Yumiko Matsubara

Coronary Artery Disease and Polymorphisms in a Receptor Mediating Shear Stress–Dependent Platelet Activation

BACKGROUND Platelets play pivotal roles in coronary thrombosis, and antiplatelet therapies are widely used for coronary artery disease (CAD). However, the effects of genetic variation in platelets on CAD are poorly understood. We have assessed the association between CAD and polymorphisms in a platelet receptor for von Willebrand factor, the glycoprotein (GP) Ib/IX complex, which mediates shear stress-dependent platelet activation. METHODS AND RESULTS Genotypes of the alpha-chain of the receptor (GP Ib alpha, 145Thr/Met) were determined in 91 patients with myocardial infarction (MI) or angina pectoris whose lesions were confirmed by coronary angiography as well as in 105 individuals from the general population with no history of angina or other heart diseases and normal resting ECGs. There was no homozygote for Met/Met in either the control or patient groups. The prevalence of the Thr/Met genotype (T/M) in all patients was not significantly different from that in the control group. However, the frequency of T/M was significantly higher in patients aged < or = 60 years (31.8%) than in control subjects aged < or = 60 years (16.0%; P<.05, odds ratio=2.5). An association was also demonstrated between CAD and the other polymorphism of GP Ib alpha, a variable number of tandem repeats of a 13-amino acid sequence, which is known to be linked to the 145Thr/Met polymorphism. There was an association between the frequency of the T/M genotype and the angiographic severity of CAD: 11.1% for Gensini score < 40 versus 50.0% for Gensini score > or = 40 (P=.0015). There was no difference in the distribution of GP Ib alpha genotypes between patients with MI and those with angina pectoris. CONCLUSIONS This study suggests that the presence of the Met allele in GP Ib alpha is a risk factor for the prevalence and severity of CAD in individuals aged < or = 60 years. The results need to be confirmed in a large-scale study of incident case subjects and matching control subjects.

Genotype distribution of estrogen receptor polymorphisms in men and postmenopausal women from healthy and coronary populations and its relation to serum lipid levels

The cardiovascular protective effects of estrogen are known to be mediated by its beneficial effects on lipid metabolism and its direct actions on the vessel wall. The latter can be mediated by a specific receptor for estrogen present on smooth muscle cells and endothelial cells. The gene for the receptor (the classic estrogen receptor [ER]) has three known polymorphisms, Pvu II, Xba I, and B-variant polymorphisms, which are reportedly associated with receptor expression and altered receptor function and with some disorders including breast cancer, hypertension, and spontaneous abortion. However, the significance of genetic variations of the ER in vascular diseases has not been reported. We have examined the association between coronary artery disease (CAD) and the three polymorphisms in ER. Genotypes (P1/P2, X1/X2, and B-wild type/B-variant type) were determined in 87 men and postmenopausal women with myocardial infarction or angina pectoris whose lesions were confirmed by coronary angiography, as well as from 94 control individuals from the general population with no coronary heart disease and normal resting ECG. For B-variant polymorphism, all individuals examined had B-wild type, which contrasts with the reported allele frequency for B-variant type (0.1) in the white population. Genotype distributions and allele frequencies of Pvu II or Xba I polymorphisms were not significantly different between control subjects and patients (P > .05 for Pvu II or Xba I genotypes; P > .05 for Pvu II or Xba I allele frequencies). When the allele frequencies were analyzed separately by sex, there was still no statistically significant difference for both polymorphisms (P > .05 for men; P > .05 for women). No association was found between the polymorphisms and the angiographic severity of CAD. Total cholesterol, triglyceride, or HDL-cholesterol levels were not significantly different among ER genotypes. These findings suggest that the three polymorphisms in ER are not associated with the prevalence and severity of CAD and that the polymorphisms are unrelated to the serum lipid levels in control subjects and patients.

