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Introduction: Patients with stroke are at a high risk of recurrence, and although they receive antiplatelet therapies such as clopidogrel for secondary prevention of non-cardioembolic stroke, the recurrence rate remains high. Three phase 3 trials (PRASTRO-I/II/III) were conducted to determine the efficacy of prasugrel in preventing recurrent stroke. Here, we performed an integrated analysis of these studies to confirm the generalizability of the PRASTRO-III findings and to supplement the small sample size of the study. Methods: Patients from PRASTRO-I, PRASTRO-II, and PRASTRO-III with ischemic stroke (large-artery atherosclerosis or small-artery occlusion) and at least one of the following were included: hypertension, dyslipidemia, diabetes mellitus, chronic kidney disease, or ischemic stroke history. The primary efficacy endpoint was the composite incidence of ischemic stroke, myocardial infarction (MI), and death from other vascular causes in the intention-to-treat population. Bleeding events (life-threatening bleeding, major bleeding, and clinically relevant bleeding) were evaluated as the primary safety endpoint. Cumulative incidences and 95% confidence intervals (CIs) were calculated for the study outcomes using the Kaplan-Meier method. Hazard ratios (HRs) and 95% CIs were calculated using the Cox regression model. Results: The data of 2,184, 274, and 230 patients from PRASTRO-I, PRASTRO-II, and PRASTRO-III, respectively, were analyzed (N = 2,688; prasugrel, N = 1,337; clopidogrel, N = 1,351). Stroke at enrollment was classified as large-artery atherosclerosis in 49.3% of patients and small-artery occlusion in 50.7% of patients. The primary efficacy endpoint composite incidence (prasugrel vs. clopidogrel) was 3.4% versus 4.3% (HR: 0.771, 95% CI: 0.522–1.138). The incidence of each component of the primary efficacy endpoint for prasugrel versus clopidogrel was 3.1% (n = 41) versus 4.1% (n = 55) for ischemic stroke, 0.3% (n = 4) versus 0.2% (n = 3) for MI, and no events of death from other vascular causes. For the primary safety endpoint, bleeding events were reported in 6.0% of patients in the prasugrel group versus 5.5% of patients in the clopidogrel group (HR: 1.074, 95% CI: 0.783–1.473). Conclusions: This integrated analysis supports the findings of PRASTRO-III. Prasugrel is a promising treatment that results in a numerical reduction in the composite incidence of ischemic stroke, MI, and death from other vascular causes in patients with ischemic stroke who are at a high risk of stroke recurrence. No major safety issues were observed for prasugrel.

There is increasing evidence that platelets participate in multiple pathophysiological processes other than thrombosis and hemostasis, such as immunity, inflammation, embryonic development, and cancer progression. A recent study revealed that heme (hemin)-activated platelets induce macrophage extracellular traps (METs) and exacerbate rhabdomyolysis-induced acute kidney injury (RAKI); however, how hemin activates platelets remains unclear. Here, we report that both C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) are platelet hemin receptors and are involved in the exacerbation of RAKI. We investigated hemin-induced platelet aggregation in humans and mice, binding of hemin to CLEC-2 and GPVI, the RAKI-associated phenotype in a mouse model, and in vitro MET formation. Using western blotting and surface plasmon resonance, we showed that hemin activates human platelets by stimulating the phosphorylation of SYK and PLCγ2 and directly binding to both CLEC-2 and GPVI. Furthermore, hemin-induced murine platelet aggregation was partially reduced in CLEC-2–depleted and FcRγ-deficient (equivalent to GPVI-deficient) platelets and almost completely inhibited in CLEC-2–depleted FcRγ-deficient (double-knockout) platelets. In addition, hemin-induced murine platelet aggregation was inhibited by the CLEC-2 inhibitor cobalt hematoporphyrin or GPVI antibody (JAQ-1). Renal dysfunction, tubular injury, and MET formation were attenuated in double-knockout RAKI mice. Furthermore, in vitro MET formation assay showed that the downstream signaling pathway of CLEC-2 and GPVI is involved in MET formation. We propose that both CLEC-2 and GPVI in platelets play an important role in RAKI development.

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.

We studied the relation between time perspective and commitment formation. These concepts are part of a complex dynamic system, in which individual transitional experiences are relevant. Individual development may proceed along different pathways. Based on theory and research we expected that this relation will firstly differ between individuals, and secondly may change within an individual with increasing age during adulthood. We expected stronger within-individual correlations in emerging adulthood than in adulthood because emerging adults have to meet the demands of a transition from school to job places while adult participants in general are either settled in a more stable life pattern with work and their own family or experience idiosyncratic life transitions. For 48 individuals we collected time series of 21 yearly measurements between age 20 and age 40. We found strong differences among these individuals in the within-individual relation between a balanced time perspective and commitment formation. As expected, the relationship in emerging adults was stronger than in later ages. These findings strengthen the notion, based on the concept of non-ergodicity, that if we want to gain knowledge about individual development, we should focus on within-individual longitudinal relations.

