Sex-related differences in the response of anti-platelet drug therapies targeting purinergic signaling pathways in sepsis.

Sepsis, a complex clinical syndrome resulting from a serious infection, is a major healthcare problem associated with high mortality. Sex-related differences in the immune response to sepsis have been proposed but the mechanism is still unknown. Purinergic signaling is a sex-specific regulatory mechanism in immune cell physiology. Our studies have shown that blocking the ADP-receptor P2Y12 but not P2Y1 receptor was protective in male mice during sepsis, but not female. We now hypothesize that there are sex-related differences in modulating P2Y12 or P2Y1 signaling pathways during sepsis. Male and female wild-type (WT), P2Y12 knock-out (KO), and P2Y1 KO mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. The P2Y12 antagonist ticagrelor or the P2Y1 antagonist MRS2279 were administered intra-peritoneally after surgery to septic male and female mice. Blood, lungs and kidneys were collected 24 hours post-surgery. Sepsis-induced changes in platelet activation, secretion and platelet interaction with immune cells were measured by flow cytometry. Neutrophil infiltration in the lung and kidney was determined by a myeloperoxidase (MPO) colorimetric assay kit. Sepsis-induced platelet activation, secretion and aggregate formation were reduced in male CLP P2Y12 KO and in female CLP P2Y1 KO mice compared with their CLP WT counterpart. Sepsis-induced MPO activity was reduced in male CLP P2Y12 KO and CLP P2Y1 KO female mice. CLP males treated with ticagrelor or MRS2279 showed a decrease in sepsis-induced MPO levels in lung and kidneys, aggregate formation, and platelet activation as compared to untreated male CLP mice. There were no differences in platelet activation, aggregate formation, and neutrophil infiltration in lung and kidney between female CLP mice and female CLP mice treated with ticagrelor or MRS2279. In human T lymphocytes, blocking P2Y1 or P2Y12 alters cell growth and secretion in vitro in a sex-dependent manner, supporting the data obtained in mice. In conclusion, targeting purinergic signaling represents a promising therapy for sepsis but drug targeting purinergic signaling is sex-specific and needs to be investigated to determine sex-related targeted therapies in sepsis.

D121 Located within the DRY Motif of P2Y12 Is Essential for P2Y12-Mediated Platelet Function.

Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. Rap1b activity was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To explore this further, we produced knock-in mice that mimic our subject. Bleeding failed to cease in homozygous KI mice during tail bleeding assays, while tail bleeding times did not differ between WT and heterozygous KI mice. Furthermore, occlusions failed to form in most homozygous KI mice following carotid artery injury via FeCl3. These data indicate that the aspartic acid residue found in the DRY motif of P2Y12 is essential for P2Y12 function.

Evidence for a PI3-kinase independent pathway in the regulation of Rap1b activation downstream of the P2Y12 receptor in platelets.

Platelet activation by adenosine diphosphate (ADP) is mediated through two G-protein-coupled receptors, P2Y1 and P2Y12, which signal through Gq and Gi, respectively. P2Y1 stimulation leads to phospholipase C activation and an increase in cytosolic calcium necessary for CalDAG-GEF1 activation. Engagement of P2Y12 inhibits adenylate cyclase, which reduces cAMP, and activation of PI3-kinase, which inhibits RASA3 resulting in sustained activated Rap1b. In this study we activated human platelets with 2-MeSADP in the presence of LY294002, a PI3-kinase inhibitor, AR-C69931MX, a P2Y12 antagonist or MRS2179, a P2Y1 antagonist. We measured the phosphorylation of Akt on Ser473 as an indicator of PI3-kinase activity. As previously shown, LY294002 and ARC69931MX abolished 2MeSADP-induced Akt phosphorylation. MRS2179 reduced ADP-induced Akt phosphorylation but did not abolish it. Rap1b activation, however, was only reduced, but not ablated, using LY294002 and was completely inhibited by ARC69931MX or MRS2179. Furthermore, 2MeSADP-induced Rap1b activation was abolished in either P2Y1 or P2Y12 null platelets. These data suggest that ADP-induced Rap1b activation requires both P2Y1 and P2Y12. In addition, although stimulation of P2Y12 results in PI3-kinase activation leading to Akt phosphorylation and Rap1b activation, Rap1b activation can occur independently of PI3-kinase downstream of P2Y12. Thus, we propose that the P2Y12 receptor can regulate Rap1b, possibly through RASA3, in a pathway independent of PI3-kinase.

