scholarly journals A Simplified and High-Throughput Chromogenic Assay for Testing Tissue Factor–Dependent Procoagulant Activity

2011 ◽  
Vol 16 (3) ◽  
pp. 295-302 ◽  
Author(s):  
Wenwen Jiang ◽  
Junping Kou ◽  
Shengtao Yuan ◽  
Li Sun ◽  
Boyang Yu
Keyword(s):  
Cardiovascular Diseases ◽  
Tissue Factor ◽  
High Throughput ◽  
Critical Role ◽  
Screening Method ◽  
Coagulation Factors ◽  
Coagulation Cascade ◽  
Chromogenic Substrate ◽  
Human Monocytes ◽  
Chromogenic Assay

Tissue factor (TF), the primary initiator of the coagulation cascade, plays a critical role in hemostasis and thrombosis, and inhibition of TF activity appears to be an attractive target for the treatment of cardiovascular diseases. However, few selective small-molecule inhibitors of TF are available, and the present assays for measuring TF activity are relatively expensive and complex. The authors present a simple and high-throughput chromogenic assay for screening TF inhibitors based on using commercial human prothrombin complex instead of purified coagulation factors, reducing the dosage, and performing with a one-stage procedure. In the optimized assay, <45 µL cell lysates was incubated with Tris-CaCl2 buffer (pH 7.3) containing human prothrombin complex at 37°C for 15 min in 96-well or 384-well plates. Tris-EDTA buffer (pH 8.4) containing chromogenic substrate Xa was then added and the absorbance measured at 405 nm. This simplified assay was more sensitive or precise than some reported methods for TF procoagulant activities. Two known active compounds (curcumin and simvastatin) inhibiting TF activity were tested by the simplified assay to validate the screening method. Furthermore, berberine and cryptotanshinone suppressed TF activity induced by lipopolysaccharides in human monocytes by this assay and might be promising new TF inhibitors.

Measuring tissue factor (factor III) activity in plasma.

1989 ◽  
Vol 35 (9) ◽  
pp. 1897-1900 ◽  
Author(s):  
C Fukuda ◽  
K Iijima ◽  
K Nakamura
Keyword(s):  
Tissue Factor ◽  
Disseminated Intravascular Coagulation ◽  
Coagulation Factors ◽  
High Plasma ◽  
Mean Value ◽  
Chromogenic Substrate ◽  
Intravascular Coagulation ◽  
Endogenous Inhibitors ◽  
Tissue Thromboplastin ◽  
The Mean

Abstract This is a method for measuring tissue factor (TF, Factor III, tissue thromboplastin) activity in plasma by using a chromogenic substrate. As pretreatment, the euglobulin fraction of plasma was prepared by removing endogenous inhibitors and heated at 60 degrees C for 3 min to remove fibrinogen. This allowed us to measure the low TF activity in plasma that could not otherwise be measured. Neither phospholipids nor coagulation factors VII, IX, X, or Xa in the samples interfere. Within-run and day-to-day reproducibility were both good. The mean value obtained by this method for normal persons was 1.02 (SD 0.91) arbitrary units/L. A markedly high plasma TF activity of 20 arb. units/L or more was observed in patients with some types of disseminated intravascular coagulation.

scholarly journals Ten Representative Saponins on Tissue Factor Expression in Human Monocytes: Structure–Activity Relationships and Molecular Docking

2020 ◽  
Vol 15 (3) ◽  
pp. 1934578X2091368
Author(s):  
Yongjiang Zeng ◽  
Xuhua He ◽  
Wenwen Jiang ◽  
Junping Kou ◽  
Boyang Yu
Keyword(s):  
Cardiovascular Disease ◽  
Molecular Docking ◽  
Tissue Factor ◽  
Inhibitory Effect ◽  
Coagulation Cascade ◽  
Human Monocytes ◽  
Messenger Ribonucleic Acid ◽  
Structure Activity Relationships ◽  
Protein Levels ◽  
Structure Activity

Saponins have significant bioactivities in treating cardiovascular disease. Whereas there is a lack of in-depth knowledge about how saponins prevent cardiovascular disease. Tissue factor (TF) is the major initiator of the coagulation cascade and plays an important role in hemostasis and thrombosis. However structure–activity relationships (SARs) of saponins inhibiting TF activity have not been discussed in detail at present. To further clarify the relationships between saponins and TF, in this study, 10 representative saponins were selected to study the inhibitory effect on TF procoagulant activity of monocytes by an improved chromogenic substrate method, and the possible SARs were preliminarily analyzed. Furthermore, molecular docking analysis suggested that 4 representative saponins had a good affinity with TF/FVIIa. In addition, a representative saponin, ruscogenin, decreased both messenger ribonucleic acid and protein levels of TF in human monocytes partly due to its downregulation of nuclear factor kappa-light-chain-enhancer of activated B cells and c-Jun N-terminal kinase pathways. In conclusion, this study provides further explanation for the cardiovascular protection of saponins, and the analysis of SARs between inhibiting TF activity and saponins will be helpful to explore the therapeutic TF inhibitors.

