Molecular mechanism of type I congenital heparin cofactor (HC) II deficiency caused by a missense mutation at reactive P2 site: HC II Tokushima.

We found a 66-year-old Japanese patient with type I congenital heparin cofactor (HC) II deficiency manifesting multiple atherosclerotic lesions. To investigate the molecular pathogenesis of our patient, we performed sequencing analysis and expressed recombinant human wild-type and mutant HC II molecules in COS-1 and CHO-K1 cells. Sequencing analysis following amplification of each of all 5 exons and its flanking region showed a single C to T transition at nucleotide position 12,854 in exon 5, which changed a Pro443 codon (CCG) to Leu codon (CTG). Because this mutation generates a new Bhv I site, the Bbv I digestion pattern of the PCR-amplified exon 5 fragments from each family member was analyzed. In all cases, the patterns were consistent with the activities and antigen levels of plasma HC I1 in those members. Transient transfection, metabolic labeling and pulse-chase experiments followed by immunoprecipitation analysis showed that the recombinant mutant HC II molecules were secreted from COS-1 cells in reduced amounts compared with the wild-type, and that an enhanced intracellular association of the mutant molecules with a chaperone, GRP78/BiP, was observed in CHO-K1 cells. Northern blot analysis indicated that the mutant HC I1 mRNA was transcribed at a similar level as that of wild-type. Immunohistochemical staining of the transfected cells revealed that COS-1 cells expressing the mutant HC II molecules were stained mainly in the perinuclear area. We conclude that the impaired secretion of the mutant HC II molecules, due to intracellular degradation, is the molecular pathogenesis of type I congenital HC II deficiency caused by a Pro443 to Leu mutation at reactive P2 site.

Histidine-rich glycoprotein (HRG) Tokushima 2: novel HRG deficiency, molecular and cellular characterization.

The proband, a 76-year-old woman, suffered from dural arteriovenous fistula. Her plasma histidine-rich glycoprotein (HRG) level was 50% of the normal level. A low level of plasma HRG was also found in her third daughter. A single nucleotide substitution of T to C was found at nucleotide position 11,438 in exon 6 of the HRG gene from the proband, converting Cys223 to Arg in the second cystatin-like domain. The same mutation was also identified in her third daughter, but not in the other four family members having normal HRG levels or in 50 unrelated healthy Japanese individuals. Expression studies in BHK cells showed that substantial intracellular degradation of the mutant occurred and only about 40% of the recombinant HRG mutant was secreted. These results indicate that congenital HRG deficiency caused by a substitution of Cys223 to Arg is hereditary in this family.

No increased risk of thrombosis in heterozygous congenital dysplasminogenemia.

To assess the risk of thrombosis in congenital dysplasminogenemia, we studied 10 unrelated families with this disorder. The probands were excluded from the analysis of data to prevent bias in the selection of subjects. Positive thrombotic histories were found in 1 of the 25 family members determined to have heterozygous congenital dysplasminogenemia and in 2 of their 41 biochemically unaffected relatives. The percentages of family members with no history of thrombosis up to a given age among subjects with and without congenital dysplasminogenemia were analyzed by the Kaplan-Meier method. No significant difference between the 2 groups was observed by generalized Wilcoxon test (P = .32) or Cox-Mantel test (P = .62). These findings suggest that heterozygous congenital dysplasminogenemia is not associated with an increased risk of thrombosis.

Intracellular degradation of histidine-rich glycoprotein mutants: tokushima-1 and 2 mutants are degraded by different proteolytic systems.

We reported the first case of a congenital histidine-rich glycoprotein deficiency (HRG Tokushima) in which substitution of Gly85 with Glu (G85E) in the first cystatin domain resulted in intracellular degradation and a low plasma level of HRG [Shigekiyo, T. et al. (1998) Blood 91, 128-133]. Recently, we identified the gene mutation of a second case of HRG deficiency as a Cys223 to Arg (C223R) mutation in the second cystatin domain. To investigate the molecular and cellular bases of these deficiencies, we expressed these HRG mutants in baby hamster kidney (BHK) cells. Pulse-chase experiments in the absence and presence of various proteinase inhibitors revealed that, while wild-type HRG was completely secreted during 4-h chase periods, both the G85E and C223R mutants were only partially secreted and primarily degraded within the cells. The intracellular degradation of the C223R mutant was almost completely inhibited in the presence of a proteasome inhibitor, lactacystin, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, resulting in increased secretion of the C223R mutant, and thus implicating the proteasome system in this degradation process. In contrast, the sum of the amounts of the G85E mutant inside and outside the cells decreased during the chase periods even in the presence of the proteasome inhibitor, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, although proteasome-specific inhibitor lactacystin and one of the cysteine protease inhibitors, E-64-d, prevented the intracellular degradation. These results suggested that intracellular degradation of G85E HRG occurred to some extent through a hitherto unknown mechanism. Similar studies involving recombinant mutants in which Gly85 or Cys223 was replaced with several other amino acids revealed that proteins with mutations leading to the destruction of the predicted b-sheet structure of the cystatin domains were eliminated by the intracellular quality control system.

