Platelet-monocyte Aggregates Research Articles

A loss of tuning of both pro-coagulant and inflammatory responses in monocytes in patients with preeclampsia

An imbalance between pro- and anti-angiogenesis is one of the leading causes of preeclampsia (PE). Monocytes, known as regulators of angiogenesis during immune responses, cooperate with platelets, but the specifics of these responses during pregnancy remain unclear. In this study, we investigated the relationship between pro-coagulant responses on monocytes [platelet activation marker CD61 as a monocyte-platelet aggregate (MPA), tissue factor (CD142), etc.], inflammatory responses [soluble CD40 ligand (sCD40L), soluble suppression of tumorigenesis-2 (sST2), etc.], and the balance of angiogenesis [soluble Fms-related receptor tyrosine kinase 1/placental growth factor (sFlt-1/PLGF) ratio]. In PE, markers of pro-coagulant and inflammatory responses were higher than those in normal pregnancy (NP). Interestingly, in NP, these markers harmonized with the sFlt-1/PLGF ratio, but not in PE. Furthermore, ex vivo examinations showed that upregulation of CD142 induced by additional platelet activation with adenosine diphosphate was diminished in PE. Conversely, low-dose aspirin, which is used as a preventive treatment for PE, could inhibit the increase of CD61 and sST2 under inflammatory stimuli and platelet activation in NP but not in PE. These results indicate that monocytes in PE upregulate basal activity and lose responsiveness to stimulation. The elevation of pro-coagulant and inflammatory responses may be mitigated by prophylaxis with low-dose aspirin. Therefore, the findings suggesting a loss of tuning of both pro-coagulant and inflammatory responses on monocytes help in understanding the pathology of PE. The harmonization between pro-coagulant responses, inflammatory responses, and angiogenesis may serve as useful indicators for the prediction and preventive treatment of PE.

The Correlation of Platelet-Monocyte Aggregate Formation and IFITM3 Gene Expression with COVID-19 Severity

Background: Platelet-leukocyte aggregates have been implicated in various infectious and inflammatory diseases. The Interferon-induced transmembrane protein 3 (IFITM3) protein plays a role in eliminating viral infections, but its role in the severity of COVID-19 is not well understood. Objectives: We aimed to investigate the correlation between IFITM3 mRNA expression and platelet-monocyte complex levels with the severity of COVID-19, as well as various inflammatory and coagulation markers. Methods: We conducted a cross-sectional study on 54 COVID-19 patients, classified into severe and mild/moderate subgroups. Demographics and laboratory findings were extracted from patients' medical records. We measured IFITM3 mRNA expression in patients' buffy coats using q-RT-PCR and used flow cytometry with CD61 and CD14 markers to measure platelet-monocyte aggregates. Results: No significant difference was found in IFITM3 mRNA expression levels or platelet-monocyte complexes between severe and mild/moderate groups (P = 0.067 and P = 0.056). Lymphocyte counts were significantly higher in the mild/moderate subgroup (21.7 ± 8.9 vs 16.3 ± 10.9, P = 0.02), while neutrophil counts were significantly higher in severe patients (78.3 ± 12.2 vs 72.3 ± 9.9, P = 0.01). Additionally, levels of CRP and LDH were significantly higher in severe COVID-19 patients (P = 0.01 and P = 0.001, respectively). A strong positive correlation was observed between the hospitalization period and CRP, CRP with neutrophils and LDH, as well as between O2 saturation and lymphocytes (P < 0.001, P = 0.0003, P = 0.002, and P = 0.005, respectively). Conclusions: Our findings suggest that IFITM3 gene expression and platelet-monocyte aggregate levels do not correlate with disease outcomes in COVID-19. However, further investigations with larger sample sizes are needed to better understand the mechanisms involved. Monitoring inflammatory and coagulation markers remains important for managing COVID-19 patients.

Monocyte Single-Cell Multimodal Profiling in Cardiovascular Disease Risk States.