scholarly journals Exosites expedite blood coagulation

2020 ◽  
Vol 295 (45) ◽  
pp. 15208-15209
Author(s):  
Maria Luiza Vilela Oliva ◽  
Ingrid Dreveny ◽  
Jonas Emsley
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Critical Role ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Coagulation Cascade ◽  
Factor X ◽  
Inhibitor Development ◽  
Prothrombinase Complex ◽  
Factor X Activation

A careful balance between active-site and exosite contributions is critically important for the specificity of many proteases, but this balance is not yet defined for some of the serine proteases that serve as coagulation factors. Basavaraj and Krishnaswamy have closed an important gap in our knowledge of coagulation factor X activation by the intrinsic Xase complex by showing that exosite binding plays a critical role in this process, which they describe as a “dock and lock.” This finding not only significantly enhances our understanding of this step in the coagulation cascade and highlights parallels with the prothrombinase complex, but will also provide a novel rationale for inhibitor development in the future.

Increased Plasma Levels of Tissue Factor Associated to Thrombomodulin Resistance in Patients with Advanced Liver Cirrhosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1094-1094 ◽  
Author(s):  
Patrick Van Dreden ◽  
Sophie Hillaire ◽  
Matthieu Grusse ◽  
Dominique François ◽  
Catherine Gorin ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Severe Disease ◽  
Coagulation Factors ◽  
Significant Negative Correlation ◽  
Lag Time ◽  
Disease Stage ◽  
Coagulation Cascade ◽  
Cirrhotic Patients

Abstract Background: Despite reduced coagulation factors, it was recently described that patients with cirrhosis have an increased thrombotic tendency. Therefore, assays predicting the risk of bleeding or thrombosis are needed. Aims: As the Tissue Factor (TF) pathway plays a major role in the initiation of the coagulation, we evaluated the equilibrium between Tissue Factor activity (TFa) and Tissue Factor Pathway Inhibitor (TFPI) and their relationship with the thrombin generation (TG) assay in 42 cirrhotic patients. TG was realized in the absence and presence of thrombomodulin (TM), since it was previously evidenced a resistance to TM in cirrhotic patients. Methods: Citrated blood samples of 42patients with confirmed cirrhosis classified according to the Child score (A,B,C), free of familial history of thromboembolism or thrombophilia and not treated by anticoagulant were analyzed. TG was triggered by 5 pM of TF in the presence/absence of thrombomodulin (4 µm), and 4 µM of phospholipids (Cat Assay, Stago,France). TFa was determined by a home-made assay, free TFPI (fTFPI) by the Asserachrom Free TFPI (Diagnostica Stago, France). Protein C (PC) and S (PS) quantified by Staclot Protein C and S assays (Diagnostica Stago, France). Results were compared to a group of 30 healthy subjects (CT). Results: Main results are summarized in the table. For TG assay, only the Lag-Time (LT) and the Endogenous Thrombin Potential (ETP) were indicated.Table 1.nTFa (pM)fTFPI (ng/mL)Thrombin generationPC (%)PC (%)without TMwith TMRatioLT (min)ETP (nM/min)LT (min)ETP (nM/min)ETP+TM/ ETP-TMCT300.24 ± 0.1114.2 ± 3.52.46 ± 0.241659 ± 2882.59 ± 0.21008 ± 2730.60 ± 0.08126 ± 1482 ± 11A120.49** ± 0.7213.8 ± 5.32.82 ± 0.551338** ± 2803.12 ± 0.64862 ± 2220.66 ± 0.1658.9*** ± 10.954.6*** ± 11.6B192.73 ** ± 5.116.5 ± 6.452.82 ± 0.711349* ± 4252.93 ± 0.6998 ± 2190.73 ** ± 0.1146.4***, ° ± 11.148.7*** ± 17.8C114.23 **, °°° ± 5.7620.3 ± 11.52.61 ± 0.611574 ± 3892.99 ± 0.681216 ± 3000.78 **, ° ± 0.0735.6***, °°° ± 1349.3*** ± 18* p< 0.05; ** p < 0.01 versus CT; ° p < 0.01 °°°; p < 0.001 versus Child A (non-parametric Mann-Whittney test) TFa was significantly (p < °0.01) increased in comparison with CT, and patients with the most severe disease (Child C) have higher levels (p < 0.001) than patients with the lowest Child score (A). In contrast, fTFPI levels were not significantly different from CT, whatever the Child score was. A significant decrease of ETP (without TM) was observed for cirrhotic patients with the lowest Child scores (A and B) in comparison with CT, whereas the lag-time (with and without TM) and ETP with TM were not different from CT. Therefore, the ratios of ETP with TM/ETP without TM increased significantly, indicating a resistance to the anticoagulant activity of TM, in relation to the severity of the disease. This resistance was in part explained by the decrease levels of PC since there was a significant negative correlation (r = - 0.35, p = 0.03) between PC levels and the ratio ETP + TM/ETP - TM. PC levels were also inversely correlated to ETP levels, in the absence (r = - 0.54, p = 0.0006) or presence of TM (r = - 0.53, p = 0.0006). PC levels were also inversely correlated to TFa levels (r = - 0.39, p = 0.01), suggesting that PC decrease could be related to a defective synthesis, but also possibly by a consumption due to the activation of the coagulation cascade by the TF. In contrast, no significant correlation were observed for PS. Conclusion: In addition toaresistance to the anticoagulant activity of the PC pathway, cirrhotic patients with the more severe disease stage (Child C) have high levels of TF activity which is not counterbalanced by TFPI. This could contribute to the higher prevalence of thrombotic disease in these patients. The origin of TF remains to be established, but could come from Kuppfer cells in reaction to the liver injury. These results may have clinical implication for the treatment or prophylaxis of thrombosis in cirrhotic patients. Disclosures Van Dreden: Diagnostica Stago: Employment.

