Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function

Background: DNA hypomethylation at the F2RL3 locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. Methods: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3,205), we explored the relationship between smoking, DNA hypomethylation at F2RL3 and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract (CSE). Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. Results: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 stimulation. In cells, CSE exposure was associated with a 4.9 to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7 (95% CI: 1.2, 2.4, p=0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. Conclusions: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk, but from any feature potentially influencing F2RL3 regulation in a similar manner.

Comparative analysis of hemostasis system state indicators in severe COVID-19

Introduction. Clinical experience in managing patients with a new coronavirus infection caused by the SARS-CoV-2 allowed to identify specific hemostasis disorders, and enables to introduce the concept of COVID-associated coagulopathy. The aim of the study was to assess the direction of coagulogram parameter changes, whole blood clotting parameters and characteristics of platelet and plasma hemostasis in patients with severe COVID-19. Materials and methods. The parameters of the hemostasis system were assessed using venous blood of 12 patients with severe COVID-19 and 16 healthy volunteers. The whole blood clotting process was investigated by low-frequency piezothromboelastography. The platelet count and indicators of spontaneous and ADP-induced platelet aggregation were estimated with the help of a laser platelet aggregation analyzer. Fibrinolytic activity of plasma, plasminogen activity, content of fibrinogen, D-dimer, PTT, APTT, PTI and INR were assessed. Results. An increased level of fibrinogen, a 6-fold increased D-dimer level, and increased PTT were found in patients with severe COVID-19. The patient platelets count was reduced by 51 % (p <0.05), spontaneous platelet aggregation remained at nearly normal level. Almost complete inhibition of ADP-induced platelet reactivity and inhibition of XIIa-dependent fibrinolysis was revealed, despite an increased by 19.3 % (p <0.05) plasminogen activity. Parameters of the whole blood coagulation process pointed a pronounced activation of platelet hemostasis, a significant intensification of the polymerization stage of clot formation and an increased intensity of clot lysis and retraction. Conclusion. The significant increase of D-dimer level and paradoxical inhibition of plasma fibrinolytic activity revealed by test of XIIa-dependent fibrinolysis (in contrast to the increased intensity of clot lysis when assessing the coagulation of whole blood) indicate the complex pathogenic mechanisms of coagulopathy caused by SARS-CoV-2 infection, and the involvement of blood cells and the vascular wall in the process of pathological thrombus formation.

Clopidogrel resistance is common in patients undergoing vascular and coronary interventions

Objectives “Clopidogrel resistance,” also defined as heightened platelet reactivity (HPR) while on clopidogrel therapy, may lead to a sub-optimal antiplatelet effect and a potential thrombotic event. There is limited literature addressing the prevalence of HPR in a large cohort of patients receiving either coronary or endovascular interventions. Methods In a large integrated healthcare system, patients with a P2Y12 reaction units (PRU) test were identified. HPR was defined as a PRU ≥ 200 during clopidogrel therapy. Vascular and coronary interventions were identified utilizing CPT codes, HPR prevalence was calculated, and Fischer’s exact test was used to determine significance. Results From an initial cohort of 2,405,957 patients (October 2014 to January 2020), we identified 3301 patients with PRU tests administered. Of these, 1789 tests had a PRU ≥ 200 (HPR overall prevalence, 54%). We then identified 1195 patients who underwent either an endovascular or coronary procedure and had a PRU measurement. This corresponded to 935 coronary and 260 endovascular interventions. In the coronary cohort, the HPR prevalence was 54% (503/935). In the vascular cohort, the HPR prevalence was 53% (137/260); there was no difference between cohorts in HPR prevalence ( p = 0.78). Conclusion “Clopidogrel resistance” or HPR was found to be present in nearly half of patients with cardiovascular disease undergoing intervention. Our data suggest HPR is more common in the cardiovascular patient population than previously appreciated. Evaluating patients for HPR is both inexpensive ($25) and rapid (< 10 min). Future randomized studies are warranted to determine whether HPR has a clinically detectable effect on revascularization outcomes.

