A monoclonal antibody-based enzyme immunoassay for human GMP-140/P-selectin.

Two hybridoma cell lines producing monoclonal antibodies WGA-1 and PL7-6, reactive only with thrombin-stimulated human platelet have been established. Both these antibodies were investigated for their specific reactivity against GMP-140, based on the amino acid composition analysis of immunopurified antigen and N terminal amino acid sequencing of its protease fragments. A two-site enzyme immunoassay for quantification of human GMP-140 was developed using WGA-1 monoclonal antibody immobilized on 96-well microplates and horseradish peroxidase-labeled PL7-6 monoclonal antibody as detector. The assay was able to measure GMP-140 in serum and plasma with a sensitivity of about 5 ng/ml and a precision better than 10%. This assay will be useful for the detection of GMP-140 derived from platelets or endothelium in biological fluids and tissue extracts.

A Glanzmann thrombasthenia-like phenotype caused by a defect in inside-out signaling through the integrin alpha(IIb)beta3.

Activation of the platelet integrin alpha(IIb)beta3, an essential step in platelet aggregation, is regulated by intracellular signal pathways (inside-out signaling). In this study, we characterize a 35-year-old Japanese female, HM, with a life-long history of mucocutaneous bleeding. HM showed a Glanzmann thrombasthenia-like phenotype with normal expression of alpha(IIb)beta3, and failure of platelet aggregation induced by various agonists. An activation-independent ligand mimic monoclonal antibody (mAb), OP-G2, and RGDS peptides bound normally to the patient's alpha(IIb)beta3, while an activating anti-beta3 mAb, AP5, induced normal aggregation of HM platelets. The nucleotide sequence of the entire coding region of the patient's alphaIIb and beta3, including the cytoplasmic domains of each subunit, revealed no abnormalities. Agonist-induced phosphorylation of platelet pleckstrin and myosin light chain was not impaired. Recently, we proposed that a Na+/Ca2+ exchanger is involved in inside-out signaling, especially in the case of chymotrypsin-induced alpha(IIb)beta3 activation (Blood 88: 2594, 1996). However, chymotrypsin-induced platelet aggregation occurred normally in patient HM. Measurement of changes in cytosolic free calcium concentration ([Ca2+]i) revealed that the plateau level of [Ca2+]i after thrombin stimulation was significantly inhibited in patient HM. Our data suggest that patient HM exhibits a Glanzmann thrombasthenia-like phenotype associated with an abnormality in inside-out signaling which would otherwise activate alpha(IIb)beta3.

Characterization of cDNA encoding full-length mouse platelet glycoprotein IX.

Glycoprotein (GP) IX is a platelet membrane 20 kD protein, which is associated with GPIbalpha, GPIb beta, and GPV to form GPIb/IX/V complex. GPIb/IX/V complex is a major receptor for von Willebrand factor, which mediates platelet adhesion and aggregation under high shear stress conditions. The relevance of this receptor for hemostasis has been implicated by a congenital bleeding disorder lacking the receptor, Bernard-Soulier syndrome. All subunits for the human receptor have been cloned and characterized. However, the function of GPIX is still elusive. To obtain further information of GPIX, we have determined a cDNA sequence of mouse GPIX (811 bp). The deduced amino-acid sequence (177aa) was 71% identical to the human GPIX protein. All cysteine residues in extracytoplasmic domain and putative N-linked glycosylation site (Asn44) were conserved. Mouse GPIX contained a leucine-rich glycoprotein sequence composed of 24 amino acids, as did human GPIX.

A normative, community-based study of Mini-Mental State in elderly adults: the effect of age and educational level.

We investigated community-based data of the Mini-Mental State Examination (MMSE) scores of elderly residents along with the effects of age and educational level. MMSE was planned for all residents over 65 years of age in a town in northern Japan. The number of elders who took the MMSE was 2,266 (90%). The score significantly declined with age and lower educational level, although no effect of sex was apparent. For the MMSE subitems, all the values except for that of naming showed effects of both age and educational level. Those screened by MMSE who fell in the range of cognitive impairment (< 24) accounted for 21.8% and those with severe cognitive impairment (< 18) constituted 6.0%. Despite the differences in language and culture, the mean scores are remarkably similar between Japan and other countries. This is the first normative, community-based study of MMSE among elderly adults in Japan.

Prevalence of senile dementia in a rural community in Japan: the Tajiri project.

