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Protein S (PS) is a member of the vitamin K-dependent protein family containing similar γ-carboxyglutamic acid (Gla) domains, although only PS has a thrombin-sensitive region (TSR), which is located between the Gla domain and the first epidermal growth factor-like domain. In this study, a novel PROS1 mutation was identified at the last nucleotide in intron C (c.260-1G>A) in a patient suffering from recurrent deep vein thrombosis associated with PS deficiency. To investigate the molecular mechanisms of PS deficiency caused by the novel PROS1 mutation, we characterized the mutant mRNA, and the secretion and function of the mutant PS molecule associated with the mutation. RT-PCR was used to detect the aberrant mRNA in the patient's platelets, the amount of which was markedly reduced and lacked the region corresponding to exon 4 coding the TSR of the PS molecule. The recombinant mutant PS lacking the TSR (TSR-lack PS) showed a markedly reduced transient expression/secretion level, 37.9% of that of wild-type (WT) PS. Activated protein C (APC) cofactor activity assay showed that TSR-lack PS had no cofactor activity. Moreover, binding assays of monoclonal antibodies recognizing the PS Gla domain and the Gla residues indicated that the bindings of TSR-lack PS to both of these antibodies were clearly weaker than those of WT PS. These findings suggest that the novel mutation leading to the absence of the TSR not only affected the secretion of mutant PS, but was also responsible for impairment of the Gla domain conformation required for the γ-carboxylation to express APC cofactor activity.

Summary Eight distinct and potentially causative mutations were identified in eight unrelated Japanese patients with protein S (PS) deficiency, by direct DNA sequencing of the protein Sa (PSa) gene-specific polymerase chain reaction products of all 15 exons and exon/intron boundaries. There were five missense mutations, including two novel mutations (Cys80Tyr and Arg314His), and three showed a major impact on the expected gene products: novel mutations of a 5-bp deletion (delCTCTG887:Cys206Stop) and a nonsense mutation (Glu208Stop), as well as a previously reported splice site (exon 10 +5 A fi G) mutation. One of the patients showed compound heterozygosity for delCTCTG887 and 732A fi G. Investigation for the cosegregation state of these two mutations with PS deficiency in the patient’s family suggested that the delCTCTG887 mutation was responsible for the abnormal phenotype and that the 732A fi G (Lys155Glu) mutation did not appear to play a key role. However, we also identified the same 732A fi G (Lys155Glu) mutation in an unrelated patient with apparent PS deficiency with severe pulmonary embolism, and found that this mutation seemed to cosegregate with a PS-deficient state in her family members. These data implied that unknown factor(s) other than the 732A fi G mutation itself might influence phenotypic expression of PS status in different individuals.

Factor V (FV) deficiency, also known as parahemophilia, is a rare bleeding disorder. Herein we investigate the first reported missense mutation associated with FV deficiency, Ala221Val, assigned as FV New Brunswick. To elucidate the molecular pathology associated with the Ala221Val substitution, the mutation was recreated in a recombinant system together with 3 FV mutants (Ala221Gly, Glu275Gln, and Cys220Ala/Cys301Ala) designed to help explain the Ala221Val phenotype. The expression pattern was analyzed by pulse-chase experiments and an FV-specific enzyme-linked immunosorbent assay (ELISA), the results suggesting the Ala221Val mutation not to interfere with the synthesis or secretion. The functional properties of the recombinant FV New Brunswick were evaluated in both plasma clotting and purified systems. The Ala221Val mutation did not affect the factor Xa (FXa) cofactor function; nor did it interfere with the activated protein C (APC)–mediated down-regulation of activated FV (FVa) activity. However, FV New Brunswick demonstrated reduced stability at 37°C due to an increased rate of dissociation of light and heavy chains of FVa. In conclusion, this in vitro study of FV New Brunswick suggests the Ala221Val mutation not to impair synthesis and expression of procoagulant activity, indicating overall proper folding of the mutant molecule. Rather, the Ala221Val substitution appears to interfere with the stability of the activated FVa mutant, the reduced stability possibly explaining the deficiency symptoms associated with the mutation.

Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme Factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIIIa. The interaction between the FVa variants and FXa and prothrombin were studied in a functional prothrombin activation assay, as well as in a direct binding assay between FVa and FXa. In both assays, the four mutants carrying a carbohydrate side chain at positions 467, 511, 652, or 1683 displayed attenuated FXa binding, whereas the prothrombin affinity was unaffected. The affinity toward FXa could be restored when the mutants were expressed in the presence of tunicamycin to inhibit glycosylation, indicating the lost FXa affinity to be caused by the added carbohydrates. The results suggested regions surrounding residues 467, 511, 652, and 1683 in FVa to be important for FXa binding. This indicates that the enzyme:cofactor assembly of the prothrombinase and the tenase complexes are homologous and provide a useful platform for further investigation of specific structural elements involved in the FVa.FXa complex assembly.

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.

The vitamin K-dependent anticoagulant protein S binds with high affinity to C4b-binding protein (C4BP), a regulator of complement. Despite the physiological importance of the complex, we have only a patchy view of the C4BP-binding site in protein S. Based on phage display experiments, protein S residues 447-460 were suggested to form part of the binding site. Several experimental approaches were now used to further elucidate the structural requirements for protein S binding to C4BP. Peptides comprising residues 447-460, 451-460, or 453-460 of protein S were found to inhibit the protein S-C4BP interaction, whereas deletion of residues 459-460 from the peptide caused complete loss of inhibition. In recombinant protein S, each of residues 447-460 was mutated to Ala, and the protein S variants were tested for binding to C4BP. The Y456A mutation reduced binding to C4BP approximately 10-fold, and a peptide corresponding to residues 447-460 of this mutant was less inhibitory than the parent peptide. A further decrease in binding was observed using a recombinant variant in which a site for N-linked glycosylation was moved from position 458 to 456 (Y456N/N458T). A monoclonal antibody (HPSf) selective for free protein S reacted poorly with the Y456A variant but reacted efficiently with the other variants. A second antibody, HPS 34, which partially inhibited the protein S-C4BP interaction, reacted poorly with several of the Ala mutants, suggesting that its epitope was located in the 451-460 region. Phage display analysis of the HPS 34 antibody further identified this region as its epitope. Taken together, our results suggest that residues 453-460 of protein S form part of a more complex binding site for C4BP. A recently developed three-dimensional model of the sex hormone-binding globulin-like region of protein S was used to analyze available experimental data.

SummaryFactor VII is a vitamin K-dependent zymogen that plays a key role in the initiation of the extrinsic pathway. A severe factor VII deficiency was identified in a 45-year old male whose plasma factor VII antigen was less than 60 ng/ml and expressed 5.2% of normal factor VII activity. DNA sequence analysis of the patient’s factor VII gene showed a thymidine to guanine transversion at nucleotide 10968 in exon VIII that results in a novel amino acid substitution of His348 to Gln. The patient was homozygous for this mutation, whereas some of his family members were heterozygous. Both wild type and mutant factor VII were transiently expressed in COS-1 cells. The level of secreted mutant factor VII antigen was only 11.0% of the level of wild type factor VII. In CHO cells stably transfected with the mutant factor VII, only 37.3% of the total labeled FVII was secreted into the conditioned media and the remainder was retained inside the cells. These data suggest this mutation leads to factor VII deficiency due to the impaired secretion of the molecule.

SummaryCongenital deficiency and dysfunction of plasminogen (PLG) are associated with a mild thrombotic tendency. To facilitate the genetic diagnosis of dysPLGemia, we combined an amplification refractory mutation system and rapid automated capillary electrophoresis. Two different fluorescence-labeled PLG-specific primers for exon XV were designed so that each DNA amplified by PCR showed fluorescence of a different wavelength. Single peaks were detected for the normal and the mutant Ala601-Thr alleles, respectively. A study of 90 normal Caucasians revealed no individuals with the mutation, whereas its gene frequency was 0.021 in Japanese. This mutation was also detected in Korean and Chinese populations at gene frequencies of 0.016 and 0.015, respectively. All of the Korean and Chinese cases with the mutation had at least one haplotype I of the PLG gene, as did most Japanese cases. The high frequency of the Ala601-Thr mutation among these Asian populations may be due to the founder effect.

