Kiyoaki Watanabe

The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress.

Exposure of platelets to shear stress leads to aggregation in the absence of exogenous agonists. We have now found that different adhesive proteins and platelet membrane glycoproteins are involved in aggregation depending on the shear stress conditions and the concentration of divalent cations in the medium. When blood is collected with trisodium citrate as anticoagulant, which causes a decrease in the levels of external ionized calcium ([Ca2+]o), platelet aggregation can be induced under low shear force (12 dyn/cm2) and is mediated by fibrinogen binding to the glycoprotein IIb-IIIa complex. Aggregates formed under these conditions are not stable, and when shear force is increased to 68 dyn/cm2, disaggregation results. By contrast, platelets from blood collected with hirudin as anticoagulant, wherein [Ca2+]o is within normal plasma levels, do not undergo low shear-induced aggregation; however, after exposure to a shear force above 80 dyn/cm2, aggregation is observed but only when von Willebrand factor is present and can interact with both its platelet binding sites, glycoprotein Ib-IX and glycoprotein IIb-IIIa. Fibrinogen is not involved in high shear-induced aggregation which, in fact, occurs normally in patients with severe afibrinogenemia. Thus, von Willebrand factor in the absence of exogenous agonists can mediate platelet aggregation in experimental conditions that may mimic the hemorheological situation of partially occluded arteries. This pathway of platelet aggregation involving only one adhesive ligand and two membrane adhesion receptors may play a relevant role in thrombogenesis.

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

Summary. 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 100000 g for 120 min at 4°C or filtration through a 0·22 μm 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.

Effects of troglitazone on fat distribution in the treatment of male type 2 diabetes

We investigated the efficacy of additional administration of 400 mg troglitazone (+T), which became available as a treatment for type 2 diabetes following the demonstration of its ability to reduce insulin resistance, in combination with diet (D + T) or sulfonylurea (S + T) therapy. Body fat area as determined by computed tomographic (CT) scanning at the umbilical level, as well as several clinical and biochemical parameters of glycemic control and lipid metabolism, were compared before and after 3 months of additional treatment with troglitazone. The body mass index (BMI) tended to increase in both groups (22.7 +/- 0.6 v 23.2 +/- 0.6 kg/m2 in D + T, nonsignificant [NS]; 22.2 +/- 0.5 v 22.3 +/- 0.5 kg/m2 in S + T, NS), while it tended to decrease in the control group (only diet therapy, 23.6 +/- 0.6 v 23.1 +/- 0.8 kg/m2, NS). Mean blood pressure ([BP] 96 +/- 3 v 89 +/- 4 mm Hg, P < .05) decreased significantly in the D + T group. Changes in the glycemic and lipid profile and leptin did not reach statistical significance. The D + T group showed a significant decline in immunoreactive insulin ([IRI] 12.4 +/- 1.2 v 8.0 +/- 1.0 microU/mL, P < .05), reflecting markedly reduced insulin resistance, as well as a significant increase in plasma insulin-like growth factor-1 ([IGF-1] 175.7 +/- 14.2 v 189.8 +/- 12.6 ng/mL, P < .05). A slight weight gain was associated with a tendency for subcutaneous fat to increase, while visceral fat decreased in both troglitazone-treated groups. The decrease in the visceral to subcutaneous fat ratio (V/S ratio) was statistically significant in the D + T group (1.09 +/- 0.11 v 0.94 +/- 0.09, P < .05), while the V/S ratio in the control group did not change. A notable finding of this study is the difference in the response to troglitazone between subcutaneous and visceral adipose tissue. It is suggested that troglitazone may exert beneficial effects by reducing visceral fat.

