Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system.

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.

[Effect of safinamide on morning-off phenomenon in elderly patients with Parkinson's disease].

AIM: Morning-off is a symptom experienced by patients with Parkinson's disease (PD), which markedly reduces patients' quality of life. The present study evaluated the effect of safinamide on morning-off in elderly PD patients. METHODS: This observational study included 30 PD patients treated with 50 or 100 mg/day of safinamide in the evening. Using patient-reported outcomes, we evaluated the effect of safinamide on daily/morning ON-time, daily/morning OFF-time, Unified Parkinson's Disease Rating Scale (UPDRS) Part III score, and non-motor symptoms. Data at baseline (treatment start) and at 4, 8, 12, and 16 weeks after baseline were recorded. RESULTS: The PD patients (75.8±7.5 years old) in this study, who tended to be older than in previous phase 2/3 or 3 studies, may represent real-world Japanese PD patients. Compared with baseline, safinamide significantly increased the daily ON-time at eight weeks and morning ON-time at four weeks. Safinamide significantly reduced the daily OFF-time and morning OFF-time at four weeks. The UPDRS Part III score was significantly reduced by 1 point at 12 weeks. Safinamide showed a tendency to reduce non-motor symptoms, such as anxiety, pain, and depressive feelings. There was no marked difference in these parameters between patients treated with 50 and 100 mg of safinamide. CONCLUSIONS: Our results suggest that safinamide administered in the evening can benefit elderly patients who experience wearing off, especially morning off, and non-motor symptoms.

Elevation of inositol pyrophosphate IP7 in the mammalian spinal cord of amyotrophic lateral sclerosis.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder associated with progressive impairment of spinal motor neurons. Continuous research endeavor is underway to fully understand the molecular mechanisms associating with this disorder. Although several studies have implied the involvement of inositol pyrophosphate IP7 in ALS, there is no direct experimental evidence proving this notion. In this study, we analyzed inositol pyrophosphate IP7 and its precursor IP6 in the mouse and human ALS biological samples to directly assess whether IP7 level and/or its metabolism are altered in ALS disease state. Methods: We used a liquid chromatography-mass spectrometry (LC-MS) protocol originally-designed for mammalian IP6 and IP7 analysis. We measured the abundance of these molecules in the central nervous system (CNS) of ALS mouse model SOD1(G93A) transgenic (TG) mice as well as postmortem spinal cord of ALS patients. Cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) from ALS patients were also analyzed to assess if IP7 status in these biofluids is associated with ALS disease state. Results: SOD1(G93A) TG mice showed significant increase of IP7 level in the spinal cord compared with control mice at the late stage of disease progression, while its level in cerebrum and cerebellum remains constant. We also observed significantly elevated IP7 level and its product-to-precursor ratio (IP7/IP6) in the postmortem spinal cord of ALS patients, suggesting enhanced enzymatic activity of IP7-synthesizing kinases in the human ALS spinal cord. In contrast, human CSF did not contain detectable level of IP6 and IP7, and neither the IP7 level nor the IP7/IP6 ratio in human PBMCs differentiated ALS patients from age-matched healthy individuals. Conclusion: By directly analyzing IP7 in the CNS of ALS mice and humans, the findings of this study provide direct evidence that IP7 level and/or the enzymatic activity of IP7-generating kinases IP6Ks are elevated in ALS spinal cord. On the other hand, this study also showed that IP7 is not suitable for biofluid-based ALS diagnosis. Further investigation is required to elucidate a role of IP7 in ALS pathology and utilize IP7 metabolism on the diagnostic application of ALS.

Inositol hexakisphosphate kinase 2 promotes cell death of anterior horn cells in the spinal cord of patients with amyotrophic lateral sclerosis.

