Platelet Activation Markers Research Articles

Very preterm birth results in later lower platelet activation markers.

Premature birth entails an adverse cardiovascular risk profile, but the underlying mechanisms are insufficiently understood. Here, we employed an unbiased cardiovascular proteomics approach to profile former very preterm-born preschoolers. This observational study investigated differences in plasma concentrations of 79 proteins, including putative cardiovascular biomarkers between very preterm- and term-born children on average 5.5 years old (53.1% male) using multiple-reaction monitoring mass spectrometry. Very preterm-born (n = 38; median gestational age 29.6 weeks) compared to term-born (n = 26; 40.2 weeks) children featured lower plasma concentrations of platelet factor 4 (PLF4; -61.6%, P < 0.0001), platelet basic protein (CXCL7; -57.8%, P < 0.0001), and hemoglobin subunit beta (-48.3%, P < 0.0001). Results remained virtually unchanged when adjusting for complete blood count parameters, including platelet count. Conversely, whole blood hemoglobin was higher (+7.62%, P < 0.0001) in preterm-born children. Very preterm birth was associated with decreased markers of platelet activation among preschoolers. These findings are consistent with reduced platelet reactivity persisting from very preterm birth to a preschool age. Former very preterm-born preschoolers featured reduced levels of platelet activation markers. While lower platelet reactivity in very preterm-born compared to term-born infants in the first days of life was established, it was unknown when, if at all, reactivity normalizes. The current study suggests that platelet hyporeactivity due to very preterm birth persists at least up to a preschool age. "Immaturity of the hemostatic system" may be a persistent sequel of preterm birth, but larger studies are needed to investigate its potential clinical implications.

Platelet disturbances correlate with endothelial cell activation in uncomplicated Plasmodium vivax malaria

Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson’s correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406–651] vs. 1029 [732–1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.

Evaluation of Immature Platelet Fraction in Lower Respiratory Tract Infections: A Retrospective Study.

IntroductionImmature platelet fraction (IPF) is a parameter of an automated hematologic analyzer and is related to platelet size and cytoplasmic RNA content. It reflects thrombopoiesis and is often used as the marker of platelet activity. IPF has been evaluated mostly in hematologic disorders and has also been evaluated in patients with gestational hypertension, sepsis, autoimmune diseases and in hospitalised patients with neutrophilia. Platelets, asides from the maintenance of hemostasis, release inflammatory mediators that can modify leukocyte and endothelial responses to various inflammatory stimuli. Lower respiratory tract infections are the leading cause of death from infections worldwide. The role of platelets in lower respiratory tract infections has been reported in many studies. IPF, which is related to platelet activation, has not been evaluated in patients with lower respiratory tract infections.MethodsThe study involved patients who fulfilled the criteria of community-acquired pneumonia (CAP) and aspiration pneumonia (AP). In addition, age and sex-matched healthy controls were involved. Whole blood samples were collected from healthy controls and from the patients on admission. The mean IPF% and C-reactive protein (CRP) levels were measured in patients with CAP, in patients with AP and in healthy controls. The mean IPF% values in patients with infection were compared to mean IPF% values in healthy controls. The mean IPF% values were compared to mean CRP levels in patients with infection. Additionally, the mean IPF% values in patients that died in the first 14 days were compared to the mean IPF% values in patients that were alive. The statistical analysis of data was performed with the Statistical Package for the Social Sciences (SPSS) for Windows, Version 13.0 (SPSS Inc, Chicago, IL). ResultsThe study population consisted of 45 patients (27 patients with CAP and 18 patients with AP), 27 males and 18 females, with a mean age of 72.11 ± 16.4 years and 39 healthy controls, 22 males and 17 females with a mean age of 64.2 ± 14.8 years. The mean CRP levels in patients with infection were 155.2±119.1 mg/dl. The mean IPF% value of patients with infection was 2.76 ± 2.27 and the mean IPF% value of controls was 1.72 ± 0.77 (p < 0.006). The IPF% value in patients with CAP was 2.55 ± 2.02 and in patients with AP 3.07 ± 2.64 (p = 0.595). The mean IPF% value in patients with infection had no linear correlation with CRP value in these patients (r = 0.076, p = 0.62). The mean IPF% value in all patients that died in the first 14 days was 3.75 ± 2.44 and the mean IPF% value in all patients alive was 2.35 ± 2.11 (p = 0.06). The mean IPF% value in patients with CAP who died in the first 14 days of hospitalisation was 5.54 ± 3.17 and in patients with CAP who were alive was 1.87 ± 0.72 (p = 0.06). The mean IPF% value in patients with AP who died was 2.63 ± 0.85 and in patients with AP who were alive was 3.41 ± 3.51 (p = 0.554).ConclusionsMean IPF% value is greater in patients with lower respiratory tract infections, including CAP and AP, compared to healthy controls. There is no linear correlation between IPF values and CRP values in patients with lower respiratory tract infections. In addition, there is a difference in mean IPF% value between patients who died in the first 14 days of hospitalisation compared to those who were alive, but not statistically significant.

