Summary:The synthesis of tissue factor pathway inhibitor (TFPI) was investigated in cloned human synovial cells and human chondrocytes. TFPI-specific DNA transcription products of these cells were isolated, and a full-length cDNA of about 1000 base pairs was amplified by reverse transcription and polymerase chain reaction. The amplified DNA was cloned into the vector pUC 18. The TFPI coding sequence was confirmed by double stranded sequencing and was identical with that previously published for human TFPI coding nucleotide sequence frorn human placental cDNA (1).The inhibitory activity of TFPI in the cell medium of cultivated human chondrocytes and cloned human synovial cells was determined by a specific chromogenic Substrate assay of factor Xa activity. The inhibitory activity of TFPI in the medium of human chondrocytes and cloned human synovial cells was 630-720 mU/10 8 cells and 1080-1665 mU/10 8 cells, respectively.In addition, TFPI activity in cell culture media of human chondrocytes and cloned human synovial cells was suppressed by a polyclonal goat anti-TFPI antibody directed against the inhibitory domain I and domain II. In the chromogenic Substrate assay, the anti-TFPl antibody completely suppressed the inhibitory activity of TFPI in the samples.

A method is described for the determination of plasma and serum glycosaminoglycans, which can be used in any laboratory equipped with an HPLC system. It is based on the sequential application of chondroitinases AC and ABC and separation of the resulting disaccharides by high-performance liquid chromatography. All reagents are commercially available. This simple and rapid separation yields an accurate quantification and an exact distribution pattern. The determination of glycosaminoglycan disaccharides is linear between 7 and 7000 μιηοΐ/ΐ with coefficients of variation between 3.0 and 7.7% for serum and between 2 and 14% for plasma. The recovery of the assay ranged from 93 to 106% for different concentrations of glycosaminoglycan disaccharides. This HPLC method may therefore be considered as a candidate reference method.

Summary: A competitive enzyme immunoassay was developed to determine chondroitin-6-sulphate in body fluids and cell cultures. The assay uses a monoclonal anti-chondroitin-6-sulphate antibody, immobilised to microtitre plates, and it involves a competitive binding reaction between chondroitih-6-sulphate in the samples and the biotinylated antigen.This assay enables the quantification of chondroitin-6-sulphate in the low concentration ränge of 16-120 g/l. The intra-assay and inter-assay coefficients of Variation are below 6.5% and 9.0%, respectively. More than 90% of chondroitin-6-sulphate was recovered when added to 0.1 mol/1 phosphate-buffered saline, an albumin solution (40 g/l in phosphäte-buffered saune) and cell culture medium (containing 100 ml/l foetal calf serum).Chondroitin-6-sulphate was also determined in sera of healthy male (n = 90) and female (n = 90) blood donors. The normal ränge was 55-169 g/l. In men the mean value was estimated at 102.2 ±37.1 g/l and in women at 98.7 i 26.4 g/l. Np age or sex dependence was observed.The urine excretion of chondroitin-6-sulphate in men (n = 16) was 44.5 ±21.1 mg/kg creatinine (mean ± Standard deviation) and in fernales (n = 10) 53.5 ± 21.3 mg/kg creatinine. The clearance rate in men was 0.41 ± 0.22 ml X min" 1 and in women €.38 ± 0.15 ml X min" 1 . No sex dependence was found.Furthermore, the enzyme immunoassay was modifted to measure the specific incorporation of a radioactively labelled precursor ([ 14 C]galactosamine) into chondroitin-6-sulphate. This modification rapidly gives Information on the cellular glycosaminoglycan synthesis in cell culture. Usmg this method our experiments with cultivated human chondrocytes showed that the synthesis of chpndroitin^o-sulphate decreased in the presence of interleukin-la (60.0% less), turnour necrosis factor a (64.4%), -interferön (21.6%) and lipopolysaccharide (53.4%).