Monocytes are a critical innate immune system cell type that serves homeostatic and immunoregulatory functions. They have been identified historically by the cell surface expression of CD14 and CD16. However, recent single-cell studies have revealed that they are much more heterogeneous than previously realized. We utilized cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing to describe the comprehensive transcriptional and phenotypic landscape of 437 126 monocytes. This high-dimensional multimodal approach identified vast phenotypic diversity and functionally distinct subsets, including IFN-responsive, MHCIIhi (major histocompatibility complex class II), monocyte-platelet aggregates, as well as nonclassical, and several subpopulations of classical monocytes. Using flow cytometry, we validated the existence of MHCII+CD275+ MHCIIhi, CD42b+ monocyte-platelet aggregates, CD16+CD99- nonclassical monocytes, and CD99+ classical monocytes. Each subpopulation exhibited unique characteristics, developmental trajectories, transcriptional regulation, and tissue distribution. In addition, alterations associated with cardiovascular disease risk factors, including race, smoking, and hyperlipidemia were identified. Moreover, the effect of hyperlipidemia was recapitulated in mouse models of elevated cholesterol. This integrative and cross-species comparative analysis provides a new perspective on the comparison of alterations in monocytes in pathological conditions and offers insights into monocyte-driven mechanisms in cardiovascular disease and the potential for monocyte subpopulation targeted therapies.

Circulating monocyte populations as biomarker for abdominal aortic aneurysms: a single-center retrospective cohort study.

Abdominal aortic aneurysm (AAA) development is driven by inflammation, in particular myeloid cells, which represent attractive biomarker candidates. Yet to date, the maximum aortic diameter is the only clinically applied predictor of AAA progression and indicator for surgical repair. We postulated that aortic inflammation is reflected in a systemic change of monocyte populations, which we investigated regarding marker potential in AAA diagnosis and prognosis. We conducted a single-center retrospective cohort study in a diagnostic setting, measuring monocyte subsets by flow cytometry in peripheral blood samples of 47 AAA patients under surveillance, matched with 25 healthy controls and 25 patients with peripheral artery disease (PAD). In a prognostic setting, we acquired longitudinal data of 60 AAA patients including aneurysm growth assessment by computed tomography at 6-month intervals. Blood levels of total monocytes, CD16+ monocytes and particularly intermediate monocytes were significantly increased in AAA patients versus healthy individuals and were also elevated compared to PAD patients. The combination of intermediate monocyte and D-dimer blood levels outperformed the individual diagnostic marker values. Additionally, the elevated concentrations of total monocytes, intermediate monocytes, and monocyte-platelet aggregates (MPA) were suited to predict rapid AAA progression over short-term periods of six months. Of note, MPA were identified as independent predictor of AAA disease progression in multivariable analysis. Circulating monocyte subsets are elevated in AAA patients and support diagnosis and prediction of aneurysm progression. Monocyte subsets and D-dimer reflect different hallmarks (inflammation and hemostasis) of AAA pathology and when combined, may serve as improved biomarker.

Association of elevated circulating monocyte-platelet aggregates with hypercoagulability in patients with nephrotic syndrome

