparticles as sources of tissue factor activity.

INTRODUCTION: Cytokine activation of endothelial cell monolayers is associated with cell detachment, microparticle shedding from plasma membranes, and phosphatidylserine appearance in the plasma membrane outer leaflets. While tissue factor expression on activated endothelial cells and microparticles is well documented, the contribution of detached endothelial cells to tissue factor activity is less clear. We studied tissue factor expression and the role of tissue factor pathway inhibitor on adherent and detached endothelial cells and on microparticles following endothelial cell activation with TNF-alpha. MATERIALS AND METHODS: Detached endothelial cells and microparticles were obtained from cultures of human umbilical vein endothelial cells by differential centrifugation of cell culture supernatant. For microparticle capture, an antibody directed against CD146 was used. Functional tissue factor activity was measured by chromogenic assay and tissue factor antigen by ELISA. Endothelial cell and microparticle morphology was examined by light and transmission electron microscopy. RESULTS: After cell activation for 22 h, functional tissue factor activity was distributed as follows: 60%, adherent endothelial cells; 35%, detached cells; and 5%, microparticles. Tissue factor protein followed a similar distribution. Cell detachment was 47%. Electron microscopy demonstrated shedding of microparticles with a diameter of 0.1-0.6 mum. Cy3-annexin V revealed increased phosphatidylserine on activated adherent endothelial cells and microparticles. Pre-incubation of adherent and detached endothelial cells and microparticles with anti-tissue factor antibody blocked factor Xa production. Pre-incubation with anti-tissue factor pathway inhibitor antibody increased tissue factor activity of adherent endothelial cells 2.8-fold, detached cells 1.4-fold, and microparticles 45-fold. CONCLUSIONS: Detached endothelial cells as well as microparticles from activated endothelial cell monolayers express tissue factor activity, and this activity on microparticles is markedly inhibited by microparticle-associated tissue factor pathway inhibitor.

Characterization of human tissue factor (TF)-specific monoclonal antibodies prepared using a rapid immunization protocol.

Tissue factor (TF) plays an important, physiological role in hemostasis. Recent studies have demonstrated the over-expression of TF in a number of solid tumor types and its pathological roles in angiogenesis and tumor metastasis. In this study, we report the development and characterization of a panel of murine MAbs that are specific for human TF, but do not inhibit TF-mediated blood coagulation. By using a modified repetitive immunizations at multiple sites (RIMMS) protocol in conjunction with an efficient hybridoma cloning procedure, anti-TF MAbs were generated within a relatively short time frame of 5-6 weeks. Following primary screening by ELISA, the binding of the MAbs to the native form of human TF was demonstrated in flow cytometry using a stable cell line expressing human TF. Several of these TF-specific MAbs did not inhibit blood coagulation in a blood coagulation assay and bound with high affinity (0.5-2 nM) to human TF in BIAcore analyses. Importantly, this study represents an independent evaluation of the RIMMS strategy for MAb generation and demonstrates that class-switched, high-affinity MAbs can be generated rapidly and reliably using RIMMS.

Progesterone increases tissue factor gene expression, procoagulant activity, and invasion in the breast cancer cell line ZR-75-1.

Progesterone in hormonal preparations increases the incidence of breast cancer. Tissue factor (TF), the initiator of the extrinsic coagulation pathway, is associated with metastasis in a wide variety of cancers. We demonstrate herein that TF mRNA and protein are up-regulated by progesterone in the breast cancer cell line ZR-75. Epidermal growth factor, also associated with increased breast cancer risk, did not regulate TF. The increase in TF is both rapid and transient; increasing after 6 h, reaching a maximum at 24 h, before decreasing to basal levels at 72 h. Sucrose gradient experiments demonstrated that TF is located in the heavy fraction of the plasma membrane, although caveolin-1 is not expressed in ZR-75. To understand the physiological implications of an increase in TF, we performed coagulation and invasion assays. An increase in TF corresponded to an increase in procoagulant activity. Furthermore, progesterone increased the invasion of ZR-75 cells through a matrigel, an effect that was blocked by an antibody against TF. Because TF expression is associated with an enhanced risk of metastasis, we postulate that the progesterone-dependent up-regulation of TF provides a survival advantage to burgeoning breast cancer cells and may contribute to the increased risk of cancer associated with combined hormone replacement therapy.

[Obtaining active extracts for use in production of soluble thromboplastin]. / Poluchenie aktivnykh ékstraktov dlia prigotovleniia tromboplastina rastvorimogo.

