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Aims: The efficacy of antiplatelet therapy may vary among different disease subtypes. Prasugrel is generally a more potent, consistent, and fast-acting platelet inhibitor than clopidogrel. This sub-analysis of the phase III comparison of PRAsugrel and clopidogrel in Japanese patients with ischemic STROke (PRASTRO-I) trial aimed to assess the differences in efficacy of these treatments for each stroke subtype. Methods: In the PRASTRO-I trial, a total of 3,753 patients with ischemic stroke were recruited from 224 centers throughout Japan and randomized (1:1) to prasugrel (3.75 mg/day) or clopidogrel (75 mg/day) for 96 weeks. For the sub-analysis, strokes were classified as large-artery atherosclerosis, small-artery occlusion (lacunar), stroke of other etiology, and stroke of undetermined etiology. The cumulative incidence of primary events (ischemic stroke, myocardial infarction, and death from other vascular cause) and hazard ratios (HRs) were calculated for each subgroup. Results: For patients with large-artery atherosclerosis, the primary event incidence was 3.8% in the prasugrel group and 4.8% in the clopidogrel group (HR 0.79; 95% confidence interval [CI] 0.45–1.41). For patients with small-artery occlusion, the incidence was 3.3% in the prasugrel group and 3.9% in the clopidogrel group (HR 0.82; 95% CI 0.45–1.50). For patients with stroke of undetermined etiology, the incidence was 4.6% in the prasugrel group and 3.0% in the clopidogrel group (HR 1.56; 95% CI 0.90–2.72). The incidence of bleeding was similar across subtypes. Conclusions: Although statistical significance was not reached, the efficacy of prasugrel was potentially different between stroke subtypes, warranting further studies.

The overall prognosis of patients with sarcoma-based cancers has changed little in the last 20 years. There is an urgent need to investigate the metastatic potential of these tumors and to develop anti-metastatic drugs. It is becoming increasingly clear that platelets play an important role in the establishment of metastasis of carcinoma cells and could be a useful therapeutic target for patients with carcinoma. However, little is known about the role of platelets in sarcoma progression. Here, we investigated how osteosarcoma progression relates to platelet function to explore the possibility of anti-platelet therapy. We found that, similar to carcinoma cells, podoplanin (also known as Aggrus)-positive osteosarcoma cells induce platelet aggregation and activation. Administration of anti-glycoprotein Ibα (GPIbα, also known as CD42b) antibody reduced the lung metastasis of osteosarcoma. The supernatant from platelets cocultured with osteosarcoma cells contained several growth factors and promoted proliferation, invasiveness, and sphere formation of osteosarcoma cells in vitro. In addition, the development of lung metastasis was highly dependent on direct interaction between osteosarcoma cells and platelets. To explore the therapeutic target, we focused on the interactions between podoplanin on osteosarcoma and C-type lectin-like receptor (CLEC)-2 on platelets. The administration of a depleting antibody against CLEC-2 efficiently suppressed osteosarcoma metastasis into the lung. We also analyzed clinical data from patient samples at primary and metastatic sites. Although GPIbα expression was similar between the two sites, there was a significant increase in podoplanin at the metastatic site compared to that in the primary site, and the level of podoplanin expression in the primary site correlated with patient prognosis. These findings suggest that blockade of interactions between platelets CLEC-2 and osteosarcoma podoplanin represent the most promising therapeutic strategy for preventing the lung metastasis of osteosarcoma. © 2020 American Society for Bone and Mineral Research.