BackgroundHypercoagulability emerges as a central pathological feature and clinical complication in nephrotic syndrome. Increased platelet activation and aggregability are closely related to hypercoagulability in nephrotic syndrome. Monocyte-platelet aggregates (MPAs) have been proposed to represent a robust biomarker of platelet activation. The aim of this study was to investigate levels of the circulating MPAs and MPAs with the different monocyte subsets to evaluate the association of MPAs with hypercoagulability in nephrotic syndrome.MethodsThirty-two patients with nephrotic syndrome were enrolled. In addition, thirty-two healthy age and sex matched adult volunteers served as healthy controls. MPAs were identified by CD14 monocytes positive for CD41a platelets. The classical (CD14 + + CD16-, CM), the intermediate (CD14 + + CD16+, IM) and the non-classical (CD14 + CD16++, NCM) monocytes, as well as subset specific MPAs, were measured by flow cytometry.ResultsPatients with nephrotic syndrome showed a higher percentage of circulating MPAs as compared with healthy controls (p < 0.001). The percentages of MPAs with CM, IM, and NCM were higher than those of healthy controls (p = 0.012, p < 0.001 and p < 0.001, respectively). Circulating MPAs showed correlations with hypoalbuminemia (r=-0.85; p < 0.001), hypercholesterolemia (r = 0.54; p < 0.001), fibrinogen (r = 0.70; p < 0.001) and D-dimer (r = 0.37; p = 0.003), but not with hypertriglyceridemia in nephrotic syndrome. The AUC for the prediction of hypercoagulability in nephrotic syndrome using MPAs was 0.79 (95% CI 0.68–0.90, p < 0.001). The sensitivity of MPAs in predicting hypercoagulability was 0.71, and the specificity was 0.78.ConclusionIncreased MPAs were correlated with hypercoagulability in nephrotic syndrome. MPAs may serve as a potential biomarker for thrombophilic or hypercoagulable state and provide novel insight into the mechanisms of anticoagulation in nephrotic syndrome.

Platelet Activation is Upregulated in Cirrhotic Patients with Portal Vein Thrombosis.

Platelet plays a key role in thrombosis formation, especially that the alteration of platelet function may influence the thrombosis development. This study aimed to investigate platelet function alterations in the formation of portal vein thrombosis (PVT) in cirrhosis. Cirrhotic patients admitted to The First Affiliated Hospital of Soochow University between October 2021 and April 2023 were recruited and divided into PVT and non-PVT groups according to radiological results. Clinical parameters and prognosis were also collected and assessed to identify potential risk factors. Flow cytometry was used to detect the expression of CD62p, CD63, monocyte-platelet aggregates (MPAs), neutrophil-platelet aggregates (NPAs), and von Willebrand factor antigen (vWF-Ag) to evaluate platelet activation and adhesion function. A total of 145 subjects were enrolled in our study including 60 cirrhotic PVT patients, 60 cirrhotic non-PVT patients, and 25 healthy volunteers. Multivariate analysis suggested that esophageal gastric varices, splenectomy, and D-dimer were independent risk factors for PVT pathogenesis in cirrhosis. The vWF-Ag expression level was reduced in the PVT group compared with the non-PVT group (p = 0.046) but was not an independent risk factor for PVT formation pathogenesis. The expression of CD41+CD62p+ and CD41+CD63+ platelets in the PVT group was significantly elevated compared with that in the non-PVT group (p < 0.05). There were no significant differences in MPAs and NPAs between the two cirrhotic groups. Subgroup analysis showed that the mean fluorescence intensity (MFI) of CD62p and CD63 was associated with portal hypertension-related complications (p = 0.008, p < 0.001), and CD63 MFI was significantly associated with thrombosis burden (p = 0.019). CD41+CD62p+ and CD41+CD63+ platelets as well as MPAs and NPAs were highly expressed in the splenectomy group compared with those in the nonsplenectomy group in cirrhotic patients (p < 0.05). Positive correlations were found between CD62p MFI and CD63 MFI, MPAs and NPAs (r = 0.642, p < 0.001; r = 0.378, p = 0.003; r = 0.430, p < 0.001). In addition, platelet counts were also correlated with MPAs (r = 0.556, p < 0.001) and NPAs (r = 0.467, p < 0.001). Cirrhotic patients with PVT had higher mortality and were more likely to experience portal hypertension-related complications in the prognostic analysis (p < 0.05). Highly activated platelet function exists in patients with cirrhosis, and platelet activation was elevated during PVT formation, suggesting that activated platelets may participate in the formation of PVT in patients with cirrhosis.