We have previously reported that inositol hexakisphosphate kinase (InsP6K)2 mediates cell death. InsP6K2 is abundantly expressed in anterior horn cells of the mammalian spinal cord. We investigated the role of InsP6K2 in spinal cords of patients with amyotrophic lateral sclerosis (ALS). Autopsy specimens of lumbar spinal cords from ten patients with sporadic ALS and five non-neurological disease patients (NNDPs) were obtained. We performed quantitative real-time PCR, immunostaining, and western blotting for InsP6K1, InsP6K2, InsP6K3, protein kinase B (Akt), casein kinase 2 (CK2), and 90-kDa heat-shock protein (HSP90). In contrast to InsP6K1 and InsP6K3 mRNA expression, InsP6K2 levels in anterior horn cells of the spinal cord were significantly increased in ALS patients compared to NNDPs. In ALS patients, InsP6K2 translocated from the nucleus to the cytoplasm. However, we observed a decrease in HSP90, CK2, and Akt activity in ALS patients compared to NNDPs. A previous study reported that InsP6K2 activity is suppressed after binding to HSP90 and subsequent phosphorylation and degradation by CK2, thus decreasing InsP6K2 activity. However, InsP7, which is generated by InsP6K2, can compete with Akt for PH domain binding. Consequently, InsP7 can inhibit Akt phosphorylation. Our results suggest that InsP6K2 is activated in the spinal cord of patients with ALS and may play an important role in ALS by inducing cell death mechanisms via Akt, CK2, and HSP90 pathways.

Author Correction: Insufficient production of IL-10 from M2 macrophages impairs in vitro endothelial progenitor cell differentiation in patients with Moyamoya disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Insufficient production of IL-10 from M2 macrophages impairs in vitro endothelial progenitor cell differentiation in patients with Moyamoya disease.

Moyamoya disease (MMD) is well known to be caused by insufficient cerebral vascular formation. However, the essential pathogenesis has not yet been identified. Using our recently developed technique of generating vasculogenic and anti-inflammatory cultures, we investigated endothelial progenitor cell (EPC) expansion and differentiation under the cytokine milieu generated by the peripheral blood mononuclear cells (PBMNCs) of the operated and non-operated MMD patients. EPC colony forming assay of the cultured PBMNCs disclosed the decline of the definitive EPC colony numbers in the both MMD patients. The level of interleukin-10 (IL-10) was lower in secretory cytokines from the cultured PBMNCs of MMD patients than that in that of controls using a cytometric bead array. The addition of human recombinant IL-10 to PBMNCs cultured from MMD patients restored the EPC colony forming potential of MMD PBMNCs. Following phorbol myristate acetate stimulation of the cultured PBMNCs, flow cytometry revealed a decrease in intracellular IL-10 storage in the main cell populations of the PBMNCs cultured from MMD patients relative to those cultured from controls. The present data provide the expected mechanism of vascular malformation in MMD pathogenesis originated from the insufficient production of IL-10 secreting cells from PBMNCs fostering EPC expansion and differentiation.

Early detection of cognitive impairment in Parkinson's disease with the use of the Wisconsin Card Sorting Test: correlations with Montreal Cognitive Assessment and smell identification test.

Cognitive function is often impaired in early Parkinson's disease (PD). The Wisconsin Card Sorting Test (WCST) is a neuropsychological test of "set-shifting" ability. To see whether WCST is useful for detecting early changes of cognitive function in PD, we examined the correlations of WCST with the Montreal Cognitive Assessment (MoCA) and the Odor Stick Identification Test (OSIT). Subjects were 48 PD patients (age 66 ± 10 years; Hoehn & Yahr stage 2.3 ± 0.8; mean duration 3.1 ± 2.5 years). WCST sub-scores for categories achieved (CA), perseverative errors of Nelson type (PEN), and difficulties of maintaining set (DMS) were evaluated. MoCA-J (Japanese version) and OSIT-J (Japanese version) were done in that order, followed by the WCST. In PD patients, CA was 2.2 ± 2.0, PEN was 7.0 ± 6.4, and DMS was 2.3 ± 2.0, and all were worse than those of age-matched normal subjects. MoCA-J scores significantly correlated with PEN. OSIT-J scores were also significantly correlated with CA and DMS. As MoCA-J and OSIT-J show high sensitivity and specificity for detecting mild cognitive impairment in PD, WCST may also be a useful supplementary diagnostic tool for early and mild cognitive impairment in PD patients.

Association of Smell Identification Deficit with Alzheimer's Disease Assessment Scale-Cognitive Subscale, Japanese Version Scores and Brain Atrophy in Patients with Dementia.