Methods of obtaining (from cadaveric human brain) the highly-active extracts to be used later as raw-materials for thromboplastin manufacturing, which is applied to determine the prothrombin (thromboplastin) time of human plasma or blood, are described in the paper. The study resulted in defining the optimal requirements to choosing the raw-materials, storing regime, centrifuging and extraction. The thus elaborated technology of soluble thromboplastin manufacturing ensures the production of a reagent with a high sensitivity to a changing level of factors VII and X and to a reducing activity of the prothrombin complex, which is achieved through the impact of indirect anticoagulants.

International multicenter international sensitivity index (ISI) calibration of a new human tissue factor thromboplastin reagent derived from cultured human cells.

The international sensitivity index (ISI) of the first working standard of Simplastin HTF, a new human tissue factor thromboplastin derived from cultured human cells, has been assessed in a calibration exercise in two Canadian and five European laboratories. Calibrations against international reference preparations (IRP) were performed for the manual method and six types of automated coagulometers that cover the majority of clotting endpoint principles in routine use. The ISI was method-dependent and varied between 1.03 and 1.29 when calibrated against rTF/95 (human IRP). The ISI was also dependent on the route of calibration. Compared with calibration against rTF/95, the ISIs obtained by calibration against RBT/90 (rabbit IRP) were on average 4.4% higher (P < 0.005). Considering the principle of 'like vs. like', the ISIs obtained by calibration against rTF/95 should be preferred.

Use of lyophilized calibrant plasmas for simplified international normalized ratio determination with a human tissue factor thromboplastin reagent derived from cultured human cells.

BACKGROUND: For monitoring of treatment with oral anticoagulants, the clotting time obtained in the prothrombin time (PT) test is transformed to the International Normalized Ratio (INR) with use of a system-specific International Sensitivity Index (ISI). The calibrant plasma procedure (CPP) is an alternative approach to INR calculation based on the use of a set of lyophilized plasmas with assigned INRs. METHODS: With the CPP, a linear relationship is established between log(PT) and log(INR), using orthogonal regression. CPP was validated for Simplastin HTF, a new human tissue factor reagent derived from cultured human cells. CPP precision was assessed as the CV of the slope of the regression line. The accuracy of the CPP was determined by comparing the INR obtained with the CPP with that obtained with the established ISI-based reference method. INRs of the calibrants were assigned by different routes: by manufacturer (consensus labeling) or by use of Simplastin HTF or International Reference Preparations (IRPs; rTF/95 or RBT/90). RESULTS: The mean CV of the CPP regression slope ranged from 1.0% (Simplastin HTF reagent-specific INR) to 2.4% (INR assigned with rTF/95). INRs calculated with the CPP were similar to those obtained with the reference method, but when the routes for assigning INRs to the calibrant plasmas were compared, the mean difference in INR between CPP and the reference method was smaller with Simplastin HTF reagent-specific values. In several (but not all) cases, this difference was significant (P <0.05, t-test). CONCLUSION: CPP can be used for local INR determination, but better precision and accuracy are obtained with reagent-specific INRs compared with INR assignment by consensus labeling or IRP.

Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein.

Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVII(a) and initiates coagulation by activating FIX and FX, ultimately resulting in thrombin formation. TF is found in adventitia of blood vessels and the lipid core of atherosclerotic plaques. In unstable coronary syndromes, plaque rupture initiates coagulation by exposing TF to blood. Biologically active TF has been detected in vessel walls and circulating blood. Elevated intravascular TF has been reported in diverse pro-thrombotic syndromes such as myocardial infarction, sepsis, anti-phospholipid syndrome and sickle-cell disease. It is unclear how TF circulates, although it may be present in pro-coagulant microparticles. We now report identification of a form of human TF generated by alternative splicing. Our studies indicate that alternatively spliced human tissue factor (asHTF) contains most of the extracellular domain of TF but lacks a transmembrane domain and terminates with a unique peptide sequence. asHTF is soluble, circulates in blood, exhibits pro-coagulant activity when exposed to phospholipids, and is incorporated into thrombi. We propose that binding of asHTF to the edge of thrombi contributes to thrombus growth by creating a surface that both initiates and propagates coagulation.

Expression of recombinant rabbit tissue factor in Pichia pastoris, and its application in a prothrombin time reagent.