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 Erythropoiesis is a complex multistep process by which erythrocytes are produced. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor almost exclusively expressed on the surface of platelets and megakaryocytes. Deletion of megakaryocyte/platelet CLEC-2 was reported to cause anemia along with thrombocytopenia in mice. PDPN-expressing stromal cells in the bone marrow (BM) were also reported to facilitate megakaryocyte expansion and maturation depending on the CLEC-2/PDPN interaction. Objectives We investigated how specific deletion of CLEC-2 in megakaryocytes/platelets leads to anemia. Methods We used flow cytometry to analyze maturation of erythroblasts, apoptotic cell death, and cell cycle distribution. CLEC-2 stimulated PDPN-expressing stromal cell-conditioned medium was analyzed by cytokine array and ELISA, and co-cultured with immature erythroblasts. Cytokine levels in serum and BM extracellular fluid were quantified by ELISA. Results We observed increased apoptosis of BM erythroblasts in megakaryocyte/platelet-specific CLEC-2 conditional knockout (Clec1bΔPLT ) mice. Moreover, PDPN-expressing stromal cells in the BM secreted insulin-like growth factor 1 (IGF-1) depending on the CLEC-2/PDPN interaction. Pretreatment with IGF-1 receptor inhibitor increased apoptosis rate and decreased the proliferation of erythroblasts in vitro. Furthermore, in Clec1bΔPLT mice, IGF-1 concentrations in serum and BM extracellular fluid were decreased, and IGF-1 replacement in Clec1bΔPLT mice attenuated anemia. Conclusions Our findings suggest that IGF-1 secretion from PDPN-expressing stromal cells by CLEC-2 stimulation positively regulates erythroblasts. This novel mechanism of erythropoiesis regulation indicates that a microenvironment consisting of megakaryocytes and PDPN-expressing stromal cells supports erythropoiesis.

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.

Tumor metastasis is the major cause of deaths in cancer patients and is modulated by intertwined stress-responsive signaling cascades. Here we demonstrate that deletion of stress-responsive apoptosis signal-regulating kinase 1 (Ask1) in platelets results in unstable hemostasis and drastic attenuation of tumor lung metastasis, both of which are attributable to platelet dysfunction. Platelet-specific deletion of Ask1 in mice leads to defects in ADP-dependent platelet aggregation, unstable hemostasis and subsequent attenuation of tumor metastasis. We also revealed that activating phosphorylation of Akt is attenuated in Ask1-deficient platelets, contrary to the previous reports suggesting that Akt is negatively regulated by ASK1. Mechanistically, ASK1-JNK/p38 axis phosphorylates an ADP receptor P2Y12 at Thr345, which is required for the ADP-dependent sustained Akt activity that is vital to normal platelet functions. Our findings offer insight into positive regulation of Akt signaling through P2Y12 phosphorylation as well as MAPK signaling in platelets by ASK1 and suggest that ASK1-JNK/p38 axis provides a new therapeutic opportunity for tumor metastasis.

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.

Megakaryopoiesis is the hierarchical differentiation of hematopoietic stem cells into megakaryocytes. Differentiating megakaryocytes undergo maturation characterized by endomitosis and produce numerous platelets through proplatelet formation. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor mainly expressed on platelets and megakaryocytes. Deletion of platelet/megakaryocyte CLEC-2 causes thrombocytopenia in mice; however, its contribution to megakaryopoiesis remains unknown. Here, we show that megakaryopoiesis is promoted through the CLEC-2/PDPN interaction in the vicinity of arterioles in the bone marrow (BM). We have also identified PDPN-expressing BM arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells. Platelet/megakaryocyte-specific CLEC-2 conditional knockout (cKO) mice showed a decrease in the number of immature megakaryocytes. CLEC-2 wild-type megakaryocyte expansion was augmented in vitro by the addition of recombinant PDPN, but not cKO megakaryocytes. Moreover, megakaryocyte colonies were colocalized with periarteriolar BM FRC-like cells in the BM. Coculture of megakaryocytes with BM FRC-like cells augmented megakaryocyte expansion, which was dependent upon the CLEC-2/PDPN interaction. Furthermore, we found that the CLEC-2/PDPN interaction induces BM FRC-like cells to secrete chemokine (C-C motif) ligand 5 (CCL5) to facilitate proplatelet formation. These observations indicate that a reciprocal interaction between CLEC-2 on megakaryocytes and PDPN on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM.