To explore the implications of the structure/ function relationships in factor XIII. a patient with severe A subunit deficiency was examined at the DNA and RNA levels. Nucleotide sequence analysis of the patient's DNA amplified by PCR revealed that the patient had a replacement of C by T in the codon for Arg260. RT-PCR analysis demonstrated that only one kind of mRNA coding for the Arg260-Cys mutation was expressed in the patient at a normal level. Another possible defective allele of the A subunit gene with a G-A polymorphism was not expressed (null allele). The substitution of Arg260 by Cys located on the interface of two A subunits would preclude the reciprocal ionic interaction (salt bridge) between Arg260 and Asp404. Molecular modelling and, for the first time, molecular mechanics calculated that Cys260 changed the local conformation of the A subunit and reduced the electrostatic interaction between two monomers, suggesting destabilization of the molecule's dimer.

SummaryWhen we compared nucleotide sequences of the 5’-flanking regions for plasminogen genes from 11 individuals, six substitutions were identified even among normal subjects. A new haplotype (termed allele II) was screened by PCR-RFLP analysis among 54 Japanese and 58 Caucasian normal subjects. The frequency of allele II was 0.787 in the Japanese and 0.560 in the Caucasians, indicating that the ratio of alleles differs between populations. Examination of 118 cases with dysplasminogenemia revealed that the Ala601-Thr mutation was present on allele I in most cases. This mutation was also associated with allele II in one-fourth of all cases, suggesting possible recombination within the plasminogen gene. Interestingly, we previously demonstrated that the expression activity of allele II was about 1.8 fold that of allele I in vitro.

SummaryGenetic analysis revealed two distinct novel splice site mutations in a compound heterozygous patient with protein S deficiency. The paternal mutation was a G-to-T transition at position -1 of the acceptor splice site of intron N (Mutation I), and the maternal mutation was a G-to-C transversion at position -1 of the donor splice site of intron C (Mutation II). Both splice site mutations decreased the mutated mRNA accumulation to the same extent, approximately 40% of the normal mRNA. However, the mutations were associated with different phenotypical expressions: the paternal mutant protein S was not detected in vivo, while the maternal mutant protein S was present in the plasma in reduced quantity. Because Mutation I caused a cryptic splicing in the mutated mRNA, resulting in a reading frameshift and premature termination, the predicted mutant protein S might be highly unstable. In contrast, Mutation II led to the substitution of Val46 by Leu, which might be much less deleterious for the synthesis, secretion and stability of the predicted mutant protein S. It was supposed that the different post-translational metabolisms produced the distinct phenotypical expressions of the mutations.

The molecular basis for a hereditary type I protein S (PS) deficiency was investigated. DNA sequence analysis in the proband showed a novel missense mutation substituting Cys (TGT) for Arg474 (CGT) that is a highly conserved amino acid residue among the related proteins. This missense mutation cosegregated with the type I PS deficiency in this family. Transient expression studies showed that the secretion of the recombinant Cys-mutant PS was markedly decreased compared with that of the recombinant wild-type PS, reproducing the observed phenotype of type I deficiency. Stable expression and pulse-chase experiments demonstrated an intracellular degradation and an impaired secretion of the recombinant Cys-mutant PS. Furthermore, the substitution of Arg474 by Ala or Glu, but not by Lys, markedly reduced the secretion of the recombinant PS mutants in transient expression studies, 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. We postulate that the loss of the highly conserved Arg474 might be responsible for the type I PS deficiency inherited in this family.

To better understand the regulation of ryudocan (syndecan-4) expression, we have determined the structural organization of the human ryudocan gene. The human ryudocan gene extends approximately 24 kilobases and is divided into five exons, which appear to be conserved in syndecan family members. Exon I encodes the signal peptide; exons II-IV, the extracellular domain; and exon V, the transmembrane and cytoplasmic domains, which are highly homologous among syndecan family members. Primer extension analysis showed that human ryudocan gene had a single transcription initiation site, located 3 bases upstream from the described cDNA [Kojima et al. (1993) BBRC 190, 814-822]. The 5'-flanking sequences of human ryudocan gene contain a TATA-like sequence as well as a variety of other potential binding sites for transcription factors, including Sp1, Ap-2, NF-kB, E-alpha H box, H4TF-2, and LBP-1, and were capable of functioning as a promoter. The determination of the human ryudocan gene structure will allow elucidation of constitutive, cell-specific, tissue-specific, and developmentally regulated expression.

SummaryBernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder which is caused by abnormal expression or function of the glycoprotein (GP) Ib/IX/V complex, a platelet major receptor for von Wille-brand factor. We studied four BSS patients in two unrelated families in which the same and novel mutation was found. Flow cytometric analysis showed that GPIX was completely absent but residual amounts of GPIbα and GPV were detectable in these patients. We analyzed all coding regions of GPIbα, GPIbβ, GPV and GPIX which were amplified from the patients’ genomic DNA by the polymerase chain reaction (PCR). In all four cases, we identified a point mutation in the GPIX coding region that changes the codon for cysteine 73 (TGT) to a codon for tyrosine (TAT). Furthermore, we confirmed by a transient expression study that the mutation caused the loss of adequate surface expression of GPIX. Since cysteine might be important for the secondary structure, this mutation of GPIX gene would lead to a dramatic conformational change of GPIX protein, resulting in the reduced surface expression. We concluded that this novel point mutation of the GPIX gene was responsible for BSS in these families

Advances in molecular biology have improved the screening for carriers and the prenatal diagnosis of many genetic disorders, including hemophilia B. For instance, based on the eight described dimorphic restriction fragment length polymorphisms within the factor IX gene, a Caucasian female has a 94% chance of being heterozygous (informative) for at least one of these markers. However, ethnic limitations of molecular genetic techniques have been found in diagnosing hemophilia B families in the Japanese and other populations. Previously, we heve demonstrated that three dinucleotide polymorphisms, which locate in the 5' flanking region at nucleotide-793 (FIX-793), in intron A at nucleotide 192 (FIX192), and in the 3' flanking region (FIXHhaI), of the factor IX gene, are present in normal Japanese. Each of these polymorphisms was able to be rapidly ascertained by the polymerase chain reaction technique.In this study, we analyzed 23 Japanese families with hemophilia B, and found that 19 families (82.6%) were heterozygous for at least one of these polymorphisms. Using these polymorphisms together with the extragenic DXS99/Sac I PFLP, which was previously reported as a useful gene marker for Japanese hemophilia B, the informative rate impproved to 87.0%. Carrier detection and the prenatal diagnosis of hemophilia B can be achieved effectively and rapidly in Japanese with these polymorphisms. In fact, we have successfully performed the prenatal diagnosis in two Japanese families with hemophilia B by using FIXHhaI or FIX192 polymorphism analysis. These polymorphisms may also be present and useful for hemophilia B carrier detection in other Oriental population.

SummaryThe genetic defect in a patient with hereditary type I protein S (PS) deficiency was investigated. All the exons and intron-exon junctions of the patient’s PS gene were amplified by PCR and subjected to heteroduplex screening. Only the PCR product of exon 4 revealed heteroduplex bands. A novel nonsense mutation, Ser62 (TCA) to Stop (TGA) was found in exon 4. RT-PCR detected the aberrant mRNA in the patient’s platelets, which was markedly reduced in amount and lacked the region of exon 4, suggesting that the nonsense mutation affected the mutated mRNA metabolism and induced exon skipping. The skipping of exon 4 causes an in-frame deletion of 29 amino acids which just construct the thrombin-sensitive region of the PS molecule. The loss of such an important domain as well as the quantitative decrease in the mutated mRNA appear to be responsible for the type I PS deficiency in this patient.

SummaryA 50-year-old Japanese man who had experienced recurrent episodes of venous thrombosis was found to have a hereditary protein S deficiency. The amount of total protein S antigen in plasma was reduced by approximately 50% in the patient and his two sons. DNA sequence analysis revealed a novel nonsense mutation, TAG for Gin 522 (CAG), in exon 14 of the protein S gene. Family studies performed by mutagenic PCR followed by restriction enzyme digestion showed that the proband and his two sons were heterozygous for this mutation. An mRNA-based analysis indicated that transcripts of the mutated allele were markedly reduced in the platelets of the affected individuals. Immunoblot analysis failed to detect the truncated mutant of protein S in the plasma or platelets of affected members. Our results demonstrated that this novel nonsense mutation was responsible for the quantitative deficiency of protein S.