Induction of functional platelets from mouse and human fibroblasts by p45NF-E2/Maf

Determinant factors leading from stem cells to megakaryocytes (MKs) and subsequently platelets have yet to be identified. We now report that a combination of nuclear factor erythroid-derived 2 p45 unit (p45NF-E2), Maf G, and Maf K can convert mouse fibroblast 3T3 cells and adult human dermal fibroblasts into MKs. To screen MK-inducing factors, gene expressions were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells known to differentiate into MKs. 3T3 cells transfected with candidate factors were cultured in a defined MK lineage induction medium. Among the tested factors, transfection with p45NF-E2/MafG/MafK lead to the highest frequency of CD41-positive cells. Adult human dermal fibroblasts transfected with these genes were cultured in MK lineage induction medium. Cultured cells had megakaryocytic features, including surface markers, ploidy, and morphology. More than 90% of MK-sized cells expressed CD41, designated induced MK (iMK). Infusion of these iMK cells into immunodeficient mice led to a time-dependent appearance of CD41-positive, platelet-sized particles. Blood samples from iMK-infused into thrombocytopenic immunodeficient mice were perfused on a collagen-coated chip, and human CD41-positive platelets were incorporated into thrombi on the chip, demonstrating their functionality. These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis.

Identification of a novel point mutation in platelet glycoprotein Ibα, Gly to Ser at residue 233, in a Japanese family with platelet-type von Willebrand disease

Summary.  Background: Interaction between platelet glycoprotein (GP)Ibα and von Willebrand factor (VWF) has critical roles in both physiological hemostasis and thrombosis. Platelet‐type von Willebrand disease (plt‐VWD) is a congenital bleeding disorder characterized by gain‐of‐function mutations of GPIbα. To date, two mutations in GPIbα, G233V and M239V, have been reported in four unrelated families with plt‐VWD. Objective: The present study aimed to determine whether G233S of GPIbα, a new mutation observed in plt‐VWD patients, causes the plt‐VWD phenotype and to examine whether conversions to other residues at this position affect VWF binding. Patients and methods: The propositus was a 3‐year‐old Japanese male. He displayed bleeding symptoms and moderate thrombocytopenia. His brother was similarly affected. Platelets from both patients were analyzed by ristocetin‐ or shear‐induced platelet aggregation. DNA sequencing was performed to analyze the GPIbα sequence. We examined the 125I‐labeled VWF binding using a series of recombinant GPIbα fragments with different residues at position 233 (G233S, G233A, G233K, and G233D) together with naturally occurring mutations previously reported in patients (G233V and M239V). Results: Platelet function analysis indicated that platelets from both patients had a typical plt‐VWD phenotype. DNA sequencing analysis showed a heterozygous mutation of Gly to Ser at residue 233 of GPIbα in both patients. The 125I‐labeled VWF binding to mutant compared with the wild type displayed three patterns, gain‐of‐function (G233S, G233V, and M239V), equivalent function (G233A), and loss‐of‐function (G233K and G233D). Conclusions: The G233S is a molecular basis of plt‐VWD, and residue 233 plays critical roles in regulating VWF binding.

Effects of VerifyNow P2Y12 test and CYP2C19*2 testing on clinical outcomes of patients with cardiovascular disease: A systematic review and meta-analysis