Postgenomic aspects of resistance to acetylsalicylic acid in patients with coronary artery disease

Aim. To study the expression level of the P-selectin gene (SELP) mRNA and the level of intercellular interaction in patients with coronary artery disease (CAD) before and after coronary artery bypass grafting (CABG) in patients with resistance to acetylsalicylic acid (ASA).Material and methods. The study included 65 patients with CAD who were scheduled to CABG. SELP expression, the platelet-leucocyte aggregate level, and the level of P-selectin expression on aggregates were determined in all participants. Resistance to ASA was defined as platelet aggregation with arachidonic acid ≥20% in at least one sample. All studies were performed before and on the 8th-10th day after CABG.Results. Among patients, 29,4% (n=30) were resistant to ASA (ASA-R) at least at one point, while 7,8% (n=8) acquired resistance after CABG. ASA-R had a higher level of SELP (0,41 [0,28; 0,48] vs 0,32 [0,23; 0,39], p=0,037), absolute content of monocyte-platelet aggregates (24,70 [9,69; 39,90] vs 10,15 [5,27; 30,63], p=0,037) and relative amount of platelet-platelet CD62P aggregates (7,37% [3,43; 35,49] vs 4,84% [0,90; 9,63], p=0,045). A negative correlation was found between SELP expression and the relative content of monocyte-platelet aggregates (ρ=-0,349, p=0,013) on days 8-10 after CABG. Mmultivariate regression analysis found that the level of SELP gene mRNA expression is a predictor of ASA resistance before CABG (odds ratio 404,48; 95% confidence interval 1,68-97461,63).Conclusion. Increased SELP expression is associated with an increase in platelet aggregation activity, which contributes to an insufficient platelet response to antithrombotic therapy in patients with CABG. The level of SELP gene mRNA expression can be considered as a predictor of ASA resistance.

Abstract 136: Protease-activating Receptor-4 Antagonism In Coronary Artery Disease

Background: BMS-986141 is a novel potent highly selective antagonist of protease-activated receptor type 4 (PAR4). PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people. Aim: To determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy. Methods: Forty-five patients with stable coronary heart disease and ten healthy volunteers completed a phase 2a open-label four-arm single-center study. Patients were allocated to one of three treatment arms for seven days: (i) ticagrelor (90 mg twice daily), (ii) aspirin (75 mg once daily) or (iii) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before, 2 and 24 hours after a single oral 4-mg dose of BMS-986141 in all participants. Results: BMS-986141 demonstrated highly selective inhibition of PAR4-agonist peptide (AP) induced platelet aggregation, p-selectin expression, and platelet-monocyte aggregate expression (p≤0.001 for all) which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (-21%, p=0.001) and in patients receiving ticagrelor alone (-28%, p=0.001), aspirin alone (-23%, p=0.018), or both in combination (-24%, p≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP-induced platelet responses (p≤0.001 for all) and total thrombus area under high shear stress conditions (p≤0.01 for all). Conclusions: PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin or their combination, in patients with stable coronary artery disease.

Circulating Monocytes Are Predictive and Responsive in Moderate-to-Severe Plaque Psoriasis Subjects Treated with Apremilast

Monocytes play a critical role in the inflammation associated with psoriasis, and their abnormalities have been reported as biomarkers of cardiovascular event risk, a psoriasis comorbidity. Monocytic cells in chronic inflammatory disorders express elevated levels of cAMP phosphodiesterase. Restoring cAMP levels using the oral cAMP phosphodiesterase-4 inhibitor, apremilast, improves clinical outcomes for a subset of patients with psoriasis. We asked whether aberrant monocyte subsets or transcriptomic pathways can function as biomarkers of psoriasis endotypes that can predict enhanced clinical responses to cAMP phosphodiesterase inhibition. A 16-week open-label study of 22 patients with monocyte flow cytometric and transcriptomic analysis was performed. Subjects with elevated hyperadhesive monocyte doublets at baseline were more likely to be responders to apremilast (P < .0001); 82% of subjects with elevated hyperadhesive monocyte doublets achieved 50% reduction in PASI compared with 46% in those without elevated doublets. We observed a significant reduction in hyperadhesive monocyte-containing doublets and monocyte-platelet aggregates, suggesting an effect of apremilast on the adhesiveness of blood monocytes during chronic inflammation. Monocyte differentially expressed gene transcripts predictive of clinical response uncovered pharmacoendotypes with distinct patterns of nucleotide metabolism, energetics, and differentiation. Further study to understand the basis of drug responsiveness and to develop an apremilast psoriasis treatment algorithm using monocyte-refined gene expression is required to validate and become practical in clinical use, offering patients a test that personalizes their likelihood of clinical response.