Coronavirus disease 2019 is diagnosed based on the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal swabs or saliva samples using reverse-transcription quantitative polymerase chain reaction. Nasopharyngeal swabs should be collected by medical professionals who are covered with full personal protective equipment (PPE), while saliva samples can be collected by patients themselves without any PPE. However, collecting saliva is difficult for people who are unable to follow instructions, including infants or unconscious patients. Owing to the high viscosity of saliva, special attention is required to handle saliva samples in laboratories. To solve these problems, we compared lingual and buccal mucosal swabs (oral swabs) with nasopharyngeal swabs and saliva samples. Among 13 patients who had a positive result for SARS-CoV-2 RNA in their nasopharyngeal swabs, 8 and 10 patients had a positive result for SARS-CoV-2 RNA in their saliva (concordance rate, 61.5%) and oral swabs (76.9%), respectively. Among the eight patients with a positive result for SARS-CoV-2 RNA in saliva, seven (87.5%) had SARS-CoV-2 detected in their oral swabs. We could not obtain saliva samples from four patients, but we found perfect concordance of SARS-CoV-2 positivity between the nasopharyngeal and oral swabs. Therefore, oral swabs can be used for SARS-CoV-2 RNA detection.

In humans, platelet count within the normal range is required for physiological hemostasis, but, adversely, platelets also support pathological thrombosis. Moreover, by releasing growth factors, they may enhance neoplastic proliferation. We hypothesize that platelet count correlates with platelet-dependent pathologies, even within the range of hemostatic competence. Because platelet production is promoted by thrombopoietin signaling through the myeloproliferative leukemia virus oncogene (cMPL), a receptor expressed on megakaryocytes, we evaluated the feasibility of selective targeting of hepatic thrombopoietin production to test this hypothesis. We synthesized murine- and primate-specific antisense oligonucleotides (THPO-ASO) that silence hepatic thrombopoietin gene (THPO) expression without blocking extrahepatic THPO. Repeated doses of THPO-ASO were administered to mice and a baboon, causing a sustained 50% decline in plasma thrombopoietin levels and platelet count within 4 weeks in both species. To investigate whether reducing platelet count within the translationally relevant hemostatic range could alter a neoplastic process, we administered THPO-ASO to 6-week-old transgenic MMTV-PyMT mice that develop early ductal atypia that progresses into cMPL-negative fatal metastatic breast cancer within 2 to 3 months. THPO-ASO treatment increased the average time to euthanasia (primary humane endpoint) at 2 cm(3) combined palpable tumor volume. Our results show that THPO-ASO reduced blood platelet count, plasma platelet factor 4, vascular endothelial growth factor, thrombopoietin levels, bone marrow megakaryocyte density, tumor growth rate, proliferation index, vascularization, platelet and macrophage content, and pulmonary metastases vs untreated controls. These findings confirm that sustained and moderate pharmacological platelet count reduction is feasible with THPO-ASO administration and can delay progression of certain platelet-dependent pathological processes within a safe hemostatic platelet count range.

Background and Purpose: High blood pressure increases bleeding risk during treatment with antithrombotic medication. The association between blood pressure levels and the risk of recurrent stroke during long-term secondary stroke prevention with thienopyridines (particularly prasugrel) has not been well studied. Methods: This was a post hoc analysis of the randomized, double-blind, multicenter PRASTRO-I trial (Comparison of Prasugrel and Clopidogrel in Japanese Patients With Ischemic Stroke-I). Patients with noncardioembolic stroke were randomly assigned (1:1) to receive prasugrel 3.75 mg/day or clopidogrel 75 mg/day for 96 to 104 weeks. Risks of any ischemic or hemorrhagic stroke, combined ischemic events, and combined bleeding events were determined based on the mean level and visit-to-visit variability, including successive variation, of systolic blood pressure (SBP) throughout the observational period. These risks were also compared between quartiles of mean SBP level and successive variation of SBP. Results: A total of 3747 patients (age 62.1±8.5 years, 797 women), with a median average SBP level during the observational period of 132.5 mm Hg, were studied. All the risks of any stroke (146 events; hazard ratio, 1.318 [95% CI, 1.094–1.583] per 10-mm Hg increase), ischemic stroke (133 events, 1.219 [1.010–1.466]), hemorrhagic stroke (13 events, 3.247 [1.660–6.296]), ischemic events (142 events, 1.219 [1.020–1.466]), and bleeding events (47 events, 1.629 [1.172–2.261]) correlated with increasing mean SBP overall. Similarly, an increased risk of these events correlated with increasing successive variation of SBP (hazard ratio, 3.078 [95% CI, 2.220–4.225] per 10-mm Hg increase; 3.051 [2.179–4.262]; 3.276 [1.172–9.092]; 2.865 [2.042–4.011]; 2.764 [1.524–5.016], respectively). Event rates did not differ between the clopidogrel and prasugrel groups within each quartile of SBP or successive variation of SBP. Conclusions: Both high mean SBP level and high visit-to-visit variability in SBP were significantly associated with the risk of recurrent stroke during long-term medication with either prasugrel or clopidogrel after stroke. Control of hypertension would be important regardless of the type of antiplatelet drugs. Registration: URL: https://www.clinicaltrials.jp ; Unique identifier: JapicCTI-111582.

Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-β depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-β signaling, thus regulating normal lung development.

Prasugrel, a novel P2Y12 receptor antagonist, has been shown to be more effective than clopidogrel for preventing cardiovascular events in patients with acute coronary syndromes undergoing percutaneous coronary intervention. We investigated the dose–response antiplatelet effects of prasugrel compared with clopidogrel in Japanese patients with non-cardioembolic stroke. The influence of cytochrome P450 (CYP) polymorphisms on the antiplatelet effects of both drugs was also compared. In this multicenter randomized active-control comparative study, patients were randomized to receive prasugrel 2.5 mg, 5 mg, or 7.5 mg (double blind) or clopidogrel 75 mg (open label) once daily for 14 days. The primary endpoint was inhibition of platelet aggregation (IPA) in response to adenosine diphosphate 20 μM within 8 h of study drug administration on day 14. Of the 66 patients randomized, data from 63 (prasugrel 2.5 mg, 5 mg, and 7.5 mg groups, n = 14, 16, and 18, respectively; clopidogrel group, n = 15) were used in the pharmacodynamic assessment. IPA (arithmetic mean ± SD) after prasugrel administration increased dose-dependently (33 ± 9%, 44 ± 11%, and 53 ± 14%, at 2.5 mg, 5 mg, and 7.5 mg, respectively) and was higher in these groups than after clopidogrel (23 ± 16%). In a subgroup of CYP2C19 intermediate metabolizers, IPA was higher in the prasugrel 5 mg and 7.5 mg groups than in the clopidogrel group. No death or serious adverse events were reported. Prasugrel was well tolerated at doses up to 7.5 mg/day and had antiplatelet effects higher than those of clopidogrel 75 mg/day. CYP2C19 polymorphisms may have reduced clopidogrel-induced IPA. Electronic supplementary material The online version of this article (10.1007/s11239-019-01926-6) contains supplementary material, which is available to authorized users.

Cholestatic liver injury leads to liver dysfunction. The available evidence suggests that platelets can either promote or reduce liver injury and fibrosis. This study focused on the functions of the C-type lectin-like receptor 2 (CLEC-2), a new special platelet receptor that binds with podoplanin-activating platelets. The role of CLEC-2 and podoplanin in cholestatic liver injury was investigated. Mice were injected intraperitoneally with weekly doses of anti-CLEC-2 antibody (2A2B10) to achieve effective CLEC-2 inhibition in their platelets. Next, left and middle hepatic bile duct ligation (BDL) procedures were performed, and mice were euthanized 1 week later (2A2B10-BDL group). In addition, mice were prepared for control groups, and relevant histological and laboratory variables were compared among these groups. The inhibition of CLEC-2 resulted in increasing hepatocellular necrosis, hepatic inflammation, and liver fibrosis. In addition, podoplanin was strongly expressed in hepatic sinusoidal endothelial cells in BDL-treated mice. Moreover, in 2A2B10-BDL mice, total plasma bile acid levels were significantly increased. In summary, podoplanin is expressed on hepatic sinusoidal endothelial cells upon BDL. Platelets bind with podoplanin via CLEC-2 and become activated. As a result, the total bile acid pool is decreased. Therefore, the CLEC-2-podoplanin interaction promotes liver protection and inhibits liver fibrosis after cholestatic liver injury.

Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.