A patient with cyclic neutropenia complicated by severe persistent neutropenia successfully delivered a healthy baby.

We describe a 24-year-old pregnant woman complicated by cyclic neutropenia (CN), who was successfully treated with granulocyte-colony stimulating factor (G-CSF). Her white blood cell (WBC) and neutrophil count fluctuated from 2,600 to 4,600/microl, and 26 to 2,530/microl, respectively. The peak neutrophil count gradually decreased as pregnancy advanced, resulting in the disappearance of its cyclicity. At 39 weeks of pregnancy when the neutrophil count became 84/microl, the patient was started on G-CSF and her neutrophil count increased to 1,550/microl on the fourth day after delivery. She delivered a healthy baby without any complications at 39 weeks of pregnancy.

HRG Tokushima: molecular and cellular characterization of histidine-rich glycoprotein (HRG) deficiency.

Previously, we found the first congenital deficiency of histidine-rich glycoprotein (HRG) in a Japanese woman with thrombosis. To elucidate the genetic basis of this deficiency, we first performed Southern blot analysis and found no gross deletion or insertion in the proband's HRG gene. We then examined the nucleotide sequences of all seven exons of the proband's HRG gene. A single nucleotide substitution, G to A at nucleotide position 429, which mutates Gly85 to Glu in the first cystatin-like domain, was found in exon 3 in 13 of 22 amplified clones. This mutation generates a unique Taq I site. Exon 3 was amplified from the proband, her family members, and 50 unrelated normal Japanese individuals, and Taq I fragmentation was examined. Fragmentation of exon 3 was observed in one allele of the genes from the proband and the family members who also have decreased plasma levels of HRG. Fifty unrelated normal Japanese individuals had a normal HRG gene, indicating that the G to A mutation is not a common polymorphism. To elucidate the identified mutation as a cause for the secretion defect of HRG in the proband's plasma, we constructed and transiently expressed the recombinant Tokushima-type HRG mutant (Gly85 to Glu) in baby hamster kidney (BHK) cells, and examined an intracellular event of the mutant protein. The results showed that only about 20% of the Tokushima-type HRG was secreted into the culture medium, and intracellular degradation of the mutant was observed. Thus, the present study strongly suggests that the HRG deficiency is caused by intracellular degradation of the Gly85 to Glu mutant of HRG in the proband.

Interferon alpha-2a therapy for disseminated intravascular coagulation in a patient with blue rubber bleb nevus syndrome. A case report.

The authors present a sixteen-year-old girl with blue rubber bleb nevus syndrome (BRBNS) associated with disseminated hemangiomas involving the skin, oral cavity, skeletal muscle, and cerebrum. Although she denied neurologic symptoms, magnetic resonance imaging of the brain demonstrated dilatated cerebral veins and the Chiari I malformation. Examination of hemostasis revealed disseminated intravascular coagulation (DIC) manifesting as Kasabach-Merritt syndrome, with the potential for life-threatening bleeding or thrombosis in the central nervous system. Since successful management of life-threatening hemangiomas with interferon alpha-2a (IFN alpha-2a) has been reported, the authors administered IFN alpha-2a with an improvement in hemostasis. These findings suggest that IFN alpha-2a therapy is beneficial for relieving the life-threatening consumptive coagulopathy associated with BRBNS.