Aorta in Pathologies May Function as an Immune Organ by Upregulating Secretomes for Immune and Vascular Cell Activation, Differentiation and Trans-Differentiation-Early Secretomes may Serve as Drivers for Trained Immunity.

To determine whether aorta becomes immune organ in pathologies, we performed transcriptomic analyses of six types of secretomic genes (SGs) in aorta and vascular cells and made the following findings: 1) 53.7% out of 21,306 human protein genes are classified into six secretomes, namely, canonical, caspase 1, caspase 4, exosome, Weibel-Palade body, and autophagy; 2) Atherosclerosis (AS), chronic kidney disease (CKD) and abdominal aortic aneurysm (AAA) modulate six secretomes in aortas; and Middle East Respiratory Syndrome Coronavirus (MERS-CoV, COVID-19 homologous) infected endothelial cells (ECs) and angiotensin-II (Ang-II) treated vascular smooth muscle cells (VSMCs) modulate six secretomes; 3) AS aortas upregulate T and B cell immune SGs; CKD aortas upregulate SGs for cardiac hypertrophy, and hepatic fibrosis; and AAA aorta upregulate SGs for neuromuscular signaling and protein catabolism; 4) Ang-II induced AAA, canonical, caspase 4, and exosome SGs have two expression peaks of high (day 7)-low (day 14)-high (day 28) patterns; 5) Elastase induced AAA aortas have more inflammatory/immune pathways than that of Ang-II induced AAA aortas; 6) Most disease-upregulated cytokines in aorta may be secreted via canonical and exosome secretomes; 7) Canonical and caspase 1 SGs play roles at early MERS-CoV infected ECs whereas caspase 4 and exosome SGs play roles in late/chronic phases; and the early upregulated canonical and caspase 1 SGs may function as drivers for trained immunity (innate immune memory); 8) Venous ECs from arteriovenous fistula (AVF) upregulate SGs in five secretomes; and 9) Increased some of 101 trained immunity genes and decreased trained tolerance regulator IRG1 participate in upregulations of SGs in atherosclerotic, Ang-II induced AAA and CKD aortas, and MERS-CoV infected ECs, but less in SGs upregulated in AVF ECs. IL-1 family cytokines, HIF1α, SET7 and mTOR, ROS regulators NRF2 and NOX2 partially regulate trained immunity genes; and NRF2 plays roles in downregulating SGs more than that of NOX2 in upregulating SGs. These results provide novel insights on the roles of aorta as immune organ in upregulating secretomes and driving immune and vascular cell differentiations in COVID-19, cardiovascular diseases, inflammations, transplantations, autoimmune diseases and cancers.

29 m6A-RNA Methylation (Epitranscriptomic) Regulators Are Regulated in 41 Diseases including Atherosclerosis and Tumors Potentially via ROS Regulation - 102 Transcriptomic Dataset Analyses.

We performed a database mining on 102 transcriptomic datasets for the expressions of 29 m6A-RNA methylation (epitranscriptomic) regulators (m6A-RMRs) in 41 diseases and cancers and made significant findings: (1) a few m6A-RMRs were upregulated; and most m6A-RMRs were downregulated in sepsis, acute respiratory distress syndrome, shock, and trauma; (2) half of 29 m6A-RMRs were downregulated in atherosclerosis; (3) inflammatory bowel disease and rheumatoid arthritis modulated m6A-RMRs more than lupus and psoriasis; (4) some organ failures shared eight upregulated m6A-RMRs; end-stage renal failure (ESRF) downregulated 85% of m6A-RMRs; (5) Middle-East respiratory syndrome coronavirus infections modulated m6A-RMRs the most among viral infections; (6) proinflammatory oxPAPC modulated m6A-RMRs more than DAMP stimulation including LPS and oxLDL; (7) upregulated m6A-RMRs were more than downregulated m6A-RMRs in cancer types; five types of cancers upregulated ≥10 m6A-RMRs; (8) proinflammatory M1 macrophages upregulated seven m6A-RMRs; (9) 86% of m6A-RMRs were differentially expressed in the six clusters of CD4+Foxp3+ immunosuppressive Treg, and 8 out of 12 Treg signatures regulated m6A-RMRs; (10) immune checkpoint receptors TIM3, TIGIT, PD-L2, and CTLA4 modulated m6A-RMRs, and inhibition of CD40 upregulated m6A-RMRs; (11) cytokines and interferons modulated m6A-RMRs; (12) NF-κB and JAK/STAT pathways upregulated more than downregulated m6A-RMRs whereas TP53, PTEN, and APC did the opposite; (13) methionine-homocysteine-methyl cycle enzyme Mthfd1 downregulated more than upregulated m6A-RMRs; (14) m6A writer RBM15 and one m6A eraser FTO, H3K4 methyltransferase MLL1, and DNA methyltransferase, DNMT1, regulated m6A-RMRs; and (15) 40 out of 165 ROS regulators were modulated by m6A eraser FTO and two m6A writers METTL3 and WTAP. Our findings shed new light on the functions of upregulated m6A-RMRs in 41 diseases and cancers, nine cellular and molecular mechanisms, novel therapeutic targets for inflammatory disorders, metabolic cardiovascular diseases, autoimmune diseases, organ failures, and cancers.