The third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events is unknown. We hypothesized that ponatinib produces an endothelial angiopathy involving excessive endothelial-associated von Willebrand factor (VWF) and secondary platelet adhesion. In wild-type mice and ApoE−/− mice on a Western diet, ultrasound molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Ibα was performed to quantify endothelial-associated VWF and platelet adhesion. After treatment of wild-type mice for 7 days, aortic molecular signal for endothelial-associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham therapy (P < .001), whereas dasatinib had no effect. In ApoE−/− mice, aortic VWF and platelet signals were two- to fourfold higher for ponatinib-treated compared with sham-treated mice (P < .05) and were significantly higher than in treated wild-type mice (P < .05). Platelet and VWF signals in ponatinib-treated mice were significantly reduced by N-acetylcysteine and completely eliminated by recombinant ADAMTS13. Ponatinib produced segmental left ventricular wall motion abnormalities in 33% of wild-type and 45% of ApoE−/− mice and corresponding patchy perfusion defects, yet coronary arteries were normal on angiography. Instead, a global microvascular angiopathy was detected by immunohistochemistry and by intravital microscopy observation of platelet aggregates and nets associated with endothelial cells and leukocytes. Our findings reveal a new form of vascular toxicity for the TKI ponatinib that involves VWF-mediated platelet adhesion and a secondary microvascular angiopathy that produces ischemic wall motion abnormalities. These processes can be mitigated by interventions known to reduce VWF multimer size.

Essentials The role of C-type lectin-like receptor-2 (CLEC-2) in cancer progression is unclear. CLEC-2-depleted mouse model is generated by using a rat anti-mouse CLEC-2 monoclonal antibody. CLEC-2 depletion inhibits hematogenous tumor metastasis of podoplanin-expressing B16F10 cells. CLEC-2 depletion prolongs cancer survival by suppressing thrombosis and inflammation. SummaryBackground C-type lectin-like receptor 2 (CLEC-2) is a platelet activation receptor of sialoglycoprotein podoplanin, which is expressed on the surface of certain types of tumor cells. CLEC-2–podoplanin interactions facilitate hematogenous tumor metastasis. However, direct evidence of the role of CLEC-2 in hematogenous metastasis and cancer progression is lacking. Objective and methods We generated immunological CLEC-2-depleted mice by using anti-mouse CLEC-2 monoclonal antibody 2A2B10 and investigated whether CLEC-2 promoted hematogenous tumor metastasis and tumor growth and exacerbated the prognosis of mice bearing podoplanin-expressing B16F10 melanoma cells. Results Our results showed that hematogenous metastasis was significantly inhibited in CLEC-2-depleted mice. B16F10 cells co-cultured with wild-type platelets, but not with CLEC-2-deficient platelets, showed increased proliferation. However, B16F10 cell proliferation was not inhibited in CLEC-2-depleted mice. Histological analysis showed that thrombus formation in tumor vessels was significantly inhibited and functional vessel density was significantly increased in CLEC-2-depleted mice. These data suggest that CLEC-2 deficiency may inhibit thrombus formation in tumor vessels and increase the density of functional vessels, thus improving oxygen and nutrient supply to tumors, indirectly promoting tumor proliferation. Furthermore, the overall survival of CLEC-2-depleted mice was significantly prolonged, which may be due to the suppression of thrombus formation in the lungs and subsequent inhibition of systemic inflammation and cachexia. Conclusions These data provide a rationale for the targeted inhibition of CLEC-2 as a new strategy for preventing hematogenous tumor metastasis and for inhibiting cancer-related thromboembolism.

