Steven D. Carson

Continuous chromogenic tissue factor assay: Comparison to clot-based assays and sensitivity established using pure tissue factor

The continuous chromogenic tissue factor assay has been compared to one-stage and two-stage coagulation assays, and its sensitivity has been established using purified human placental tissue factor. Log-log plots of the clot-times versus A405/min2 are linear over the mutually useful ranges of tissue factor concentration. Using human placental tissue factor reconstituted into phosphatidylserine-phosphatidylcholine (30:70) vesicles, the chromogenic assay quantitatively detected tissue factor in dilutions containing from 3 femtomoles to less than 10 attomoles of protein.

Tissue factor activity in hela cells measured with a continuous chromogenic assay and elisa reader

Tissue factor activity expressed by Hela cells cultured in 96-well plates has been quantitated in situ using a continuous spectrophotometric assay. Following the assay, cells assayed without physical disruption remained as viable as cells not subjected to the assay. Very little (or no) tissue factor was expressed in nondisrupted cells relative to that available in cells disrupted by freeze-thawing and sonication. Total tissue factor activity (that available in disrupted cells) decreased not as a simple function of time after subculturing, but was inversely related to cell density.

Computerized analysis of enzyme cascade reactions using continuous rate data obtained with an ELISA reader

Two programs have been written which permit analysis of multiple continuous-rate enzyme-cascade assays conducted with the use of an ELISA spectrophotometer and a synthetic chromogenic substrate. Because the product of the first reaction functions as the enzyme in the second reaction, production of chromophore continuously accelerates and it is the rate of acceleration which serves to measure the rate of the initial reaction in the system. The first program determines the rate of acceleration using linear regression to analyze the reaction curves as a function of the square of time. The second program, using a Simplex algorithm, determines the parameters which establish the assay standard curve by fitting the rate data to the Hill equation. Used together, these programs facilitate the analysis of many kinetic experiments conducted simultaneously.

Regional assignment of human tissue factor gene (F3) to chromosome 1p21-p22

Tissue factor, or coagulation factor III, is a membrane-bound glycoprotein and acts as a cofactor for factor VII-dependent initiation of blood coagulation. The tissue factor gene (F3) was previously assigned to human chromosome 1, region p21-pter. The present report has further refined the mapping position to 1p21-p22 using a cDNA probe for the tissue factor gene and in situ hybridization to metaphase chromosomes.

Rapid chromatographic purification of apolipoproteins A-I and A-II from human plasma.

Rapid, large-scale isolation of human apolipoproteins A-I and A-II has been accomplished using two chromatographic procedures. The apolipoproteins adsorbed from plasma onto a column of phenyl-Sepharose are eluted with increasing propylene glycol concentrations. Apolipoproteins A-I and A-II can be resolved by elution with a linear 0 to 80% propylene glycol gradient. Homogeneous preparations of apo A-I and A-II are obtained following gel filtration in 3M guanidinium chloride.

Cadmium binding to human α2-macroglobulin

alpha 2-Macroglobulin (alpha 2M) is one of the major cadmium-binding proteins of human plasma. As determined with equilibrium dialysis, alpha 2M bound 4.6 (+/- 0.7) mol Cd2+ per mol protein with an apparent dissociation constant of (9.6 (+/- 5.0] X 10(-7) M. Methylamine-modified alpha 2M (alpha 2M-Me) had a similar affinity for Cd2+ (Kd,app = 5.3 X 10(-7) M), but fewer binding sites. Cadmium produced a small increase in the amidolytic activity of trypsin in the presence of alpha 2M and soybean trypsin inhibitor. Using the binding parameters determined from the equilibrium dialysis studies, the Cd2+ concentration which produced a half-maximal increase in amidolytic activity corresponded to saturation of all Cd2+-binding sites in one-half of the alpha 2M molecules. From these results, a model is proposed in which one Cd2+-binding site is present in each of the four polypeptide chains which compose alpha 2M.

Tissue factor (coagulation factor III) is present in placental microvilli and cofractionates with microvilli membrane proteins

We have demonstrated that tissue factor is present in preparations of placental microvilli. The similarity of the procoagulant activities of microvilli and placental homogenates indicate that tissue factor is neither deficient nor enriched in microvilli relative to the bulk of placental tissue. On the basis of solubility in Triton X-100 and ammonium sulphate, and its affinity for phenyl-Sepharose, microvilli tissue factor is indistinguishable from that in whole placental homogenates.

Selective immunoprecipitate identification using monoclonal anti-rabbit IgG

A monoclonal mouse antibody directed against rabbit IgG has been conjugated with horseradish peroxidase and used to identify immunoprecipitates which contain rabbit antibodies. By combining a specific rabbit antisera with a general antiserum from another species (e.g., goat antiserum against human serum), immunoprecipitates containing the antigen(s) recognized by the rabbit antibodies have been selectively identified by colorimetric development of peroxidase activity. Since the monoclonal antibody is specific for rabbit IgG and nonprecipitating, the peroxidase conjugate can be included in the agarose with the primary antisera.