Ryudocan, a heparan sulfate proteoglycan, was isolated from human endothelium-like EAhy926 cells by a combination of ion-exchange and immunoaffinity chromatography. Purified human ryudocan has biochemical properties similar to those of rat ryudocan isolated from microvascular endothelial cells. Human ryudocan contains only heparan sulfate (HS) glycosaminoglycan chains along with a core protein with an apparent molecular mass of 30 kDa. We evaluated the interactions between purified human ryudocan and several extracellular ligands by using a solid-phase binding assay. We found that basic fibroblast growth factor (bFGF), midkine (MK), and tissue factor pathway inhibitor (TFPI) exhibit significant ryudocan binding. Heparitinase (but not chondroitin ABC lyase) treatment destroyed the ability of ryudocan binding to bFGF, MK, and TFPI. Heparin and HS, but not chondroitin sulfate, inhibited such ryudocan binding. Thus, the HS chains of ryudocan appear to be responsible for its binding to bFGF, MK, and TFPI. The apparent dissociation constants for purified ryudocan were as follows: bFGF, 0.50 nM; MK, 0.30 nM; and TFPI, 0.74 nM. Immunohistochemical analysis revealed that ryudocan was expressed in fibrous connective tissues, peripheral nerve tissues, and placental trophoblasts. These findings suggest that ryudocan may possess multiple biological functions, such as bFGF modulation, neurite growth promotion, and anticoagulation, via HS-binding effectors in the cellular microenvironment.

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To investigate the molecular mechanisms of the quantitative factor V (FV) deficiency associated with the FV R2 haplotype, 4 missense mutations, Met385Thr, His1299Arg, Met1736Val, and Asp2194Gly, identified in the R2 haplotype allele, were analyzed by in vitro expression studies. The FV variant carrying all 4 mutations showed a markedly lower steady-state expression level than wild-type FV because of low synthesis rate and impaired secretion of the mutant protein. The Asp2194Gly mutation was found to play a key role in the impaired secretion of the mutant FV by interfering with its transport from the endoplasmic reticulum to the Golgi complex. The deleterious effect of the Asp2194Gly mutation was shown to be dominant among the 4 mutations. The Met385Thr mutation and His1299Arg mutation had no effect on steady-state expression levels, but the secretion rates of the mutant proteins were moderately decreased by these mutations. The His1299Arg mutation partially impaired glycosylation in the C-terminal part of the B-domain of the mutant FV, which was supposed to affect the secretion rate, but not the steady-state expression level. It was also suggested that the Met385Thr mutation partially impairs posttranslational modification of the mutant FV without affecting the steady-state expression level. No deleterious effect of the Met1736Val mutation was observed in terms of expression and intracellular processing. Our in vitro data strongly suggest that the naturally existing R2 haplotype mutant FV, which carries all 4 mutations, has the potential to result in quantitative FV deficiency in vivo owing to impaired expression of the mutant protein when the Asp2194Gly mutation is present. IntroductionFactor V (FV) is a plasma glycoprotein that plays key roles in blood coagulation. 1 FV is a single-peptide protein consisting of multiple domains, A1-A2-B-A3-C1-C2, whose domain structure is similar to that of factor VIII (FVIII). 1 In the blood coagulation cascade, FV is proteolytically activated by thrombin or activated factor X (FXa). 1 The B-domain is released upon activation, whereas the heavy chain (A1-A2) and light chain (A3-C1-C2) form a noncovalently bound heterodimeric molecule, activated FV (FVa). 1 FVa binds to FXa and serves as its cofactor in the prothrombinase complex that converts prothrombin to the active enzyme thrombin. 1 FV also functions as an anticoagulant cofactor in the protein C pathway. 2 Activated protein C (APC) downregulates blood coagulation by inactivating both FVa and activated FVIII (FVIIIa). 2 FV and protein S serve as synergistic cofactors in the APC-mediated inactivation of FVIIIa. 2 In human plasma, FV exists in 2 isoforms, designated FV1 and FV2, that have slightly different molecular weights because of partial N-linked glycosylation in the C2-domain. 3 FV1 and FV2 have different characteristics in terms of procoagulant activity, inactivation by APC, and anticoagulant function in the protein C pathway. 4 Consequently, FV1 has the overall potential to generate more thrombin than FV2.Molecular...

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