PAR4 Antagonism in Patients With Coronary Artery Disease Receiving Antiplatelet Therapies.

BMS-986141 is a novel potent highly selective antagonist of PAR (protease-activated receptor) type 4. PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people. We sought to determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy. Forty-five patients with stable coronary heart disease and 10 healthy volunteers completed a phase 2a open-label 4-arm single-center study. Patients were allocated to 1 of 3 treatment arms for 7 days: (1) ticagrelor (90 mg BID), (2) aspirin (75 mg QD), or (3) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before and 2 and 24 hours after a single oral 4-mg dose of BMS-986141 on the first study visit day in all participants. BMS-986141 demonstrated highly selective inhibition of PAR4-AP (agonist peptide)-induced platelet aggregation, P-selectin expression, and platelet-monocyte aggregate expression (P≤0.001 for all), which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (-21%; P=0.001) and in patients receiving ticagrelor alone (-28%; P=0.001), aspirin alone (-23%; P=0.018), or both in combination (-24%; P≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP-induced platelet responses (P≤0.001 for all) and total thrombus area under high shear stress conditions (P≤0.01 for all). PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin, or their combination, in patients with stable coronary heart disease. URL: https://www.clinicaltrials.gov; Unique identifier: NCT05093790.

Persistent complement dysregulation with signs of thromboinflammation in active Long Covid.

Long Covid is a debilitating condition of unknown etiology. We performed multimodal proteomics analyses of blood serum from COVID-19 patients followed up to 12 months after confirmed severe acute respiratory syndrome coronavirus 2 infection. Analysis of >6500 proteins in 268 longitudinal samples revealed dysregulated activation of the complement system, an innate immune protection and homeostasis mechanism, in individuals experiencing Long Covid. Thus, active Long Covid was characterized by terminal complement system dysregulation and ongoing activation of the alternative and classical complement pathways, the latter associated with increased antibody titers against several herpesviruses possibly stimulating this pathway. Moreover, markers of hemolysis, tissue injury, platelet activation, and monocyte-platelet aggregates were increased in Long Covid. Machine learning confirmed complement and thromboinflammatory proteins as top biomarkers, warranting diagnostic and therapeutic interrogation of these systems.

Toll-like receptor expression and functional behavior in platelets from patients with systemic lupus erythematosus

BackgroundMultiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. Materials and MethodsThirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. ResultsIn resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. ConclusionOur study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.

The rapid change of shear rate gradient is beneficial to platelet activation

Fluid shear plays a key role in hemostasis and thrombosis, and the purpose of this study was to investigate the effect of shear gradient change rate (SGCR) on platelet reactivity and von Willebrand factor (vWF) activity and its mechanism. In this study, we developed a set of microfluidic chips capable of generating different shear gradients and simulated the shear rate distribution in the flow field by COMSOL Multiphysics software. Molecular markers of platelet activation (P-selectin, activated GPIIb/IIIa, phosphatidylserine exposure, and monocyte-platelet aggregate formation) were analyzed by flow cytometry. Platelet aggregation induced by shear gradient was studied by a microfluidic experimental platform, and plasma vWF ristocetin cofactor (vWF: RCO) activity was investigated by flow cytometry. The expression of p-Akt was studied by Western blotting. The results showed that the faster the SGCR, the higher the expression of platelet p-Akt, and the stronger the platelet reactivity and vWF activity. This indicates that fluid shear stress can activate platelets and vWF in a shear gradient-dependent manner through the PI3K/AKT signal pathway, and the faster the SGCR, the more significant the activation effect.

