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

Abstract

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.

Atheroprotective Effects and Molecular Targets of Tanshinones Derived From Herbal Medicine Danshen

“…149,150 Mechanistic studies indicated that CTS decreased the level of pro-inflammatory cytokines ICAM1, VCAM1, MCP-1, and TF while restoring eNOS-derived NO production in endothelial cells. [149][150][151][152] In line with this evidence, CTS also triggered eNOS-dependent NO production in endothelial cells via an ERα-dependent pathway. 153 Interestingly, CTS mediates anti-inflammatory effects in SMCs by inhibiting iNOSdependent NO production via an ERβ-dependent pathway.…”

“…In agreement with this finding, two independent studies have validated that CTS suppressed both TNF‐α‐ and oxLDL‐induced endothelial hyperpermeability and monocyte adhesion to endothelial cells . Mechanistic studies indicated that CTS decreased the level of pro‐inflammatory cytokines ICAM1, VCAM1, MCP‐1, and TF while restoring eNOS‐derived NO production in endothelial cells . In line with this evidence, CTS also triggered eNOS‐dependent NO production in endothelial cells via an ERα‐dependent pathway .…”

“…The anti‐inflammatory effects of CTS may be associated with inhibitory effects on pro‐inflammatory transcriptional factors, including NF‐ƙB, AP‐1 and TLR4 . CTS also has thrombo‐protective effects since CTS suppressed LPS‐induced TF activity in human monocytes by high‐throughput chromogenic assay . Based on the constituent abundance, structural similarity and anti‐atherosclerotic profiles of TSN and CTS, it is plausible that both compounds are important bioactive constituents that have anti‐oxidant and anti‐inflammatory effects accountable for the atheroprotective effects of Danshen.…”

“…156,157 CTS also has thrombo-protective effects since CTS suppressed LPS-induced TF activity in human monocytes by high-throughput chromogenic assay. 152 Based on the constituent abundance, structural similarity and anti-atherosclerotic profiles of TSN and CTS, it is plausible that both compounds are important bioactive constituents that have anti-oxidant and antiinflammatory effects accountable for the atheroprotective effects of Danshen. The therapeutic effect of CTS in patients with atherosclerotic CVDs remains to be investigated.…”

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

“…In order to evaluate the feasibility of saponins as TF inhibitor and explore the SARs, TF procoagulation activity was measured using improved chromogenic substrate methods. 18 As shown in Figure 2, 7 compounds including ruscogenin, diosgenin, pennogenin, parissaponin Pe, pm 13 (pennogenin-…”