Spontaneous Platelet Aggregation in Blood Is Mediated by FcγRIIA Stimulation of Bruton’s Tyrosine Kinase

High platelet reactivity leading to spontaneous platelet aggregation (SPA) is a hallmark of cardiovascular diseases; however, the mechanism underlying SPA remains obscure. Platelet aggregation in stirred hirudin-anticoagulated blood was measured by multiple electrode aggregometry (MEA) for 10 min. SPA started after a delay of 2–3 min. In our cohort of healthy blood donors (n = 118), nine donors (8%) with high SPA (>250 AU*min) were detected. Pre-incubation of blood with two different antibodies against the platelet Fc-receptor (anti-FcγRIIA, CD32a) significantly reduced high SPA by 86%. High but not normal SPA was dose-dependently and significantly reduced by blocking Fc of human IgG with a specific antibody. SPA was completely abrogated by blood pre-incubation with the reversible Btk-inhibitor (BTKi) fenebrutinib (50 nM), and 3 h after intake of the irreversible BTKi ibrutinib (280 mg) by healthy volunteers. Increased SPA was associated with higher platelet GPVI reactivity. Anti-platelet factor 4 (PF4)/polyanion IgG complexes were excluded as activators of the platelet Fc-receptor. Our results indicate that high SPA in blood is due to platelet FcγRIIA stimulation by unidentified IgG complexes and mediated by Btk activation. The relevance of our findings for SPA as possible risk factor of cardiovascular diseases and pathogenic factor contributing to certain autoimmune diseases is discussed.

Impact of COVID-19 disease on platelet reactivity and association with inflammatory parameters

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the coronavirus disease 2019 (COVID-19) pandemic. Aside from the pulmonary manifestations, COVID-19 is associated with increased risk of venous and arterial thrombotic complications. The actual impact of SARS-CoV-2 infection on platelet reactivity and whether this is mediated by a hyperinflammatory status has not been fully elucidated to date. Objective To evaluate platelet reactivity in COVID-19 patients compared to healthy subjects and to assess the association between platelet reactivity and levels of inflammatory biomarkers among COVID-19 patients. Methods This prospective observational investigation included COVID-19 patients admitted into a tertiary care hospital and adult healthy volunteers, all of them not receiving any antiplatelet therapy. Subjects were classified in three groups: 1) Healthy subjects (HS group); 2) COVID-19 patients in a pulmonary phase (viral pneumonia and bilateral infiltrates) but without meeting criteria for systemic hyperinflammation (C19-Pulm group); and 3) COVID-19 patients in a hyperinflammation phase (C19-Infl group) meeting at least 2 of the following criteria: CRP>100mg/l, D-dimer >1000mcg/l, LDH>400U/l, ferritin>1000ng/ml, IL-6>70ng/l. Blood samples for platelet function testing and quantification of inflammatory parkers were collected at a single visit. Platelet function was measured with multiple electrode aggregometry using ADP (MEA-ADP, primary endpoint), arachidonic acid (AA) and thrombin receptor activating peptide (TRAP) as stimuli. Unadjusted analyses are presented. Results A total of 60 patients were included in the present investigation (20 in each group). A significantly greater platelet reactivity, measured with MEA-ADP, was observed in both groups of COVID-patients compared to healthy subjects (HS: 634,9±53,5, C19-Pulm: 919,9±53,5 and C19-Infl: 931,6±53,5 AU*min; p for C19-Pulm vs. HS <0,001, p for C19-Infl vs. HS <0,001, p for C19-Pulm vs. C19-Infl 0,878; Figure 1). Parallel findings were found when using AA as stimulus for platelet aggregation showing greater platelet aggregation in COVID-19 patients compared to healthy subjects, but numerical differences were not statistically significant when using TRAP. Among COVID-19 patients, when stratified by IL-6 levels splitted into tertiles, greater platelet reactivity was observed in patients with higher IL-6 concentrations (mid and upper tertile together) compared to those with values in the lower tertile, as assessed with MEA-ADP (lower tertile: 829,0±75,8, mid and upper tertile: 1028,7±56,2; p=0,043); a similar trend was observed with AA and TRAP as stimuli. Conclusion Patients with severe COVID-19 disease have greater platelet reactivity than healthy subjects. Increased IL-6 levels might be associated with the observed heightened platelet reactivity among COVID-19 patients. FUNDunding Acknowledgement Type of funding sources: None. Figure 1

Bleeding Complication in a Patient with Concomitant Use of Rivaroxaban and Saffron Supplement: A Case Report