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.

This study explores how identity development relates to time beliefs in the transition to adulthood. Time belief was evaluated by delay of gratification, unconcern for the future, and present-mindfulness. Longitudinal data (N = 232) were analyzed at ages 24, 27, and 30 using structural equation modeling. Commitment was positively related to delay of gratification and negatively to unconcern for the future, and exploration was positively related to present-mindfulness. These results suggest that a future-oriented attitude, including delay of gratification and concern for the future, as well as openness to experiences provided by present-mindfulness, contribute to the development of identity in the transition to young adulthood.

This study explored how sharing past and future life events among late adolescents and their parents influenced the quality of their own time perspectives. Triads ( N =104) of female students and their parents described three important life events from their past and future. The results showed that adolescents who shared past and future life events predicted a positive impact on their time perspective, whereas parents displayed a more complex dynamism that included the impact of sharing the past on the future and vice versa. It is suggested that the impact of sharing past and future life events varies according to the age group.

The platelet activation receptor C-type lectin-like receptor 2 (CLEC-2) interacts with podoplanin on the surface of certain types of tumor cells, and this interaction facilitates tumor metastasis. CLEC-2 is also involved in thrombus formation and its stabilization. Because CLEC-2–depleted mice are protected from experimental lung metastasis and thrombus formation and do not show increased bleeding time, CLEC-2 may serve as a good target for antimetastatic or antithrombotic drugs. We screened 6770 compounds for their capability to inhibit CLEC-2–podoplanin binding using an enzyme-linked immunosorbent assay. In the first screening round, 63 compounds were identified and further evaluated by flow cytometry using CLEC-2–expressing cells. We identified protoporphyrin IX (H2-PP) as the most potent inhibitor and modified its hematoporphyrin moiety to be complexed with cobalt (cobalt hematoporphyrin [Co-HP]), which resulted in an inhibitory potency much stronger than that of H2-PP. Surface plasmon resonance analysis and molecular docking study showed that Co-HP binds directly to CLEC-2 at N120, N210, and K211, previously unknown podoplanin-binding sites; this binding was confirmed by analysis of CLEC-2 mutants with alterations in N120 and/or K211. Co-HP at a concentration of 1.53 μM inhibited platelet aggregation mediated through CLEC-2, but not that mediated through other receptors. IV administration of Co-HP to mice significantly inhibited hematogenous metastasis of podoplanin-expressing B16F10 cells to the lung as well as in vivo arterial and venous thrombosis, without a significant increase in tail-bleeding time. Thus, Co-HP may be a promising molecule for antimetastatic and antiplatelet treatment that does not cause bleeding tendency.

C-type lectin-like receptor 2 (CLEC-2) has been identified on the surface of platelets as a receptor for a platelet activating snake venom, rhodocytin/aggretin. CLEC-2 belongs to a C-type lectin superfamily and binds to a sialoglycoprotein, podoplanin, in vivo. Platelets play a crucial role in hemostasis and thrombosis, but recent studies have uncovered multiple roles of platelets beyond hemostasis in physiology and pathology. The interaction between platelet CLEC-2 and podoplanin is the key to several roles of platelets beyond hemostasis. The spatial and temporal expression patterns of podoplanin regulate vascular/lymphatic development, maintenance of vascular integrity, tissue regeneration, and some pathological processes including tumor metastasis and thromboinflammation. CLEC-2 facilitates blood/lymphatic vessel separation during embryonic development by binding to podoplanin on lymphatic endothelial cells. The leakage of platelets from hyperpermeable vessels for maintaining vascular integrity during inflammation depends on CLEC-2. During wound healing, the expression of podoplanin in keratinocytes is upregulated, which helps in the process. Podoplanin is expressed on the surface of tumor cells and facilitates hematogenous metastasis by inducing platelet aggregation through CLEC-2. During thrombotic processes, such as development of deep vein thrombosis, podoplanin is upregulated on unknown cells in the vessel wall in the area of inflammation, facilitates thrombus formation, and promotes further inflammation by binding to CLEC-2. In this article, the roles of platelets beyond hemostasis are comprehensively reviewed.

