S100A8/A9 drives the formation of procoagulant platelets through GPIbα.

S100A8/A9, also known as "calprotectin" or "MRP8/14," is an alarmin primarily secreted by activated myeloid cells with antimicrobial, proinflammatory, and prothrombotic properties. Increased plasma levels of S100A8/A9 in thrombo-inflammatory diseases are associated with thrombotic complications. We assessed the presence of S100A8/A9 in the plasma and lung autopsies from patients with COVID-19 and investigated the molecular mechanism by which S100A8/A9 affects platelet function and thrombosis. S100A8/A9 plasma levels were increased in patients with COVID-19 and sustained high levels during hospitalization correlated with poor outcomes. Heterodimeric S100A8/A9 was mainly detected in neutrophils and deposited on the vessel wall in COVID-19 lung autopsies. Immobilization of S100A8/A9 with collagen accelerated the formation of a fibrin-rich network after perfusion of recalcified blood at venous shear. In vitro, platelets adhered and partially spread on S100A8/A9, leading to the formation of distinct populations of either P-selectin or phosphatidylserine (PS)-positive platelets. By using washed platelets, soluble S100A8/A9 induced PS exposure but failed to induce platelet aggregation, despite GPIIb/IIIa activation and alpha-granule secretion. We identified GPIbα as the receptor for S100A8/A9 on platelets inducing the formation of procoagulant platelets with a supporting role for CD36. The effect of S100A8/A9 on platelets was abolished by recombinant GPIbα ectodomain, platelets from a patient with Bernard-Soulier syndrome with GPIb-IX-V deficiency, and platelets from mice deficient in the extracellular domain of GPIbα. We identified the S100A8/A9-GPIbα axis as a novel targetable prothrombotic pathway inducing procoagulant platelets and fibrin formation, in particular in diseases associated with high levels of S100A8/A9, such as COVID-19.

Structural characterization of a novel GPVI-nanobody complex reveals a biologically active domain-swapped GPVI dimer.

Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C' loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.

A Proteomics-Based Assessment of Inflammation Signatures in Endotoxemia.

We have previously shown that multimers of plasma pentraxin-3 (PTX3) were predictive of survival in patients with sepsis. To characterize the release kinetics and cellular source of plasma protein changes in sepsis, serial samples were obtained from healthy volunteers (n = 10; three time points) injected with low-dose endotoxin (lipopolysaccharide [LPS]) and analyzed using data-independent acquisition MS. The human plasma proteome response was compared with an LPS-induced endotoxemia model in mice. Proteomic analysis of human plasma revealed a rapid neutrophil degranulation signature, followed by a rise in acute phase proteins. Changes in circulating PTX3 correlated with increases in neutrophil-derived proteins following LPS injection. Time course analysis of the plasma proteome in mice showed a time-dependent increase in multimeric PTX3, alongside increases in neutrophil-derived myeloperoxidase (MPO) upon LPS treatment. The mechanisms of oxidation-induced multimerization of PTX3 were explored in two genetic mouse models: MPO global knock-out (KO) mice and LysM Cre Nox2 KO mice, in which NADPH oxidase 2 (Nox2) is only deficient in myeloid cells. Nox2 is the enzyme responsible for the oxidative burst in neutrophils. Increases in plasma multimeric PTX3 were not significantly different between wildtype and MPO or LysM Cre Nox2 KO mice. Thus, PTX3 may already be stored and released in a multimeric form. Through in vivo neutrophil depletion and multiplexed vascular proteomics, PTX3 multimer deposition within the aorta was confirmed to be neutrophil dependent. Proteomic analysis of aortas from LPS-injected mice returned PTX3 as the most upregulated protein, where multimeric PTX3 was deposited as early as 2 h post-LPS along with other neutrophil-derived proteins. In conclusion, the rise in multimeric PTX3 upon LPS injection correlates with neutrophil-related protein changes in plasma and aortas. MPO and myeloid Nox2 are not required for the multimerization of PTX3; instead, neutrophil extravasation is responsible for the LPS-induced deposition of multimeric PTX3 in the aorta.