INTRODUCTION: Olfactory dysfunction is commonly associated with Alzheimer's disease (AD) and may be related to disorder of the central olfactory processing system. In this work, therefore, we examined the relationships between olfactory changes and the most affected cognitive domain or degree of brain atrophy in patients with AD and mild cognitive impairment (MCI). METHODS: The subjects were 55 AD patients and 27 MCI patients. Smell identification tests were performed using Odor Stick Identification Test for Japanese -(OSIT-J). The severity and nature of cognitive dysfunctions were evaluated using the AD Assessment Scale-cognitive subscale, Japanese version (ADAS-Jcog). MRI with voxel-based specific regional analysis system for AD software was used for evaluation of brain atrophy. RESULTS: -OSIT-J scores were significantly correlated with total -ADAS-Jcog scores, as well as with ADAS-Jcog subscale items of word recall task, orientation (memory domain) and ideational praxis. Smell identification deficit was proportional to the degree of atrophy of the medial temporal lobe. CONCLUSION: Smell identification deficit in AD/MCI is strongly associated with the memory domain of cognitive function and with atrophy of the medial temporal lobe.

Characteristic deterioration of ADAS-Jcog subscale scores and correlations with regional cerebral blood flow reductions in Alzheimer's disease.

The Alzheimer Disease Assessment Scale (Japanese version) cognitive subscale (ADAS-Jcog) is composed of a number of subscale tasks. However, it is not clear which subscale tasks are most susceptible to impairment in Alzheimer's disease (AD) or what is the relationship between reduction in regional cerebral blood flow (rCBF) and decreased ADAS-Jcog scores. Subjects were 32 AD patients, aged 52-86 years. We examined the relationship between subscale tasks that showed marked score changes and brain regions that showed reduced rCBF over a 2-year period. rCBF was measured by single-photon emission computed tomography (SPECT) with technetium-99m ethyl cysteinate dimer (99mTc-ECD), and the SPECT imaging data were analyzed with the easy Z-score imaging system (eZIS) and voxel-based stereotactic extraction estimation (vbSEE) methods. Total score of ADAS-Jcog deteriorated from 19.5 ± 7.0 to 35.7 ± 15.2 after 2 years. Subscale scores were significantly worse in all fields, particularly in orientation, word recall, remembering test instructions, commands, constructional praxis, and ideational praxis, in that order. Significant correlations were found between (1) word recall and commands and rCBF in the left middle temporal lobe, (2) naming objects/fingers and rCBF in the left temporal (middle, inferior) lobe, and (3) constructional and ideational praxis and rCBF in the right parietal (superior, inferior) lobe, temporal (superior, middle) lobe, angular gyrus, and cingulate gyrus. We identified the brain regions associated with specifically impaired subscales of ADAS-Jcog during progressive deterioration of AD over 2 years.

123I-Meta-iodobenzylguanidine (MIBG) myocardial scintigraphy in patients showing scans without evidence of dopaminergic deficits (SWEDDs).

OBJECTIVE: Scans without evidence of dopaminergic deficits (SWEDDs) in dopamine transporter single-photon emission computed tomography (DAT-SPECT) are found in 3.6-19.6% of patients with clinically suspected Parkinson's disease (PD). We investigated whether combined use of 123I-meta-iodobenzylguanidine (MIBG) myocardial scintigraphy would be helpful to differentiate PD among SWEDDs patients. PATIENTS AND METHODS: 145 patients with clinically suspected PD underwent both DAT-SPECT and MIBG myocardial scintigraphy. Striatal binding ratio (SBR) of DAT-SPECT and heart-to-mediastinal (H/M) ratio and washout rate (WR) of MIBG myocardial scintigraphy were calculated. RESULTS: Among 18 SWEDDs patients (12.4%), 11 were finally diagnosed with PD based on follow-up for at least two years after the DAT-SPECT and MIGB myocardial scintigraphy scans. Among the latter group, 8 patients showed an H/M ratio of less than 2.2, and 9 showed WR above 30%. CONCLUSION: Our results indicate that the combination of low H/M ratio and high WR of MIBG myocardial scintigraphy of SWEDDs patients may be helpful for detection of PD patients.

Dual Therapy with Aspirin and Cilostazol May Improve Platelet Aggregation in Noncardioembolic Stroke Patients: A Pilot Study.

