Cardiovascular risk factors are similar in late premenopausal and early postmenopausal women, matched by age and body composition, with the exception that postmenopausal women have higher high- and low-density lipoprotein-cholesterol levels. A 3-month intervention of high-intensity aerobic training reduces risk factors for type 2 diabetes and cardiovascular disease to a similar extent in late premenopausal and early postmenopausal women.

Background The risk of atherothrombotic events increases after the menopause. Regular physical activity has been shown to reduce platelet reactivity in younger women, but it is unknown how regular exercise affects platelet function after the menopause. Objectives To examine the effects of regular aerobic exercise in late premenopausal and recent postmenopausal women by testing basal platelet reactivity and platelet sensitivity to prostacyclin and nitric oxide. Methods Twenty-five sedentary, but healthy, late premenopausal and 24 matched recently postmenopausal women, mean (95% confidence interval) 49.1 (48.2-49.9) and 53.7 (52.5-55.0) years old, participated in an intervention study: 3-month high-intensity supervised aerobic spinning-cycle training (1 h, × 3/week). Basal platelet reactivity was analyzed in platelet-rich plasma from venous blood as agonist-induced % aggregation. In a subgroup of 13 premenopausal and 14 postmenopausal women, platelet reactivity was tested ex vivo after femoral arterial infusion of prostacyclin, acetylcholine, a cyclooxygenase inhibitor, and after acute one-leg knee extensor exercise. Results Basal platelet reactivity (%aggregation) to TRAP-6 (1 μm) was higher in the postmenopausal, 59% (50-68), than the premenopausal women, 45% (35-55). Exercise training reduced basal platelet reactivity to collagen (1 μg mL ) in the premenopausal women only: from 63% (55-71%) to 51% (41-62%). After the training intervention, platelet aggregation was more inhibited by the arterial prostacyclin infusion and the acute exercise in both premenopausal and postmenopausal women. Conclusions These results highlight previously unknown cardioprotective aspects of regular aerobic exercise in premenopausal and postmenopausal women, improving their regulation of platelet reactivity through an increased platelet sensitivity to prostacyclin, which may counterbalance the increased atherothrombotic risk associated with the menopause.

Rationale: Data on the molecular mechanisms that regulate platelet-pulmonary endothelial adhesion under conditions of hypoxia are lacking, but may have important therapeutic implications.Conclusions: The NEDD9-P-selectin protein-protein interaction is a modifiable target with which to inhibit platelet-pulmonary endothelial adhesion and thromboembolic vascular remodeling, with potential therapeutic implications for patients with disorders of increased hypoxia signaling pathways, including CTEPH.

The current guidelines following an acute coronary syndrome recommend dual‐antiplatelet therapy (DAPT) (aspirin plus a P2Y12 antagonist) alongside lifestyle modifications, including more regular physical activity. It is currently unknown whether regular exercise affects the pharmacology of DAPT. Aim To explore how exercise‐induced improvements in vascular and platelet function affect the efficacy of DAPT, in a cross‐sectional study of men with different physical activity levels (training status). Methods A total of 42 healthy, normal‐weight, middle‐aged men were divided into 3 groups: untrained, moderately trained and well‐trained. Their platelet reactivity (agonist‐induced % aggregation) was investigated in platelet‐rich plasma at rest and after inhibition with aspirin and ticagrelor and/or prostacyclin and nitric oxide added to the blood in vitro, and after physiological tests of vascular function; passive movement of the leg, flow‐mediated dilation and one‐leg knee‐extensor exercise. Vascular function of the femoral artery (changes in arterial blood flow) was assessed by ultrasound Doppler. Results Platelets from the well‐trained subjects had lower basal reactivity, a higher sensitivity to the anti‐aggregatory effects of prostacyclin and were more potently inhibited by DAPT compared to the untrained subjects. The moderately trained and well‐trained subjects had a superior vascular function compared to untrained subjects, and their platelets were more inhibited by the passive movement, flow‐mediated dilation and one‐leg knee‐extensor exercise. Discussion A habitually active lifestyle leads to an increased platelet sensitivity to pharmacological and physiological platelet inhibitors. We suggest that physical activity habits (training status) should be considered when personalizing and optimizing antithrombotic treatment strategies.