Two-Year Outcomes After Minimally Invasive Surfactant Therapy in Preterm Infants: Follow-Up of the OPTIMIST-A Randomized Clinical Trial.

Importance: The long-term effects of surfactant administration via a thin catheter (minimally invasive surfactant therapy [MIST]) in preterm infants with respiratory distress syndrome remain to be definitively clarified. Objective: To examine the effect of MIST on death or neurodevelopmental disability (NDD) at 2 years' corrected age. Design, Setting, and Participants: Follow-up study of a randomized clinical trial with blinding of clinicians and outcome assessors conducted in 33 tertiary-level neonatal intensive care units in 11 countries. The trial included 486 infants with a gestational age of 25 to 28 weeks supported with continuous positive airway pressure (CPAP). Collection of follow-up data at 2 years' corrected age was completed on December 9, 2022. Interventions: Infants assigned to MIST (n = 242) received exogenous surfactant (200 mg/kg poractant alfa) via a thin catheter; those assigned to the control group (n = 244) received sham treatment. Main Outcomes and Measures: The key secondary outcome of death or moderate to severe NDD was assessed at 2 years' corrected age. Other secondary outcomes included components of this composite outcome, as well as hospitalizations for respiratory illness and parent-reported wheezing or breathing difficulty in the first 2 years. Results: Among the 486 infants randomized, 453 had follow-up data available (median gestation, 27.3 weeks; 228 females [50.3%]); data on the key secondary outcome were available in 434 infants. Death or NDD occurred in 78 infants (36.3%) in the MIST group and 79 (36.1%) in the control group (risk difference, 0% [95% CI, -7.6% to 7.7%]; relative risk [RR], 1.0 [95% CI, 0.81-1.24]); components of this outcome did not differ significantly between groups. Secondary respiratory outcomes favored the MIST group. Hospitalization with respiratory illness occurred in 49 infants (25.1%) in the MIST group vs 78 (38.2%) in the control group (RR, 0.66 [95% CI, 0.54-0.81]) and parent-reported wheezing or breathing difficulty in 73 (40.6%) vs 104 (53.6%), respectively (RR, 0.76 [95% CI, 0.63-0.90]). Conclusions and Relevance: In this follow-up study of a randomized clinical trial of preterm infants with respiratory distress syndrome supported with CPAP, MIST compared with sham treatment did not reduce the incidence of death or NDD by 2 years of age. However, infants who received MIST had lower rates of adverse respiratory outcomes during their first 2 years of life. Trial Registration: anzctr.org.au Identifier: ACTRN12611000916943.

Antithrombotic Effects of Fostamatinib in Combination with Conventional Antiplatelet Drugs.

New antithrombotic medications with less effect on haemostasis are needed for the long-term treatment of acute coronary syndromes (ACS). The platelet receptor glycoprotein VI (GPVI) is critical in atherothrombosis, mediating platelet activation at atherosclerotic plaque. The inhibition of spleen tyrosine kinase (Syk) has been shown to block GPVI-mediated platelet function. The aim of our study was to investigate if the Syk inhibitor fostamatinib could be repurposed as an antiplatelet drug, either alone or in combination with conventional antiplatelet therapy. The effect of the active metabolite of fostamatinib (R406) was assessed on platelet activation and function induced by atherosclerotic plaque and a range of agonists in the presence and absence of the commonly used antiplatelet agents aspirin and ticagrelor. The effects were determined ex vivo using blood from healthy volunteers and aspirin- and ticagrelor-treated patients with ACS. Fostamatinib was also assessed in murine models of thrombosis. R406 mildly inhibited platelet responses induced by atherosclerotic plaque homogenate, likely due to GPVI inhibition. The anti-GPVI effects of R406 were amplified by the commonly-used antiplatelet medications aspirin and ticagrelor; however, the effects of R406 were concentration-dependent and diminished in the presence of plasma proteins, which may explain why fostamatinib did not significantly inhibit thrombosis in murine models. For the first time, we demonstrate that the Syk inhibitor R406 provides mild inhibition of platelet responses induced by atherosclerotic plaque and that this is mildly amplified by aspirin and ticagrelor.