Identifying Novel Coagulation Biomarkers for Thrombosis in Patients with Lymphoma

CONCLUSION Thromboembolic events are a common complication of lymphoma, influenced by a number of factors, including status of the disease, patient characteristics and treatment modalities. Recent studies demonstrated presence of hemostatic dysregulation in lymphoma patients with the possible involvement of tissue factor (TF) in the development of thrombotic events; however, more data are needed to link coagulation biomarkers' levels with clinical data and increased thrombotic risk. TF initiates blood coagulation and expressed by nonvascular cells plays a major role in hemostasis, while TF expressed by vascular cells induces intravascular thrombosis. In this study, we aimed to profile coagulation biomarkers and determine the roles of neutrophils, monocytes, platelets and endothelial cells and their interactions in thrombus formation in patients with lymphoma. Patients with newly diagnosed lymphoma were prospectively recruited and followed for all thromboembolic events from the time of diagnosis until 3 months after the last cycle of therapy. Peripheral blood samples were obtained before hematological treatment initiation from 127 patients with lymphoma. The coagulation factors profile (Fibrinogen, P-selectin, FVIII, Thrombin and TF) was measured by enzyme-linked immunosorbent assay (ELISA), while TF expression and monocyte-platelet aggregates were determined by flow cytometry. The isolation, quantification and flow cytometry profiling of extracellular vesicles (EVs) ≤1.0 µm have been done according to the protocol developed by Institute for Medical Research, Belgrade, Serbia. Thromboembolic events were diagnosed objectively based on clinical examination, radiographic studies and laboratory evaluation. The mean patients' age was 55±16 years (range, 18-86 years); 44.9% were males. Most patients (58.2%) had advanced stage disease (III/IV), and 35.4% had ECOG performance status ≥1. More than half of the patients had aggressive forms of lymphoma (64.6%), mostly diffuse large B-cell lymphoma (DLBCL). Thrombosis occurred in 14.3% of patients with lymphoma (18/127), predominantly in patients with aggressive (18.3%) vs. indolent forms (6.8%). Total number of thrombosis was 22 (4 patients had more than one thrombosis site), and venous thrombosis was the most prevalent (72.7%). ThroLy score ≥2 had 72.2% of patients with thrombosis, in contrast to 37% of patients without thrombosis. The median value of TF was higher in patients with thrombosis compared to those without thrombosis (224.41 [93.1-446.0] (median [IQR]) vs. 136.43 [109.4-193.4], p=0.036). The concentration of the other assessed coagulation related cells and biomarkers were not significantly different between patients who developed thrombosis during follow-up and those who did not. In multivariate regression analysis, ThroLy score ≥2 (OR=5.505, 95%CI 1.505‒20.100, p=0.01), TF (OR=1.005, 95%CI 1.002‒1.009, p=0.003), and aggressive form of lymphoma (OR=4.680, 95%CI 1.132‒19.356, p=0.033), were independent prognostic factors for thrombosis occurrence. CD11b +CD16 + neutrophils were increased 8-fold in aggressive (DLBCL) compared to indolent form of lymphoma (p&amp;lt;0.001), as were 2-fold CD61 + platelets and 3-fold CD31 + endothelial cells labelled with TF (CD142 +, p&amp;lt;0.05) procoagulant EVs. The monocyte-platelet aggregates (CD14 +CD45 +CD41 +) were increased 5-fold in DLBCL patients compared to the healthy controls (p&amp;lt;0.001). The presented results support the use of ThroLy score for differentiating the sub-groups of lymphoma patients who are at increased risk for thrombotic events, while profiling coagulation biomarkers in different lymphoma populations may aid in understanding the pathophysiologic mechanisms underlying the thromboembolic events. Special emphasis should be placed on the assessment of TF which might be used as a coagulation biomarker in addition to clinical prediction scores.

