Antiphospholipid syndrome (APS) is a thromboinflammatory disorder in which antiphospholipid antibodies (aPL), particularly anti-β2GPI, initiate a complex, graded platelet activation that integrates immune signaling with coagulation. Clinical and multiparametric flow-cytometric evidence demonstrates a sustained prothrombotic platelet endotype characterized by enrichment of tissue factor-positive (TFpos)-platelets, an increase in the number of TFpos-platelet-leukocyte heteroaggregates, and in the expression of classical adhesion markers (P selectin, activated αIIbβ3) despite anticoagulation. These abnormalities correlate with high-risk aPL profiles and likely contribute to residual thrombotic risk not captured by conventional coagulation biomarkers. Pathogenic anti-β2GPI - especially Domain 1 specific-IgG - induce a selective, early procoagulant TFpos-phenotype in the absence of full adhesive activation. This phenotype reflects peripheral ApoER2-dependent signaling rather than bone marrow-driven megakaryocyte programming. Inflammatory mediators, notably IL6, act as indispensable amplifiers, converting the TF-only phenotype into a broader thromboinflammatory program involving P-selectin expression, integrin activation, phosphatidylserine exposure, and formation of multicellular immunothrombotic units. Omics-based profiling corroborates a chronically primed platelet state with dysregulated inhibitory checkpoints (CD73-adenosine-cAMP axis), enhanced ADP/P2Y12 signaling, and membrane remodeling conducive to procoagulant differentiation. TFpos-platelets emerge as a mechanistically grounded biomarker candidate of thrombotic propensity and a potential therapeutic target. Ex vivo data show distinct pharmacological sensitivity since aspirin and P2Y12 inhibition attenuate both adhesive and TF-dependent procoagulant programs, whereas hydroxychloroquine selectively modulates classical activation markers but not platelet-associated TF. These observations delineate separable adhesive vs. procoagulant platelet modules and support a precision medicine framework in which quantification and targeted suppression of TFpos-platelets may attenuate the basal prothrombotic milieu and mitigate APS-related thrombotic risk.

Tissue Factor (TF)pos-platelets represent a subset of the platelet population. Recently, this subset has been shown to predict cardiovascular mortality in patients with coronary artery disease, establishing it as a biomarker useful for thrombotic risk stratification. Accurate quantification of TFpos-platelets by flow cytometry requires strict standardization of pre-analytical handling, staining procedures, and instrument settings, particularly in multicenter studies where technical variability may affect their measurement. This protocol describes a harmonized workflow for whole-blood flow cytometry assessment of circulating TFpos-platelets, designed to ensure reproducibility across laboratories with different technical infrastructures. The procedure includes standardized blood collection and whole-blood fixation to preserve the in vivo platelet phenotype. Two different methods for sample preparation are provided according to local laboratory capabilities: shipment of fixed samples to the Core Laboratory for centralized staining, acquisition, and analysis for centers without flow cytometry facilities, or local staining and acquisition for centers equipped with flow cytometry instrumentation and trained personnel. The assessment of the percentage of TFpos-platelets is achieved by direct labeling with anti-TF Star Fluor 488 and anti-CD41 PerCP-Cy5.5 antibodies to identify the target protein and the platelet population marker, respectively. Flow cytometer harmonization is achieved either through a shared acquisition template for identical cytometer models or through bead-based alignment for different platforms. The workflow further incorporates a standardized gating approach and centralized data analysis to enable reliable comparison of TFpos-platelet measurements across sites. Representative results demonstrated that this workflow supports reproducible quantification of TFpos-platelets across the validated multicenter acquisition settings. This protocol may facilitate broader application of TFpos-platelet assessment in thrombotic risk stratification and support wider standardization of platelet flow cytometry in translational and clinical research.

A subset of circulating human platelets stores Tissue Factor (TF) intracellularly, the key activator of the blood coagulation cascade and thrombus formation. Upon platelet activation, TF is exposed on the cell membrane, where it binds to FVII, ultimately leading to thrombin generation. Considering that (1) levels of TF-positive platelets increase in various clinical settings, contributing to the patient's prothrombotic phenotype, and (2) different drugs can modulate platelet-associated TF expression, a standardized method for assessing TF-positive platelets is valuable, as its evaluation has been controversial in the past. Here, we outline a protocol for measuring the percentage of TF-positive platelets using flow cytometry in whole blood and platelet-rich plasma (PRP)/washed platelets. This protocol aims to provide detailed instructions for quantifying the percentage of TF-positive platelets by assessing the protein (1) intracellularly in resting conditions, and (2) on the cell surface, in both resting and activated conditions. The first section provides essential information for correctly performing blood withdrawal to ensure that pre-analytical procedures do not affect the results. Next, the protocol focuses on sample preparation and labeling procedures for flow cytometry analysis. Detailed steps for cell stimulation, labeling, fixation, and permeabilization -- where necessary -- are outlined. Finally, instructions for flow cytometry settings to correctly identify the platelet population and analyze TF-positive events are described. Lastly, the method includes the procedure for preparing PRP if TF-positive platelets are to be measured in isolated platelets. Since only a subset of platelets contains TF, it is important to ensure that these platelets are not lost during the centrifugation steps required to obtain PRP.