Salmonella-induced thrombi in mice develop asynchronously in the spleen and liver and are not effective bacterial traps.

Thrombosis is a frequent, life-threatening complication of systemic infection associated with multiple organ damage. We have previously described a novel mechanism of inflammation-driven thrombosis induced by Salmonella Typhimurium infection of mice. Thrombosis in the liver develops 7 days after infection, persisting after the infection resolves, and is monocytic cell dependent. Unexpectedly, thrombosis was not prominent in the spleen at this time, despite carrying a similar bacterial burden as the liver. In this study, we show that thrombosis does occur in the spleen but with strikingly accelerated kinetics compared with the liver, being evident by 24 hours and resolving rapidly thereafter. The distinct kinetics of thrombosis and bacterial burden provides a test of the hypothesis that thrombi form in healthy vessels to trap or remove bacteria from the circulation, often termed immunothrombosis. Remarkably, despite bacteria being detected throughout infected spleens and livers in the early days of infection, immunohistological analysis of tissue sections show that thrombi contain very low numbers of bacteria. In contrast, bacteria are present throughout platelet aggregates induced by Salmonella in vitro. Therefore, we show that thrombosis develops with organ-specific kinetics and challenge the universality of immunothrombosis as a mechanism to capture bacteria in vivo.

Novel antiplatelet targets in the treatment of acute coronary syndromes.

Acute coronary syndromes (ACS) are a global cause of mortality and morbidity that affect millions of lives worldwide. Following atherosclerotic plaque rupture, platelet activation and aggregation are the two major elements that initiate thrombus formation inside a coronary artery, which can obstruct blood flow and cause myocardial ischemia; ergo, antiplatelet therapy forms a major part of the treatment strategy for ACS. Patients with ACS routinely receive dual antiplatelet therapy (DAPT), which consists of aspirin and a platelet P2Y12 inhibitor to both treat and prevent atherothrombosis. Use of platelet glycoprotein (GP) IIb/IIIa inhibitors is now limited due to the risk of severe bleeding and thrombocytopenia. Thus, administration of GPIIb/IIIa inhibitors is generally restricted to bail out thrombotic events associated with PCI. Furthermore, current antiplatelet medications mainly rely on thromboxane A2 and P2Y12 inhibition, which have broad-acting effects on platelets and are known to cause bleeding, which especially limits the long-term use of these agents. In addition, not all ACS patients treated with current antiplatelet treatments are protected from recurrence of arterial thrombosis, since many platelet mechanisms and activation pathways remain uninhibited by current antiplatelet therapy. Pharmacological antagonism of novel targets involved in platelet function could shape future antiplatelet therapies that could ultimately lead to more effective or safer therapeutic approaches. In this article, we focus on inhibitors of promising targets that have not yet been introduced into clinical practice, including inhibitors of GPVI, protease-activated receptor (PAR)-4, GPIb, 5-hydroxytryptamine receptor subtype 2A (5-HT2A), protein disulfide isomerase, P-selectin and phosphoinositide 3-kinase ß.

Novel antiplatelet strategies targeting GPVI, CLEC-2 and tyrosine kinases.

Antiplatelet medications comprise the cornerstone of treatment for diseases that involve arterial thrombosis, including acute coronary syndromes (ACS), stroke and peripheral arterial disease. However, antiplatelet medications may cause bleeding and, furthermore, thrombotic events may still recur despite treatment. The interaction of collagen with GPVI receptors on the surface of platelets has been identified as one of the major players in the pathophysiology of arterial thrombosis that occurs following atherosclerotic plaque rupture. Promisingly, GPVI deficiency in humans appears to have a minimal impact on bleeding. These findings together suggest that targeting platelet GPVI may provide a novel treatment strategy that provides additional antithrombotic efficacy with minimal disruption of normal hemostasis compared to conventional antiplatelet medications. CLEC-2 is gaining interest as a therapeutic target for a variety of thrombo-inflammatory disorders including deep vein thrombosis (DVT) with treatment also predicted to cause minimal disruption to hemostasis. GPVI and CLEC-2 signal through Src, Syk and Tec family tyrosine kinases, providing additional strategies for inhibiting both receptors. In this review, we summarize the evidence regarding GPVI and CLEC-2 and strategies for inhibiting these receptors to inhibit platelet recruitment and activation in thrombotic diseases.