Non-severe burn injury causes sustained platelet hyperreactivity

Individuals who present to a hospital for treatment of a burn of any magnitude are more frequently hospitalised for ischemic heart disease, even decades after injury. Blood platelets are key mediators of cardiovascular disease. To investigate platelet involvement in post-burn cardiovascular risk, platelet reactivity was assessed in patients at 2- and 6-weeks after non-severe (TBSA < 20%) burn injury, and in a murine model 30 days after 8% TBSA full-thickness burn injury. Platelets were stimulated with canonical agonists and function reported by GPIIb/IIIa PAC1-binding site, CD62P expression, and formation of monocyte-platelet aggregates. In vivo thrombosis in a modified Folts model of vascular injury was assessed. Burn survivors had elevated frequencies of circulating monocyte-platelet aggregates, and platelets were hyperreactive, primarily to collagen stimulation. Burn plasma did not cause hyper-reactivity when incubated with control platelets. Platelets from burn injured mice also demonstrated increased response to collagen peptides but did not show any change in thrombosis following vascular injury. This study demonstrates the persistence of a small but significant platelet hyperreactivity following burn injury. Although our data does not suggest this heightened platelet sensitivity modulates thrombosis following vascular injury, the contribution of sub-clinical platelet hyperreactivity to accelerating atherogenesis merits further investigation.

Evaluating the impact of transient shear stress on platelet activation, adhesion, and aggregation with microfluidic chip technique.

When nonphysiological stenosis occurs, the transient high shear stress formed in vessels increases the risk of thrombosis and is a potential factor for cardiovascular diseases. But the platelet adhesion and aggregation behavior at nonphysiological post-stenosis and its affecting factors are not fully understood yet. In this experiment, platelet aggregation on collagen and fibrinogen at different shear stresses and different hematocrits were observed by microfluidic technology. Platelet activation (P-selectin, glycoprotein IIb/IIIa) and monocyte-platelet aggregate (MPA) levels under different shear stresses were analyzed by flow cytometry. On fibrinogen, platelets aggregate more at higher shear stress conditions. While on collagen, it becomes more difficult for platelets to form stable aggregation at higher shear stress conditions. If platelets adhere initially at low shear stress, stable platelet aggregation can be formed at subsequent high shear stress. Moreover, when the shear stress increases, platelet activity markers (P-selectin, glycoprotein IIb/IIIa and MPAs) increase significantly. Hematocrit affects the degree of platelet aggregation, and the influence of hematocrit is obvious at high shear stress. Transient high shear stress (46 ms) can effectively activate platelets. Platelet aggregation behavior was different for coated fibrinogen and collagen protein. Stable platelet adhesion at post-stenosis is more dependent on fibrinogen and platelet aggregation is stable on both fibrinogen and collagen. Hematocrit can significantly affect the formation of platelet aggregation.

Platelets exacerbate cardiovascular inflammation in a murine model of Kawasaki disease vasculitis.

Kawasaki disease (KD) is the leading cause of acquired heart disease among children. Increased platelet counts and activation are observed during the course of KD, and elevated platelet counts are associated with higher risks of developing intravenous immunoglobulin resistance and coronary artery aneurysms. However, the role of platelets in KD pathogenesis remains unclear. Here, we analyzed transcriptomics data generated from the whole blood of patients with KD and discovered changes in the expression of platelet-related genes during acute KD. In the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis, LCWE injection increased platelet counts and the formation of monocyte-platelet aggregates (MPAs), upregulated the concentration of soluble P-selectin, and increased circulating thrombopoietin and interleukin 6 (IL-6). Furthermore, platelet counts correlated with the severity of cardiovascular inflammation. Genetic depletion of platelets (Mpl-/- mice) or treatment with an anti-CD42b antibody significantly reduced LCWE-induced cardiovascular lesions. Furthermore, in the mouse model, platelets promoted vascular inflammation via the formation of MPAs, which likely amplified IL-1B production. Altogether, our results indicate that platelet activation exacerbates the development of cardiovascular lesions in a murine model of KD vasculitis. These findings enhance our understanding of KD vasculitis pathogenesis and highlight MPAs, which are known to enhance IL-1B production, as a potential therapeutic target for this disorder.