Background Direct oral anticoagulants (DOACs) carry a lower potential risk of food/herb and drug interactions compared with oral vitamin K antagonists. However, as a new class of medications some of these interactions have not been fully known. Case presentation: A 64-year old male with a medical history of non-valvular atrial fibrillation presented to the emergency department with a complaint of acute onset epistaxis and bleeding gums following the concomitant use of rivaroxaban and saffron supplement. Rivaroxaban plasma concentration was 54 ng/ml with a post-intake delay of 17 hours. The results of laboratory tests were unremarkable except for platelet function tests. Whole blood multiple electrode aggregometry was performed to assess platelet function. Area under the aggregation curve (AUC) values were 83 and 51 aggregation unit (AU)*min by arachidonic acid and adenosine diphosphate-induced platelet aggregation tests, respectively. As the patient had not taken any antiplatelet medication, platelet dysfunction was greatly attributed to the saffron supplement. The patient was immediately admitted to hospital and received local hemostatic measures and tranexamic acid. Moreover, saffron was discontinued permanently and rivaroxaban was paused for 24 hours. The bleeding stopped a few hours later and the patient was discharged after 2 days in a good general condition. Subsequently, he was followed up at 4, 8, and 12-week intervals. He was in a stable clinical condition with no bleeding complications. The patient was advised to consult with his doctor or pharmacist before taking any supplement or herbal medicine to ensure possible interactions. Conclusions It seems that coadministration of DOACs and saffron supplements should be avoided due to the potential drug-herbal interactions and possible risk of subsequent bleeding complications. However, further studies are needed to confirm the findings and assess the clinical significance.

Human -Synuclein Inhibits Platelets Aggregation in vitro by Interfering with the -Thrombin/Protease-Activated Receptor 1 Functional Axis

-Synuclein (Syn) is a small (140 amino acids) disordered, acidic (pI: 4.7) protein, highly conserved in vertebrates and implicated in the pathogenesis of Parkinsons disease (PD), a neurodegenerative disease characterized by the deposition of Syn amyloid fibrils in dopaminergic neurons. Beyond the central nervous system, significant expression of Syn has also been measured in the blood (~1 M), where platelets are the main cellular hosts of Syn. Although the pathological implication of Syn in PD is widely accepted, the physiological role of blood Syn is still elusive. Starting from the notion that platelets are either the major cellular reservoir of Syn in the blood and, concomitantly, act as key players in hemostasis, being activated also by -thrombin (T) via cleavage of protease-activated receptors (PARs), we decided to investigate the possibility that Syn could modulate platelet activation by interfering with the T-PAR functional axis. Using multiple electrode aggregometry, i.e. a fast and specific platelet-function-testing method, as well as steady-state fluorescence spectroscopy, surface plasmon resonance, and fluorescence microscopy, we show here that monomeric Syn functions as a negative regulator of T-mediated platelets activation. Syn acts either directly, via competitive inhibition of PAR1 activation by T and TRAP6 agonist, and indirectly, by scavenging T on the platelet plasma membrane. A simple electrostatic model of Syn platelet antiaggregating effect is proposed and the possible role of the protein at the interplay of amyloidosis and thrombosis is discussed.

The Pre- and Postoperative Prevalence and Risk Factors of ASA Nonresponse in Vascular Surgery

Background An antiplatelet therapy with acetylsalicylic acid (ASA) is prescribed in the prevention of cardiovascular events, but around 24% of ASA takers are resistant to the treatment. Aim In this prospective, observational cohort study, we aimed to identify the prevalence and risk factors of ASA nonresponse in patients who underwent vascular surgery. Methods The study was conducted in the University hospital in Frankfurt am Main. In total, 70 patients were pre-treated with 100 mg of ASA per day and underwent either elective carotid thromboendarterectomy, femoral thromboendarterectomy or endovascular aneurysm repair of the abdominal aorta. The platelet function was measured on the first preoperative and the second or fourth postoperative day with the multiple electrode aggregometry by in-vitro stimulation with arachidonic acid (ASPItest) and thrombin receptor activating peptide 6 (TRAPtest). The primary end point was the in-vitro induced platelet aggregation in the ASPItest. If the ASPItest amounted ≥400 AU × min, the patients were categorized as ASA nonresponders. Results The total prevalence of ASA nonresponse in our study was 20% preoperatively and 35.7% postoperatively (p = 0.005). As significant predictors for ASA nonresponse, we demonstrated the area under the aggregation curve in the TRAPtest preoperatively (p = 0.04) and postoperatively (p = 0.02), and the two comorbidities arterial hypertension ( P < .001; rho 0.44) and diabetes mellitus (p = 0.04; rho 0.39), which are already well known to be associated with ASA nonresponse. Conclusion In conclusion, data of the study indicate a high incidence of perioperative, laboratory ASA nonresponse in patients undergoing vascular surgery.