Effect of Minimally Invasive Surfactant Therapy vs Sham Treatment on Death or Bronchopulmonary Dysplasia in Preterm Infants With Respiratory Distress Syndrome: The OPTIMIST-A Randomized Clinical Trial.

Importance: The benefits of surfactant administration via a thin catheter (minimally invasive surfactant therapy [MIST]) in preterm infants with respiratory distress syndrome are uncertain. Objective: To examine the effect of selective application of MIST at a low fraction of inspired oxygen threshold on survival without bronchopulmonary dysplasia (BPD). Design, Setting, and Participants: Randomized clinical trial including 485 preterm infants with a gestational age of 25 to 28 weeks who were supported with continuous positive airway pressure (CPAP) and required a fraction of inspired oxygen of 0.30 or greater within 6 hours of birth. The trial was conducted at 33 tertiary-level neonatal intensive care units around the world, with blinding of the clinicians and outcome assessors. Enrollment took place between December 16, 2011, and March 26, 2020; follow-up was completed on December 2, 2020. Interventions: Infants were randomized to the MIST group (n = 241) and received exogenous surfactant (200 mg/kg of poractant alfa) via a thin catheter or to the control group (n = 244) and received a sham (control) treatment; CPAP was continued thereafter in both groups unless specified intubation criteria were met. Main Outcomes and Measures: The primary outcome was the composite of death or physiological BPD assessed at 36 weeks' postmenstrual age. The components of the primary outcome (death prior to 36 weeks' postmenstrual age and BPD at 36 weeks' postmenstrual age) also were considered separately. Results: Among the 485 infants randomized (median gestational age, 27.3 weeks; 241 [49.7%] female), all completed follow-up. Death or BPD occurred in 105 infants (43.6%) in the MIST group and 121 (49.6%) in the control group (risk difference [RD], -6.3% [95% CI, -14.2% to 1.6%]; relative risk [RR], 0.87 [95% CI, 0.74 to 1.03]; P = .10). Incidence of death before 36 weeks' postmenstrual age did not differ significantly between groups (24 [10.0%] in MIST vs 19 [7.8%] in control; RD, 2.1% [95% CI, -3.6% to 7.8%]; RR, 1.27 [95% CI, 0.63 to 2.57]; P = .51), but incidence of BPD in survivors to 36 weeks' postmenstrual age was lower in the MIST group (81/217 [37.3%] vs 102/225 [45.3%] in the control group; RD, -7.8% [95% CI, -14.9% to -0.7%]; RR, 0.83 [95% CI, 0.70 to 0.98]; P = .03). Serious adverse events occurred in 10.3% of infants in the MIST group and 11.1% in the control group. Conclusions and Relevance: Among preterm infants with respiratory distress syndrome supported with CPAP, minimally invasive surfactant therapy compared with sham (control) treatment did not significantly reduce the incidence of the composite outcome of death or bronchopulmonary dysplasia at 36 weeks' postmenstrual age. However, given the statistical uncertainty reflected in the 95% CI, a clinically important effect cannot be excluded. Trial Registration: anzctr.org.au Identifier: ACTRN12611000916943.

Offset of ticagrelor prior to coronary artery bypass graft surgery for acute coronary syndromes: effects on platelet function and cellular adenosine uptake.