Antisense oligonucleotides (ASOs) are DNA-based, disease-modifying drugs. Clinical trials with 2'-O-methoxyethyl (2’MOE) ASOs have reported dose- and sequencespecific lowering of platelet counts according to two phenotypes. Phenotype 1 is a moderate (but not clinically severe) drop in platelet count. Phenotype 2 is rare, severe thrombocytopenia. This paper will focus on the underlying cause of the more common Phenotype 1, investigating the effects of ASOs on platelet production and platelet function. Five phosphorothioate ASOs were included: three 2’MOE sequences; 487660 (no effects on platelet count), 104838 (associated with Phenotype 1), 501861 (effects unknown) and two CpG sequences; 120704 and ODN 2395 (known to activate platelets). Human cord blood-derived megakaryocytes were treated with these ASOs to study effects on proplatelet production. Platelet activation (surface P-selectin) and platelet-leukocyte aggregates (PLAs) were analyzed in ASO-treated blood from healthy human volunteers. None of the ASOs inhibited proplatelet production from human megakaryocytes. All the ASOs were shown to bind to the platelet receptor glycoprotein VI (GPVI, KD ~0.2-1.5μM). CpG ASOs had the highest affinity to GPVI and the most potent platelet activating effects and PLA formation. 2’MOE ASO 487660 had no detectable platelet effects, while 2’MOE ASOs 104838 and 501861 triggered moderate platelet activation and SYK-dependent formation of PLAs. Donors with higher platelet GPVI levels had larger ASO-induced platelet activation. Sequence-dependent ASO-induced platelet activation and PLAs may explain Phenotype 1- moderate drops in platelet count. Platelet GPVI levels could be useful as a screening tool to identify patients at higher risk of ASO-induced platelet side effects.

Endothelial mechanotransduction is important for vascular function but alterations and activation of vascular mechanosensory proteins have not been investigated in humans. In endothelial cell culture, simple sugars effectively impair mechanosensor proteins. To study mechanosensor- and vascular function in humans, 12 young healthy male subjects supplemented their diet with 3 × 75 g sucrose day for 14 days in a randomized cross-over design. Before and after the intervention period, the hyperaemic response to passive lower leg movement and active knee extensor exercise was determined by ultrasound doppler. A muscle biopsy was obtained from the thigh muscle before and after acute passive leg movement to allow assessment of protein amounts and the phosphorylation status of mechanosensory proteins and NADPH oxidase. The sucrose intervention led to a reduced flow response to passive movement (by 17 ± 2%) and to 12 W of active exercise (by 9 ± 1%), indicating impaired vascular function. A reduced flow response to passive and active exercise was paralleled by a significant up-regulation of platelet endothelial cell adhesion molecule (PECAM-1), endothelial nitric oxide synthase, NADPH oxidase and the Rho family GTPase Rac1 protein expression in the muscle tissue, as well as an increased basal phosphorylation status of vascular endothelial growth factor receptor 2 and a reduced phosphorylation status of PECAM-1. The phosphorylation status was not acutely altered with passive leg movement. These findings indicate that a regular intake of high levels of sucrose can impair vascular mechanotransduction and increase the oxidative stress potential, and suggest that dietary excessive sugar intake may contribute to the development of vascular disease.

