BACKGROUND: ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y ₁ and P2Y ₁₂ receptors in ADP-induced TF exposure; (2) modulation of TF ^pos -platelets in anti-P2Y ₁₂ –treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets. METHODS: The effects of P2Y ₁ or P2Y ₁₂ antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y ₁₂ ^−/− , and patients with gray platelet syndrome. Ex vivo, P2Y ₁₂ inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization. RESULTS: In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TF ^pos -platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y ₁₂ inhibition only (−81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y ₁₂ is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TF ^pos -platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TF ^pos -platelets similar to the poor-responder group. Indeed, a stronger P2Y ₁₂ inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TF ^pos -platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome. CONCLUSIONS: Data show that TF expression is regulated by P2Y ₁₂ and not P2Y ₁ ; P2Y ₁₂ antagonists downregulate the percentage of TF ^pos -platelets. In clopidogrel good-responder patients, assessment of TF ^pos -platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.

Studies exploring alterations in blood coagulation and platelet activation induced by COVID-19 vaccines are not concordant. We aimed to assess the impact of four COVID-19 vaccines on platelet activation, coagulation, and inflammation considering also the immunization dose and the history of SARS-CoV-2 infection. TREASURE study enrolled 368 consecutive subjects (161 receiving viral vector vaccines -ChAdOx1-S/Vaxzevria or Janssen- and 207 receiving mRNA vaccines -Comirnaty/Pfizer-BioNTech or Spikevax/Moderna). Blood was collected the day before and 8 ± 2 days after the vaccination. Platelet activation markers (P-selectin, aGPIIbIIIa and Tissue Factor expression; number of platelet-monocyte and -granulocyte aggregates) and microvesicle release were analyzed by flow cytometry. Platelet thrombin generation (TG) capacity was measured using the Calibrated Automated Thrombogram. Plasma coagulation and inflammation markers and immune response were evaluated by ELISA. Vaccination did not induce platelet activation and microvesicle release. IL-6 and CRP levels (+30%), D-dimer, fibrinogen and F₁₊₂ (+13%, +3.7%, +4.3%, respectively) but not TAT levels significantly increased upon immunization with all four vaccines, with no difference among them and between first and second dose. An overall minor post-vaccination reduction of aPC, TM and TFPI, all possibly related to endothelial function, was observed. No anti-PF4 seroconversion was observed. This study showed that the four COVID-19 vaccines administered to a large population sample induce a transient inflammatory response, with no onset of platelet activation. The minor changes in clotting activation and endothelial function might be potentially involved at a population level in explaining the very rare venous thromboembolic complications of COVID-19 vaccination.

The relative contribution of small (sEVs) and large extracellular vesicles (lEVs) to the total plasma procoagulant potential is not yet well defined. Thus, we compared total and TF^pos-sEVs and -lEVs isolated from healthy subjects and COVID-19 patients during the acute phase of the infection and after symptom remission in terms of (1) vesicle enumeration using nanoparticle tracking assay, imaging flow cytometry, and TF immunofluorescence localization in a single-vesicle analysis using microarrays; (2) cellular origin; and (3) TF-dependent Xa generation capacity, as well as assessing the contribution of the TF inhibitor, TFPI. In healthy subjects, the plasma concentration of CD9/CD63/CD81^pos sEVs was 30 times greater than that of calcein^pos lEVs, and both were mainly released by platelets. Compared to lEVs, the levels of TF^pos-sEVs were 2-fold higher. The TF-dependent Xa generation capacity of lEVs was three times greater than that of sEVs, with the latter being hindered by TFPI. Compared to HSs, the amounts of total and TF^pos-sEVs and -lEVs were significantly greater in acute COVID-19 patients, which reverted to the physiological values at the 6-month follow-up. Interestingly, the FXa generation of lEVs only significantly increased during acute infection, with that of sEV being similar to that of HSs. Thus, in both healthy subjects and COVID-19 patients, the TF-dependent procoagulant potential is mostly sustained by large vesicles.

Background: Migraine is a chronic neurovascular disorder with a multifactorial aetiology. A strong relationship links migraine with aura (MHA) and patent foramen ovale (PFO), with migraine regression after PFO closure. Increased platelet aggregation and oxidative stress were documented in migraineurs. Current guidelines suggest the use of aspirin or P2Y12-antagonists. To date, however, no clear mechanisms connecting MHA to PFO has been demonstrated. Purpose: To perform a comprehensive analysis of platelet activation, inflammation and oxidative stress status in 78 aspirin-treated MHA-patients before (T0) and 6-months after (T1) PFO closure (LEARNER Study-NCT03521193-clinicaltrials.gov). Primary endpoint was migraine regression rate in relation to these parameters. Methods: P-selectinpos-, activated-glycoproteinIIbIIIa (aGPIIbIIIa)pos-, Tissue Factor (TF)pos-, reactive oxygen species (ROS)pos-platelets, platelet-leukocyte aggregates (PLA) and microvesicles (MVs) were evaluated by flow cytometry; thrombin generation (TG) by Calibrated Automated Thrombogram (CAT) assay; oxidative stress status by mass spectrometry. Twelve aspirin-treated-healthy subjects (HS) were enrolled for comparison. To test the effect of PFO patients' plasma on platelet activation, blood from HS was plasma-depleted and replaced with a pool of plasma from PFO patients. Results: Migraine resolution occurred in 69.7%, significant reduction in 27%, while no effect was observed in 2 patients (3.2%). Only ROSpos-platelets, and TFpos-platelets and -MVs were significantly higher at T0, sustaining a TG capacity that was associated with an altered blood GSSG/GSH (Oxidized/Reduced Glutathione) ratio. This phenotype reverted to HS levels at T1 (Fig. 1). MHA-PFO plasma, added to HS blood, mirrored the in vivo platelet activation and N-acetylcysteine blunted it. In vitro GSSG treatment of HS platelets reproduced the in vivo condition. Aspirin had little effect on the platelet prothrombotic phenotype which was effectively inhibited by a P2Y12-antagonist (Fig.2). Conclusions: MHA-PFO patients show a platelet-associated prothrombotic phenotype, characterized by a marked thrombin generation capacity sustained by an elevated number of platelets and MVs expressing a functionally active Tissue Factor and sustained by altered oxidative stress status. This phenotype, not fully controlled by aspirin but by P2Y12-antagonism, reverted after PFO closure together with a complete migraine remission. Funding Acknowledgement: Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health