A novel role for endoplasmic reticulum protein 46 (ERp46) in platelet function and arterial thrombosis in mice.

Although several members of protein disulfide isomerase (PDI) family support thrombosis, other PDI family members with the CXYC motif remain uninvestigated. ERp46 has 3 CGHC redox-active sites and a radically different molecular architecture than other PDIs. Expression of ERp46 on the platelet surface increased with thrombin stimulation. An anti-ERp46 antibody inhibited platelet aggregation, adenosine triphosphate (ATP) release, and αIIbß3 activation. ERp46 protein potentiated αIIbß3 activation, platelet aggregation, and ATP release, whereas inactive ERp46 inhibited these processes. ERp46 knockout mice had prolonged tail-bleeding times and decreased platelet accumulation in thrombosis models that was rescued by infusion of ERp46. ERp46-deficient platelets had decreased αIIbß3 activation, platelet aggregation, ATP release, and P-selectin expression. The defects were reversed by wild-type ERp46 and partially reversed by ERp46 containing any of the 3 active sites. Platelet aggregation stimulated by an αIIbß3-activating peptide was inhibited by the anti-ERp46 antibody and was decreased in ERp46-deficient platelets. ERp46 bound tightly to αIIbß3 by surface plasmon resonance but poorly to platelets lacking αIIbß3 and physically associated with αIIbß3 upon platelet activation. ERp46 mediated clot retraction and platelet spreading. ERp46 more strongly reduced disulfide bonds in the ß3 subunit than other PDIs and in contrast to PDI, generated thiols in ß3 independently of fibrinogen. ERp46 cleaved the Cys473-Cys503 disulfide bond in ß3, implicating a target for ERp46. Finally, ERp46-deficient platelets have decreased thiols in ß3, implying that ERp46 cleaves disulfide bonds in platelets. In conclusion, ERp46 is critical for platelet function and thrombosis and facilitates αIIbß3 activation by targeting disulfide bonds.

Protease-activated receptor 4 causes Akt phosphorylation independently of PI3 kinase pathways.

PI-3 Kinase plays an important role in platelet activation mainly through regulation of RASA3. Akt phosphorylation is an indicator for the activity of PI3 kinase. The aim of this study is to characterize the pathways leading to Akt phosphorylation in platelets. We performed concentration response curves of LY294002, a pan-PI3 kinase inhibitor, on platelet aggregation and Akt phosphorylation, in washed human and mouse platelets. At concentrations as low as 3.12 µM, LY294002 abolished Akt phosphorylation induced by 2MeSADP and SFLLRN, but not by AYPGKF. It required much higher concentrations of LY294002 (12.5-25 µM) to abolish AYPGKF-induced Akt phosphorylation, both in wild type and P2Y12 null mouse platelets. We propose that 3.12 µM LY294002 is sufficient to inhibit PI3 kinase isoforms in platelets and higher concentrations might inhibit other pathways regulating Akt phosphorylation by AYPGKF. We conclude that Protease-activated receptor 4 (PAR4) might cause Akt phosphorylation through pathways distinctly different from those of Protease-activated receptor 1 (PAR1).