Platelet Measurements and Type 2 Diabetes: Investigations in Two Population-Based Cohorts.

Type 2 diabetes is a major risk factor for cardiovascular disease. Given the contribution of platelets to atherothrombosis—which in turn is a major contributor to cardiac events, there may be cause to consider platelet function in management of diabetes. Despite the large body of research concerning the role of platelets in cardiovascular complications of type 2 diabetes, evidence from population-based studies of platelet aggregation in diabetes is limited. Mean Platelet Volume (MPV), a cell trait partially associated with markers of platelet activity, is more commonly available. We investigated the association of metabolic syndrome and diabetes with platelet aggregation to three physiological agonists, ADP, collagen, and epinephrine, in the Framingham Heart Study Offspring cohort. We further examined the relationship between MPV measured with Beckman Coulter LH750 instruments and self-reported diabetes as well as MPV and diabetes medication in the UK BioBank cohort, performing the largest such analysis to date. Increased platelet aggregation associated with prevalent diabetes was observed for low concentration epinephrine (0.1 μM) alone and only in analyses of participants stratified either by male sex and/or having metabolic syndrome. Other agonists and concentrations were not significant for prevalent diabetes, or in opposite direction to the main hypothesis (i.e., they showed lower platelet aggregation associated with diabetes). After a median of 18.1 years follow-up, no platelet aggregation trait was associated with increased risk of diabetes (n = 344 cases). As expected, increased MPV was significantly associated with diabetes (β = 0.0976; P = 8.62 × 10−33). Interestingly, sex-stratified analyses indicated the association of MPV with diabetes is markedly stronger in males (β = 0.1232; P = 1.00 × 10−31) than females (β = 0.0514; P = 7.37 × 10−5). Among diabetes medications increased MPV was associated with Insulin (β = 0.1341; P = 1.38 × 10−11) and decreased MPV with both Metformin (β = 0.0763; P = 1.99 × 10−6) as well as the sulphonylureas (β = 0.0559; P = 0.0034). Each drug showed the same direction of effect in both sexes, however, the association with MPV was nearly twice as great or more in women compared to men. In conclusion, platelet function as measured by aggregation to ADP, collagen, or epinephrine does not appear to be consistently associated with diabetes, however, MPV is robustly associated suggesting future work may focus on how MPV segments pre-diabetics and diabetics for risk prediction.

Increased monocyte-platelet aggregates and monocyte-endothelial adhesion in healthy individuals with vitamin D deficiency.

Vitamin D deficiency is a major public health problem worldwide, linked to several chronic diseases including cardiovascular diseases. While immunomodulatory effects of vitamin D on monocytes have been reported in cardiovascular and metabolic diseases, there is limited understanding on monocyte phenotype in healthy individuals with suboptimal vitamin D levels and without any clinical diseases. In this work, we performed label-free, microfluidic isolation of monocytes, and characterized their functional phenotype using flow cytometry and in vitro vascular models in healthy subjects with (n=7) and without vitamin D deficiency (n=16). Vitamin D deficient (VitD-Def) subjects (25(OH)D3 level<26ng/mL) expressed significant downregulation of vitamin D receptor (VDR) on monocytes as compared to controls (P<.0001), and VDR expression was well-associated with serum 25(OH)D3 levels. Increased monocyte-platelet aggregates (MPA), a marker for platelet activation, were also observed in VitD-Def subjects (P<.05) which suggests a pro-inflammatory monocyte phenotype. Monocyte adhesion to endothelial cells, an early-stage atherosclerosis event, was also higher in VitD-Def individuals, and inversely correlated to serum 25(OH)D3 level (P<.05). Taken together, these results indicate the pro-inflammatory state and atherogenic potential of monocytes in VitD-Def healthy subjects, and propound the use of vitamin D supplementation as a prospective immunomodulatory and anti-inflammatory therapy in atherosclerosis.