Relationship between platelet aggregation and stroke risk after percutaneous coronary intervention: a PENDULUM analysis

In patients undergoing percutaneous coronary intervention (PCI) with a stent, high on-treatment platelet reactivity may be associated with an increased risk of stroke. This post hoc analysis of the PENDULUM registry compared the risk of post-PCI stroke according to on-treatment P2Y12 reaction unit (PRU) values. Patients aged ≥ 20 years who underwent PCI were stratified by baseline PRU (at 12 and 48 h post-PCI) as either high (HPR, > 208), optimal (OPR, > 85 to ≤ 208), or low on-treatment platelet reactivity (LPR, ≤ 85). The incidences of non-fatal ischemic and non-ischemic stroke through to 12 months post-PCI were recorded. Almost all enrolled patients (6102/6267 [97.4%]) had a risk factor for ischemic stroke, and most were receiving dual antiplatelet therapy. Of the 5906 patients with PRU data (HPR, n = 2227; OPR, n = 3002; LPR, n = 677), 47 had a non-fatal stroke post-PCI (cumulative incidence: 0.68%, ischemic; 0.18%, non-ischemic stroke). Patients with a non-fatal ischemic stroke event had statistically significantly higher post-PCI PRU values versus those without an event (P = 0.037). The incidence of non-fatal non-ischemic stroke was not related to PRU value. When the patients were stratified by PRU ≤ 153 versus > 153 at 12–48 h post-PCI, a significant difference was observed in the cumulative incidence of non-fatal stroke at 12 months (P = 0.044). We found that patients with ischemic stroke tended to have higher PRU values at 12–48 h after PCI versus those without ischemic stroke.Clinical trial registration: UMIN000020332.

The Changes of Blood Platelet Reactivity in the Presence of Crude Extracts Isolated from Leaves and Twigs of Elaeagnus Rhamnoides (L.) A. Nelson Measuring in Whole Blood

Uncontrolled blood platelet activation is an important risk factor of cardiovascular disease (CVDs). Various studies on phenolic compounds indicate that they have a protective effect on the cardiovascular system through different mechanisms, including the reduction of blood platelet activation. One of the plants that is particularly rich in phenolic compounds is sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson). The aim of the present study in vitro was to determine the anti-platelet properties of crude extracts isolated from leaves and twigs of E. rhamnoides (L.) A. Nelson in whole blood using flow cytometric and total thrombus-formation analysis system (T-TAS). The aim of our study was also analyze of blood platelet proteoms in the presence of different sea buckthorn extracts. A significant new finding is a decrease surface expression of P-selectin on blood platelets stimulated by 10 µM ADP and 10 µg/mL collagen, and a decrease surface expression of GPIIb/IIIa active complex on non-activated platelets and platelets stimulated by 10 µM ADP and 10 µg/mL collagen in the presence of sea buckthorn leaf extract (especially at the concentration 50 µg/mL). The twig extract also displayed antiplatelet potential. However, this activity was higher in the leaf extract than in the twig extract in whole blood. In addition, our present findings clearly demonstrate that investigated plant extracts have anticoagulant properties (measured by T-TAS). Therefore, the two tested extracts may be promising candidates for the natural anti-platelet and anticoagulant supplements.

Analysis The Effect of Gene and Platelet Testing Guide Dual Anti-Upgrade Therapy After PCI in Patients With ACS

Objective To analyze whether CYP2C19 gene and platelet testing guide ACS patients PCI benefit from postoperative dual antiplatelet escalation therapy. Methods Selecting ACS patients with 209 routine PCI surgery from January 2018 to January 2019 in Department of Cardiology, Third Affiliated Hospital of Guangzhou Medical University. Preoperative administration of aspirin 300mg and clopidogrel 600mg, and continued administration of clopidogrel 75mg/d and aspirin 100mg/d after operation. Genotype and light transmittance aggregation (LTA) was detected by gene chip 24 h after operation. According to genotype the remaining patients were divided into non loss of function (Non-LOF) alleles group Extensive-metabolisms (EMs) type, loss of function (LOF) alleles group as Intermediate metabolic (IMs) type and Poor-metabolisms (PMs) type. Define the maximum platelet aggregation rate (MPA)≥46% as hyperplatelet reactivity (HPR). The LOF group consisted of 23 patients who had both HPR and proclopidogrel and were upgraded to tigrillo for further treatment.The remaining patients without HPR who continued to be treated with clopidogrel comprised 90 patients in the LOF group without upgrade and 95 patients in the non-LOF group who continued to be treated with clopidogrel.Major adverse cardiovascular events (MACE) were recorded in the follow-up period of 1 year, and the incidence of MACE in the three groups was compared to determine whether gene and platelet detection could guide the benefit of dual anti-platelet upgrade therapy in ACS patients after PCI. Results There were 26 cases occurred during follow-up MACE, among which the incidence of unstable angina recurrence and overall MACE in the LOF allele-not-up group was the highest and significantly different compared with the Non-LOF allele group (P<0.05), there was no significant difference compared with the LOF allele-up group (0.05). while there was no significant difference between the Non-LOF allele and the LOF allele upgrading group (P>0.05). Conclusions Gene is an important factor in the difference of platelet reactivity and is associated with MACE. Upgraded treatments for high-risk patients screened for gene and platelet testing did not benefit.