Effects of Recombinant SARS-CoV-2 Spike Protein Variants on Platelet Morphology and Activation.

Platelets are central elements of hemostasis and also play a pivotal role in the pathogenesis of thrombosis in coronavirus disease 2019. This study was planned to investigate the effects of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant spike protein variants on platelet morphology and activation. Citrated whole blood collected from ostensibly healthy subjects was challenged with saline (control sample) and with 2 and 20 ng/mL final concentration of SARS-CoV-2 recombinant spike protein of Ancestral, Alpha, Delta, and Omicron variants. Platelet count was found to be decreased with all SARS-CoV-2 recombinant spike protein variants and concentrations tested, achieving the lowest values with 20 ng/mL Delta recombinant spike protein. The mean platelet volume increased in all samples irrespective of SARS-CoV-2 recombinant spike protein variants and concentrations tested, but especially using Delta and Alpha recombinant spike proteins. The values of both platelet function analyzer-200 collagen-adenosine diphosphate and collagen-epinephrine increased in all samples irrespective of SARS-CoV-2 recombinant spike protein variants and concentrations tested, and thus reflecting platelet exhaustion, and displaying again higher increases with Delta and Alpha recombinant spike proteins. Most samples where SARS-CoV-2 recombinant spike proteins were added were flagged as containing platelet clumps. Morphological analysis revealed the presence of a considerable number of activated platelets, platelet clumps, platelet-monocyte, and platelet-neutrophils aggregates, especially in samples spiked with Alpha and Delta recombinant spike proteins at 20 ng/mL. These results provide support to the evidence that SARS-CoV-2 is capable of activating platelets through its spike protein, though such effect varies depending on different spike protein variants.

Platelet-monocyte aggregates: molecular mediators of thromboinflammation.

Platelets, key facilitators of primary hemostasis and thrombosis, have emerged as crucial cellular mediators of innate immunity and inflammation. Exemplified by their ability to alter the phenotype and function of monocytes, activated platelets bind to circulating monocytes to form monocyte-platelet aggregates (MPA). The platelet-monocyte axis has emerged as a key mechanism connecting thrombosis and inflammation. MPA are elevated across the spectrum of inflammatory and autoimmune disorders, including cardiovascular disease, systemic lupus erythematosus (SLE), and COVID-19, and are positively associated with disease severity. These clinical disorders are all characterized by an increased risk of thromboembolic complications. Intriguingly, monocytes in contact with platelets become proinflammatory and procoagulant, highlighting that this interaction is a central element of thromboinflammation.

Platelets reprogram monocyte functions by secreting MMP-9 to benefit postoperative outcomes following acute aortic dissection

Platelets have a great ability to modulate immune responses. Monocyte-platelet aggregates (MPAs) are associated with the pathogenesis of cardiac disease. Notably, a low preoperative platelet count often indicates poor postoperative recovery following acute aortic dissection (AAD). The functions of platelets and MPAs in AAD, however, remain poorly understood. We found that, despite decreased platelet counts, platelets were also activated in AAD patients, with significant alterations in immune-modulating mediators. Of interest, monocytes in AAD patients had a suppressed immune status, which was correlated with poor outcomes following surgery. Interestingly, platelets preferentially aggregated with monocytes, and the levels of MPAs were related to recovery after surgical repair in AAD patients. Platelets restored suppressed monocyte functions in AAD patients by forming aggregates and partly by secreting matrix metalloproteinase-9 (MMP-9). Thus, the results point to a previously unknown mechanism for platelets involving monocyte reprogramming, which may improve postoperative outcomes following complex cardiovascular surgery.