Endotheliopathy in COVID-19-associated coagulopathy: evidence from a single-centre, cross-sectional study

SummaryBackgroundAn important feature of severe acute respiratory syndrome coronavirus 2 pathogenesis is COVID-19-associated coagulopathy, characterised by increased thrombotic and microvascular complications. Previous studies have suggested a role for endothelial cell injury in COVID-19-associated coagulopathy. To determine whether endotheliopathy is involved in COVID-19-associated coagulopathy pathogenesis, we assessed markers of endothelial cell and platelet activation in critically and non-critically ill patients admitted to the hospital with COVID-19.MethodsIn this single-centre cross-sectional study, hospitalised adult (≥18 years) patients with laboratory-confirmed COVID-19 were identified in the medical intensive care unit (ICU) or a specialised non-ICU COVID-19 floor in our hospital. Asymptomatic, non-hospitalised controls were recruited as a comparator group for biomarkers that did not have a reference range. We assessed markers of endothelial cell and platelet activation, including von Willebrand Factor (VWF) antigen, soluble thrombomodulin, soluble P-selectin, and soluble CD40 ligand, as well as coagulation factors, endogenous anticoagulants, and fibrinolytic enzymes. We compared the level of each marker in ICU patients, non-ICU patients, and controls, where applicable. We assessed correlations between these laboratory results with clinical outcomes, including hospital discharge and mortality. Kaplan–Meier analysis was used to further explore the association between biochemical markers and survival.Findings68 patients with COVID-19 were included in the study from April 13 to April 24, 2020, including 48 ICU and 20 non-ICU patients, as well as 13 non-hospitalised, asymptomatic controls. Markers of endothelial cell and platelet activation were significantly elevated in ICU patients compared with non-ICU patients, including VWF antigen (mean 565% [SD 199] in ICU patients vs 278% [133] in non-ICU patients; p<0·0001) and soluble P-selectin (15·9 ng/mL [4·8] vs 11·2 ng/mL [3·1]; p=0·0014). VWF antigen concentrations were also elevated above the normal range in 16 (80%) of 20 non-ICU patients. We found mortality to be significantly correlated with VWF antigen (r = 0·38; p=0·0022) and soluble thrombomodulin (r = 0·38; p=0·0078) among all patients. In all patients, soluble thrombomodulin concentrations greater than 3·26 ng/mL were associated with lower rates of hospital discharge (22 [88%] of 25 patients with low concentrations vs 13 [52%] of 25 patients with high concentrations; p=0·0050) and lower likelihood of survival on Kaplan–Meier analysis (hazard ratio 5·9, 95% CI 1·9–18·4; p=0·0087).InterpretationOur findings show that endotheliopathy is present in COVID-19 and is likely to be associated with critical illness and death. Early identification of endotheliopathy and strategies to mitigate its progression might improve outcomes in COVID-19.FundingThis work was supported by a gift donation from Jack Levin to the Benign Hematology programme at Yale, and the National Institutes of Health.

Serum concentration of selected biochemical markers of endothelial dysfunction and inflammation in patients with the varying activity of inflammatory bowel disease.