During the course of structural gene analysis of a family with type III protein S deficiency, we found a novel DNA polymorphism: an A or G variation at nucleotide 732 in exon 6 of the PS-α gene. This A to G mutation would lead to a substitution of Lys155 by Glu in the second EGF domain. Linkage study with restriction enzyme analysis using mutagenic PCR strategy showed that the same mutation was also present in three other members of the patient's family and two individuals from an unrelated kindred, while they all had normal amounts of both immunological and functional PS levels. Restriction enzyme analysis of 182 normal Japanese genomic samples showed that 1.65% of normal population were heterozygotes for this variant allele. These findings suggest that this substitution in exon 6 is not responsible for the type III protein S deficiency but a phenotypically neutral polymorphism. Hereby we designate this polymorphism as PS-732.

Two photosensitive uranyl complexes have been prepared and examined by a single-crystal X-ray diffraction study. Both compounds crystallize in the monoclinic system. In bis(β-ethoxypropionato)dioxouranium(VI), uranyl ions are equatorially coordinated by six carboxylate oxygen atoms, forming one-dimensional infinite chains. In bis(ethoxyacetato)dioxouranium(VI), uranyl ions are equatorially coordinated by four carboxylate and one ethoxy oxygen atoms, forming two-dimensional network of uranyl-ligand infinite chains.

A number of arylbiurets were prepared and evaluated as antiinflammatory and analgesic agents by using the carrageenan paw edema and acetic acid stretching tests. Among them, the antiinflammatory activity of 1,3-dimethyl-5-phenylbiuret (7), l-ethyl-3-methyl-5-phenylbiuret (11), and 1,1,3-trimethyl-5-phenylbiuret (13) were found to be more potent than phenylbutazone. The analgesic activity of 7 and of 5-(4-chlorophenyl)-1,13-trimethylphenylbiuret (16) is higher than that of aminopyrine. Synthese, antiinflammatorische und analgetische Wirkungen von Arylbiuret-Derivaten Einige Arylbiuret-Derivate wurden synthetisch dargestellt und im Carrageenin-induzierten Rattenpfoten-Odem und Essigsaure-induzierten Writhing-Test bewertet. Die entzundungshemmende Wirkung von 1,3-Dimethyl-5-phenylbiuret (7), l-Ethyl-3-methyl-5-phenylbiuret (11) und 1,1,3-Trimethyl-5-phenylbiuret (13) war starker als die von Phenylbutazon. 7 und 5-(4-Chlorphenyl)-1,1,3-trimethylphenylbiuret (16) zeigten starkere analgetische Wirkung als Aminopyrin.

Bernard-Soulier syndrome (BSS) is a rare bleeding disorder characterized by giant platelets, thrombocytopenia, and prolonged bleeding time. It is caused by abnormalities in the glycoprotein (GP) Ib/IX/V complex, the receptor for von Willebrand factor (vWF). Most cases of BSS described so far involve quantitative rather than qualitative defects in the complex. In this study, we investigated the effects of two naturally occurring mutations in the GPIbbeta gene, C122S and 443delG, on the expression of the GPIb/IX complex identified in a variant type of BSS in which the platelets had severely reduced GPIbalpha ( approximately 10%) and less markedly reduced GPIbbeta and GPIX ( approximately 20%) expression. Immunoblot analysis showed the absence of non-reduced GPIb (GPIbalpha/GPIbbeta) in the patient's platelets. Transient transfection experiments in 293T cells revealed the expression of GPIbbeta Ser122 polypeptide and absence of GPIbbeta 443delG polypeptide. Although no disulfide-linked association was observed between GPIbbeta Ser122 and GPIbalpha, GPIbbeta Ser122 was non-covalently associated with both GPIbalpha and GPIX subunits on the cell surface when cotransfected with wild-type GPIbalpha and GPIX. Chinese hamster ovary cells stably expressing GPIbalpha/Ibbeta Ser122/IX had the ability to bind soluble vWF and to aggregate in the presence of ristocetin. These results suggest that despite disruption of the disulfide linkage between GPIbalpha and GPIbbeta, GPIb/IX is formed, but its stability may be impaired, resulting in low levels of the complex on the platelet membranes.