A novel missense mutation in the endoglin gene in hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystem vascular dysplasia and recurrent hemorrhage. Recent investigation has mapped one of the responsible genes for HHT to chromosome 9q33-q34; subsequently, nine different mutations have been identified in the endoglin gene, which encodes a transforming growth factor beta (TGF-beta) binding protein, in nine unrelated families with HHT. We examined the endoglin gene in a Japanese patient with HHT and her family members. Using PCR-SSCP analysis followed by sequencing, we identified a C to A missense mutation in exon 4 which changed an Ala160 codon(GCT) to an Asp160 codon (GAT). Since this mutation destroys one of three Fnu4H 1 sites in exon 4, the Fnu4H I digestion patterns of the PCR-amplified exon 4 fragments from each family member were analyzed. In affected members, the restriction patterns were all consistent with a phenotype of HHT. PCR-amplified exon 4 fragments from 150 normal individuals were also analyzed by allele-specific oligonucleotide hybridization analysis. As a result, the mutation was not found in any of them. We conclude that the C to A mutation in exon 4 of the endoglin gene in this proband is responsible for the occurrence of HHT in this family.

Molecular pathogenesis of type I congenital plasminogen deficiency: expression of recombinant human mutant plasminogens in mammalian cells.

We previously reported the genetic abnormality in a Japanese family with type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation. To characterize the molecular pathogenesis of the disease in this family, we expressed recombinant human wild-type and mutant (rS572P) plasminogens in COS-1 cells. Activation-resistant wild-type and mutant plasminogen stable transfectants in CHO-K1 cells also were established. Transient transfection and metabolic labeling experiments followed by immunoprecipitation analysis showed that the mutant plasminogen was secreted from COS-1 cells in reduced amounts, compared with the wild type. Endo H digestion of the wild-type and mutant plasminogen showed no shift in their migrations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, indicating that both contain complex type oligosaccharide structures and could therefore be secreted. Furthermore, the secretion of activation-resistant mutant plasminogen was significantly reduced. Pulse-chase experiments and Northern blot analysis showed that the impaired secretion of the mutant plasminogen was the consequence of the accumulation of the mutant protein inside the cells but not of reduced plasminogen mRNA. Immunocytochemical staining of stable transfectants also revealed that CHO-K1 cells expressing the activation-resistant mutant plasminogen stained mainly in the perinuclear area, suggesting delayed processing of the mutant protein in the intracellular transport pathway. We conclude that the impaired secretion of mutant plasminogen, due to intracellular accumulation, is the molecular pathogenesis of type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation.

A case of dermatomyositis complicated by thrombotic thrombocytopenic purpura.

A 60-year-old man with dermatomyositis was admitted to our hospital because of dyspnea and hypertension. He had high fever and convulsive seizures after admission. Laboratory examinations showed hemolytic anemia, thrombocytopenia, and renal failure. A clinical diagnosis of thrombotic thrombocytopenic purpura (TTP) was made. He failed to respond to plasma exchange therapy, pulse therapy with methylprednisolone, high-dose gamma-globulin therapy, and antiplatelet therapies with ticlopidine, dipyridamole and a prostacyclin analog of beraprost sodium. He died on his 17th day in hospital. Autopsy examination revealed widespread microthrombi in his kidneys, lungs, spleen, and intestine. Only seven cases of dermatomyositis or polymyositis complicated by TTP have been cited in the literature. TTP was fatal in 6 of these 7 cases. Early diagnosis and prompt treatment may improve the outcome of TTP patients with dermatomyositis. Dermatologists should keep in mind that TTP occasionally arises as a serious complication of dermatomyositis.

An in vitro system for identifying agents capable of changing serum lipoprotein(a) concentration by regulating the transcriptional activity of the apolipoprotein(a) gene promoter.

A high serum concentration of lipoprotein(a) [Lp(a)] is a significant and independent risk factor for cardiovascular disease. We examined the effects of agents on the transcriptional activity of the apolipoprotein(a) [apo(a)] gene promoter and determined whether drugs identified by this assay would affect the serum concentration of Lp(a) in vivo. All-trans-retinoic acid (ATRA) and interleukin-6 increased the transcriptional activity of the apo(a) gene promoter 2.1- and 2.5-fold, respectively, whereas danazol reduced activity to 76% of the control value. Triiodothyronine had no effect on transcriptional activity. Treatment of two acute promyelocytic leukemia patients with ATRA induced maximal 2.7- and 3.2-fold increases in serum Lp(a) concentrations, respectively. Thus, the in vitro luciferase assay system is capable of identifying agents that affect the serum concentration of Lp(a) and thus may prove beneficial in the screening of new drugs for treatment of individuals with high serum Lp(a) concentrations.

Increase in plasma thrombomodulin level in patients with vibration syndrome.