Endothelial dysfunction leads to an increased expression of cell adhesion molecules, leukocyte diapedesis, vascular smooth‑muscle tone, excessive permeability of vascular walls, and increased procoagulant activity. We investigated whether serum levels of several endothelial and platelet activation markers correlated with disease activity in patients with inflammatory bowel disease (IBD). This study included 56 patients with ulcerative colitis, 66 with Crohn disease, and 40 healthy controls. We measured the complete blood count and levels of fibrinogen, C‑reactive protein, albumin, interleukin 6, tumor necrosis factor α, E‑selectin, P‑selectin, monocyte chemoattractant protein 1 (MCP‑1), soluble CD40 ligand (sCD40L), and microparticles. There were no significant differences in the median levels of E‑selectin, P‑selectin, MCP‑1, sCD40L, and microparticles between patients with active IBD, those with inactive IBD, and healthy controls. The clinical disease activity assessed with the Mayo scale in the ulcerative‑colitis group was weakly, positively correlated with sCD40L (R = 0.32, P = 0.02), P‑selectin (R = 0.32, P = 0.02), and inflammatory marker levels. The clinical disease activity index in the Crohn disease group was positively correlated with the markers of inflammation yet not with the markers of endothelial activity. E‑selectin, P‑selectin, sCD40L, MCP‑1, and microparticle levels do not significantly differ between patients with the varying activity of IBD. However, due to the observed correlations, further studies of a larger patient group should be conducted to confirm our observations.

006 The association of platelet activation markers, neutrophil extracellular traps and anti-mitochondrial autoantibodies with cutaneous manifestations in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is an autoimmune disease associated with platelet activation, formation of neutrophil extracellular traps (NETs) and development of anti-mitochondrial autoantibodies (AMA). In the current study, we investigated the clinical utility of these markers and autoantibodies in cutaneous lupus manifestations. Clinical data on cutaneous manifestations were obtained in a large SLE cohort (n=73) through review of SLE questionnaires, SLICC Classification Criteria and revised ACR Criteria for SLE.

Hemoglobin-bound platelets correlate with the increased platelet activity in hemoglobin E/β-thalassemia.

An increase in platelet activity is a contributing factor to vascular complications in hemoglobin E/β-thalassemia (HbE/β-thal). Plasma-free hemoglobin (Hb) increases in HbE/β-thal patients and correlates with platelet activation, but the levels of Hb-bound platelets have never been reported. In this study, we aimed to investigate the levels of Hb-bound platelets and its association with platelet activity in HbE/β-thal patients. Hb-bound platelets were measured by flow cytometry in 22 healthy subjects and 26 HbE/β-thal patients (16 nonsplenectomized and 10 splenectomized HbE/β-thal patients). Plasma Hb was measured by the chemiluminescence method based on the consumption of nitric oxide (NO) by Hb. Expression of P-selectin and activated glycoprotein (aGP) IIb/IIIa on platelets was measured by flow cytometry as a marker of platelet activity. Both nonsplenectomized and splenectomized HbE/β-thal patients had higher levels of Hb-bound platelets and plasma Hb than healthy subjects. In vitro incubation of dialyzed Hb from patients with platelets of healthy subjects caused an increase in Hb-bound platelets, which was partially inhibited by anti-GPIbα antibody. Plasma Hb positively correlated with Hb-bound platelets. Platelet P-selectin expression at baseline and in response to adenosine diphosphate (ADP, 1µM) stimulation was higher in nonsplenectomized and splenectomized HbE/β-thal patients than healthy subjects. The ADP-induced aGPIIb/IIIa expression on platelets was also higher in HbE/β-thal patients than healthy subjects. Hb-bound platelets correlated with baseline P-selectin expression and ADP-induced P-selectin expression. HbE/β-thal patients have increased Hb-bound platelets, which is associated with increased baseline platelet activation and reactivity.

Additive effect of erythropoietin use on exercise-induced endothelial activation and hypercoagulability in athletes