Patent Foramen Ovale (PFO) is a congenital cardiac anomaly, anatomically persistent in approximately 25% of the adult population. While traditionally associated with paradoxical embolism and cryptogenic stroke, increasing evidence suggests a functional link between PFO and migraine with aura. However, the biomechanical mechanisms underlying these associations remain poorly defined, particularly regarding the role of PFO morphology in modulating local hemodynamics and red blood cell (RBC) mechanical stress. This study employs computational fluid dynamics (CFD) combined with Lagrangian particle tracking to assess the impact of PFO tunnel geometry on flow behavior and RBC loading across eight representative morphologies. Velocity fields, wall shear stress (WSS), and particle-level stress histories were computed under physiologically calibrated boundary conditions replicating Valsalva-induced shunting. Results reveal a dichotomy between elongated/narrow and short/wide morphotypes, with the former exhibiting jet-like flows, higher WSS, and significantly elevated RBC stress metrics (up to 31 Pa and 0.49 Pa·s of stress accumulation). The length-to-mean-quadratic-diameter ratio ([Formula: see text]) emerged as a strong predictor of mechanical exposure ([Formula: see text]), while outlet diameter correlated with potential systemic desaturation. This dual-scale analysis reveals a mechanistic connection between pathological stress levels and tunnel geometry, identifying [Formula: see text] as a candidate index for future imaging-based stratification of PFO-related clinical risk.

Thrombotic risk stratification in coronary artery disease (CAD) patients is an unmet need. CAD patients show increased platelet activation, but its prognostic relevance remains unexplored. We aimed to assess the prognostic value of platelet-activation markers on mortality in CAD patients. Surface expression of platelet-associated activated GPIIbIIIa, P-selectin, tissue factor (TF), and platelet–leukocyte aggregate was analyzed in 527 CAD patients (acute coronary syndromes [ACS, n = 149] and chronic coronary syndrome [CCS, n = 378]) by whole-blood flow-cytometry and plasma F1 + 2 by ELISA. With COX regression model 5-year survival analysis from all-cause (AC) and cardiovascular (CV) mortality was performed. Cross-validated cut-off of TFpos platelets was calculated by Euclidean distance method. AC and CV mortality rates were 9.7 and 6.5%, respectively. Among the biomarkers evaluated, only TF independently predicted AC mortality (hazard ratio [HR] =2.02, p = 0.042) also after adjustment for CAD presentation. ACS and CCS patients with TFpos platelets >4% (the best cut-off value for all-cause mortality prediction) had the highest levels of F1 + 2 and a worse prognosis for AC and CV mortality (HR = 1.91; p = 0.018 and HR = 2.51; p = 0.005; respectively) than those with <4% TFpos platelets. Interestingly, patients on dual antiplatelet therapy (n = 246, 46.8%) responder to P2Y12 inhibitors with TFpos platelets >4% had the highest risk for AC mortality (HR = 4.11; p = 0.0215) and CV mortality (HR = 6.88; p = 0.0408). In these patients, TFpos platelet levels outperformed a clinical model in CV mortality prediction (net reclassification improvement = 0.436, p < 0.001). Platelet TF predicted AC (HR = 3.03; p = 0.012) and CV mortality (HR = 3.56; p = 0.008) in aspirin-only treated patients also (n = 239, 45.3%). The percentage of circulating TFpos platelets may serve as an independent predictor of AC and CV mortality in CAD patients on antiplatelet therapy.