C242T Polymorphism of NADPH Oxidase p22 PHOX Gene and Ischemic Cerebrovascular Disease in the Japanese Population

BACKGROUND AND PURPOSE Superoxide has been implicated in the pathogenesis of ischemic stroke and atherosclerosis. NADPH oxidase, a major source of superoxide generation in neutrophils and the vascular system, plays a critical role in ischemic injury and atherogenesis. Recently, an association between the C242T polymorphism of p22 PHOX, an essential component of NADPH oxidase, and coronary artery disease (CAD) has been reported in several studies. To investigate the relationship between the C242T polymorphism of p22 PHOX and ischemic cerebrovascular disease (CVD), we conducted a case-control study. METHODS We recruited 226 CVD patients (atherothrombotic infarction, lacunar infarction, and transient ischemic attack) and 301 control subjects and analyzed C242T polymorphism of p22 PHOX by detection of restriction fragment length polymorphism. RESULTS The TC+TT genotype frequencies in the CVD group and control group were 21.7% and 13.3%, respectively, and the prevalence of the TC+TT genotype was significantly higher in the CVD patients (chi(2)=6.477, P=0.01, OR 1.81, 95% CI 1.15 to 2.86). Analysis by CVD subtypes showed that the OR for the TC+TT genotype was higher in the CVD patients with atherothrombotic infarction than in those with lacunar infarction and transient ischemic attack. CONCLUSIONS The C242T polymorphism of the NADPH oxidase p22 PHOX gene is a novel pathogenetic risk factor for CVD.

A Thermodynamic Property Model for Fluid-Phase Propane

A fundamental equation of state for propane (R-290), formulated in terms of the non-dimensional Helmholtz free energy, is presented. It was developed based on selected reliable measurements for pressure-volume-temperature (PVT), isochoric and isobaric heat capacities, speed of sound, and the saturation properties which were all converted to ITS-90. Supplementary input data calculated from a virial equation for the vapor-phase PVT properties at lower temperatures and other correlations for the saturated vapor pressures and saturated vapor- and liquid-densities have also been used. The present equation of state includes 19 terms in the residual part and represents most of the reliable experimental data accurately in the range of validity from 85.48 K (the triple point temperature) to 623 K, at pressures to 103 MPa, and at densities to 741 kg·m−3. The smooth behavior of the derived thermodynamic properties in the entire fluid phase is demonstrated. In addition, graphical and statistical comparisons between experimental data and the available thermodynamic models, including the present one, showed that the present model can provide a physically sound representation of all the thermodynamic properties of engineering importance.

Polymorphism in the promoter of lipopolysaccharide receptor CD14 and ischemic cerebrovascular disease

Background and Purpose A growing amount of evidence suggests that infectious and inflammatory processes may be involved in the initiation of arteriosclerosis, but the mechanisms are conceivably multifactorial and complex. Two European groups have recently demonstrated that a C(−260)→T polymorphism in the promoter of the CD14 lipopolysaccharide receptor may be a risk factor for coronary artery disease (CAD). The T allele of this polymorphism reportedly increases the expression of CD14 and may be involved in atherogenesis. In the present study we investigated a possible association between the C(−260)→T polymorphism in the CD14 promoter and the occurrence of symptomatic ischemic cerebrovascular disease (CVD). Methods Genotype frequencies of the C(−260)→T polymorphism in the CD14 promoter were determined in 235 patients with CVD, as confirmed by brain CT and/or MRI, and 309 age- and sex-matched control subjects. Results The distribution of genotypes was as follows: CVD patients, T/ T 24.3%, C/ T 53.2%, and C/ C 22.6%; controls, T/ T 26.9%, C/ T 50.2%, and C/ C 23.0%. There was no significant difference between the CD14 promoter genotypes of the CVD patients and the controls (&khgr;2=0.601, P =0.741). We also measured the concentration of serum soluble CD14 and the density of membranous CD14 on monocytes in the CVD patients, but the polymorphism was not associated with either the concentration of soluble CD14 or the density of membranous CD14 (P =0.358, P =0.238, respectively). Conclusions Our results indicate that the C(−260)→T polymorphism in the CD14 promoter is not associated with an increased risk for CVD.