PurposeRecombinant human erythropoietin (rHuEPO) is known to increase thrombotic risk in patients and might have similar effects in athletes abusing the drug. rHuEPO is prohibited by anti-doping legislation, but this risk has not been investigated thoroughly. This analysis was designed to evaluate whether rHuEPO impacts hemostatic profile and endothelial and platelet activation markers in trained subjects, and whether the combination with exercise affects exercise induced alterations.MethodsThis double-blind, randomized, placebo-controlled trial enrolled healthy, trained male cyclists aged 18–50 years. Participants were randomly allocated (1:1) to receive subcutaneous injections of rHuEPO (epoetin-β; mean dose 6000 IU per week) or placebo (0.9% NaCl) for 8 weeks. Subjects performed five maximal exercise tests and a road race, coagulation and endothelial/platelet markers were measured at rest and directly after each exercise effort.ResultsrHuEPO increased P-selectin (+ 7.8% (1.5–14.5), p = 0.02) and E-selectin (+ 8.6% (2.0–15.7), p = 0.01) levels at rest. Maximal exercise tests significantly influenced all measured coagulation and endothelial/platelet markers, and in the rHuEPO group maximal exercise tests led to 15.3% ((7.0–24.3%), p = 0.0004) higher E-selectin and 32.1% ((4.6–66.8%), p = 0.0207) higher Platelet factor 4 (PF4) levels compared to the placebo group.ConclusionIn conclusion, rHuEPO treatment resulted in elevated E- and P-selectin levels in trained cyclists, indicating enhanced endothelial activation and/or platelet reactivity. Exercise itself induces hypercoagulability, and the combination of rHuEPO and exercise increased E-selectin and PF4 levels more than either intervention alone. Based on this, exercise potentially increases thrombotic risk, a risk that might be enhanced in combination with rHuEPO use.

Mean platelet volume and mechanical thrombectomy

Background and purposeMean Platelet Volume (MPV) is a marker of platelet activity and it is an independent predictor for long-term outcome in stroke patients. The aim of this study was to evaluate the association between baseline MPV value and clinical outcome at 90-days in anterior circulation stroke and large vessel occlusion (LVO) patients submitted to mechanical thrombectomy (MT). MethodsWe conducted a prospective observational cohort study in acute ischemic stroke (AIS) patients submitted to MT between January 2017 and May 2018. MPV was measured at admission. Patients were initially stratified into two groups according to the mean MPV level. We also compared groups that were stratified according to the MPV cut-off obtained by Peng F et al (10,4 fL) and performed analyses among MPV terciles. ResultsA total of 129 patients were included. Mean level of MPV was 10,9 fL. Patients with embolic stroke of undetermined source (ESUS) had significantly higher rates of good outcome at 3 months compared with large-artery atherosclerotic disease and cardioembolism [(82,9%) vs (78,3%) vs (55,2%); p=0,009]. There were no statistically significant differences in the mean MPV value (p=0,222), successful recanalization (p=0,464) and mortality (p=0,343) when evaluated for all TOAST etiologies. There were no statistically significant differences between the two groups according to the MPV level (10,4 and 10,9 fL) or between the terciles (lowest tertile <10,3 fL, median 10,3 – 11,3 fL, highest >11,3fL) concerning functional outcome at 3 months (p=0,357; p=0,24 and p=0,558, respectively), successful recanalization (p=0,108; p=0,582 and p=0,899, respectively) or mortality at 3 months (p=0,465; p=0,061 and p=0,484, respectively). ConclusionOur study did not find an association between elevated MPV and worse outcome at 3 months in patients with acute anterior circulation stroke and LVO treated with MT. Since ischemic strokes have different pathophysiologic mechanisms, MPV may have distinct prognostic value according to each stroke etiology.

The microvesicle/CD36 complex triggers a prothrombotic phenotype in patients with non-valvular atrial fibrillation.

The mechanisms responsible for platelet activation, the prothrombotic state, in non‐valvular atrial fibrillation (NVAF) are still obscure. Microvesicles (MVs) can transfer various messages to target cells and may be helpful for exploring the detailed mechanisms. We aimed to investigate the possible mechanisms by which proatherogenic factors of NVAF contribute to platelet activation. Two hundred and ten patients with NVAF were stratified as being at ‘low to moderate risk’ or ‘high risk’ for stroke according to the CHADS2 score. Levels of platelet‐derived MVs (PMVs) and platelet activation were examined. CD36‐positive or CD36‐deficient human platelets were stimulated by MVs isolated from NVAF patients with or without various inhibitors in vitro. Levels of PMVs and platelet activation markers enhanced significantly in high‐risk patients. The MVs isolated from plasma of NVAF patients bound to platelet CD36 and activated platelets by phosphorylating the mitogen‐activated protein kinase 4/Jun N‐terminal kinase 2 (MKK4/JNK2) pathways. However, CD36 deficiency protected against MV‐induced activation of platelets. We reveal a possible mechanism of platelet activation in NVAF and suggest that the platelet CD36 might be an effective target in preventing the prothrombotic state in NVAF.