Tissue Factor: A Critical Role in Inflammation and Cancer

2007 ◽  
Vol 9 (2) ◽  
pp. 97-107 ◽  
Author(s):  
Bashir A. Lwaleed ◽  
Alan J. Cooper ◽  
David Voegeli ◽  
Kathryn Getliffe
Keyword(s):  
Tissue Factor ◽  
Vascular Endothelium ◽  
Critical Role ◽  
Vascular Diseases ◽  
The Body ◽  
Coagulation Cascade ◽  
Enzymatic Reactions ◽  
Blood Proteins ◽  
Disease States ◽  
Enzyme Cascade

A series of coordinated enzymatic reactions takes place in the body whenever blood clots. The major physiological initiator of these reactions is a membrane-bound glycoprotein known as tissue factor (TF), which is normally separated from the bloodstream by the vascular endothelium. Bleeding, caused by injury or tissue damage, activates a complex enzyme cascade as TF becomes exposed to the bloodstream. In disease states, leukocytes or the vascular endothelium may abnormally express TF to cause intravascular coagulation. The blood-coagulation cascade is also relevant to diseases such as hemophilia, in which patients are deficient in blood proteins necessary for clotting, and is linked to vascular diseases such as heart attack and stroke, in which clotting can lead to the occlusion of blood vessels. Coagulation is also activated in inflammation and cancer. In this article, we discuss characteristics of TF and review its role in inflammation and cancer.

scholarly journals Coagulative Disorders in Critically Ill COVID-19 Patients with Acute Distress Respiratory Syndrome: A Critical Review

2021 ◽  
Vol 10 (1) ◽  
pp. 140
Author(s):  
Chiara Robba ◽  
Denise Battaglini ◽  
Lorenzo Ball ◽  
Alberto Valbusa ◽  
Italo Porto ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Critically Ill ◽  
Clinical Manifestations ◽  
Factor Xa ◽  
Protein S ◽  
Coagulation Factors ◽  
Coagulation Cascade ◽  
Acute Distress ◽  
Estimated Risk ◽  
Tissue Factor Pathway

In critically ill patients with acute respiratory distress syndrome (ARDS) coronavirus disease 2019 (COVID-19), a high incidence of thromboembolic and hemorrhagic events is reported. COVID-19 may lead to impairment of the coagulation cascade, with an imbalance in platelet function and the regulatory mechanisms of coagulation and fibrinolysis. Clinical manifestations vary from a rise in laboratory markers and subclinical microthrombi to thromboembolic events, bleeding, and disseminated intravascular coagulation. After an inflammatory trigger, the mechanism for activation of the coagulation cascade in COVID-19 is the tissue factor pathway, which causes endotoxin and tumor necrosis factor-mediated production of interleukins and platelet activation. The consequent massive infiltration of activated platelets may be responsible for inflammatory infiltrates in the endothelial space, as well as thrombocytopenia. The variety of clinical presentations of the coagulopathy confronts the clinician with the difficult questions of whether and how to provide optimal supportive care. In addition to coagulation tests, advanced laboratory tests such as protein C, protein S, antithrombin, tissue factor pathway inhibitors, D-dimers, activated factor Xa, and quantification of specific coagulation factors can be useful, as can thromboelastography or thromboelastometry. Treatment should be tailored, focusing on the estimated risk of bleeding and thrombosis. The aim of this review is to explore the pathophysiology and clinical evidence of coagulation disorders in severe ARDS-related COVID-19 patients.