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Introduction Antisense oligonucleotides (ASOs) are a new class of single-stranded DNA based drugs that hold great therapeutic promise for their disease modifying potential in a wide range of genetic diseases. Preclinical toxicology studies in monkeys, as well as late stage clinical trials in humans, have upon repeated dosing, reported events of ASO sequence-specific lowering of platelet counts (mild to severe thrombocytopenia) (Henry et al. Nucleic acid therapeutics 2017). The underlying cause of this platelet decrease is still unclear in humans. We have investigated if the thrombocytopenia associated with ASOs is due to either impaired platelet production and/or destruction of platelets (clearance) due to increased platelet reactivity (activation/aggregation status). Preliminary data from mouse derived fetal liver megakaryocytes suggest that pro-platelet production does not seem to be reduced by ASOs and hence in the current study we hypothesized that the ASO-induced thrombocytopenia is due to increased clearance of platelets from the circulation. Methods In the current study we explored how ASOs affect platelet aggregation in platelet rich plasma (PRP) and platelet-leukocyte aggregates in whole blood (WB) obtained from healthy volunteers after informed consent. PRP or WB was treated with a clinically relevant concentration of ASO (5µM) corresponding to expected maximum plasma concentration levels, or a 20x-supra-therapeutic concentration (100µM). Four ASOs were tested: two CpG-rich phosphorothioate deoxyoligonucleotide (PS ODN) sequences: 818290 and 120704, and two non-CpG 2'-MOE containing sequences: 104838 and 501861. 818290 was included as a positive control since it has been shown to cause direct platelet activation (Flierl et al. JEM 2015). 104838 have been reported to cause moderate, dose dependent drops in platelet counts in monkeys and humans, with platelet sequestration in the liver and spleen (Narayanan PK, et al. Toxicol Sci. 2018). 501861 has triggered sporadic severe thrombocytopenia in select monkeys. ASO treated PRP was analyzed for platelet aggregation using 96-well optimul aggregometry (Lordkipanidzé et al. Blood 2014) in the presence of vehicle (PBS) or 6 concentrations of thrombin receptor activating peptide-6 (0.08-80µM,TRAP6). In a separate experiment, PRP was incubated with ASOs plus the spleen tyrosine kinase (Syk) inhibitor PRT-060318 (10µM). ASO treated WB was incubated with fluorescently labelled CD41/61 antibody to label platelets and a leukocyte-specific antibody CD14, and platelet-leukocyte aggregates were analyzed by FACS according to (Gerrits et al. Curr. Protoc. 2016). Results The two non-CpG rich 2'-MOE ASO sequences 104838 and 501861 did not affect platelet aggregation at either concentration (5µM + 100µM) (Figure 1 A+B). Whilst the two CpG-rich PS ODN ASOs (818290 and 120704) triggered spontaneous platelet aggregation in PRP at 100µM (Figure 1 C+D), that was normalized by co-incubating these ASOs with a Syk inhibitor (Figure 2). 5µM of ASO treatment triggered a significant increase in platelet-leukocyte aggregates in WB for all the ASOs tested (Figure 3). Conclusion We have shown that the two CpG-rich PS ODN ASOs (818290 and 120704) triggered spontaneous platelet aggregation in PRP at 100µM. This effect was inhibited by a Syk inhibitor. 818290 has previously been identified to activate platelets through a Syk-dependent, GPVI receptor mediated mechanism (Flierl et al. JEM 2015). Here, we report for the first time that the aggregatory effects of 120704 have been identified to be Syk dependent as well, possibly through a similar interaction with platelet GPVI receptors. We have also presented novel data that therapeutically relevant concentrations of all the ASOs tested cause an increase in platelet-leukocyte aggregates in WB. Based on these data we highlight the importance of screening ASOs in multi-cellular assays, not just PRP, since there was no effect of the ASOs 104838 or 501861 on platelet aggregation. Enhanced formation of platelet-leukocyte aggregates could be one contributing factor for increased platelet clearance, explaining ASO-induced thrombocytopenia. Further investigation into the ASO-induced interactions between platelets and immune cells are warranted. Defining the mechanisms by which ASO-based drugs cause low platelet count may yield strategies to manage this drug-induced thrombocytopenia in patients. Disclosures Thon: Platelet Biogenesis: Employment, Equity Ownership, Other: Co-founder, Patents & Royalties. Henry:Ionis Pharmaceuticals: Employment. Narayanan:Ionis Pharmaceuticals: Employment. Italiano:Platelet Biogenesis: Equity Ownership, Other: Co-founder, Patents & Royalties.