To determine whether endothelial cells are injured in vibration syndrome, we measured plasma levels of thrombomodulin (TM) in 100 patients with this syndrome using one-step sandwich enzyme immunoassay. Plasma level of TM in patients with vibration syndrome was significantly higher than that in normal control (p < 0.0001). There was no significant difference in the plasma TM level between patients with vibration syndrome and those with collagen disease. Plasma TM concentration in chain-saw operators was significantly higher than that in rock-drill operators (p < 0.05). Plasma TM value did not significantly differ between patients with vibration-induced white finger (VWF) and those without VWF. These results suggest that endothelial injury is present in patients with vibration syndrome, the degree of endothelial injury in patients with vibration syndrome equals that in patients with collagen disease, and the endothelial injury in chain-saw operators is greater than that in rock-drill operators. However, there was no difference in the degree of endothelial injury between patients with VWF and those without VWF.

Takayasu's arteritis associated with antiphospholipid antibodies. Report of two cases.

The authors describe 2 patients with Takayasu's arteritis in whom lupus anticoagulant was positive and the titer of anticardiolipin antibody was elevated. One patient developed diffusely stenotic and occlusive changes in the multiple larger arteries. Histology of the small-sized arteries in another patient showed occlusive vasculitis without thrombosis, in addition to the findings in large-sized arteries compatible with Takayasu's disease. These findings are uncommon in Takayasu's arteritis. These findings suggest that antiphospholipid antibodies may have contributed to the pathogenesis of the extensive vasculopathy and may have triggered vasculitis in these patients.

A novel missense mutation in two families with congenital plasminogen deficiency: identification of an Ala675 to Thr675 substitution.

We used a polymerase chain reaction (PCR) strategy and restriction fragment polymorphism analysis to evaluate all 19 exons of the plasminogen (PLG) gene in a Japanese patient with congenital PLG deficiency and her family members (family C). Sequence analysis following amplification of each exon and its flanking regions showed a single G to A transition in exon 17, resulting in the conversion of an Ala675 codon (GCT) to Thr675 codon (ACT). Since this mutation generates a new Mae III site, the Mae III digestion patterns of the PCR-amplified exon 17 fragments from each family member were analyzed. In all cases, the patterns correlated with the activities and antigen levels of plasma PLG in those members. The identical G to A transition in the same codon of exon 17 was detected by a Mae III digestion experiment in another proband and her family members with congenital PLG deficiency (family K). Furthermore, 20 normal individuals examined had no Mae III restriction site at this location. We conclude that a G to A transition in exon 17 is responsible for the congenital PLG deficiency inherited in these two Japanese families.

[Isolated iliac artery aneurysms associated with disseminated intravascular coagulation (DIC)].

An 84-year-old man was referred to our hospital on December 3, 1993, because of excessive bleeding after tooth extraction. Coagulation studies revealed thrombocytopenia (2.2 x 10(4)/microliter), increased serum levels of FDP-E (3172 ng/ml) and D dimer (42.6 micrograms/ml), and increased plasma levels of thrombin-atithrombin III complex (48.0 ng/ml) and plasmin-alpha 2-plasmin inhibitor complex (6.8 micrograms/ml). Computed tomography showed bilateral common iliac artery aneurysms. A diagnosis of DIC due to bilateral common iliac artery aneurysms was made. Heparin (10,000 U/day) administered to treat DIC resulted in good control, and operative repair of the aneurysms was successful. This is the second reported case of isolated iliac artery aneurysms associated with DIC.

Congenital deficiency of histidine-rich glycoprotein: failure to identify abnormalities in routine laboratory assays of hemostatic function, immunologic function, and trace elements.

Histidine-rich glycoprotein (HRGP) has many biologic activities, but its physiologic function is still unclear. To show the physiologic function of HRGP, we studied five patients with congenital HRGP deficiency. Hemostatic screening tests, activities of natural anticoagulants and fibrinolytic proteins, markers of thrombin and plasmin generation, plasma levels of platelet-specific proteins, thrombin times with various concentrations of bovine thrombin, prolongation of thrombin time after addition of heparin or demartan sulfate, and contact activation of blood coagulation were normal or nearly normal in these patients. Serum concentrations of immunoglobulin, functional activity of the classical and the alternative pathway of complement, lymphocyte subsets, and serum concentrations of soluble interleukin-2 receptor were approximately normal in all patients, and serum concentrations of copper and zinc were completely normal. These results suggest that the physiologic functions of HRGP are limited when compared with its biologic activities. However, because the patients examined had plasma HRGP levels of 20% to 35% of normal, it is possible that 20% of normal HRGP level is sufficient for its physiologic functions.