Association Between Platelet Glycoprotein Ibα Genotype and Ischemic Cerebrovascular Disease

BACKGROUND AND PURPOSE Platelets play pivotal roles in the development of ischemic cerebrovascular disease (CVD). The platelet glycoprotein (GP) Ib/IX/V complex is a receptor for von Willebrand factor, which plays a major role in the initial phase of platelet activation under high shear stress conditions. This study was designed to investigate the association between a genetic variation of this receptor and the prevalence of CVD. METHODS Two hundred patients with ischemic CVD, as confirmed by brain CT and/or MRI, and 317 age- and sex-matched control subjects without clinical evidence of CVD or cardiovascular disease were analyzed for their genotype frequencies of the (145)Thr/Met dimorphism of the alpha-chain of GPIb (GPIbalpha). RESULTS Genotypes with (145)Met (T/M and M/M) were more frequently found in the CVD patients (26.5%) than in control subjects (14.2%, P=0.0005). The genotype effect was more obvious in those <60 years of age or without acquired cardiovascular risk factors. The odds ratio for nonsmoking women <60 years of age was 10. 6 (95% confidence intervals, 2.2 to 51.7). Although the number of patients studied was small (n=24), transient ischemic attack showed the highest odds ratio (4.3, P=0.0004), followed by lacunar infarction (OR=2.2, P=0.0024) and atherothrombotic infarction (OR=1. 5, P=0.3143). Logistic regression analysis revealed that the presence of Met-allele was independently associated with CVD. CONCLUSIONS Our study suggests that the platelet GPIbalpha genotype is a genetic risk factor for ischemic CVD.

Automated measurement of reticulated platelets in estimating thrombopoiesis

Abstract: We described a fully automated measurement of reticulated platelets using a fluorescent dye, auramine O, and a reticulocyte counter, the R‐3000, equipped with special software. Reproducibility and linearity were shown to be good. In the normal subjects studied (n = 60), the mean value for reticulated platelets was 0.98% ± 0.41% and the mean absolute count was 2.12 ± 0.69 × 109/l. The absolute count for reticulated platelets was significantly lower (p < 0.05) in patients with reduced thrombopoiesis as seen in acute myeloblastic leukemia, aplastic anemia or chemotherapy‐induced thrombocytopenia and it was elevated (p < 0.05) in essential thrombocythemia and in chronic myelocytic leukemia with thrombocytosis. All 20 patients with chronic idiopathic thrombocytopenic purpura had a high percentage of reticulated platelets. The percentage of reticulated platelets was significantly increased (p < 0.05) in patients with impaired thrombopoiesis despite the reduction in the absolute count. In 2 leukemic patients, an apparent rise was noticed in the percentage of reticulated platelets which preceded by several days a progressive increase in the platelet count at the recovery phase of thrombocytopenia. The results suggest that an automated measurement of reticulated platelets can be applied to routine laboratories for clinical use.

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.

Cholesteryl ester transfer protein polymorphism associated with macroangiopathy in japanese patients with type 2 diabetes

A polymorphism in the gene for cholesteryl ester transfer protein (CETP) has been reported to be associated with serum cholesterol levels and risk for atherosclerotic vascular diseases, and to clarify the relationship between the gene polymorphism for CETP and macroangiopathy in diabetes mellitus, a cross-sectional study was performed. The subjects of the study were182 Japanese (age: 59.6+/-8.6 years) with type 2 diabetes and no signs of renal dysfunction, 24 of whom had macroangiopathy, and 158 of whom did not. The genotype of the subjects for the TaqIB polymorphism of CETP in intron one was analyzed by using polymerase chain reaction - restriction fragment length polymorphism. Serum CETP levels were significantly higher in the B1/B1 genotype than in the other genotypes (P<0.05). The serum CETP levels were correlated with the serum LDL cholesterol levels (P<0.01), but not with the HDL cholesterol levels. Macroangiopathy was more frequently observed in subjects with the B1/B1 genotype than in the other genotypes (odds ratio=2.953, 95% confidence interval=1.250-6.977, P=0.0136). Logistic regression analysis revealed that the CETP genotype was independently associated with macroangiopathy. The exact mechanism underlying the association remains unknown, but differences in serum CETP levels may be involved.