The VerifyNow P2Y12 test is a whole-blood, light transmission-based optical detection assay that measures adenosine diphosphate-induced platelet aggregation in a cartridge containing fibrinogen-coated beads. The usefulness of this device to assess the effect of clopidogrel on platelet function was recently highlighted for predicting cardiovascular (CV) events. We therefore performed a meta-analysis to analyze whether adverse clinical outcomes of patients with CV disease increase in association with high on-treatment platelet reactivity (HPR) using the VerifyNow P2Y12 test. We also investigated the effect of the CYP2C19*2 polymorphism on the occurrence of CV events among clopidogrel-treated patients. Relevant studies were searched for in the PubMed database and Cochrane Central Register of Controlled Trials database, and selected if they included data about platelet function assessed using the VerifyNow P2Y12 test or CYP2C19*2 polymorphism in clopidogrel-treated patients with CV disease, and the occurrence of CV events within 6–12 months. In our meta-analysis, 4817 subjects from eight studies and 5307 subjects from seven studies were included for review of platelet function and CYP2C19*2 polymorphism, respectively. Overall, 2237 (46.4%) patients had HPR and these patients had significantly higher odds of CV events compared to patients without HPR (odds ratio(OR) = 3.05, 95% confidence interval: 2.33–3.98, p < 0.001). Heterogeneity among studies was not significant (Cochrans Q-test, p = 0.80 and I2 = 0%). In the secondary investigation, 1926 (36.3%) patients had at least one CYP2C19*2 allele. Heterogeneity in the OR of the CV events between the CYP2C19*2 allele carriers and non-carriers was observed among the seven studies (Cochrans Q-test, p = 0.01 and I2 = 56.5%), and the range of the ORs among the seven studies was 0.58–16.6. In conclusion, HPR assessed by VerifyNow P2Y12 test was associated with increased adverse clinical outcomes of clopidogrel-treated patients with CV disease.

Generation of megakaryocytes and platelets from human subcutaneous adipose tissues.

Recent advances in regenerative medicine have created a broad spectrum of stem cell research. Among them, tissue stem cell regulations are important issues to clarify the molecular mechanism of differentiation. Adipose tissues have been shown to contain abundant preadipocytes, which are multipotent to differentiate into cells including adipocytes, chondrocytes, and osteoblasts. In this study, we have first shown that megakaryocytes and platelets can be generated from adipocyte precursor cells. Human adipocyte precursor cells were cultured in conditioned media for 12 days to differentiate adipocytes, followed by 12 days of culture in media containing thrombopoietin. The ultrastructures of adipocyte precursor cell- and bone marrow CD34-positive cell-derived megakaryocytes and platelets were similar. In addition, adipocyte precursor cell-derived platelets exhibited surface expression of P-selectin and bound fibrinogen upon stimulation with platelet agonists, suggesting that these platelets were functional. This is the first demonstration that human subcutaneous adipocyte precursor cells can generate megakaryocyte and functional platelets in an in vitro culture system.

Studies of a microchip flow-chamber system to characterize whole blood thrombogenicity in healthy individuals

INTRODUCTION A whole blood flow-chamber system, the Total Thrombus-formation Analysis System (T-TAS), was developed for quantitative analysis of platelet thrombus formation (PTF) using microchips with thrombogenic surfaces (collagen, PL chip; collagen plus tissue thromboplastin, AR chip) under shear stress conditions. We evaluated the usefulness of the T-TAS for assessing individual thrombogenicity compared with other platelet function tests. MATERIALS AND METHODS Blood samples from 31 healthy volunteers were applied to the T-TAS to measure PTF starting time (T10: time to reach 10 kPa), occlusion time (T60 for PL chip; T80 for AR chip), and area under the curve (AUC10, area under curve until 10 min for PL chip; AUC30, 30 min for AR chip) under various shear rates (1000, 1500, 2000s(-1) for PL chip; 300 s(-1) for AR chip). Platelet functions were also tested using platelet aggregometry, the PFA-100 (collagen and epinephrine [C/EPI], collagen and adenosine diphosphate [C/ADP]), and the VerifyNow P2Y12 assay. RESULTS Individual pressure waveforms, including PTF starting and ending points, varied among healthy subjects. In the PL chip, T10 and AUC10 showed a shear-dependent correlation with C/EPI or C/ADP. VerifyNow P2Y12 values were not significantly associated with the parameters of the T-TAS. Platelet counts were correlated with all AR measurements, and mostly with PL measurements. CONCLUSION The results of the T-TAS were associated with those of the PFA-100 in many respects, indicating that its characteristics are related to shear-induced PTF. The T-TAS showed few correlations with platelet aggregometry and the VerifyNow P2Y12 assay. The T-TAS may allow for the measurement of comprehensive parameters of individual thrombogenicity under whole blood flow conditions.