The protein tyrosine phosphatase PTPN7 is a negative regulator of ERK activation and thromboxane generation in platelets.

Protein tyrosine phosphatase nonreceptor type 7 (PTPN7), also called hematopoietic protein tyrosine phosphatase, controls extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase in T lymphocytes. Because ERK1/2 plays an important role in regulating thromboxane A2 (TXA2) generation in platelets, we investigated the function of PTPN7 in these cells. Using immunoblot analysis, we detected PTPN7 in both human and mouse platelets but not in PTPN7-null mice. PTPN7 KO mouse platelets exhibited increased platelet functional responses, including aggregation, dense granule secretion, and TXA2 generation, compared with platelets from WT littermates, upon stimulation with both G protein-coupled receptor (GPCR) and glycoprotein VI (GPVI) agonists. Using the GPCR agonist AYPGKF in the presence of the COX inhibitor indomethacin, we found that PTPN7 KO mouse platelets aggregated and secreted to the same extent as WT platelets, suggesting that elevated TXA2 is responsible for the potentiation of platelet functional responses in PTPN7-KO platelets. Phosphorylation of ERK1/2 was also elevated in PTPN7 KO platelets. Stimulation of platelets with the GPVI agonist collagen-related peptide along with the COX inhibitor indomethacin did not result in phosphorylation of ERK1/2, indicating that GPVI-mediated ERK phosphorylation occurs through TXA2 Although bleeding times did not significantly differ between PTPN7-null and WT mice, time to death was significantly faster in PTPN7-null mice than in WT mice in a pulmonary thromboembolism model. We conclude that PTPN7 regulates platelet functional responses downstream of GPCR agonists, but not GPVI agonists, through inhibition of ERK activation and thromboxane generation.

Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice.

BACKGROUND AND OBJECTIVE: CD45 is a receptor protein tyrosine phosphatase present on the surface of all hematopoietic cells except for erythrocytes and platelets. Proteomics studies, however, have demonstrated the presence of a CD45 c-terminal catalytic peptide in platelets. Therefore, we investigated the functional role of this truncated isoform of CD45 in platelets, which contains the c-terminal catalytic domain but lacks the extracellular region. METHODS AND RESULTS: We used an antibody specific to the c-terminus of CD45 to confirm the presence of a truncated CD45 isoform in platelets. We also examined ex vivo and in vivo platelet function using CD45 knockout (KO) mice. Aggregation and secretion mediated by the glycoprotein VI (GPVI) receptor was impaired in CD45 KO platelets. Consequently, CD45 KO mice had impaired hemostasis indicated by increased tail bleeding times. Also, using a model of pulmonary embolism we showed that CD45 KO mice had defective in vivo thrombus formation. Next, we investigated whether or not the truncated isoform of CD45 had a role in GPVI signaling. The full-length isoform of CD45 is known to regulate Src family kinase (SFK) activation in lymphocytes. We find a similar role for the truncated isoform of CD45 in platelets. SFK activation was impaired downstream of the GPVI receptor in the CD45 KO murine platelets. Consequently, Syk, PLCγ2, and pleckstrin phosphorylations were also impaired in CD45 KO murine platelets. CONCLUSION: We conclude that the truncated CD45 isoform regulates GPVI-mediated signaling and platelet functional responses by regulating SFK activation.

Role of Protein Kinase C-delta in regulating platelet activation and platelet-leukocyte interaction during sepsis.