Platelet hyperactivation in multiple myeloma is also evident in patients with premalignant monoclonal gammopathy of undetermined significance

Thrombotic events are common in patients with multiple myeloma (MM), smouldering myeloma (SM) and monoclonal gammopathy of undetermined significance (MGUS). Previous studies have indicated platelet hyperactivation as a feature of thrombotic risk in MM, but there is a dearth of data in MGUS. In the present study, multiparameter analysis of platelet activation and responsiveness was investigated by flow cytometry in patients with MGUS, SM/MM and healthy controls (HCs). The median platelet surface CD63 levels, annexin V and PAC-1 antibody (specific for activated integrin αIIbβ3) binding were significantly elevated in patients with MGUS versus the HCs. These markers were also elevated in SM/MM, but not significantly. In all, 74% of MGUS and 38% of SM/MM patients had one or more elevated marker of platelet activation, compared to 19% of the HCs. Marker-specific hyporesponsiveness of platelets to agonist [adenosine diphosphate (ADP), thrombin receptor-activating peptide 6] stimulation in vitro was observed, with significantly reduced surface levels of P-selectin in response to ADP in patients with MGUS. Platelet-leucocyte aggregates were not altered in patients, while platelet-associated immunoglobulins were elevated in a subset of patients. Overall, we found that platelet hyperactivation is prevalent in both MGUS and SM/MM patients and is potentially related to hyporesponsiveness. These observations suggest that further investigation of the predictive and prognostic value of platelet hyperactivation in such patients is warranted.

Platelets Endocytose Viral Particles and Are Activated via TLR (Toll-Like Receptor) Signaling.

Thrombocytopenia is associated with many viral infections suggesting virions interact with and affect platelets. Consistently, viral particles are seen inside platelets, and platelet activation markers are detected in viremic patients. In this article, we sought mechanistic insights into these virion/platelet interactions by examining how platelets endocytose, traffic, and are activated by a model virion. Approach and Results: Using fluorescently tagged HIV-1 pseudovirions, 3-dimensional structured illumination microscopy, and transgenic mouse models, we probed the interactions between platelets and virions. Mouse platelets used known endocytic machinery, that is, dynamin, VAMP (vesicle-associated membrane protein)-3, and Arf6 (ADP-ribosylation factor 6), to take up and traffic HIV-1 pseudovirions. Endocytosed HIV-1 pseudovirions trafficked through early (Rab4+) and late endosomes (Rab7+), and then to an LC3+ (microtubule-associated protein 1A/1B-light chain 3) compartment. Incubation with virions induced IRAK4 (interleukin 1 receptor-associated kinase 4), Akt (protein kinase B), and IKK (IκB kinase) activation, granule secretion, and platelet-leukocyte aggregate formation. This activation required TLRs (Toll-like receptors) and MyD88 (myeloid differentiation primary response protein 88) but was less extensive and slower than activation with thrombin. In vivo, HIV-1 pseudovirions injection led to virion uptake and platelet activation, as measured by IKK activation, platelet-leukocyte aggregate formation, and mild thrombocytopenia. All were decreased in VAMP-3-/- and, megakaryocyte/platelet-specific, Arf6-/- mice. Similar platelet activation profiles (increased platelet-leukocyte aggregates, plasma platelet factor 4, and phospho-IκBα) were detected in newly diagnosed and antiretroviral therapy-controlled HIV-1+ patients. Collectively, our data provide mechanistic insights into the cell biology of how platelets endocytose and process virions. We propose a mechanism by which platelets sample the circulation and respond to potential pathogens that they take up.

Primary Hemostasis in Chronic Liver Disease and Cirrhosis: What Did We Learn over the Past Decade?