Eight distinct and potentially causative mutations were identified in eight unrelated Japanese patients with protein S (PS) deficiency, by direct DNA sequencing of the protein Salpha (PSalpha) gene-specific polymerase chain reaction products of all 15 exons and exon/intron boundaries. There were five missense mutations, including two novel mutations (Cys80Tyr and Arg314His), and three showed a major impact on the expected gene products: novel mutations of a 5-bp deletion (delCTCTG887:Cys206Stop) and a nonsense mutation (Glu208Stop), as well as a previously reported splice site (exon 10 +5 A--G) mutation. One of the patients showed compound heterozygosity for delCTCTG887 and 732A--G. Investigation for the cosegregation state of these two mutations with PS deficiency in the patient's family suggested that the delCTCTG887 mutation was responsible for the abnormal phenotype and that the 732A--G (Lys155Glu) mutation did not appear to play a key role. However, we also identified the same 732A--G (Lys155Glu) mutation in an unrelated patient with apparent PS deficiency with severe pulmonary embolism, and found that this mutation seemed to cosegregate with a PS-deficient state in her family members. These data implied that unknown factor(s) other than the 732A--G mutation itself might influence phenotypic expression of PS status in different individuals.

Activated coagulation factor V (FVa) is a cofactor of activated factor X (FXa) in prothrombin activation. FVa is composed of a light chain (LC) and a heavy chain (HC) that are noncovalently associated in a calcium-dependent manner. We constructed a recombinant FV Asp111Asn/Asp112Asn mutant (rFV-NN) to abolish calcium binding to a potential calcium-binding site in FVa in order to study the specific role of these residues in the expression of FVa activity. Whereas thrombin-activated recombinant FV wild type (rFV-wt) presented with stable FVa activity, incubation of rFV-NN with thrombin resulted in a temporary increase in FVa activity, which was rapidly lost upon prolonged incubation. Loss of FVa activity was most likely due to dissociation of HC and LC since, upon chromatography of rFVa-NN on a SP-Sepharose column, the HC did not bind significantly to the resin whereas the LC bound and could be eluted at high ionic strength. In contrast, rFVa-wt adhered to the column, and both the HC and LC coeluted at high ionic strength. In the presence of phospholipid vesicles, the loss of rFVa-NN activity was partially prevented by FXa, active site inhibited FXa, and prothombin in a dose-dependent manner. We conclude that the introduced amino acid substitutions result in a loss of the high-affinity (calcium-dependent) interaction of the HC and LC of FVa. We propose that the introduced substitutions disrupt the calcium-binding site in FV, thereby yielding a FV molecule that rapidly loses activity following thrombin-catalyzed activation most likely via dissociation of the HC and LC.

The D2194G mutation is responsible for increased levels of FV1 in carriers of the factor V R2 haplotype

We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.

SummaryWe have identified, in two unrelated patients, factor X deficiency that we have designated factor X Nagoya 1 and Nagoya 2, respectively. The proband with factor X Nagoya 1 showed factor X activity level of 3% and factor X antigen level

Ryudocan, a ubiquitous heparan sulfate proteoglycan, is a member of the syndecan family of cell surface proteoglycans. The full-length cDNA encoding the murine ryudocan core protein has now been cloned and sequenced. The deduced primary structure of mouse ryudocan, including the three glycosaminoglycan attachment sites in the extracellular domain as well as the transmembrane and cytoplasmic regions, is highly similar to those of the rat, human, and chicken proteins. Northern analysis detected a 2.7-kb transcript in all mouse tissues examined, with the highest concentrations apparent in liver, kidney, and lung. The mouse ryudocan gene was shown to span approximately 19.7 kb of genomic DNA and to contain five exons, with an intron-exon organization identical to that of the human gene. The promoter region of the mouse gene contains various cis-acting elements, including a TATA-like box and a GC box as well as potential binding sites for the transcription factors NF-IL6, MyoD, GATA, C/EBP, AP-2, NF-kappaB, AP-1, and Sp1. Transient transfection experiments with a construct containing the 690 bp upstream of the transcription start site fused to a luciferase reporter gene showed functional promoter activity. Deletion analysis suggested that the proximal promoter region including the TATA-like box, the GC box, and other Sp1 binding sites was required for full transcriptional activity. These findings will be useful for the study of ryudocan gene regulation and the generation of mice with targeted disruption of the gene.