Sepsis is characterized by an intense systemic inflammatory response activating a cascade of proinflammatory events resulting in leukocyte dysregulation and host tissue damage. The lung is particularly susceptible to systemic inflammation, leading to acute lung injury. Key to inflammation-induced lung damage is the excessive migration of neutrophils across the vascular endothelium. The mechanisms which regulate neutrophil activation and migration in sepsis are not well defined but there is growing evidence that platelets are actively involved and play a key role in microvascular permeability and neutrophil-mediated organ damage. We previously identified PKC-delta (PKCδ) as a critical regulator of the inflammatory response in sepsis and demonstrated PKCδ inhibition was lung protective. However, the role of PKCδ in sepsis-induced platelet activation and platelet-leukocyte interactions is not known. In this study, rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Following surgeries, a PKCδ inhibitor (200µg/kg) or vehicle (PBS) was administered intra-tracheally. At 24 hours post-surgeries, lung tissue, BAL fluid, and blood samples were collected. While sepsis caused thrombocytopenia, the remaining circulating platelets were activated as demonstrated by increased p-selectin expression, elevated plasma PF4, and enhanced platelet-leukocyte aggregate formation compared to Sham animals. Platelet activation was associated with increased platelet PKCδ activity. Inhibition of PKCδ attenuated sepsis-induced platelet activation, secretion and aggregate formation. Sepsis-induced thrombocytopenia was also significantly reduced and circulating platelet numbers were similar to sham animals. In the lung, sepsis induced significant influx of platelets and neutrophils and the development of lung injury. Administration of the PKCδ inhibitor decreased platelet and neutrophil influx, and was lung protective. Thus, PKCδ inhibition modulated platelet activity both locally and systemically, decreased neutrophil influx into the lung, and was lung protective. We demonstrate for the first time that PKCδ plays an important role in platelet activation and platelet-neutrophil interaction during sepsis.

PKC-epsilon deficiency alters progenitor cell populations in favor of megakaryopoiesis.

BACKGROUND: It has long been postulated that Protein Kinase C (PKC) is an important regulator of megakaryopoiesis. Recent contributions to the literature have outlined the functions of several individual PKC isoforms with regard to megakaryocyte differentiation and platelet production. However, the exact role of PKCε remains elusive. OBJECTIVE: To delineate the role of PKCε in megakaryopoiesis. APPROACH AND RESULTS: We used a PKCε knockout mouse model to examine the effect of PKCε deficiency on platelet mass, megakaryocyte mass, and bone marrow progenitor cell distribution. We also investigated platelet recovery in PKCε null mice and TPO-mediated signaling in PKCε null megakaryocytes. PKCε null mice have higher platelet counts due to increased platelet production compared to WT littermate controls (p<0.05, n = 8). Furthermore, PKCε null mice have more bone marrow megakaryocyte progenitor cells than WT littermate control mice. Additionally, thrombopoietin-mediated signaling is perturbed in PKCε null mice as Akt and ERK1/2 phosphorylation are enhanced in PKCε null megakaryocytes stimulated with thrombopoietin. Finally, in response to immune-induced thrombocytopenia, PKCε null mice recovered faster and had higher rebound thrombocytosis than WT littermate control mice. CONCLUSIONS: Enhanced platelet recovery could be due to an increase in megakaryocyte progenitor cells found in PKCε null mice as well as enhanced thrombopoietin-mediated signaling observed in PKCε deficient megakaryocytes. These data suggest that PKCε is a negative regulator of megakaryopoiesis.

Gq pathway regulates proximal C-type lectin-like receptor-2 (CLEC-2) signaling in platelets.

Platelets play a key role in the physiological hemostasis or pathological process of thrombosis. Rhodocytin, an agonist of the C-type lectin-like receptor-2 (CLEC-2), elicits powerful platelet activation signals in conjunction with Src family kinases (SFKs), spleen tyrosine kinase (Syk), and phospholipase γ2 (PLCγ2). Previous reports have shown that rhodocytin-induced platelet aggregation depends on secondary mediators such as thromboxane A2 (TxA2) and ADP, which are agonists for G-protein-coupled receptors (GPCRs) on platelets. How the secondary mediators regulate CLEC-2-mediated platelet activation in terms of signaling is not clearly defined. In this study, we report that CLEC-2-induced Syk and PLCγ2 phosphorylation is potentiated by TxA2 and that TxA2 plays a critical role in the most proximal event of CLEC-2 signaling, i.e. the CLEC-2 receptor tyrosine phosphorylation. We show that the activation of other GPCRs, such as the ADP receptors and protease-activated receptors, can also potentiate CLEC-2 signaling. By using the specific Gq inhibitor, UBO-QIC, or Gq knock-out murine platelets, we demonstrate that Gq signaling, but not other G-proteins, is essential for GPCR-induced potentiation of Syk phosphorylation downstream of CLEC-2. We further elucidated the signaling downstream of Gq and identified an important role for the PLCß-PKCα pathway, possibly regulating activation of SFKs, which are crucial for initiation of CLEC-2 signaling. Together, these results provide evidence for novel Gq-PLCß-PKCα-mediated regulation of proximal CLEC-2 signaling by Gq-coupled receptors.