Essentials We generated recombinant rhodocytin that could aggregate platelets via CLEC-2. Recombinant wild-type rhodocytin formed heterooctamer with four α- and β-subunits. Asp 4 in α-subunit of rhodocytin was required for binding to CLEC-2. Inhibitory mutant of rhodocytin blocked podoplanin-dependent hematogenous metastasis. Summary Background Rhodocytin, a disulfide-linked heterodimeric C-type lectin from Calloselasma rhodostoma consisting of α-subunits and β-subunits, induces platelet aggregation through C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation. Objective To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti-CLEC-2 drug based on the findings. Methods We used Chinese hamster ovary cells to express recombinant rhodocytin (wild-type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC-2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN-induced metastasis in an experimental lung metastasis mouse model. Results Functional WT] rhodocytin (αWTβWT) was obtained by coexpression of both subunits. Asp4 in α-subunits of rhodocytin was required for CLEC-2 binding. αWTβWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTβK53A/R56A), forming a heterotetramer, bound to CLEC-2 without inducing platelet aggregation, and blocked CLEC-2-PDPN interaction-dependent platelet aggregation and experimental lung metastasis. Conclusion These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC-2.

Background: Under normal conditions, endothelial cells (ECs) govern blood flow dynamics including providing a barrier between blood and tissue and regulating platelet aggregation and thrombin generation in the bloodstream. In turn, blood components, primarily platelets and coagulation factors such as thrombin, regulate EC barrier integrity. The breakdown of EC barrier function is a hallmark of a variety of vascular diseases. In sepsis, for example, the dysfunction of vascular ECs has been correlated with poorer outcomes due to hemorrhage and multi-organ failure associated with consumption of platelets and coagulation factors into clots within the microcirculation, a condition termed disseminated intravascular coagulation (DIC). Aim: Develop an endothelialized flow chamber to study the platelet-endothelium interface. Methods and Results: We developed a 3D-chamber with a perfuseable cylindrical microvessel embedded in an extracellular matrix (ECM) material. This model allows for the study of the role of thrombin generation and platelet aggregation in endothelial barrier leak development and repair in healthy as well as inflamed microvessels. Incorporation of subendothelial matrix proteins in these 3D-microvessel devices expands the capacity of the microfluidic studies to investigate blood cell extravasation and enables the control of physical parameters such as transmural pressure and interstitial flow through the ECM. Conclusion: This model may provide insight into the pathophysiology of different disease states and serve as an expedient platform for therapy design and testing. The platelet-endothelium interface under shear flow. Diagram ( A ) and an experimental prototype ( B ) of a 3D-perfuseable device. Microvessel phenotype (following treatment with vehicle or 10 ng/mL TNFα) pre- and post- perfusion with recalcified whole blood for 33 min as visualized by differential interference contrast, DIC, ( C ) and fluorescence microscopy ( D ).

Background: ADAMTS13 cleaves and inactivates von Willebrand factor (VWF), which binds collagen, facilitating platelet adhesion under vascular injury. But is still uncertain how ADAMTS13 activity is regulated. Thrombin and plasmin have been shown to cleave ADAMTS13. Based on the fact that elevated levels of FXI is an independent risk factor for deep vein thrombosis and ischemic stroke, we hypothesize that FXIa inactivates ADAMTS13 leading to platelet aggregation and thrombus formation. Aim: To determine the functional role of inactivation of ADAMTS13 by FXIa. Methods and Results: Recombinant ADAMTS13 (250 nM) was incubated with FXIa (50 nM) for increasing times (0-3 hours) at 37 o C before being analyzed by western blot using an anti-ADAMTS13 antibody against the two CUB domains (C-terminal) or against the metalloproteinase (MET) domain (N-terminal). Our results show that FXIa caused the disappearance of the ADAMTS13 band (~200 kDa) and the appearance of a band at ~150 kDa when the samples were analyzed with the anti-MET antibody and a ~50 kDa band when the samples were analyzed with the anti-CUB antibody. The presence of aprotinin, which inhibits FXIa activity, blocked the degradation of ADAMTS13. Kallikrein or FXIIa were unable to cleave ADAMTS13. Using a cell surface immunoassay we observed that after incubation with FXIa, the detection of the CUB domain from ADAMTS13 was lost from endothelial cells surface. The incubation of ADAMTS13 with FXIa caused an increase in ADAMTS13 activity as measured by a fluorogenic substrate (FRETS). Conclusion: ADAMTS13 circulates in a closed conformation, which is maintained by a CUB-spacer domain binding interaction. ADAMTS13 becomes conformationally activated through interaction of its CUBs domains with VWF. Here we show that FXIa-mediated deletion of ADAMTS13-CUB domains enhances its capacity to cleave FRETS and blocks the interaction with VWF. Our results suggest that FXIa may limit ADAMTS13-mediated VWF inactivation.