Objective Some previous studies have found clinical benefit of dual antiplatelet therapy with aspirin and cilostazol for prevention of secondary stroke, but the physiological mechanism involved remains unknown. We aimed to clarify the effects of aspirin/cilostazol therapy on the platelet and endothelial functions of patients with acute noncardioembolic ischemic stroke, in comparison to patients who were treated with aspirin alone. Methods The present randomized prospective pilot study enrolled 24 patients within a week after the onset of noncardioembolic ischemic stroke. The patients were randomly allocated to receive aspirin (100 mg/day) (A group; 11 patients) or cilostazol (200 mg/day) plus aspirin (100 mg/day) (CA group; 13 patients). We measured platelet aggregation, platelet activation, and the thrombomodulin (TM), highly sensitive C-reactive protein (hs-CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and von Willebrand (vWF) antigen levels and vWF activity over a 4-week period after enrollment. Results There was no significant difference in the platelet functions of the A and CA groups. However, the platelet aggregation induced by adenosine diphosphate (ADP) was decreased at 2 and 4 weeks (p<0.05) after treatment in comparison to the pre-treatment values in the CA group, but not in the A group. Platelet activation, and the hs-CRP, TM, ICAM-1, VCAM-1 and vWF values did not significantly decrease after treatment in either group. Conclusion Although there were no significant differences in platelet aggregation, platelet activation or the endothelial biomarker levels of the A and CA groups, dual therapy with aspirin and cilostazol inhibited platelet aggregation in comparison to the pre-treatment values, similarly to patients who received aspirin alone. This may suggest the clinical usefulness of dual therapy with aspirin and cilostazol in the treatment of patients with noncardioembolic ischemic stroke.

Cilostazol protects against microvascular brain injury in a rat model of type 2 diabetes.

Cilostazol, a pluripotent phosphodiesterase III-specific inhibitor with anti-platelet and vasculogenic effects, is useful for preventing recurrent brain vascular events, particularly in stroke patients with diabetes mellitus (DM). However, it is unclear whether cilostazol affects autoregulatory responses in small cerebral arteries. Thus, we investigated the effect of cilostazol on diabetic brain vasculopathy in a model of type II DM using male OLETF rats. OLETF rats were treated with either cilostazol (CG) or vehicle (VG) and subjected to microangiography with monochromatic synchrotron radiation to investigate middle cerebral artery (MCA) vasoreactivity following an injection of acetylcholine (Ach). Ach administration led to MCA diameter contraction in the VG, but MCA dilation in the CG. We also evaluated morphological changes in the small intracranial vessels and found that in the CG, the endothelial cell structure in the small artery was not destroyed. Moreover, protein levels of phosphorylated endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were higher in each evaluated brain region in CG rats vs. VG rats. Our results suggest that cilostazol could potentially improve autoregulatory responses in the small cerebral arteries by increasing eNOS phosphorylation and VEGF expression in DM, and thus, may act as a neurovascular protectant.

Clinical Utility of Platelet Function Testing Following Non-Cardioembolic Stroke.

OBJECTIVE: To verify the usefulness in selection of antiplatelet agent based on the platelet functional assays on the secondary prevention of ischemic stroke. METHODS: Platelet functional assays were performed twice for acute ischemic stroke patients at hospitalization and 1 month after. An antiplatelet agent was administered based on the results of initial assay. The alterations of platelet aggregation by antiplatelet agent were evaluated in the second assay, and the patients were subsequently divided into inhibited and invariance groups. The relationship between incidence of recurrent ischemic or hemorrhagic stroke and the alterations of platelet aggregation by each selected antiplatelet agent was assessed. RESULTS: Of the 585 consecutive patients, 124 were enrolled in the present study. Recurrent ischemic stroke was seen in 6 (5.3%) and 2 (18.2%) patients in the inhibited and invariance groups during the study period, respectively. In patients who were observed for more than 12 months, recurrent ischemic stroke was seen in 4 (5.0%) and 2 (33.3%) patients in the inhibited and invariance groups, respectively (p = 0.009). CONCLUSIONS: We indicated that selection of the optimum antiplatelet agent based on the platelet functional assays for each individual patient may contribute to a reduction in the incidence of recurrence of ischemic stroke.

Effect of single nucleotide polymorphisms of the prostacyclin receptor gene on platelet activation in Japanese healthy subjects and patients with cerebral infarction.