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

BACKGROUND: Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. METHODS: We measured P2Y12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-κB pathway in regulating P2Y12 receptor expression in megakaryocytes. RESULTS: Platelet P2Y12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y12 expression correlates with ADP-induced platelet aggregation (r=0.89, P<0.01). P2Y12 in platelets from patients with diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, P<0.05). Using a FeCl3-injury mesenteric arteriole thrombosis model in rats and an arteriovenous shunt thrombosis model in rats, we found that the inverse agonist AR-C78511 has greater antithrombotic effects on GK rats with diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P<0.01). We also found that a pathway involving high glucose-reactive oxygen species-nuclear factor-κB increases platelet P2Y12 receptor expression in diabetes mellitus. CONCLUSIONS: Platelet P2Y12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus.

Hyperglycaemia suppresses microRNA expression in platelets to increase P2RY12 and SELP levels in type 2 diabetes mellitus.

Megakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analysed the expression of platelet and circulating miR-223, miR-26b, miR-126 and miR-140 that might be altered with their target mRNAs in type 2 diabetes mellitus (DM2). MiRNAs were isolated from leukocyte-depleted platelets and plasma samples obtained from 28 obese DM2, 19 non-DM obese and 23 healthy individuals. The effect of hyperglycaemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycaemic conditions after 8 hours up to four weeks. Quantitation of mature miRNA, pre-miRNAs and target mRNA levels (P2RY12 and SELP) were measured by RT-qPCR. To prove the association of miR-26b and miR-140 with SELP (P-selectin) mRNA level, overexpression or inhibition of these miRNAs in MEG-01 MKs was performed using mimics or anti-miRNAs, respectively. The contribution of calpain substrate Dicer to modulation of miRNAs was studied by calpain inhibition. Platelet activation was evaluated via surface P-selectin by flow cytometry. Mature and pre-forms of investigated miRNAs were significantly reduced in DM2, and platelet P2RY12 and SELP mRNA levels were elevated by two-fold at increased platelet activation compared to controls. Significantly blunted miRNA expressions were observed by hyperglycaemia in MEG-01 and K562-MK cells versus baseline values, while the manipulation of miR-26b and miR-140 expression affected SELP mRNA level. Calpeptin pretreatment restored miRNA levels in hyperglycaemic MKs. Overall, miR-223, miR-26b, miR-126 and miR-140 are expressed at a lower level in platelets and MKs in DM2 causing upregulation of P2RY12 and SELP mRNAs that may contribute to adverse platelet function.

Chronic Kidney Disease Induces Inflammatory CD40+ Monocyte Differentiation via Homocysteine Elevation and DNA Hypomethylation.

RATIONALE: Patients with chronic kidney disease (CKD) develop hyperhomocysteinemia and have a higher cardiovascular mortality than those without hyperhomocysteinemia by 10-fold. OBJECTIVE: We investigated monocyte differentiation in human CKD and cardiovascular disease (CVD). METHODS AND RESULTS: We identified CD40 as a CKD-related monocyte activation gene using CKD-monocyte -mRNA array analysis and classified CD40 monocyte (CD40+CD14+) as a stronger inflammatory subset than the intermediate monocyte (CD14++CD16+) subset. We recruited 27 patients with CVD/CKD and 14 healthy subjects and found that CD40/CD40 classical/CD40 intermediate monocyte (CD40+CD14+/CD40+CD14++CD16-/CD40+CD14++CD16+), plasma homocysteine, S-adenosylhomocysteine, and S-adenosylmethionine levels were higher in CVD and further elevated in CVD+CKD. CD40 and CD40 intermediate subsets were positively correlated with plasma/cellular homocysteine levels, S-adenosylhomocysteine and S-adenosylmethionine but negatively correlated with estimated glomerular filtration rate. Hyperhomocysteinemia was established as a likely mediator for CKD-induced CD40 intermediate monocyte, and reduced S-adenosylhomocysteine/S-adenosylmethionine was established for CKD-induced CD40/CD40 intermediate monocyte. Soluble CD40 ligand, tumor necrosis factor (TNF)-α/interleukin (IL)-6/interferon (IFN)-γ levels were elevated in CVD/CKD. CKD serum/homocysteine/CD40L/increased TNF-α/IL-6/IFN-γ-induced CD40/CD40 intermediate monocyte in peripheral blood monocyte. Homocysteine and CKD serum-induced CD40 monocyte were prevented by neutralizing antibodies against CD40L/TNF-α/IL-6. DNA hypomethylation was found on nuclear factor-κB consensus element in CD40 promoter in white blood cells from patients with CKD with lower S-adenosylmethionine / S-adenosylhomocysteine ratios. Finally, homocysteine inhibited DNA methyltransferase-1 activity and promoted CD40 intermediate monocyte differentiation, which was reversed by folic acid in peripheral blood monocyte. CONCLUSIONS: CD40 monocyte is a novel inflammatory monocyte subset that appears to be a biomarker for CKD severity. Hyperhomocysteinemia mediates CD40 monocyte differentiation via soluble CD40 ligand induction and CD40 DNA hypomethylation in CKD.