Protein C Nagoya, an elongated variant of the human protein C, is retained and degraded within the cells in which it is produced (Yamamoto et al, J Clin Invest 90:2439, 1992). To determine the subcellular localization of the protein C Nagoya, the recombinant protein C bearing this mutation was expressed in Chinese hamster ovary (CHO) cells. The mutant protein C was not secreted from the cells and remained susceptible to endo-beta-N-acetylglucosaminidase H (endo H). Immunoelectron microscopy indicated that protein C Nagoya was retained in the endoplasmic reticulum (ER), whereas wild-type protein C was observed in both the ER and the Golgi apparatus. Metabolic radiolabeling with [35S] methionine in combination with chemical cross- linking showed that the protein C Nagoya existed in the ER as a complex with 78-kD glucose-regulated protein (GRP78) and 94-kD glucose- regulated protein (GRP94). Because both GRP78 and GRP94 associate to a far lesser degree with wild-type protein C than with protein C Nagoya, our data suggest that both stress proteins function as molecular chaperones and work in concert with the folding and assembly of protein C. These findings extend our understanding the molecular pathogenesis of protein C deficiency.

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.

SummaryType IIA von Willebrand disease (vWD) is the most common type II vWD and is characterized by the selective loss of large and intermediate sized multimers. One explanation for this disorder has been postulated to be a qualitative defect in von Willebrand factor (vWF) which results in increased susceptibility to proteolysis at the bond between residues Tyr842 and Met843. Four missense mutations that may cause type IIA vWD have recently been identified near the cleavage site. We analyzed the molecular basis for type IIA vWD in six patients. A 512 bp DNA sequence spanning the proteolytic cleavage site was targeted for PCR amplification and sequencing. We exploited a difference in restriction sites between the vWF gene and the pseudogene and have designed allele-specific oligomer used with PCR to distinguish these two genes. Three candidate missense mutations; Ser743 (TCG) → Leu (TTG), Leu799 (CTG) → Pro (CCG), and Arg834 (CGG) → Trp (TGG) were identified in 4 out of 6 patients. The amino acid substitution at Arg834 has been reported previously, but the other substitutions at Ser743 and Leu799 are novel candidate mutations locating 99 and 43 amino acids to the N-terminal side of the cleavage site, respectively. Our results indicate that amino acid substitutions located relatively distant from the cleavage site may also be involved in type IIA vWD.

A number of arylbiurets were prepared and evaluated as antiinflammatory and analgesic agents by using the carrageenan paw edema and acetic acid stretching tests. Among them, the antiinflammatory activity of 1,3-dimethyl-5-phenylbiuret (7), l-ethyl-3-methyl-5-phenylbiuret (11), and 1,1,3-trimethyl-5-phenylbiuret (13) were found to be more potent than phenylbutazone. The analgesic activity of 7 and of 5-(4-chlorophenyl)-1,13-trimethylphenylbiuret (16) is higher than that of aminopyrine. Synthese, antiinflammatorische und analgetische Wirkungen von Arylbiuret-Derivaten Einige Arylbiuret-Derivate wurden synthetisch dargestellt und im Carrageenin-induzierten Rattenpfoten-Odem und Essigsaure-induzierten Writhing-Test bewertet. Die entzundungshemmende Wirkung von 1,3-Dimethyl-5-phenylbiuret (7), l-Ethyl-3-methyl-5-phenylbiuret (11) und 1,1,3-Trimethyl-5-phenylbiuret (13) war starker als die von Phenylbutazon. 7 und 5-(4-Chlorphenyl)-1,1,3-trimethylphenylbiuret (16) zeigten starkere analgetische Wirkung als Aminopyrin.

We genetically screened patients with hereditary protein S deficiency for heterozygosity of protein S mRNA using PCR-RFLP for Pro626 polymorphism. All patients who showed hemizygous state of protein S mRNA, characterized by markedly decreased levels of one allele, had a phenotype of type I protein S deficiency. A putative mutation, such as a nonsense or splice site mutation, in the silent alleles may have affected the mutated mRNA metabolism and reduced the mutated mRNA accumulation, and consequently resulted in type I protein S deficiency in these patients. We also applied this mRNA-based analysis to family studies in hereditary protein S deficiency. In a family with type I protein S deficiency, all affected individuals showed a loss of one allele at the mRNA level and the silent allele cosegregated with the disease phenotype. Detection of hemizygous expression of protein S mRNA provided direct evidence for type I protein S deficiency without further precise genetic analysis. Our findings indicate that this mRNA-based analysis can be a useful strategy for genetic screening and family studies in hereditary protein S deficiency.