Changes in primary hemostasis have been described in patients with chronic liver disease (CLD) and cirrhosis and are still subject to ongoing debate. Thrombocytopenia is common and multifactorial. Numerous studies also reported platelet dysfunction. In spite of these changes, primary hemostasis seems to be balanced. Patients with CLD and cirrhosis can suffer from both hemorrhagic and thrombotic complications. Variceal bleeding is the major hemorrhagic complication and is mainly determined by high portal pressure. Non portal hypertension-related bleeding due to hemostatic failure is uncommon. Thrombocytopenia can complicate management of invasive procedures in CLD patients. Recently, oral thrombopoietin agonists have been approved to raise platelets before invasive procedures. In this review we aim to bundle literature, published over the past decade, discussing primary hemostasis in CLD and cirrhosis including (1) platelet count and the role of thrombopoietin (TPO) agonists, (2) platelet function tests and markers of platelet activation, (3) von Willebrand factor and (4) global hemostasis tests.

Platelet Activation via Shear Stress Exposure Induces a Differing Pattern of Biomarkers of Activation versus Biochemical Agonists

Implantable cardiovascular therapeutic devices, while hemodynamically effective, remain limited by thrombosis. A driver of device-associated thrombosis is shear-mediated platelet activation (SMPA). Underlying mechanisms of SMPA, as well as useful biomarkers able to detect and discriminate mechanical versus biochemical platelet activation, are poorly defined. We hypothesized that SMPA induces a differing pattern of biomarkers compared with biochemical agonists. Gel-filtered human platelets were subjected to mechanical activation via either uniform constant or dynamic shear; or to biochemical activation by adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP-6), thrombin, collagen, epinephrine, or arachidonic acid. Markers of platelet activation (P-selectin, integrin αIIbβ3 activation) and apoptosis (mitochondrial membrane potential, caspase 3 activation, and phosphatidylserine externalization [PSE]) were examined using flow cytometry. Platelet procoagulant activity was detected by chromogenic assay measuring thrombin generation. Contribution of platelet calcium flux in SMPA was tested employing calcium chelators, ethylenediaminetetraacetic acid (EDTA), and BAPTA-AM. Platelet exposure to continuous shear stress, but not biochemical agonists, resulted in a dramatic increase of PSE and procoagulant activity, while no integrin αIIbβ3 activation occurred, and P-selectin levels remained barely elevated. SMPA was associated with dissipation of mitochondrial membrane potential, but no caspase 3 activation was observed. Shear-mediated PSE was significantly decreased by chelation of extracellular calcium with EDTA, while intracellular calcium depletion with BAPTA-AM had no significant effect. In contrast, biochemical agonists ADP, TRAP-6, arachidonic acid, and thrombin were potent inducers of αIIbβ3 activation and/or P-selectin exposure. This differing pattern of biomarkers seen for SMPA for continuous uniform shear was replicated in platelets exposed to dynamic shear stress via circulation through a ventricular assist device-propelled circulatory loop. Elevated shear stress, but not biochemical agonists, induces a differing pattern of platelet biomarkers-with enhanced PSE and thrombin generation on the platelet surface. This differential biomarker phenotype of SMPA offers the potential for early detection and discrimination from that mediated by biochemical agonists.

Detection of Platelet-Activating Antibodies Associated with Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet-activating antibodies that in most instances recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays (i.e., functional assays) are more specific than immunoassays, since they are able to discern clinically relevant heparin-induced antibodies. All functional assays used for HIT diagnosis share the same principle, as they assess the ability of serum/plasma from suspected HIT patients to activate fresh platelets from healthy donors in the presence of several concentrations of heparin. Depending on the assay, donors’ platelets are stimulated either in whole blood (WB), platelet-rich plasma (PRP), or in a buffer medium (washed platelets, WP). In addition, the activation endpoint studied varies from one assay to another: platelet aggregation, membrane expression of markers of platelet activation, release of platelet granules. Tests with WP are more sensitive and serotonin release assay (SRA) is considered to be the current gold standard, but functional assays suffer from certain limitations regarding their sensitivity, specificity, complexity, and/or accessibility. However, the strict adherence to adequate preanalytical conditions, the use of selected platelet donors and the inclusion of positive and negative controls in each run are key points that ensure their performances.

Profiling the endothelial function using both peripheral artery tonometry (EndoPAT) and Laser Doppler Flowmetry (LD) - Complementary studies or waste of time?