TULA-2 Protein Phosphatase Suppresses Activation of Syk through the GPVI Platelet Receptor for Collagen by Dephosphorylating Tyr(P)346, a Regulatory Site of Syk.

Protein-tyrosine phosphatase TULA-2 has been shown to regulate receptor signaling in several cell types, including platelets. Platelets are critical for maintaining vascular integrity; this function is mediated by platelet aggregation in response to recognition of the exposed basement membrane collagen by the GPVI receptor, which is non-covalently associated with the signal-transducing FcRγ polypeptide chain. Our previous studies suggested that TULA-2 plays an important role in negatively regulating signaling through GPVI-FcRγ and indicated that the tyrosine-protein kinase Syk is a key target of the regulatory action of TULA-2 in platelets. However, the molecular basis of the down-regulatory effect of TULA-2 on Syk activation via FcRγ remained unclear. In this study, we demonstrate that suppression of Syk activation by TULA-2 is mediated, to a substantial degree, by dephosphorylation of Tyr(P)346, a regulatory site of Syk, which becomes phosphorylated soon after receptor ligation and plays a critical role in initiating the process that yields fully activated Syk. TULA-2 is capable of dephosphorylating Tyr(P)346 with high efficiency, thus controlling the overall activation of Syk, but is less efficient in dephosphorylating other regulatory sites of this kinase. Therefore, dephosphorylation of Tyr(P)346 may be considered an important "checkpoint" in the regulation of Syk activation process. Putative biological functions of TULA-2-mediated dephosphorylation of Tyr(P)346 may include deactivation of receptor-activated Syk or suppression of Syk activation by suboptimal stimulation.

Nuclear factor-κB regulates expression of platelet phospholipase C-ß2 (PLCB2).

Phospholipase C (PLC)-ß2 (gene PLCB2) is a critical regulator of platelet responses upon activation. Mechanisms regulating of PLC-ß2 expression in platelets/MKs are unknown. Our studies in a patient with platelet PLC-ß2 deficiency revealed the PLCB2 coding sequence to be normal and decreased platelet PLC-ß2 mRNA, suggesting a defect in transcriptional regulation. PLCB2 5'- upstream region of the patient revealed a heterozygous 13 bp deletion (-1645/-1633 bp) encompassing a consensus sequence for nuclear factor-κB (NF-κB). This was subsequently detected in three of 50 healthy subjects. To understand the mechanisms regulating PLC-ß2, we studied the effect of this variation in the PLCB2. Gel-shift studies using nuclear extracts from human erythroleukaemia (HEL) cells or recombinant p65 showed NF-κB binding to oligonucleotide with NF-κB site; in luciferase reporter studies its deletion reduced PLCB2 promoter activity. PLCB2 expression was decreased by siRNA knockdown of NF-κB p65 subunit and increased by p65 overexpression. By immunoblotting platelet PLC-ß2 in 17 healthy subjects correlated with p65 (r=0.76, p=0.0005). These studies provide the first evidence that NF-κB regulates MK/platelet PLC-ß2 expression. This interaction is important because of the major role of PLC-ß2 in platelet activation and of NF-κB in processes, including inflammation and atherosclerosis, where both are intimately involved.

P2Y12 Receptor Modulates Sepsis-Induced Inflammation.

