Katsuhiko Nawa

Purification and subunit structure of hepatocyte growth factor from rat platelets

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four‐step procedure: stimulation of its release from platelets by thrombin, cation‐exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin‐Sepharose CL‐6B chromatography, and reverse‐phase HPLC on a C4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a beat‐ and acid‐labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS‐PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS‐PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet‐derived growth factor (PDGF).

Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin

We constructed a human soluble thrombomodulin (sTM) expression vector using the RSV promoter. Recombinant sTM (rsTM) was expressed in CHO cells and was recovered from culture medium by ion exchange chromatography. Two active fractions, designated as rsTM alpha (low salt elution) and rsTM beta (high salt elution), were detected and further purified by immunoaffinity chromatography. Purified rsTM beta contained bound chondroitin-4-sulfate as judged by HPLC detection of the chondroitinase ABC and AC I digestion product, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose. The apparent Kd values for thrombin of alpha and beta were 7.4 and 1.4 nM respectively. RsTM beta was more effective at inhibition of thrombin clotting activity and had antithrombin III-dependent anticoagulant activity which was not possessed by rsTM alpha. Both anticoagulant activities were lost after chondroitinase treatment of rsTM beta.

The cDNA cloning and mRNA expression of rat protein C

We cloned a cDNA coding for rat protein C, which provides hybridization probes for the detection of protein C mRNA in several tissues. The cloned cDNA was 1543 bp long and contained a single open reading frame of 1383 nucleotides. The proposed rat protein C precursor contained 461 amino acid residues: a 41 amino acid preproleader sequence, and light (155 amino acids) and heavy (263 amino acids) chains joined by a Lys-Arg dipeptide. Northern blot analysis showed that the rat protein C mRNA was expressed not only in the liver, but also in the kidney.

Monoclonal antibodies against human thrombomodulin whose epitope is located in epidermal growth factor-like domains

Thrombomodulin (TM) on endothelial cells is a glycoprotein that functions as a cofactor for thrombin-catalyzed activation of protein C. The structural requirement for thrombin binding and cofactor activity were investigated using monoclonal antibodies (moAbs) against TM and site-directed mutagenesis of recombinant human soluble TM (rsTM). Results showed that moAb 2A2 inhibited thrombin binding to rsTM and also abolished its functions as a cofactor in thrombin-catalyzed activation of protein C and as an anticoagulant by modifying thrombin-induced fibrinogen clotting and platelet aggregation, moAb 1F2 did not affect its activity as an anticoagulant, but inhibited its cofactor activity, and moAb 10A3 did not inhibit either activity. Epitope analysis was carried out by site directed mutagenesis of rsTM expressed in CHO cells. Some proteins with mutations within the second disulfide loop of the fourth EGF-like domain showed reduced affinity for moAb 1F2, but retained cofactor activity. These results suggest that the epitope of moAb 1F2 includes the second disulfide loop of the fourth EGF-like domain, which is close to a region required for cofactor activity. Mutant proteins of the third disulfide loop of the fifth EGF-like domain showed loss of interaction with moAb 2A2. Thus the epitope of moAb 2A2 may include the third disulfide loop of the fifth EGF-like domain. Furthermore, replacement of Asn-439 by Gln decreased the cofactor activity and anticoagulant activity, and resulted in low affinity for either moAb 1F2 or 2A2, suggesting that Asn-439, which is located in the second disulfide loop of the sixth EGF-like domain, is critical for determining the functional conformation of the EGF-like domains 4-6.