Benzhydryl 2 beta-[(1,2,3-triazol-1-yl)methyl]-2 alpha-methylpenam- 3 alpha-carboxylate 1,1-dioxide was prepared by heating benzhydryl 2 beta-(azidomethyl)-2 alpha-methylpenam-3 alpha-carboxylate 1,1-dioxide with (trimethylsilyl)acetylene. The ester group was removed by hydrogenolysis to give sodium 2 beta-[(1,2,3-triazol-1-yl)methyl]-2 alpha-methylpenam-3 alpha-carboxylate 1,1-dioxide (3i, YTR-830), which was found to be a potent inhibitor of various bacterial beta-lactamases. A series of related compounds was prepared in a similar way, and all of these compounds show excellent beta-lactamase inhibitory properties.

The thermolysis of trichloroethyl 6-phenoxyacetamido penicillanate sulfoxide and ethyl 2-mercaptoacetate in toluene gave the unsym-azetidinone disulfide(β,γ-isomer) in 65% yield. With added catalytic amounts of N,N-dimethylaniline the α,β-isomer was the major product (60%), along with a new β-lactam cleaved product, 8 (5–6%).

Etude de la reduction deshalogenante des halogeno-6- et dihalogeno-6,6 penicillanates d'alkyle: selon le reactif, les derives bromes conduisent aux penicillanates d'alkyle (A) et/ou aux halogeno-6 dimethyl-2,2 oxo-7 tetrahydro-2,3,4,7 thiazepine-1,4 carboxylates-3 (B); l'oxydation du sulfure en sulfoxyde ou sulfone stabilise le systeme A et previent la transposition en B; les derives diiodes conduisent principalement aux composes B

2,5-Dimercapto-1,3,4-thiadiazole and 2,4-dimercaptopyrimidine were used to trap sulfenic acids from penicillin sulfoxides.

2-Aklylidene-4-arylidene-1, 3-oxathiolan-5-one (III-1a-m) and 2, 4-diarylidene-1, 3-oxathiolan-5-one (III-2a-i) derivatives were synthesized by treating β-aryl-α-mercaptoacrylic acids (I) with alkanoic acid anhydrides (II) or by treating α-acylthio-β-arylacrylic acids (V) with thionyl chloride in dimethylformamide. Basic hydrolysis and methanolysis of III-1 and III-2 in the presence of lithium hydroxide easily occurred to give the corresponding ring-cleaved products, the carboxylic acid (I and II) and the ester (VII and VIII), respectively. The catalytic hydrogenation of the two olefinic bonds of III-2 in the presence of 10% palladium charcoal proceeded easily without ring cleavage to give 1, 3-oxathiolan-5-one (IXa-e) derivatives. The oxidation of III-1 and III-2 with m-chloroperbenzoic acid afforded the corresponding 1, 3-oxathiolan-5-one S-oxide (Xa, b) derivatives.

La decomposition thermique d'oxydes-1 de penicillines conduit a des oxo-2 sulfeno-4 azetidineacetates-1; en presence de derives du benzenethiols, on obtient des aryldithio-2 α-isopropylidene oxo-4 azetidineacetates-1

2-Aklylidene-4-arylidene-1, 3-oxathiolan-5-one (III-1a-m) and 2, 4-diarylidene-1, 3-oxathiolan-5-one (III-2a-i) derivatives were synthesized by treating β-aryl-α-mercaptoacrylic acids (I) with alkanoic acid anhydrides (II) or by treating α-acylthio-β-arylacrylic acids (V) with thionyl chloride in dimethylformamide. Basic hydrolysis and methanolysis of III-1 and III-2 in the presence of lithium hydroxide easily occurred to give the corresponding ring-cleaved products, the carboxylic acid (I and II) and the ester (VII and VIII), respectively. The catalytic hydrogenation of the two olefinic bonds of III-2 in the presence of 10% palladium charcoal proceeded easily without ring cleavage to give 1, 3-oxathiolan-5-one (IXa-e) derivatives. The oxidation of III-1 and III-2 with m-chloroperbenzoic acid afforded the corresponding 1, 3-oxathiolan-5-one S-oxide (Xa, b) derivatives.