Ticagrelor is an antagonist of both platelet adenosine diphosphate (ADP) receptor P2Y12 and equilibrative nucleoside transporter-1. Optimal timing of ticagrelor cessation prior to coronary artery bypass grafting (CABG) remains unclear. We characterized the offset of ticagrelor's effects on platelets and cellular adenosine uptake in ticagrelor-treated patients (n = 13) awaiting CABG. Blood was drawn prior to CABG at multiple timepoints 2 to 120 (h) after the last dose of ticagrelor. Platelet function (n = 13) was assessed with multiple electrode aggregometry (MEA), expressed as arbitrary units (U) derived from area-under-the-curve (AUC) in response to ADP, and inhibition of adenosine uptake by high-performance liquid chromatography (n = 7). Mean±SD AUC was 20.3 ± 8.2 U (2 h post-ticagrelor), 33.0 ± 18.3U (24 h), 56.6 ± 30.6U (48 h), 61.4 ± 20.2U (72 h), 82.8 ± 24.2U (96 h) and 96.0 ± 15.3U (120 h). There was a significant difference between 72 h and 120 h (p = .007), but not between 96 h and 120 h (p > .99). By 96 h, all patients had AUC >31U, an accepted cutoff below which surgical bleeding risk is increased. Adenosine uptake showed no significant differences between the timepoints. These data suggest it takes 4 days for platelet reactivity to recover sufficiently after cessation of ticagrelor to avoid the excess risk of CABG-related bleeding. Discontinuing ticagrelor had no measurable effect on cellular adenosine uptake.

Data-driven discovery and validation of circulating blood-based biomarkers associated with prevalent atrial fibrillation.

AIMS: Undetected atrial fibrillation (AF) is a major health concern. Blood biomarkers associated with AF could simplify patient selection for screening and further inform ongoing research towards stratified prevention and treatment of AF. METHODS AND RESULTS: Forty common cardiovascular biomarkers were quantified in 638 consecutive patients referred to hospital [mean ± standard deviation age 70 ± 12 years, 398 (62%) male, 294 (46%) with AF] with known AF or ≥2 CHA2DS2-VASc risk factors. Paroxysmal or silent AF was ruled out by 7-day ECG monitoring. Logistic regression with forward selection and machine learning algorithms were used to determine clinical risk factors, imaging parameters, and biomarkers associated with AF. Atrial fibrillation was significantly associated with age [bootstrapped odds ratio (OR) per year = 1.060, 95% confidence interval (1.04-1.10); P = 0.001], male sex [OR = 2.022 (1.28-3.56); P = 0.008], body mass index [BMI, OR per unit = 1.060 (1.02-1.12); P = 0.003], elevated brain natriuretic peptide [BNP, OR per fold change = 1.293 (1.11-1.63); P = 0.002], elevated fibroblast growth factor-23 [FGF-23, OR = 1.667 (1.36-2.34); P = 0.001], and reduced TNF-related apoptosis-induced ligand-receptor 2 [TRAIL-R2, OR = 0.242 (0.14-0.32); P = 0.001], but not other biomarkers. Biomarkers improved the prediction of AF compared with clinical risk factors alone (net reclassification improvement = 0.178; P < 0.001). Both logistic regression and machine learning predicted AF well during validation [area under the receiver-operator curve = 0.684 (0.62-0.75) and 0.697 (0.63-0.76), respectively]. CONCLUSION: Three simple clinical risk factors (age, sex, and BMI) and two biomarkers (elevated BNP and elevated FGF-23) identify patients with AF. Further research is warranted to elucidate FGF-23 dependent mechanisms of AF.

Circulating MicroRNA Levels Indicate Platelet and Leukocyte Activation in Endotoxemia Despite Platelet P2Y12 Inhibition.

There is evidence for the effects of platelet inhibition on innate immune activation. Circulating microRNAs (miRNAs) have been implicated as markers of platelet and leukocyte activation. In the present study, we assessed the effects of P2Y12 inhibitors on platelet and leukocyte miRNAs during endotoxemia. Healthy volunteers were randomly assigned to receive oral ticagrelor (n = 10), clopidogrel (n = 8) or no drug (n = 8) for one week, followed by an intravenous bolus of 2 ng/kg endotoxin. Serum was collected at baseline, after one week of antiplatelet treatment and 6 and 24 h after endotoxin administration. MiRNAs were screened using LNA-based qPCR, followed by TaqMan-qPCR validation of candidates. Clinical validation was performed in 41 sepsis patients. Platelet-enriched miR-197, miR-223 and miR-223* were decreased in volunteers following antiplatelet therapy. Endotoxin increased platelet miRNAs, whilst the opposite effect was seen for leukocyte-enriched miR-150. Neither of these endotoxin-mediated effects were altered by P2Y12 inhibitors. Sepsis patients with fatal outcomes (n = 12) had reduced miR-150 levels compared with survivors (n = 29). In conclusion, we show that miR-150 is downregulated in experimental endotoxemia and can predict survival in sepsis but is unaffected by P2Y12 inhibition. While P2Y12 inhibition reduces platelet-associated miRNAs in healthy volunteers, it fails to attenuate the response of platelet miRNAs to endotoxemia.