Endothelial dysfunction (ED) plays a key role in developing of cardiovascular diseases and is an important predictor of future cardiovascular events. Nevertheless, there is no established method assessing endothelial function in general population. The most popular protocol includes the ultrasound-flow-mediated-dilation, but its repeatability is operator-dependent. We intended to compare the two other operator-independent methods assessing endothelial function – the EndoPAT and Laser Doppler flowmetry (LD), and we endeavored to place them on current individual profile of biochemical cardiovascular risk and endothelial function. A total of 61 clinically healthy subjects (aged 29 ± 1y) were investigated. The blood was collected for conventional cardiovascular risk markers, the NO-pathway metabolites (ADMA, L-arginine, SDMA), oxidative-stress-markers (MDA, thiol-index) as well as endothelial and platelet activation markers (sICAM1, sVCAM1, PAI-1, sE-selectin, sP-selectin, VEGF). Subsequently, all participants underwent examination by both EndoPAT and LD. There was a poor correlation between EndoPAT and LD results. No significant differences between participants with preserved and impaired endothelial function regarding endothelial activation nor cardiovascular risk markers were observed. Both methods assess endothelial function independently from the profile of endothelial pro/anti-inflammatory status and conventional risk factors, therefore further prospective studies are needed in order to verify their additional value in the cardiovascular risk stratification.

Circulating MicroRNA Levels Indicate Platelet and Leukocyte Activation in Endotoxemia Despite Platelet P2Y12 Inhibition.

There is evidence for the effects of platelet inhibition on innate immune activation. Circulating microRNAs (miRNAs) have been implicated as markers of platelet and leukocyte activation. In the present study, we assessed the effects of P2Y12 inhibitors on platelet and leukocyte miRNAs during endotoxemia. Healthy volunteers were randomly assigned to receive oral ticagrelor (n = 10), clopidogrel (n = 8) or no drug (n = 8) for one week, followed by an intravenous bolus of 2 ng/kg endotoxin. Serum was collected at baseline, after one week of antiplatelet treatment and 6 and 24 h after endotoxin administration. MiRNAs were screened using LNA-based qPCR, followed by TaqMan-qPCR validation of candidates. Clinical validation was performed in 41 sepsis patients. Platelet-enriched miR-197, miR-223 and miR-223* were decreased in volunteers following antiplatelet therapy. Endotoxin increased platelet miRNAs, whilst the opposite effect was seen for leukocyte-enriched miR-150. Neither of these endotoxin-mediated effects were altered by P2Y12 inhibitors. Sepsis patients with fatal outcomes (n = 12) had reduced miR-150 levels compared with survivors (n = 29). In conclusion, we show that miR-150 is downregulated in experimental endotoxemia and can predict survival in sepsis but is unaffected by P2Y12 inhibition. While P2Y12 inhibition reduces platelet-associated miRNAs in healthy volunteers, it fails to attenuate the response of platelet miRNAs to endotoxemia.

The association between urinary levels of 1,3-butadiene metabolites, cardiovascular risk factors, microparticles, and oxidative stress products in adolescents and young adults

1,3-Butadiene (BD) is a synthetic colorless gas used in the production of synthetic rubber and polymers. Exposure to BD has been reported to increase oxidative stress and accelerate atherosclerosis in vitro and in animal studies. In occupational studies, BD exposure has been linked to cardiovascular disease (CVD). However, no previous research has been reported on whether BD exposure is associated with CVD risk factors and oxidative stress in the general population. We recruited 853 young participants to study the correlation between urinary levels of the BD metabolite, N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA), CVD risk factors, serum levels of endothelial microparticles and platelet microparticles, and the urinary levels of 8-hydroxydeoxyguanosine (8-OHdG). The results showed the DHBMA levels were positively correlated with low-density lipoprotein-C, carotid intima-media thickness (CIMT), CD31+/CD42a − counts (endothelial apoptosis markers), and urinary 8-OHdG levels. Moreover, DHBMA levels were negatively correlated with CD62 P counts (platelet activation marker). The correlation between DHBMA, CIMT, and 8-OHdG was more evident when the levels of CD31+/CD42a − or CD62 P were above 50%. In conclusion, we reported that the urinary levels of DHBMA were associated with the lipid profile, CIMT, microparticles, and marker of oxidative stress in this young population. Future studies on BD exposure and atherosclerosis are needed.