During the developmental stages, when the primary lymph sac arises from the cardinal vein, podoplanin in lymphatic endothelial cells (LECs) activates platelets by binding to CLEC-2, which facilitates blood/lymphatic vessel separation. One of the mechanisms for this process is that granule contents from activated platelets inhibit the migration and proliferation of LECs, thereby facilitating separation of the vessels. Angiogenesis consists of several steps, including activation of endothelial cells (ECs), migration to the site of the vessel branch, proliferation, and formation of tube-like structures. Granule contents released from activated platelets, including VEGF and FGF, are required for EC proliferation. On the other hand, direct contact between platelets and ECs via platelet membrane proteins, including GPIbα and αIIbβ3, prompts angiogenesis by stabilizing developing vessels. Platelets also play a crucial role in maintaining the vascular integrity of high endothelial venules in lymph nodes and inflammatory vessels via the podoplanin receptor CLEC-2 and the collagen receptor glycoprotein VI. Platelets also regulate cerebrovascular patterning and integrity during development via CLEC-2 and podoplanin in neuroepithelial cells. Thus, platelets play a role, not only in thrombosis and hemostasis, but also in lymphangiogenesis, angiogenesis, and maintenance of vascular integrity.

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.

Essentials Regeneration role of C-type lectin receptor-2 (CLEC-2) after 70% hepatectomy (HPx) was investigated. Wild-type or CLEC-2 deleted from platelets of chimeric mice (flKO) underwent HPx. The liver/body weight ratio was significantly lower in the flKO than in the wild-type. CLEC-2 plays an essential role in liver regeneration after HPx. SummaryBackground and aim The aim of the present study was to investigate the role of C-type lectin receptor (CLEC)-2 in liver regeneration following partial liver resection in mice. Materials and methods Irradiated chimeric mice transplanted with fetal liver cells from wild-type (WT) mice, CLEC-2-deleted (KO) mice or mice with CLEC-2 deleted specifically from platelets (flKO) were generated. Mice underwent 70% partial hepatectomy (PH). Immunohistochemical staining was performed to investigate the expression of the endogenous ligand for CLEC-2, podoplanin. The accumulation of platelets in the liver was also quantified. The hepatic expression of the IL-6/gp130 and STAT3, Akt and ERK1/2 was also examined. Results The liver/body weight ratio and expression of all cell proliferation markers were significantly lower in the flKO group than in the WT group. The expression of phosphorylated (p) Akt and pERK1/2 was similar in the WT and flKO groups. On the other hand, the expression of pSTAT3 and IL-6 was significantly stronger in the WT group than in the flKO group. The expression of podoplanin was detected in the hepatic sinusoids of both groups. However, the extent to which platelets accumulated in hepatic sinusoids was significantly less in the flKO group than in the WT group. Conclusion CLEC-2 was involved in hepatic regeneration after liver resection and CLEC-2-related liver regeneration was attributed to the interaction between platelets and sinusoidal endothelial cells.

The platelet receptor CLEC-2 is involved in thrombosis/hemostasis, but its ligand, podoplanin, is expressed only in advanced atherosclerotic lesions. We investigated CLEC-2 ligands in vessel walls. Recombinant CLEC-2 bound to early atherosclerotic lesions and normal arterial walls, co-localizing with vascular smooth muscle cells (VSMCs). Flow cytometry and immunocytochemistry showed that recombinant CLEC-2, but not an anti-podoplanin antibody, bound to VSMCs, suggesting that CLEC-2 ligands other than podoplanin are present in VSMCs. VSMCs stimulated platelet granule release and supported thrombus formation under flow, dependent on CLEC-2. The time to occlusion in a FeCl3-induced animal thrombosis model was significantly prolonged in the absence of CLEC-2. Because the internal elastic lamina was lacerated in our FeCl3-induced model, we assume that the interaction between CLEC-2 and its ligands in VSMCs induces thrombus formation. Protein arrays and Biacore analysis were used to identify S100A13 as a CLEC-2 ligand in VSMCs. However, S100A13 is not responsible for the above-described VSMC-induced platelet activation, because S100A13 is not expressed on the surface of normal VSMCs. S100A13 was released upon oxidative stress and expressed in the luminal area of atherosclerotic lesions. Suspended S100A13 did not activate platelets, but immobilized S100A13 significantly increased thrombus formation on collagen-coated surfaces. Taken together, we proposed that VSMCs stimulate platelets through CLEC-2, possibly leading to thrombus formation after plaque erosion and stent implantation, where VSMCs are exposed to blood flow. Furthermore, we identified S100A13 as one of the ligands on VSMCs.