Cerebral infarction (CI) is a complex multifactorial disorder that is thought to result from the interaction of various environmental factors and an individual's genetic make-up, including genes associated with platelet activation. In order to clarify whether single nucleotide polymorphisms (SNPs) of the prostacyclin receptor (IP) gene affects platelet activation in ischemic stroke, we investigated the relationship between platelet function and genetic polymorphism of the coding sequence of the IP gene in 64 Japanese patients with CI and 54 healthy subjects. We determined the entire nucleotide sequence of the IP gene in healthy Japanese subjects, and found that an adenine (A) to cytosine (C) substitution at base 984 (A984C) in exon 3 is the most frequent SNP. Using flow cytometry, the power-transformed mean percentage of PAC-1-positive platelets, [PAC-1](1/3), was significantly higher in healthy subjects with the C/C genotype than in healthy subjects with the A/A genotype (p ≤ 0.05), although there was no significant difference in patients with CI between these two genotypes. Furthermore, we genotyped 158 control patients and 106 patients with CI. The homozygous C/C genotype was more frequently found in the CI group (46.2%) than in the healthy control group (17.1%; p < 0.001). The present report is the first to show an association between the A984C polymorphism of the IP gene and platelet activation in Japanese subjects. This polymorphism may be clinically significant in disorders in which prostacyclin plays a key role, such as CI.

Appearance of WBC-platelet complex in acute ischemic stroke, predominantly in atherothrombotic infarction.

AIM: Platelet aggregates with white blood cells (WBC-platelet complex) have recently been proposed as a marker of activated platelets, in addition to well-known molecular markers. We aimed to investigate the colocalization of activated platelets and WBC-platelet complex by means of flow cytometry, in patients with ischemic stroke. METHODS: Eighty-six patients with cerebral infarction (CI) in the acute phase (58 males, 28 females; 65±14 years old) and 62 non-CI controls (23 males, 39 females; 53±14 years old) were registered. The appearance of WBC-platelet complex was quantified using 3-color flow cytometry. RESULTS: The appearance rate of WBC-platelet complex was significantly higher in the CI group than in the controls. The appearance rate of WBC-platelet complex was significantly higher in atherothrombotic infarction (AT) than in lacunar infarction (LA) (p < 0.05). Furthermore, positive rates of both monocyte-platelet complex and granulocyte-platelet complex, but not lymphocyte-platelet complex, were significantly higher in the AT group than in the controls. CONCLUSION: We concluded that WBC-platelet complex, especially involving monocytes and granulocytes, is a novel marker of platelet activation in the acute phase of ischemic stroke, mainly in AT.

Platelet aggregates detected by a conventional hematology analyzer method is a risk factor for stroke or a predictive factor in patients with chronic-stage cerebral infarction.

Assessment of platelet activation and/or function is important for primary and secondary prevention of vascular events. To test the hypothesis that determination of platelet aggregation in patients with chronic-stage cerebral infarction (CI) provides a simple measure of risk for ischemic stroke, we used a conventional hematology analyzer to detect aggregates and to assess the efficacy of antiplatelet agents in preventing spontaneous aggregate formation. Platelet aggregates were measured in citrated blood from 142 magnetic resonance imaging confirmed CI patients and 97 controls without CI (nonstroke). Aggregates were detected in 1 of 36 (2.8%) nonstroke subjects without risk factors, but in 24 of 52 (46.2%) nonstroke subjects with risk factors (odds ratio [OR], 3.32; 95% confidence interval [CI], 1.10-10.00), in 21 of 35 (60.0%) nonstroke subjects with a predictive factor (carotid artery intima-media thickness [IMT] >1.1 mm) (OR, 9.13; 95% CI, 2.70-30.48), and in 31 of 63 (49.2%) CI patients who had not received antiplatelet therapy (OR, 2.16; 95% CI, 1.12-4.17). After adjusting for other risk factors, the appearance of platelet aggregates was correlated only with IMT ≥1.1 mm. The rate of appearance of platelet aggregates was 0.33-fold lower in patients on antiplatelet therapy compared with those not on antiplatelet therapy (24.1%; 19 of 79 CI patients). Patients with platelet aggregates in the blood are considered at high risk for ischemic stroke, because the appearance of aggregates is associated with increased IMT. Our method is suitable for screening platelet function in high-risk patients and for examining the efficacy of antiplatelet agents.