Atherothrombosis, the primary driver of acute cardiovascular (CV) events, is characterized by the activation of three key pathophysiological pathways: platelets, coagulation, and inflammation. Dual antiplatelet therapy (DAPT) is the current standard of care for patients with acute coronary syndrome, providing significant reductions in cardiovascular (CV) events, albeit with an associated increased risk of bleeding. However, the high residual risk of recurrent events among these patients highlights the need for alternative strategies to treat and prevent atherothrombosis. To this extent, several approaches aimed at targeting atherothrombosis have been proposed. Among these, a strategy of dual-pathway inhibition simultaneously targeting platelets, using single or DAPT, and coagulation, using a low-dose anticoagulant such as rivaroxaban 2.5 mg twice daily, has shown to reduce CV events but at the expense of increased bleeding. Targeting inflammatory pathways has the potential to be a highly effective strategy to tackle atherothrombosis without increasing bleeding risk. Several anti-inflammatory agents have been tested in patients with coronary artery disease, but to date only colchicine is approved for secondary prevention on top of standard care, including antiplatelet therapy. However, many aspects of colchicine’s mechanism of action, including its antiplatelet effects and how it synergizes with antiplatelet therapy, remain unclear. In this review, we summarize the available clinical and pre-clinical evidence on the antiplatelet effects of colchicine and its synergistic interactions with antiplatelet therapy, highlighting their potential role in addressing atherothrombosis.

Anti-phospholipid antibodies (aPL) mediate platelet- and leukocyte-interaction with damaged endothelium, contributing to anti-phospholipid syndrome (APS) vasculopathy. This study aimed to understand the mechanisms sustaining the pro-adhesive/-thrombotic platelet phenotype and the in vitro effects of different drugs. We included 34 primary APS (PAPS) patients and 12 healthy subjects (HS). All patients had medium/high aPL levels with vascular/obstetric symptoms according to the 2023 ACR/EULAR classification. In vivo, we evaluated by flowcytometry platelet activation markers (P-selectin, activated GPIIbIIIa [aGPIIbIII], tissue factor [TF], ApoER2 and β2GPI expression and platelet-monocyte and -granulocyte aggregates [PMA and PGA]). In vitro, the impact of antiplatelet and anti-inflammatory drugs on platelet activation induced by different aPL subpopulations was investigated. PAPS patients exhibited greater percentages of circulating ApoER2pos-, P-selectinpos-, aGPIIbIIIapos-, TFpos-platelets, and TFpos-platelet-leukocyte aggregates. In vitro, HS blood incubation with PAPS plasma fully reproduced the activation found in vivo. While anti-β2GPI-Domain(D)1, but not anti-D4,5, immunoglobulin (Ig)G upregulated platelet TF expression only, the addition of interleukin (IL)-6 also induced P-selectin and aGPIIbIIIa upregulation. An IL-6 receptor-blocking monoclonal antibody prevented the pro-adhesive/-coagulant platelet phenotype and the formation of platelet-leukocyte aggregates mediated by PAPS plasma or by total IgG plus exogenous IL-6. While aspirin and P2Y12 inhibitor fully inhibited platelet activation, hydroxychloroquine (HCQ) did not blunt TF expression. PAPS patients exhibit circulating pro-adhesive/-coagulant (TF-positive) platelets and platelet-leukocyte aggregates mediated by β2GPI-D1-dependent IgG and an inflammatory trigger. While aspirin and P2Y12 inhibitor significantly inhibited the aPL-mediated P-Selectin and TF upregulation, HCQ affected the adhesion phenotype only, and might not be adequate to prevent platelet-mediated thrombosis.

Frailty is an age-related syndrome commonly associated with different comorbidities, and its occurrence is particularly frequent in patients with Alzheimer's disease (AD). A persisting low-grade inflammation has been suggested to favor the onset of both AD and frailty. Besides their role in hemostasis and thrombosis, blood platelets are true inflammatory cells, and their direct contribution to the onset and progression of AD has been documented. In this work, we investigated whether platelet hyperreactivity and pro-oxidative functions are implicated in the development of frailty in a mouse model of AD, the APP23 mice. Assessment of 31 specific clinical signs of deterioration in mice at 3, 9, and 18 months of age demonstrated that the development of frailty was significantly more pronounced in the APP23 mice compared to wild-type littermates. In 18-month-old APP23 mice, a significant platelet hyperreactivity was detected as shown by a significantly stronger platelet aggregation in response to submaximal stimulation of both collagen and thrombin receptors. Moreover, the pro-inflammatory function of platelets, evaluated as circulating and agonist-induced platelet-neutrophil aggregate formation, was significantly increased in aged APP23 mice compared to wild-type littermates. Platelet hyperreactivity was partially prevented by prolonged treatment with the anti-oxidant agent Tempol, which reduced both agonist-induced aggregation and platelet-neutrophil aggregate formation. Importantly, prolonged treatment of APP23 mice with Tempol significantly reduced also the frailty index score in 18-month-old animals. These results outline the possible beneficial effect of an anti-oxidant treatment in hampering platelet hyperreactivity and preventing the onset of frailty associated to AD.

