PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 4014693

Enzyme-linked coagulation assay: a clot-based, solid-phase assay for thrombin.

G J Doellgast·H Rothberger
View original source ↗Export metadata ↓

Abstract

A new, solid-phase microtiter plate assay for thrombin has been developed, using fibrinogen bound to wells of a microtiter plate and peroxidase-fibrinogen in solution as an indicator system. When small amounts of thrombin are added to the mixture, peroxidase-fibrin and plate-bound fibrin are formed, and the peroxidase-fibrin binds to the plate-bound fibrin. The amount of peroxidase-fibrin binding is proportional to the thrombin concentration and time of incubation. Using this assay, thrombin was measured at concentrations as low as 0.25 ng/ml (0.006 nM) in 150 microliter of sample. In the presence of the specific inhibitors benzamidine and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, the thrombin activity is reduced, at relative concentrations of inhibitors consistent with their affinities and mechanisms of action. The enzyme-linked coagulation assay is generally useful as a highly sensitive and convenient alternative to conventional "clot-based" tests of coagulation.

Explore related subjects

Amino Acid Chloromethyl KetonesBenzamidinesBlood CoagulationFibrinHumansMethodsPeroxidasesThrombinJournal ArticleResearch Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

G J Doellgast, H Rothberger. 1985. Enzyme-linked coagulation assay: a clot-based, solid-phase assay for thrombin.. https://doi.org/10.1016/0003-2697(85)90310-0

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

PPARgamma inhibitors reduce tubulin protein levels by a PPARgamma, PPARdelta and proteasome-independent mechanism, resulting in cell cycle arrest, apoptosis and reduced metastasis of colorectal carcinoma cells.

The nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma) has been identified as an important therapeutic target in murine models of colorectal cancer (CRC). To examine whether PPARgamma inhibition has therapeutic effects in late-stage CRC, the effects of PPARgamma inhibitors on CRC cell survival were examined in CRC cell lines and a murine CRC model. Low doses (0.1-1 microM) of PPARgamma inhibitors (T0070907, GW9662 and BADGE) did not affect cell survival, while higher doses (10-100 microM) of all 3 PPARgamma inhibitors caused caspase-dependent apoptosis in HT-29, Caco-2 and LoVo CRC cell lines. Apoptosis was preceded by altered cell morphology, and this alteration was not prevented by caspase inhibition. PPARgamma inhibitors also caused dual G and M cell cycle arrest, which was not required for apoptosis or for morphologic alterations. Furthermore, PPARgamma inhibitors triggered loss of the microtubule network. Notably, unlike other standard antimicrotubule agents, PPARgamma inhibitors caused microtubule loss by regulating tubulin post-transcriptionally rather than by altering microtubule polymerization or dynamics. Proteasome inhibition by epoxomicin was unable to prevent tubulin loss. siRNA-mediated reduction of PPARgamma and PPARdelta proteins did not replicate the effects of PPARgamma inhibitors or interfere with the inhibitors' effects on apoptosis, cell cycle or tubulin. PPARgamma inhibitors also reduced CRC cell migration and invasion in assays in vitro and reduced both the number and size of metastases in a HT-29/SCID xenograft metastatic model of CRC. These results suggest that PPARgamma inhibitors are a novel potential antimicrotubule therapy for CRC that acts by directly reducing microtubule precursors.

Amino Acid Chloromethyl Ketones↗
PubMed2007

Antiviral effects of pan-caspase inhibitors on the replication of coxsackievirus B3.

The induction of apoptosis during coxsackievirus B3 (CVB3) infection is well documented. In order to study whether the inhibition of apoptosis has an impact on CVB3 replication, the pan-caspase inhibitor Z-VAD-FMK was used. The decreased CVB3 replication is based on reduced accumulation of both viral RNA and viral proteins. These effects are due to an inhibitory influence of Z-VAD-FMK on the proteolytic activity of the CVB3 proteases 2A and 3C, which was demonstrated by using the target protein poly(A)-binding protein (PABP). The antiviral effect of the structurally different pan-caspase inhibitor Q-VD-OPH was independently of the viral protease inhibition and resulted in suppression of virus progeny production and impaired release of newly produced CVB3 from infected cells. A delayed release of cytochrome c into the cytoplasm was detected in Q-VD-OPH-treated CVB3-infected cells pointing to an involvement of caspases in the initial steps of mitochondrial membrane-permeabilization.

Amino Acid Chloromethyl Ketones↗
PubMed2007

Chondrocyte apoptosis is not essential for cartilage calcification: evidence from an in vitro avian model.

The calcification of cartilage is an essential step in the process of normal bone growth through endochondral ossification. Chondrocyte apoptosis is generally observed prior to the transition of calcified cartilage to bone. There are, however, contradictory reports in the literature as to whether chondrocyte apoptosis is a precursor to cartilage calcification, a co-event, or occurs after calcification. The purpose of this study was to test the hypothesis that chondrocyte apoptosis is not a requirement for initial calcification using a cell culture system that mimics endochondral ossification. Mesenchymal stem cells harvested from Stages 21-23 chick limb buds were plated as micro-mass cultures in the presence of 4 mM inorganic phosphate (mineralizing conditions). The cultures were treated with either an apoptosis inhibitor or stimulator and compared to un-treated controls before the start of calcification on day 7. Inhibition of apoptosis with the caspase inhibitor Z-Val-Ala-Asp (O-Me)-fluoromethylketone (Z-VAD-fmk) caused no decreases in calcification as indicated by radioactive calcium uptake or Fourier transform infrared (FT-IR) analysis of mineral properties. When apoptosis was inhibited, the cultures showed more robust histological features (including more intense staining for proteoglycans, and more intact cells within the nodules as well as along the periphery of the cells as compared to untreated controls), more proliferation as noted by bromo-deoxyuridine (BrdU) labeling, decreases in terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end labeling (TUNEL) staining, and fewer apoptotic bodies in electron microscopy. Stimulation of apoptosis with 40-120 nM staurosporine prior to the onset of calcification resulted in inhibition of calcium accretion, with the extent of total calcium uptake significantly decreased, the amount of matrix deposition impaired, and the formation of abnormal mineral crystals. These results indicate that chondrocyte apoptosis is not a pre-requisite for calcification in this culture system.

Amino Acid Chloromethyl Ketones↗