Angiotensin converting enzyme insertion/deletion polymorphism is associated with plasma antigen levels of plasminogen activator inhibitor-1 in healthy Japanese population

Plasminogen activator inhibitor-1 (PAI-1) has a central role in the regulation of the fibrinolytic enzyme system. An elevated plasma PAI-1 level is associated with thrombotic disorders. In vitro and in vivo studies indicate that the renin-angiotensin system is involved in the regulation of PAI-1. A 287-bp insertion/deletion (I/D) polymorphism in the gene-encoding angiotensin converting enzyme (ACE) is associated with cardiovascular disorders. We evaluated the association between the ACE I/D polymorphism and plasma PAI-1 antigen levels in 110 healthy Japanese male subjects. Subjects with the D-allele of the gene-encoding ACE had higher levels of PAI-1 (26.3 +/- 14.7 ng/ml, mean +/- standard deviation) compared with those without (21.0 +/- 12.0; P = 0.0491). A multiple linear regression model with independent variables (age, body-mass index, total cholesterol level, triglyceride level, ACE I/D genotype, and PAI-1 genotype due to a single guanine I/D polymorphism in the PAI-1 gene) demonstrated that the triglyceride level (P = 0.0059) and ACE I/D genotype (P = 0.0372) were independent predictors of plasma PAI-1 antigen levels in a subset of the subjects without diabetes mellitus that were not taking lipid-lowering drugs. These findings suggest that the ACE I/D polymorphism is a genetic factor for the regulation of plasma PAI-1 antigen levels in the healthy Japanese population.

Culture of Megakaryocytes and Platelets from Subcutaneous Adipose Tissue and a Preadipocyte Cell Line

The molecular mechanisms whereby stem cells develop into platelet-producing megakaryocytes (MKs) are not yet fully understood. Within this chapter we describe a two-step in vitro culture system in which MKs and platelets are generated from primary subcutaneous adipose tissues and the preadipocyte cell line 3T3L1. The cells are first cultured in an adipocyte induction medium for 10-12 days, followed by 8-14 days culture in a MK differentiation medium. Adipose tissue-derived MKs and platelets display a number of morphological and functional characteristics (e.g., secretory granules, open canalicular membranes) comparable with the native cell type. The use of subcutaneous adipose tissue to produce a large number of platelets is advantageous because this tissue is easily obtained and available in large quantities. Thus, this in vitro culture system may prove useful in both regenerative medicine, but it may also be used in understanding fundamental research questions within MK and platelet research, including further elucidation of the pathways that cause cells to differentiate along the MK lineage ultimately leading to platelet production.

Platelet glycoprotein Ib alpha polymorphisms affect the interaction with von Willebrand factor under flow conditions

Interaction of platelet glycoprotein (GP) Ibα with von Willebrand factor (VWF) is essential for thrombus formation, particularly under high shear conditions. Previous case–control studies indicated that two GPIbα polymorphisms, 145Thr/Met and/or variable number (1–4) tandem repeats of 13 amino‐acid sequences, are associated with arterial thrombosis. The 145Met‐allele and the 3R‐ or 4R‐allele is associated with increased risk. However, there is little clear experimental data to support this association. To elucidate the functional effects of these polymorphisms, we prepared recombinant GPIbα fragments and tested them in vitro. The dissociation constants of ristocetin‐induced 125I‐labelled VWF binding to two forms of soluble recombinant GPIbα [1His–302Ala, either 145Thr (145T) or 145Met (145M)] were not different. Four types of Chinese hamster ovary cells expressing full‐length GPIbαβ/IX, 145T with one repeat (T1R), 145M with one repeat (M1R), 145T with four repeats (T4R), and 145M with four repeats (M4R), were prepared, and cell interactions with immobilized‐VWF were examined under various shear conditions. The cell rolling velocity of M4R under a shear condition of 114/s was significantly slower than that of T1R. Intermediate values were obtained with M1R and T4R. The results suggest that M4R interacts more strongly with VWF under flow conditions.