OBJECTIVE: Platelets modulate hemostasis and immune responses via interactions with immune cells through secretion of immunemodulators and cell-cell interactions. The P2Y12 receptor mediates ADP-induced aggregation and secretion in platelets. APPROACH AND RESULTS: Using a mouse model of intra-abdominal sepsis and acute lung injury, we investigated the role of the P2Y12 receptor in neutrophil migration and lung inflammation in P2Y12 null mice and in mice pretreated with the P2Y12 antagonist clopidogrel. Our data show a decrease in circulating white blood cells and a decrease in platelet activation and platelet-leukocyte interactions in treated mice compared with untreated mice. Additionally, lung injury and platelet sequestration were diminished in clopidogrel-treated mice compared with their untreated septic littermates. Similar results were observed in P2Y12 null mice: platelet activation and platelet-leukocyte aggregates were decreased in septic P2Y12 null mice compared with wild-type mice. P2Y12 null mice were refractory to lung injury compared with wild-type mice. Finally, to evaluate P2Y12-independent effects of clopidogrel, we pretreated P2Y12 null mice. Interestingly, the number of circulating neutrophils was reduced in treated septic P2Y12 null mice, suggesting neutrophils as a target for clopidogrel pleiotropic effects. No difference was observed in P2Y1 null mice during sepsis, indicating that the P2Y12 receptor is responsible for the effects. CONCLUSIONS: P2Y12 null mice are refractory to sepsis-induced lung injury, suggesting a key role for activated platelets and the P2Y12 receptor during sepsis.

Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway.

Interleukin-17 (IL-17)-secreting T helper 17 cells were recently identified as a CD4(+) T helper subset and implicated in various inflammatory and autoimmune diseases. The issues of whether and by what mechanism hyperlipidemic stress induces IL-17A to activate aortic endothelial cells (ECs) and enhance monocyte adhesion remained largely unknown. Using biochemical, immunological, microarray, experimental data mining analysis, and pathological approaches focused on primary human and mouse aortic ECs (HAECs and MAECs) and our newly generated apolipoprotein E (ApoE)(-/-)/IL-17A(-/-) mice, we report the following new findings. 1) The hyperlipidemia stimulus oxidized low density lipoprotein up-regulated IL-17 receptor(s) in HAECs and MAECs. 2) IL-17A activated HAECs and increased human monocyte adhesion in vitro. 3) A deficiency of IL-17A reduced leukocyte adhesion to endothelium in vivo. 3) IL-17A activated HAECs and MAECs via up-regulation of proinflammatory cytokines IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine CXC motif ligand 1 (CXCL1), and CXCL2. 4) IL-17A activated ECs specifically via the p38 mitogen-activated protein kinases (MAPK) pathway; the inhibition of p38 MAPK in ECs attenuated IL-17A-mediated activation by ameliorating the expression of the aforementioned proinflammatory cytokines, chemokines, and EC adhesion molecules including intercellular adhesion molecule 1. Taken together, our results demonstrate for the first time that IL-17A activates aortic ECs specifically via p38 MAPK pathway.

Protease-activated receptor 4 deficiency offers cardioprotection after acute ischemia reperfusion injury.

Protease-activated receptor (PAR)4 is a low affinity thrombin receptor with less understood function relative to PAR1. PAR4 is involved in platelet activation and hemostasis, but its specific actions on myocyte growth and cardiac function remain unknown. This study examined the role of PAR4 deficiency on cardioprotection after myocardial ischemia-reperfusion (IR) injury in mice. When challenged by in vivo or ex vivo IR, PAR4 knockout (KO) mice exhibited increased tolerance to injury, which was manifest as reduced infarct size and a more robust functional recovery compared to wild-type mice. PAR4 KO mice also showed reduced cardiomyocyte apoptosis and putative signaling shifts in survival pathways in response to IR. Inhibition of PAR4 expression in isolated cardiomyocytes by shRNA offered protection against thrombin and PAR4-agonist peptide-induced apoptosis, while overexpression of wild-type PAR4 significantly enhanced the susceptibility of cardiomyocytes to apoptosis, even under low thrombin concentrations. Further studies implicate Src- and epidermal growth factor receptor-dependent activation of JNK on the proapoptotic effect of PAR4 in cardiomyocytes. These findings reveal a pivotal role for PAR4 as a regulator of cardiomyocyte survival and point to PAR4 inhibition as a therapeutic target offering cardioprotection after acute IR injury.