Tissue factor (TF), or thromboplastin, is a cell membrane-associated glycoprotein composed, in full length, of cytoplasmic, transmembrane, and extracellular domains. It functions as a cofactor in a complex with factor VII (FVII), generating activated factor VII (FVIIa) and initiating blood coagulation. The prothrombin time (PT) assay uses TF as the in vitro activator of coagulation under defined conditions, and it is primarily used to diagnose and manage the extrinsic-pathway factor defficiencies. To overcome the limitations of natural-source TF, we have expressed the mature full-length recombinant rabbit TF (rRTF) protein in Pichia pastoris. Isolation, by purification by immobilized metal-affinity chromatography, of full-length rRTF was facilitated by engineering a (His)(6) tail on its C-terminus, which maximizes the selection of rRTF with intact transmembrane and cytoplasmic domains, critical for proper activity. A PT reagent that incorporates this purified rRTF has performance characteristics similar to those of PT reagents made with natural TF as indicated in method comparison studies, and shows lot-to-lot consistency and reproducibility.

Thromboplastin Bilbao: reproducibility and sensitivity of a Spanish thromboplastin.

Prothrombin time (PT) is the control test for oral anticoagulant therapy as well as the screening test for defects of the extrinsic pathway of coagulation. Its responsiveness to decreased extrinsic clotting factors depends on the source and type of tissue factor thromboplastin extract. In 1994, a rabbit brain thromboplastin - Thromboplastin Bilbao (TBi) - was introduced as a replacement for a human brain preparation used since 1983, with the aim of establishing a national standard. The purpose of this study was to check the reproducibility, the inter-assay/intra-assay accuracy and the stability of this reagent under temperature changes and over time. A method modified from Frei et al. [World Health Organisation Regional Publications, Eastern Mediterranean Series, Alexandria, 1995] was used for the preparation of thromboplastin extract. Thirty-five batches of human TBi were prepared from 1983 to 1988, while from 1993 to 1999 13 batches of rabbit TBi were produced. The inter-assay reproducibility of rabbit TBi exhibited a coefficient of variation (CV) of 1.07-1.57% for normal plasma and of 1.25-2.56% for anticoagulated plasma. The intra-assay CV was 0.06-1.30% for normal plasma and 1.23-2.66% for anticoagulated plasma. The stability of the reagent to temperature changes and time was also estimated, with similar results for the two thromboplastins. As a result of the Oral Anticoagulant Treatment Quality Assessment Scheme in the Basque Country, an in-house rabbit thromboplastin with good sensitivity and reproducibility was developed.

Production of extracellular domain of human tissue factor using maltose-binding protein fusion system.

Making use of the physiological process of coagulation as an anti-tumor effector function may be beneficial in various coagulation-mediated diseases. Preclinical and clinical studies with novel tissue factor targeting constructs require that efficient procedures for preparing large quantities of pure truncated TF (tTF) become available. In this study, we described a simple and rapid on-column method for purifying large quantities of human tTF from Escherichia coli. The coding region of extracellular domain of tissue factor was linked to the 3(')-end of maltose-binding protein (MBP) gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tTF was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 21.5% of the total soluble protein in E. coli, allowing approximately 15mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-TF monoclonal antibody and the activity was confirmed by chromogenic assay. This MBP-fusion system permits large-scale functional expression and purification of recombinant soluble proteins, providing a basis for the future study of structure and function of tTF.

Continuous annular chromatography: General characterization and application for the isolation of recombinant protein drugs.

Isolation of recombinant protein drugs from cell culture supernatant is usually performed in batch mode, even if the fermentation process itself is continuous. As a novel approach, continuous separation techniques like continuous annular chromatography (CAC) can be used for continuous isolation. The potential of CAC for industrial application is demonstrated by continuous isolation of rFVIII from cell culture supernatant in pilot scale (i.e., 144-288 l/day). Thirty-fold concentration can be achieved at 94% yield, while purity is increased 3-5-fold. For this a batch direct feed ion exchange chromatography method was adapted to a commercial preparative CAC system (P-CAC). A headspace loading technique was used to maximize the concentration factor, while buffer incompatibility problems were addressed by a specifically modified inlet geometry. To allow sterile on-line coupling to FVIII-producing perfusion fermenters, an autoclavable pilot scale P-CAC prototype was developed. General characterization of P-CAC revealed a current limitation of the technology, i.e., variations in the outlet flow rates of up to +/-20%. These flow variations are shown to be caused mainly by a nonuniform annular resin bed and in turn result in "peak wobbling," i.e., the slight variation of peak position (up to +/-4 degrees ) and shape (e.g., A(s) = 0.9-1.4) as a specific function of column position. Some additional peak broadening, although less significant, is caused by a "peak oscillation" effect that results from the necessary segmentation of flow into discrete outlets. Both effects are only measurable if peaks are either monitored continuously or at least measured at multiple column positions. For isolation processes, these nonideal flow phenomena mean that more outlet streams have to be collected in order to achieve maximum yield and thus the achievable concentration factor is somewhat lower than the theoretical maximum.