A simplified and low-cost one-stage chromogenic assay for tissue factor dependent procoagulant activity of endothelial cells

Pouplard, Claire; Reverdiau, Pascale; Piquemal, R.; Watier, Hervé; Lebranchu, Yvon; Bardos, P.; Gruel, Yves

doi:10.1016/0049-3848(95)00208-1

Cited by 11 publications

(12 citation statements)

References 17 publications

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“…3 In these conditions, a significant increase in the generation of FXa was measured (P ϭ .006; paired t test) ( Figure 1A), supporting TF expression on the basis of the high specificity of the assay. 17 In contrast, no significant monocyte PCA was measured when normal plasma was added to PBMCs in similar conditions. When tested alone, plasma from the 5 patients with HIT did not induce significant monocyte PCA; thus, factor Xa generation remained below 0.1 mU Xa/well, with values identical to those measured when PBMCs were incubated with normal plasma.…”

Section: Resultsmentioning

confidence: 92%

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Induction of monocyte tissue factor expression by antibodies to heparin–platelet factor 4 complexes developed in heparin-induced thrombocytopenia

Pouplard

Iochmann

Renard

et al. 2001

Blood

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196

136

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The pathogenesis of thrombosis in heparin-induced thrombocytopenia (HIT) was studied by investigating whether antibodies to heparin-platelet factor 4 (H-PF4) induced tissue factor (TF) synthesis by monocytes. Plasma from 5 patients with HIT containing IgG to H-PF4 was incubated with peripheral blood mononuclear cells without or with purified PF4 and heparin. Significant TF-dependent procoagulant activity (PCA) expressed by monocytes, measured with a factor Xabased chromogenic assay, was induced after incubation of each HIT plasma sample. This monocyte PCA required the presence of PF4 and was inhibited by high concentrations of heparin. Furthermore, purified HIT IgG added to whole blood with PF4 and heparin also provoked significant synthesis of TF mRNA by monocytes, demonstrated by RT-PCR, and this effect was not observed with normal IgG. These findings strongly support the hypothesis that antibodies to PF4 developed in HIT trigger the production of tissue factor by monocytes, and this effect could account in vivo for hypercoagulability and thrombotic complications in affected patients. IntroductionHeparin-induced thrombocytopenia (HIT) is a common complication of unfractionated heparin (UFH) therapy, often associated with arterial or venous thrombosis. 1 Such thrombocytopenia results from platelet activation caused by antibodies specific for complexes composed of heparin and platelet factor 4 (FP4). [2][3][4][5] The platelet activation involves the binding of HIT IgG to specific Fc receptors (Fc␥RIIA), 6 partly explaining why HIT differs from most other types of drug-induced immune thrombocytopenia, which are mainly complicated by hemorrhage.The most frequent manifestation in HIT is thrombosis, sometimes associated with disseminated intravascular coagulation, and one of the mechanisms proposed to explain the hypercoagulability in HIT is the production of phospholipid platelet microparticles after platelet activation. 7 However, HIT antibodies might also bind to endothelial cells and trigger the synthesis of tissue factor (TF), 4,8,9 a transmembrane glycoprotein that initiates the coagulation pathway. 10,11 TF can be produced by monocytes when activated by lipopolysaccharides, cytokines, or immune complexes, 12 and it can be triggered in the presence of activated platelets. 13 We therefore investigated whether antibodies to PF4 developed in patients with HIT to induce the synthesis of TF by human monocytes. Study design Plasma and purification of immunoglobulin GPlasma from 5 patients with definite HIT complicated by venous (n ϭ 5) and arterial (n ϭ 1) thromboses was studied. Every sample contained significant levels of antibodies to H-PF4 complexes (mean A 492 , 1.7; range, 1.0-2.1) (Asserachrom HPIA, Stago; Asnières, France) 14 able to induce heparin-dependent platelet activation in serotonin release assay (SRA). 15 Normal human plasma without antibodies to H-PF4 complexes (mean A 492 Ͻ 0.2) was collected from 5 healthy volunteers not receiving heparin. All samples were heated at 56°C for 1 hour, centrifuged ...

