A novel splice site mutation in intron C of PROS1 leads to markedly reduced mutant mRNA level, absence of thrombin-sensitive region, and impaired secretion and cofactor activity of mutant protein S.

Protein S (PS) is a member of the vitamin K-dependent protein family containing similar gamma-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 gamma-carboxylation to express APC cofactor activity.

Establishment of a stroma-dependent human acute myelomonocytic leukemia cell line, NAMO-2, with FLT3 tandem duplication.

We have established a stroma-dependent myelomonocytic cell line, NAMO-2, with FLT3 internal tandem duplication (FLT3/ITD). Leukemia cells from a patient with acute myelomonocytic leukemia were administered to form subcutaneous tumors in nude mice, which were maintained successively, although we failed to establish continuously growing cells from the original leukemia cell culture. In the cultures of cells from subcutaneous tumors, there were stroma cells that had originated from the nude mice and showed continuous growth. The leukemia cells showed continuous growth dependent on this stroma, and this cell line was named NAMO-2. Detection of FLT3/ITD by the reverse transcriptase polymerase chain reaction (PCR) and genomic PCR showed that NAMO-2 was homozygous for FLT3/ITD. Constitutive activation of FLT3 was detected by Western blotting, and the phosphorylation of Akt, MEK, and STAT5 was also observed. FLT3 kinase inhibitor AG1296 specifically inhibited cell growth. NAMO-2 provides a useful tool to analyze adherence-dependent survival signaling of leukemia with FLT3/ITD and a model for the screening of FLT3 kinase inhibitors.

Molecular genetic analysis of a variant Bernard-Soulier syndrome due to compound heterozygosity for two novel glycoprotein Ibbeta mutations.

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.

Identification of protein Salpha gene mutations including four novel mutations in eight unrelated patients with protein S deficiency.

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.

Functional properties of recombinant factor V mutated in a potential calcium-binding site.

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.

Factor V New Brunswick: Ala221Val associated with FV deficiency reproduced in vitro and functionally characterized.

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 degrees 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.

Defining the factor Xa-binding site on factor Va by site-directed glycosylation.

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.

Molecular basis of quantitative factor V deficiency associated with factor V R2 haplotype.

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.

Structural requirements of anticoagulant protein S for its binding to the complement regulator C4b-binding protein.

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.