Knowledge of the prevalence of dementia in different age groups is needed for the planning of a health policy. This study shows the prevalence of dementia and magnetic resonance imaging (MRI) findings in elderly people aged 65 years and over, living in the town of Tajiri in the northern part of Japan. They were shown by two cognitive screening tests, the Mini-Mental State examination (MMS) and the Dementia Screening Test (DST) and medical diagnosis. Two subject groups were assessed, those who completed both tests (Subjects I, n=2066) and those from among the 200 'MRI-administered subjects' who were interviewed and diagnosed (Subjects II, n=170). For Subjects I, there were 6.3 and 10.2% 'dementia range' according to the severe and mild criteria, respectively. As for Subjects II, 9.4% were clinically diagnosed as having dementia. They met the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) criteria of probable Alzheimer's disease (AD) or possible AD with cerebrovascular disease. The estimated prevalence rate of dementia was 8.0%. Visual ratings of brain atrophy using MRI disclosed two distribution patterns. The 'continuous' pattern of the frontal and temporal lobes atrophy suggest that both are affected by the aging process, while a 'discontinuous' pattern of the hippocampal atrophy could indicate a pathologic background such as early changes of AD.

Depressive symptoms and associated factors in a cognitively normal elderly population: the Tajiri Project.

BACKGROUND AND OBJECTIVE: Since depression is one of the main problems of elderly subjects, it is important to examine the prevalence of this condition and to identify associated factors. METHODS: A total of 1525 cognitively normal subjects aged 65 years and over in the town of Tajiri, a typical agricultural town in Japan, were analysed. Their MMSE (mini-mental state examination) scores were 24 or over. Depressive state was assessed by Zung's SDS (self-rating depression scale) with a comprehensive interview to examine ADL, demographics and symptoms associated with illness, etc. The prevalence of depression was calculated. To determine the factors associated with depression, the t-test and the Chi-square test were used. To examine the relative strength of each factor, logistic regression analysis was performed. RESULTS: The ratio of the depressive subjects was 6.4%, lower than those of previous reports, probably due to the effect of excluding dementia subjects. The ratio for older females aged 80 years and over was 14.3%, which was significantly higher than that of the males. Among socio-demographic factors, sex, age, number of children and perception of economic status, were significantly related. For health status and ADL, such factors as perception of health and medical history of heart disease and rheumatism were related. For familial and social status, factors such as daily activity and several conversation abilities were related. The logistic regression analysis indicated that perception of health and daily activity were associated. CONCLUSIONS: In this study, we isolated some factors related to depression in a cognitively normal population. Knowledge of such factors is important for appropriate mental care of aged subjects.

Soluble P-selectin is present in normal circulation and its plasma level is elevated in patients with thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome.

P-selectin is an integral membrane glycoprotein stored in the secretory granules of platelets and endothelial cells. To determine whether soluble P-selectin may be present in the circulation of healthy humans, we used a sandwich immunoassay to assess citrated plasma from 50 subjects. P-selectin was present in concentrations ranging from 19 to 521 ng/ml (mean +/- SD = 121 +/- 84 ng/ml). The apparent molecular weight of P-selectin immunoisolated from platelet-poor plasma was similar to that of the detergent-soluble form isolated from platelet membrane. Plasma levels of P-selectin were unaffected by the following procedures: (1) drawing of blood in the presence of protease inhibitors; (2) stimulation of platelet-rich plasma with aggregating agents; (3) ultracentrifugation at 100,000 g for 120 min at 4 degrees C or filtration through a 0.22 micron membrane; or (4) preincubation of platelet-poor plasma with immobilized anti-platelet glycoprotein Ib monoclonal antibodies. It appeared that plasma P-selectin did not result from the in vitro activation of platelets, nor was it derived from platelet microparticles. We also found that plasma P-selectin levels were significantly elevated in patients with thrombotic thrombocytopenic purpura (12 patients, 332 +/- 184 ng/ml, P < 0.001) and haemolytic uraemic syndrome (17 patients, 297 +/- 191 ng/ml, P < 0.0001), as compared to the normal levels. Thus, these data should facilitate the study of the pathophysiological significance of circulating P-selectin.

A family with hereditary factor X deficiency with a point mutation Gla32 to Gln in the Gla domain (factor X Tokyo).

We report a new family with hereditary factor X deficiency. The propositus had a markedly prolonged prothrombin time, a mild prolongation of activated partial thromboplastin time and a clotting time activated by Russell's viper venom. Factor X activity in plasma was 3 u/dl (normal range 56-138 u/dl). Factor X antigen level was 61 u/dl. Molecular analysis revealed a homozygous mutation, Glu (GAG) to Gln (CAG) at residue 32 which normally undergoes gamma-carboxylation within the gamma-carboxyglutamic acid rich domain. The genotypes of family members completely correlated with their factor X activities. It is suggested that the Glu32 to Gln mutation is the molecular basis for the abnormal factor X in this family.

Novel point mutations in the alphaIIb subunit (Phe289-->Ser, Glu324-->Lys and Gln747-->Pro) causing thrombasthenic phenotypes in four Japanese patients.

