Molecular Biological Analysis for Mechanism of Thombosis Regulation and Its APlication for Clinical Desease.

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To determine the subcellular localization of the protein C (PC) Nagoya, an elongated variant of the human PC,the recombinant PC bearing this mutation was expressed in Chinese hamster ovary cells. Immunoelectron microscopy indicated that PC Nagoya was relained in the ER,whereas wild type PC was observed in both the ER and the Golgi apparatus. Metabolic radiolabeling with [35S] methionine in combination with chemical cross-linking revealed that the PC Nagoya existed in the ER as an complex with GRP78 and GRP94. Because both GRP78 and GRP94 associate far lesser degree with wild type PC than with PC Nagoya, our data suggest that both stress proteins function as molecular chaperones, and work in concert with the folding and assembly of PC.DNA sequence analysis in the proband with a hereditary type I protein S (PS) deficiency showed a novel missense mutation substituting Cys (TGT) for Arg474 (CGT). Stable expression and pulse-chase experiments demonstrated an intracellular degradation and an impaired secretion of the recombinant Cys-mutant PS.Furthermore, the substitution of Arg 474 by Ala or Clu, but not Lys. markedly reduced the secretion of the recombinant PS mutant, suggesting that a positively charged basic amino acid might be needed at residue 474 and might play a key role in the protein structure and conformation of the sex hormone binding globulin-homology domain of the PS molecule.It was found that basic fibroblast growth factor (bFGF), midkine (MK), and tissue factor pathway inhibitor (TFPI) exhibited significant ryudocan binding through its heparan sulfate chains. Immuno-histochemical analysis revealed that ryudocan was expressed in peripheral nerve tissues, fibrous connective tissues, and placental trophoblasts. These observations suggest that ryudocan may possess multiple biologic functions, such as bFGF modulation, neurite growth promotion, and anticoagulation, via heparan sulfate binding effectors present in the cellular microenvironment.