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Section: Resultsmentioning

confidence: 92%

“…Procoagulant activity of monocytes was then evaluated by an amidolytic assay adapted from a previously described procedure, and the results were expressed as mU factor Xa/well. 17 …”

Section: Chromogenic Assay For Procoagulant Activity Of Monocytesmentioning

confidence: 99%

Induction of monocyte tissue factor expression by antibodies to heparin–platelet factor 4 complexes developed in heparin-induced thrombocytopenia

Pouplard

Iochmann

Renard

et al. 2001

Blood

Self Cite

196

136

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“…1b) showed that the oD 405 increased in proportion to the concentrations of factor Xa chromogenic substrate at the range of 0.0625-1 mm, and 0.5 mm chromogenic substrate provided higher absorbance, which was elevated slightly above this concentration, similar to the previous report. 17 as for the influence of various concentrations of prothrombin complex, human monocytes were incubated in the absence (normal) or in the presence of lps (100 ng/ml) (model) for 5 h, and indicated concentrations of prothrombin complex (300 iu) were tested. the results showed that the difference in formation of tf between the normal and model group was significant at the concentration of prothrombin complex at 10 g/l.…”

Section: Optimization Of Tf Chromogenic Assay Based On Prothrombin Comentioning

confidence: 99%

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

et al. 2011

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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.

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“…26 Cells were washed and resuspended in Trisbuffered saline (0.05 M Tris-HCl, 0.2 M NaCl, pH 8.4) at 10 6 cells/mL and and pooled citrated plasma from fibrinogen-null mice containing 4 units/mL of human hirudin (Sigma, St Louis, MO). The reaction was quenched in 0.25M EDTA, and FXa generation was measured using the chromogenic substrate S2222 (Chromogenix, Milano, Italy).…”

Section: Tf Tm and Tfpi Functional Assaysmentioning

confidence: 99%

Fetomaternal cross talk in the placental vascular bed: control of coagulation by trophoblast cells

Sood

Kalloway

Mast

et al. 2006

Blood

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IntroductionIn the hemochorial type of placentation observed in humans and mice, fetal nutrition involves the direct uptake of nutrients by zygote-derived trophoblast cells from circulating maternal blood. The required placental morphology is achieved through a highly regulated process of trophoblast differentiation coupled with remodeling of maternal and fetal vasculature. As a consequence, in contrast to all other vascular beds in which the blood vessel endothelium is the principal gatekeeper between tissue and blood, the terminal vascular space of the placenta is lined by trophoblast cells. 1,2 Trophoblast cells are genetically distinct from the maternal vascular endothelium and are derived from a different developmental lineage than endothelial cells. 3 In all nonplacental vascular beds, normal endothelium proactively suppresses the activity of the coagulation system, thereby maintaining a nonthrombogenic surface. A survey of existing data suggests that trophoblast cells also produce endothelial regulators of hemostasis, such as thrombomodulin (TM), endothelial protein C receptor (EPCR), and tissue factor pathway inhibitor (TFPI). [4][5][6][7][8] Such findings indicate that trophoblast cells might exhibit an endothelial cell-like ability to partake in the regulation of hemostasis at the fetomaternal interface. Indeed, the term "endothelial mimicry" has been coined to describe a process of remodeling of the maternal arteries, during which so-called "endovascular" trophoblast cells replace the maternal endothelium in these blood vessels and switch their expression from epithelial to endothelial adhesion receptor repertoire. [9][10][11] It is unknown whether trophoblast cells acquire anticoagulant gene expression in a temporally and spatially controlled manner similar to that described for the subset of endovascular trophoblast cells or whether the acquisition of an endothelial cell-like anticoagulant phenotype is a cell type-defining feature of trophoblast cells in general.The placenta also is a rich source of the initiator of coagulation, tissue factor (TF). TF antigen and procoagulant activity are detected in mouse giant and labrynthine trophoblasts and on human syncytiotrophoblast membranes. [12][13][14][15] With the exception of angiogenic endothelium, and in endothelium subjected to inflammatory and thrombotic stimuli, TF expression is strictly excluded from endothelial cells. Proinflammatory cytokines, ligands for Tollreceptors, and the principal coagulation protease, thrombin, induce TF expression in cultured endothelial cells, evoke increased production of endothelial-leukocyte adhesion receptors, and simultaneously suppress the expression of anticoagulant gene products. This transition from a noncoagulant and antiadhesive phenotype to a state of enhanced coagulation and leukocyte interactions has been termed "endothelial activation" and appears to reflect a principal switch in a concerted gene-expression program. 16 In contrast, trophoblast cells constitutively express TF, thus exhibiting, even under ...

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A simplified and low-cost one-stage chromogenic assay for tissue factor dependent procoagulant activity of endothelial cells

Cited by 11 publications

References 17 publications

Induction of monocyte tissue factor expression by antibodies to heparin–platelet factor 4 complexes developed in heparin-induced thrombocytopenia

Induction of monocyte tissue factor expression by antibodies to heparin–platelet factor 4 complexes developed in heparin-induced thrombocytopenia

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

Fetomaternal cross talk in the placental vascular bed: control of coagulation by trophoblast cells

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