Study of Two Dose Regimens of Ticagrelor Compared With Clopidogrel in Patients Undergoing Percutaneous Coronary Intervention for Stable Coronary Artery Disease.

BACKGROUND: Ticagrelor has superior efficacy to clopidogrel in the management of acute coronary syndromes but has not been assessed in patients undergoing percutaneous coronary intervention for stable coronary artery disease. We compared the pharmacodynamic effects of ticagrelor and clopidogrel in this stable population. METHODS: One hundred eighty aspirin-treated stable coronary artery disease patients, who were planned to undergo elective percutaneous coronary intervention in a single center, were randomized 1:1:1 to either a standard clopidogrel regimen or 1 of 2 regimens of ticagrelor, either 90 mg (T90) or 60 mg twice daily (T60), both with a 180 mg loading dose. Cellular adenosine uptake was assessed, at the time of the procedure and pre- and postdose at 1 month, by adding adenosine 1 µmol/L to aliquots of anticoagulated whole blood and mixing with a stop solution at 0, 15, 30, and 60 seconds, then measuring residual plasma adenosine concentration by high-performance liquid chromatography. Systemic plasma adenosine concentration and platelet reactivity were assessed at the same timepoints. High-sensitivity troponin T was measured pre- and 18 to 24 hours postpercutaneous coronary intervention. RESULTS: One hundred seventy-four patients underwent an invasive procedure, of whom 162 received percutaneous coronary intervention (mean age 65 years, 18% female, 21% with diabetes mellitus). No effect on in vitro adenosine uptake was seen postdose at 1 month for either ticagrelor dose compared with clopidogrel (residual adenosine at 15 seconds, mean±SD: clopidogrel 0.274±0.101 µmol/L; T90 0.278±0.134 µmol/L; T60 0.288±0.149 µmol/L; P=0.37). Similarly, no effect of ticagrelor on in vitro adenosine uptake was seen at other timepoints, nor was plasma adenosine concentration affected (all P>0.1). Both maintenance doses of ticagrelor achieved more potent and consistent platelet inhibition than clopidogrel (VerifyNow P2Y12 reaction units, 1 month, mean±SD: predose, T60: 62±47, T90: 40±38, clopidogrel 181±44; postdose, T60: 34±30, T90: 24±21, clopidogrel 159±57; all P<0.0001 for ticagrelor versus clopidogrel). High platelet reactivity was markedly less with both T60 and T90 compared with clopidogrel (VerifyNow P2Y12 reaction units>208, 1 month postdose: 0%, 0%, and 21%, respectively). Median (interquartile range) high-sensitivity troponin T increased 16.9 (6.5-46.9) ng/L for clopidogrel, 22.4 (5.5-53.8) ng/L for T60, and 17.7 (8.1-43.5) ng/L for T90 (P=0.95). There was a trend toward less dyspnea with T60 versus T90 (7.1% versus 19.0%; P=0.09). CONCLUSIONS: Maintenance therapy with T60 or T90 had no detectable effect on cellular adenosine uptake at 1 month, nor was there any effect on systemic plasma adenosine levels. Both regimens of ticagrelor achieved greater and more consistent platelet inhibition than clopidogrel but did not appear to affect troponin release after percutaneous coronary intervention. CLINICAL TRIAL REGISTRATION: URL: https://www. CLINICALTRIALS: gov. Unique identifier: NCT02327624.

Novel Risk Markers and Risk Assessments for Cardiovascular Disease.