Significant relationship between platelet activation and intra-media thickness of the carotid artery in patients with ischemic cerebrovascular disease.

INTRODUCTION: We have studied the relationship between the ratio of activated platelets and the thickness of intima and media of the carotid artery in ischemic CVD patients in the chronic stage. METHODS: Platelet activation was assessed by means of flow cytometry of whole blood using activation-dependent monoclonal antibodies (MoAb). Forty-one MRI-proven normative subjects and 55 patients with a history of ischemic CVD were examined. The intima-media thickness of the carotid artery was measured by using B-mode ultrasound in all subjects. RESULTS: The appearance rates of PAC-1-positive and CD62P-positive platelets (%) were increased in ischemic CVD patients compared with those in controls (p<0.0001, p<0.001, respectively) The patients and controls were divided into those with atherosclerosis (Ath+), defined as intima-media thickness 1.1 mm, and those without (Ath-). There was no significant difference of PAC-1-positive platelets between the Ath- and Ath+ subgroups in either group, but there was increase in Ath- ischemic CVD patients versus Ath- control subjects (p<0.01), and in Ath+ patients versus Ath+ controls (p<0.05). CD62-positive platelets in the Ath+ subgroup were significantly increased versus the Ath- subgroup in both the controls (p<0.001) and ischemic CVD patients (p<0.05), and there was also an increase in Ath- patients versus Ath- controls (p<0.05). CONCLUSION: Platelet activation markers were increased in patients with ischemic CVD compared with controls. A significant relationship was found between increased CD62-P-positive platelets and carotid artery abnormalities in both controls and ischemic CVD patients, suggesting that platelet activation may be a potential marker for atherosclerosis.

Antiplatelet effects of a Kampo medicine, Orengedokuto.

INTRODUCTION: Orengedokuto, a Kampo medicine, is used to treat patients with hypertension and cerebrovascular disease in Japan. One of its effects is thought to be antiplatelet action, but the mechanisms involved are unclear. Therefore, we investigated the effects of Orengedokuto on platelet aggregation and activation. METHODS: Platelet aggregation was determined by measuring light transmission (optical density [OD]) and light scattering intensity. Platelet activation was assessed by flow-cytometric determination of PAC-1+ and CD62P+ platelets. For the in vitro study, blood samples were obtained from 45 patients with cerebral infarction (chronic stage) and 27 magnetic resonance imaging-proven control subjects. Furthermore, Orengedokuto was administered to 10 patients with cerebral infarction for up to 12 months, and its effects on platelet aggregation and activation were evaluated. RESULTS: Orengedokuto dose-dependently inhibited agonist-induced platelet aggregation. In vitro, the OD% (mean +/- SD) after stimulation with 2 mumol/L adenosine diphosphate was decreased from 54.4 +/- 21.0 to 16.0 +/- 14.2 (P < .01) in patients, and from 56.8 +/- 26.9 to 19.8 +/- 18.9 (P < .01) in control subjects (1000 mug/mL Orengedokuto). Similarly, the OD% after 0.5 mug/mL collagen stimulation was decreased from 98.3 +/- 37.0 to 24.8 +/- 27.9 (P < .01) in patients, and from 79.2 +/- 13.8 to 21.5 +/- 21.9 (P < .01) in the control subjects. In the clinical study, platelet aggregation was also significantly decreased (P < .05). Agonist-induced platelet activation was not significantly inhibited by Orengedokuto in vitro, but spontaneous platelet activation in patients after oral administration was reduced from 31.0 +/- 18.3 to 14.2 +/- 8.7 (PAC-1+) and from 11.3 +/- 7.8 to 5.1 +/- 3.9 (CD62+) (P < .05). CONCLUSION: Our results demonstrated inhibitory activity of Orengedokuto on both platelet aggregation and activation at a clinically relevant dose.

Activated platelets in transient global amnesia and TIA.

To assess whether platelets are activated in transient global amnesia (TGA) and TIA, blood samples were analyzed by fluorescence-activated cytoscan using antibodies specific for platelet fibrinogen receptor (PAC1) and P-selectin (CD62P). Samples from TIA contained high levels of CD62P compared with age-matched control subjects, whereas those from TGA did not. The authors suggest that activated platelets are involved in brain ischemia, whereas ischemia appears not to be associated with most TGA.