Background Given the role of platelets in coronary artery disease (CAD), assessment of a soluble platelet-activation marker may be useful to improve thrombotic risk stratification. Objectives This study aimed to perform a meta-analysis investigating the association between levels of 14 such markers associated with CAD. Methods PubMed, Web of Science, and Excerpta Medica dataBASE (EMBASE) were searched until November 2024. The primary end point was the difference in levels of 11-dehydro-thromboxane B2, 2,3-dinor-thromboxane B2, β-thromboglobulin, soluble CD40L (sCD40L), glycocalicin, glycoprotein (GP)V, GPVI, matrix metalloproteinase (MMP)-9 and MMP-2, platelet factor (PF)4, soluble (s) P-selectin, SCUBE1, serotonin and thrombospondin (TSP)-1 between patients with CAD and healthy subjects (HSs) in plasma and/or serum. When possible, patients with CAD were stratified into acute coronary syndrome (ACS) and chronic coronary disease. Standardized mean difference (SMD) was calculated. Results Due to heterogeneity in the assessed studies, meta-analysis was performed for sCD40L, soluble GPV, MMP-9, PF4, sP-selectin, SCUBE1, and TSP-1. All markers but TSP-1 were significantly elevated in patients with CAD compared with HSs. Differences in sCD40L and SCUBE1 were statistically significant only when studies that assessed plasma were combined with those that assessed serum. When compared with HSs, the differences were bigger in patients with ACS than patients with chronic coronary disease for MMP-9 (SMD, 2.49 vs 0.49), PF4 (SMD, 2.01 vs 0.96), and sP-selectin (SMD, 1.81 vs 0.63). Publication bias was identified for sCD40L and, in ACS, for sP-selectin and PF4. Conclusion The increased levels of sCD40L, soluble GPV, MMP-9, PF4, sP-selectin, and SCUBE1 in patients with CAD compared with HSs provide a rationale for designing new studies to address the potential of such molecules as biomarkers for thrombotic risk stratification.

Background: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19, or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen, but only a few studies have compared some of these assays. Objectives: The International Society on Thrombosis and Haemostasis Scientific and Standardization Committee Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity, and reproducibility of these assays. Methods: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without lipopolysaccharide stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. Results: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared with antigen assays. In addition, there was a large intra-assay and interassay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared with assays that isolated EVs by high-speed centrifugation. Conclusion: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

In the present study, we provide evidence on the potential mechanisms involved in the residual pulmonary impairment described in long COVID syndrome. Data highlight that lung damage is significantly associated with a proinflammatory platelet phenotype, characterized mainly by the formation of platelet-leukocyte aggregates. In ex vivo experiments, long COVID plasma reproduces the platelet activation observed in vivo and highlights low-grade inflammation as a potential underpinning mechanism, exploiting a synergistic activity between C-reactive protein and subthreshold concentrations of interleukin 6. The platelet-activated phenotype is blunted by anti-inflammatory and antiplatelet drugs, suggesting a potential therapeutic option in this clinical setting.

Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbβ3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.

Background Endothelial dysfunction plays a central role in the pathophysiology of COVID-19 and is closely linked to the severity and mortality of the disease. The inflammatory response to SARS-CoV-2 infection can alter the capacity of the endothelium to regulate vascular tone, immune responses, and the balance between anti-thrombotic and pro-thrombotic properties. However, the specific endothelial pathways altered during COVID-19 still need to be fully understood. Objective In this study, we sought to identify molecular changes in endothelial cells induced by circulating factors characteristic of COVID-19. Methods and results To this aim, we cultured endothelial cells with sera from patients with COVID-19 or non-COVID-19 pneumonia. Through transcriptomic analysis, we were able to identify a distinctive endothelial phenotype that is induced by sera from COVID-19 patients. We confirmed and expanded this observation in vitro by showing that COVID-19 serum alters functional properties of endothelial cells leading to increased apoptosis, loss of barrier integrity, and hypercoagulability. Furthermore, we demonstrated that these endothelial dysfunctions are mediated by protease-activated receptor 2 (PAR-2), as predicted by transcriptome network analysis validated by in vitro functional assays. Conclusion Our findings provide the rationale for further studies to evaluate whether targeting PAR-2 may be a clinically effective strategy to counteract endothelial dysfunction in COVID-19.