The use of risk markers has transformed cardiovascular medicine, exemplified by the routine assessment of troponin, for both diagnosis and assessment of prognosis in patients with chest pain. Clinical risk factors form the basis for risk assessment of cardiovascular disease and the addition of biochemical, cellular, and imaging parameters offers further refinement. Identifying novel risk factors may allow greater risk stratification and a steady, but gradual progression toward precision medicine. Indeed, the generation of data in this area of research is explosive and when combined with new technologies and techniques provides the potential for more refined, targeted approaches to cardiovascular medicine. Although discussing the most recent developments in this field, this review article aims to strike a balance between novelty and validity by focusing on recent large sample-size studies that have been validated in a separate cohort in most cases. Risk markers related to atherosclerosis, thrombosis, inflammation, cardiac injury, and fibrosis are introduced in the context of their pathophysiology. Rapidly developing new areas, such as assessment of micro-RNA, are also explored. Subsequently the prognostic ability of these risk markers in coronary artery disease, heart failure, and atrial fibrillation is discussed in detail.

Genetic identification of SNP markers linked to a new grape phylloxera resistant locus in Vitis cinerea for marker-assisted selection.

BACKGROUND: Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a major insect pest that negatively impacts commercial grapevine performance worldwide. Consequently, the use of phylloxera resistant rootstocks is an essential component of vineyard management. However, the majority of commercially available rootstocks used in viticulture production provide limited levels of grape phylloxera resistance, in part due to the adaptation of phylloxera biotypes to different Vitis species. Therefore, there is pressing need to develop new rootstocks better adapted to specific grape growing regions with complete resistance to grape phylloxera biotypes. RESULTS: Grapevine rootstock breeding material, including an accession of Vitis cinerea and V. aestivalis, DRX55 ([M. rotundifolia x V. vinifera] x open pollinated) and MS27-31 (M. rotundifolia specific hybrid), provided complete resistance to grape phylloxera in potted plant assays. To map the genetic factor(s) of grape phylloxera resistance, a F1 V. cinerea x V. vinifera Riesling population was screened for resistance. Heritability analysis indicates that the V. cinerea accession contained a single allele referred as RESISTANCE TO DAKTULOSPHAIRA VITIFOLIAE 2 (RDV2) that confers grape phylloxera resistance. Using genetic maps constructed with pseudo-testcross markers for V. cinerea and Riesling, a single phylloxera resistance locus was identified in V. cinerea. After validating SNPs at the RDV2 locus, interval and linkage mapping showed that grape phylloxera resistance mapped to linkage group 14 at position 16.7 cM. CONCLUSION: The mapping of RDV2 and the validation of markers linked to grape phylloxera resistance provides the basis to breed new rootstocks via marker-assisted selection that improve vineyard performance.

Inhibition of Btk by Btk-specific concentrations of ibrutinib and acalabrutinib delays but does not block platelet aggregation mediated by glycoprotein VI.

Ibrutinib and acalabrutinib are irreversible inhibitors of Bruton tyrosine kinase used in the treatment of B-cell malignancies. They bind irreversibly to cysteine 481 of Bruton tyrosine kinase, blocking autophosphorylation on tyrosine 223 and phosphorylation of downstream substrates including phospholipase C-γ2. In the present study, we demonstrate that concentrations of ibrutinib and acalabrutinib that block Bruton tyrosine kinase activity, as shown by loss of phosphorylation at tyrosine 223 and phospholipase C-γ2, delay but do not block aggregation in response to a maximally-effective concentration of collagen-related peptide or collagen. In contrast, 10- to 20-fold higher concentrations of ibrutinib or acalabrutinib block platelet aggregation in response to glycoprotein VI agonists. Ex vivo studies on patients treated with ibrutinib, but not acalabrutinib, showed a reduction of platelet aggregation in response to collagen-related peptide indicating that the clinical dose of ibrutinib but not acalabrutinib is supramaximal for Bruton tyrosine kinase blockade. Unexpectedly, low concentrations of ibrutinib inhibited aggregation in response to collagen-related peptide in patients deficient in Bruton tyrosine kinase. The increased bleeding seen with ibrutinib over acalabrutinib is due to off-target actions of ibrutinib that occur because of unfavorable pharmacodynamics.