Platelets have long been proposed to play a role in fostering neoplasias, ranging from tissue repair to cancer, but clinical evidence remains limited. Since available therapautics antagonizing hemostatic platelet functions lead to bleeding, the concept of targeting platelets in the context of cancer prevention or treatment has not been pursued in controlled clinical trials. To address this, we developed a novel antiplatelet strategy by partial inhibition of thrombopoiesis. We hypothesized that if platelets played a key role in fostering neoplastic malignancies, controlled platelet count reduction within a safe range would slow platelet-dependent cancer progression without adversely affecting hemostasis. Since the MPL ligand megakaryocyte growth and development factor (thrombopoietin, MGDF) is required for maintenance of normal platelet count, and the liver is responsible only for a portion of thrombopoietin synthesis, we tested this hypothesis by targeted knock-down of hepatic thrombopoietin synthesis using an antisense oligonucleotide (ASO) in mammary cancer-prone transgenic mice. We investigated whether partial reduction of thrombopoietin sythesis through ASO knock-down was hemostatically safe and impacted kinetics or penetrance of neoplastic progression in de novo mammary carcinogenesis in MMTV-PyMT mice. We designed and generated thrombopoietin ASO-s (TPO ASO) to achieve about 50% platelet count reduction in mice by inhibition of hepatic thrombopoietin gene expression. Then MMTV-PyMT transgenic mice were subcutaneously administered one of the effective TPO ASO-s, with treatment initiated at 40 days of age and before appearance of palpable (>2.0 mm) mammary tumors. We assessed the impact of saturating dose TPO ASO, including platelet count, plasma TPO level, and number of megakaryocytes in the femoral bone marrow. Mammary tumor growth, pulmonary metastases, and overall survival (fixed end-point study based on total tumor burden) of TPO ASO-treated mice were compared with untreated controls. At study endpoints, mammary carcinomas, platelet deposition, intra-tumoral vessel density, and Ki-67 positive cells were quantitatively evaluated. As anticipated from the drug screen, TPO ASO treatment at saturating effect reduced plasma thrombopoietin levels and blood platelet count by ~50% within 4 weeks, and the number of megakaryocytes in femoral bone marrow. TPO ASO also suppressed primary tumor growth (attached figure) and impaired development of pulmonary metastases (p In summary, we found that TPO ASO reduces thrombopoietin levels and platelet count within the hemostatically safe range and significantly slows neoplastic progression of spontaneous mammary carcinomas in MMTV-PyMT mice. Overall, pharmacological knockdown of hepatic TPO synthesis is a new antiplatelet strategy that may be reasonably safe and effective in various platelet-driven disorders, including cancer. Figure Figure. Disclosures Revenko: Ionis Pharmaceuticals, Inc: Employment, Other: Intellectual property rights. Monia:Ionis Pharmaceuticals, Inc: Employment, Other: Intellectual property rights. Gruber:Aronora, Inc: Employment, Other: Intellectual property rights.

Energy management evolved as a part of industrial automation in the industry plants now clears the wall of the plants, and extends its coverage to the regional and to the global scale by enhancing ICT infrastructure. By connecting intelligent sensors, control devices and mobile terminals to the ubiquitous network, energy demand can be monitored and controlled from anywhere at anytime. This idea extends to a concept of smart grid developed in the power distribution system or to a concept of smart community in the social infrastructure domain. Measurement and control technologies developed in the industrial automation will enable raising energy efficiency of commercial and transportation sectors equivalent to that of the industry sector. Since the oil crisis era in the 70's, Japanese industries have kept growing economically without increasing energy consumption. Measurement and control technologies that have supported such harmonious economic growth now come into the next stage coordinated with ICT technologies, and build the foundations of so-called Green IT. The successful outcome of advanced Green IT is expected to be transferred to the emerging countries especially Asian countries. It will finally contribute to achieving sustainable low carbon society worldwide.

A puerperant woman, who was previously healthy and had no disease known to affect bone metabolism, experienced lower back pain and lumbar vertebral fractures during lactation. Both bone formation markers and resorption markers were markedly elevated. Bone mineral density of the lumbar spine as measured by dual energy X-ray absorptiometry was extremely low. She stopped lactation through the use of bromocriptine because of the large volume of milk secretion. After treatment with calcitonin injections and the use of a corset, her back pain gradually disappeared. This case appears to be postpregnancy osteoporosis.

Radial force of a centrifugal pump was measured, using a pump which had a device for measuring the radial force and a volute casing with an interchangeable tongue. The authors obtained some knowledge about the following items and discussed them: (1) The effect of the cross-sectional area and shape of volute tongue on the pump characteristic and radial force. (2) The characteristic of dynamic component of radial force. (3) The effect of cavitation on the radial force.