scholarly journals Coagulation abnormalities in SARS-CoV-2 infection: overexpression tissue factor

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Zahra Eslamifar ◽  
Mahin Behzadifard ◽  
Masoud Soleimani ◽  
Saba Behzadifard
Keyword(s):  
Tissue Factor ◽  
Proinflammatory Cytokines ◽  
Viral Infections ◽  
Multiorgan Failure ◽  
Critical Role ◽  
Coagulation Cascade ◽  
Coagulation System ◽  
Alveolar Cells ◽  
Increased Risk ◽  
Life Threatening

AbstractAmong the pathways and mediators that may be dysregulated in COVID-19 infection, there are proinflammatory cytokines, lymphocyte apoptosis, and the coagulation cascade. Venous and arterial thromboembolisms also are frequent in COVID-19 patients with the increased risk of some life-threatening complications such as pulmonary embolism, myocardial infarction, and ischemic stroke. In this regard, overproduction of proinflammatory cytokines such as IL-6, IL-1β, and TNF-α induce cytokine storms, increase the risk of clot formation, platelet activation, and multiorgan failure that may eventually lead to death among these patients. Surface S protein of SARS-CoV-2 binds to its target transmembrane receptor, named as angiotensin converting enzyme 2 (ACE2(, on various cells such as lymphocyte, alveolar cells, monocytes/macrophages, and platelets. Notably, the activation of the coagulation cascade occurs through tissue factor (TF)/FVIIa-initiated hemostasis. Accordingly, TF plays the major role in the activation of coagulation system during viral infection. In viral infections, the related coagulopathy multiple factors such as inflammatory cytokines and viral specific TLRs are involved, which consequently induce TF expression aberrantly. SARS-COV-2 may directly infect monocytes/ macrophages. In addition, TF expression/release from these cells may play a critical role in the development of COVID-19 coagulopathy. In this regard, the use of TF- VIIa complex inhibitor may reduce the cytokine storm and mortality among COVID-19 patients.

Thrombin selectively induces transcription of genes in human monocytes involved in inflammation and wound healing

2014 ◽  
Vol 112 (11) ◽  
pp. 992-1001 ◽  
Author(s):  
Gustavo Bruges ◽  
Gustavo Crespo ◽  
Victor Salazar ◽  
Pierre-Antoine Deglesne ◽  
Heike Schneider ◽  
...  
Keyword(s):  
Wound Healing ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Coagulation Cascade ◽  
Main Role ◽  
Human Monocytes ◽  
Gene Encoding ◽  
Blocking Antibody ◽  
Microarray Experiments

SummaryThrombin is essential for blood coagulation but functions also as a mediator of cellular signalling. Gene expression microarray experiments in human monocytes revealed thrombin-induced upregulation of a limited subset of genes, which are almost exclusively involved in inflammation and wound healing. Among these, the expression of F3 gene encoding for tissue factor (TF) was enhanced indicating that this physiological initiator of coagulation cascade may create a feed-forward loop to enhance blood coagulation. Activation of protease-activated receptor type 1 (PAR1) was shown to play a main role in promoting TF expression. Moreover, thrombin induced phosphorylation of ERK1/2, an event that is required for expression of thrombin-regulated genes. Thrombin also increased the expression of TF at the protein level in monocytes as evidenced by Western blot and immunostaining. Furthermore, FXa generation induced by thrombin-stimulated monocytes was abolished by a TF blocking antibody and therefore it is entirely attributable to the expression of tissue factor. This cellular activity of thrombin provides a new molecular link between coagulation, inflammation and wound healing.

Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Human Factor ◽  
Solid Phase ◽  
Three Dimensional ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Dimensional Structure ◽  
Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

Amidolytic Detection of Prothrombin Activation Products after SDS-Gel Electrophoresis

1989 ◽  
Vol 61 (03) ◽  
pp. 386-391 ◽  
Author(s):  
Guido Tans ◽  
Truus Janssen-Claessen ◽  
Jan Rosing
Keyword(s):  
Gel Electrophoresis ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Product Formation ◽  
Chromogenic Substrate ◽  
Nitrocellulose Membrane ◽  
Activation Products ◽  
Factor Xia ◽  
Prothrombin Activation

SummaryIn this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation.The detection limit of the so-called “amidoblot” is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting.It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

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