Fast Phenomics in Vineyards: Development of GRover, the Grapevine Rover, and LiDAR for Assessing Grapevine Traits in the Field.

This paper introduces GRover (the grapevine rover), an adaptable mobile platform for the deployment and testing of proximal imaging sensors in vineyards for the non-destructive assessment of trunk and cordon volume and pruning weight. A SICK LMS-400 light detection and ranging (LiDAR) radar mounted on GRover was capable of producing precise (±3 mm) 3D point clouds of vine rows. Vineyard scans of the grapevine variety Shiraz grown under different management systems at two separate locations have demonstrated that GRover is able to successfully reproduce a variety of vine structures. Correlations of pruning weight and vine wood (trunk and cordon) volume with LiDAR scans have resulted in high coefficients of determination (R² = 0.91 for pruning weight; 0.76 for wood volume). This is the first time that a LiDAR of this type has been extensively tested in vineyards. Its high scanning rate, eye safe laser and ability to distinguish tissue types make it an appealing option for further development to offer breeders, and potentially growers, quantified measurements of traits that otherwise would be difficult to determine.

Platelet P2Y12 Inhibitors Reduce Systemic Inflammation and Its Prothrombotic Effects in an Experimental Human Model.

OBJECTIVE: Clinical studies suggest that platelet P2Y12 inhibitors reduce mortality from sepsis, although the underlying mechanisms have not been clearly defined in vivo. We hypothesized that P2Y12 inhibitors may improve survival from sepsis by suppressing systemic inflammation and its prothrombotic effects. We therefore determined whether clopidogrel and the novel, more potent P2Y12 inhibitor, ticagrelor, modify these responses in an experimental human model. APPROACH AND RESULTS: We randomized 30 healthy volunteers to ticagrelor (n=10), clopidogrel (n=10), or no antiplatelet medication (controls; n=10). We examined the effect of P2Y12 inhibition on systemic inflammation, which was induced by intravenous injection of Escherichia coli endotoxin. Both P2Y12 inhibitors significantly reduced platelet-monocyte aggregate formation and peak levels of major proinflammatory cytokines, including tumor necrosis factor α, interleukin-6, and chemokine (C-C motif) ligand 2. In contrast to clopidogrel, ticagrelor also significantly reduced peak levels of IL-8 and growth colony-stimulating factor and increased peak levels of the anti-inflammatory cytokine IL-10. In addition, ticagrelor altered leukocyte trafficking. Both P2Y12 inhibitors suppressed D-dimer generation and scanning electron microscopy revealed that ticagrelor also suppressed prothrombotic changes in fibrin clot ultrastructure. CONCLUSIONS: Potent inhibition of multiple inflammatory and prothrombotic mechanisms by P2Y12 inhibitors demonstrates critical importance of platelets as central orchestrators of systemic inflammation induced by bacterial endotoxin. This provides novel mechanistic insight into the lower mortality associated with P2Y12 inhibitors in patients with sepsis in clinical studies.

The future of P2Y12 receptor antagonists.

Platelet P2Y12 inhibitors have become a central component of the treatment strategy for patients with atherothrombosis due to the importance of platelet P2Y12 receptors in arterial thrombosis. P2Y12 inhibitors effectively reduce the risk of adverse cardiovascular events in patients with acute coronary syndromes (ACS) and patients undergoing percutaneous coronary intervention (PCI). However, despite this, patients with ACS continue to suffer from recurrent atherothrombosis and an increased risk of mortality. In addition, P2Y12 inhibitors increase the risk of bleeding, thereby limiting their clinical benefit. It is therefore clear that further optimizations are needed in the pharmacology and treatment strategies of P2Y12 inhibitors. The objective of these optimizations is to maximize cardiovascular benefit whilst minimizing adverse effects on haemostasis. This review article summarizes the most successful recent strategies in P2Y12 inhibition in order to identify the optimizations and developments that are most likely to be successful in the future.