We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the alphaIIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)-289 to Ser (TCT) mutation (F289S) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 2 3, a region previously not associated with GT, resulting in a Gln (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on alphaII(b)beta3 surface expression, the wild-type alphaIIb cDNA or mutant alphaIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type beta3 cDNA. Flow cytometric analysis using an anti-alphaII(b)beta3 complex antibody revealed that 50.6% of CHO cells with wild-type alphaII(b)beta3 expressed complexes, whereas only 1 6%, 7.7% and 31.3% of cells, with IIb(F289S)beta3, alphaIIb(E324K)beta3 and alphaIIb(Q747P)beta3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the alphaIIb subunit may hamper surface expression of the alphaII(b)beta3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.

Elderly Japanese emigrants to Brazil before World War II: II. Prevalence of senile dementia.

BACKGROUND: We previously showed the prevalence of dementia in the town of Tajiri (Miyagi Prefecture, Japan), and found it to be 8.0%. The first population-based study on dementia in Brazil (Catanduva) disclosed the prevalence as being 7.1%. To evaluate the effects of environment on development of dementia, elderly Japanese immigrants living in Brazil were examined. Brazil is the country with the largest number of Japanese immigrants. METHODS: All immigrants aged 65 years and over from Miyagi Prefecture, living in the four cities of the São Paulo Metropolitan area were targeted (n = 192). We were able to examine 166 subjects (86.5%). The diagnosis of dementia was based on the DSM-IV with the severity assessed by the CDR (clinical dementia rating) scales. The cognitive ability screening instrument (CASI) was used for neuropsychological assessment. RESULTS: Thirteen subjects were diagnosed with dementia, CDR 1-3, the prevalence being 7.8%. Older subjects suffered more from dementia, and, paradoxically, the more highly educated subjects also suffered more. All the CASI items, except for long-term memory and visual construction, significantly deteriorated in the CDR 0.5 group compared with the CDR 0 group. COMMENTS: The prevalence of dementia was not thought to be affected by environmental factors. A paradoxically higher rate of dementia in the more educated subjects was probably due to the historical problems of the immigrants. Intact CASI item long-term memory in the CDR 0.5 group indicated that suspected dementia patients could maintain this function. This is the first epidemiological study on dementia in elderly Japanese immigrants in Brazil.

Elderly Japanese emigrants to Brazil before World War II: I. Clinical profiles based on specific historical background.

OBJECTIVE: To research the demographic and clinical profiles of elderly Japanese emigrants, who arrived in Brazil before World War II, in order to give them appropriate psychogeriatric care. DESIGN: Elderly Japanese immigrants aged 65 years and over, belonging to the Miyagi Association in the São Paulo Metropolitan Area, were targeted. They emigrated from Miyagi Prefecture to Brazil and are now living in the area. We were able to interview 166 respondents. All data were gathered using standardized interview methods covering (a) free interview about the immigration history, (b) demographics, and (c) physical status. RESULTS: Through the free interview, we found their immigration histories, which affected their clinical profiles. The mean age and educational level were 77.5 years and 6.3 years, respectively. Sixty per cent of them immigrated when they were younger than 14. Ninety-four per cent of them still keep Japanese nationality. Fifty-seven per cent of them usually use Japanese, while 10% of them use Portuguese. Although their emigration histories were hard, 76% of them perceived their health as being excellent or relatively good. The percentages of subjects with histories of disease were hypertension, 52.5%; cardiac disease, 20.8%; diabetes mellitus, 24.2%; and hyperlipidemia, 25.0%, which were affected by the Brazilian environment. CONCLUSION: The elderly Japanese who emigrated to Brazil before World War II have a unique historical and demographic background. Their clinical profiles cannot be fully understood without knowing their histories. They definitely need high quality international psychogeriatric care.

Three novel integrin beta3 subunit missense mutations (H280P, C560F, and G579S) in thrombasthenia, including one (H280P) prevalent in Japanese patients.

We analyzed three unrelated Japanese patients with type II Glanzmann thrombasthenia (GT) for associated mutations. Polymerase chain reaction and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the integrin beta3 subunit gene (His (CAT)-280 to Pro (CCT), Cys (TGT)-560 to Phe (TTT), and Gly(GGC)-579 to Ser(AGC)). Interestingly, the three unrelated patients all had the H280P mutation; one was homozygous and the other two heterozygous for this mutation. Ectopic expression of wild type and mutant complexes in Chinese hamster ovary cells revealed decreased surface expression of the mutated alphaIIbbeta3 complexes, thus demonstrating that these mutations may result in the mild GT phenotypes. The identification of three unrelated patients having the same mutation (H280P) suggests that this mutation might be prevalent in the Japanese thrombasthenic population.