Effects of Polyphosphate on Leukocyte Function.

Factor eight inhibitory bypassing agent (FEIBA) is used as a therapeutic option in haemophilia patients who have developed inhibitors. The measurement of thrombin generation has been applied to monitor the efficacy of FEIBA. However, a major concern about the clinical use of FEIBA is whether or not an increase in thrombin activity causes subsequent platelet activation and risk of thrombosis. Our aim is to evaluate whether FEIBA causes platelet and leucocyte activation in haemophilia patients with inhibitors. We evaluated the effects of FEIBA on platelet and leucocyte activity in correlation with thrombin generation. Initially, an in vitro study was conducted to evaluate the effects of FEIBA on platelet and leucocyte activity (using flow cytometry) using peripheral blood from normal volunteers. We then performed an ex vivo study looking at the effect of FEIBA on the above parameters in two haemophiliacs with high-titre inhibitors. A parallel study was also carried out ex vivo to evaluate thrombin generation using a thrombinoscope. FEIBA did not cause platelet or leucocyte activation in either the in vitro or ex vivo studies but showed a predictable increase in thrombin generation. Our study is the first one to address the effect of FEIBA on platelet and leucocyte function. We found no evidence of ‘systemic’ platelet activation. The findings suggest that whilst FEIBA improves global haemostasis, platelet activation is likely to be contained to the site of injury and systemic platelet activation, a previously feared consequence of FEIBA infusion that that may have contributed to thrombotic risk is absent.

Platelet and leucocyte activity are important in the acute development of thrombosis and in the pathogenesis of ischaemic vascular disease. Dan Shen Di Wan (DS, Cardiotonic Pill or Dantonic® Pill) is one of the most commonly used Chinese herbal formulations for treating patients with atherosclerotic disease in China and several Asian countries. We studied the effect of DS on platelet and leucocyte function and compared the effects with conventional antiplatelet agents, cangrelor (ADP P2Y12 receptor antagonist) and aspirin (acetyl salicylic acid, ASA). Measurements were made by platelet aggregation (%) and activation (CD62P %), platelet-monocyte conjugate formation (P/M, CD42a median fluorescence, mf), platelet-neutrophil conjugate formation (P/N, mf), and leucocyte activation (CD11b median fluorescence on monocytes and neutrophils, mf) in response to 3.3 µmol/L adenosine diphosphate (ADP), 1.0 µmol/L platelet activating factor (PAF), 5.0 µmol/L adrenaline and 0.5 µg/mL collagen. We also evaluated the effect of its main component, water soluble extract of salvia miltiorrhiza (SME) on intracellular calcium mobilization in platelets triggered by 10 µmol/L ADP, 10 µmol/L PAF, 2 µg/mL collagen and 15 µmol/L thrombin receptor activating peptide (TRAP). Overall DS showed inhibition of platelet aggregation, platelet activation, platelet-leucocyte conjugate formation and leucocyte activation in response to all the agonists apart from adrenaline (all p < 0.01). DS showed inhibition of platelet aggregation and leucocyte activation equivalent to cangrelor 100 nmol/L and ASA 100 µmol/L. SME dose-dependently inhibited intracellular calcium mobilization in platelets following stimulation with all the platelet agonists with maximum effective at 0.36 mg/mL (all p < 0.01). When used at 0.18 mg/mL its inhibitory effect was equivalent to cangrelor and ASA. We conclude that DS is a potential inhibitor of both platelet and leucocyte activation.

Genetic common ground between allergic and autoimmune disease: The role of cytotoxic T-lymphocyte antigen 4

There is growing interest in possible beneficial effects of specific dietary components on cardiovascular health. Platelets and leukocytes contribute to arterial thrombosis and to inflammatory processes. Previous studies performed in vitro have demonstrated inhibition of platelet function by (-)-epicatechin and (+)-catechin, flavan-3-ols (flavanols) that are present in several foods including some cocoas. Also, some modest inhibition of platelet function has been observed ex vivo after the consumption of flavanol-containing cocoa products by healthy adults. So far there are no reports of effects of cocoa flavanols on leukocytes. This paper summarizes 2 recent investigations. The first was a study of the effects of cocoa flavanols on platelet and leukocyte function in vitro. The second was a study of the effects of consumption of a flavanol-rich cocoa beverage by healthy adults on platelet and leukocyte function ex vivo. Measurements were made of platelet aggregation, platelet-monocyte conjugate formation (P/M), platelet-neutrophil conjugate formation (P/N), platelet activation (CD62P on monocytes and neutrophils), and leukocyte activation (CD11b on monocytes and neutrophils) in response to collagen and/or arachidonic acid. In the in vitro study several cocoa flavanols and their metabolites were shown to inhibit platelet aggregation, P/M, P/N, and platelet activation. Their effects were similar to those of aspirin and the effects of a cocoa flavanol and aspirin did not seem to be additive. There was also inhibition of monocyte and neutrophil activation by flavanols, but this was not replicated by aspirin. 4'-O-methyl-epicatechin, 1 of the known metabolites of the cocoa flavanol (-)-epicatechin, was consistently effective as an inhibitor of platelet and leukocyte activation. The consumption of a flavanol-rich cocoa beverage also resulted in significant inhibition of platelet aggregation, P/M and P/N, and platelet activation induced by collagen. The inhibitory effects were related to their flavanol content. There was also inhibition of monocyte and neutrophil activation, but here it was concluded that cocoa constituents other than flavanols may contribute to the inhibition that was observed. It can be concluded that cocoa flavanols, their metabolites and possibly other cocoa constituents can modulate the activity of platelets and leukocytes in vitro and ex vivo. The research suggests that the consumption of certain cocoa products may provide a dietary approach to maintaining or improving cardiovascular health.

Platelet and leukocyte activation, atherosclerosis and inflammation in European and South Asian men.

Polyphosphate elicits pro‐inflammatory responses that are counteracted by activated protein C in both cellular and animal models

Clinical studies suggest that stent design and surface texture are responsible for differences in biocompatibility of metallic endovascular stents. A simple in vitro experimental setup was established to test stent-induced degree of platelet and leukocyte activation and platelet-leukocyte aggregation by flow cytometry. Heparin-coated tantalum stents and gold-coated and uncoated stainless steel stents were tested. Stents were implanted into silicone tubes and exposed to blood from healthy volunteers. Platelet and leukocyte activation and percentage of leukocyte-platelet aggregates were determined in a whole-blood assay by subsequent staining for activation-associated antigens (CD41a, CD42b, CD62p, and fibrinogen binding) and leukocyte antigens (CD14 and CD45) and flow cytometric analysis. Blood taken directly after venous puncture or exposed to the silicone tube alone was used as negative controls. Positive control was in vitro stimulation with thrombin receptor activating peptide (TRAP-6). Low degree of platelet activation and significant increase in monocyte- and neutrophil-platelet aggregation were observed in blood exposed to stents (P < 0.05). In addition, leukocyte activation was induced as measured by increased CD45 and CD14 expression. Heparin coated stents continuously induced less platelet activation and leukocyte-platelet aggregation than uncoated stainless steel stents of the same length and shorter stents of the same structure. Stent surface coating and texture plays a role in platelet and leukocyte activation and leukocyte-platelet aggregation. Using this simple in vitro assay and whole blood and flow cytometry, it seems possible to differentiate stents by their potency to activate platelets and/or leukocytes. This assay could be applied for improving the biocompatibility of coronary stents.

Patients diagnosed with essential thrombocythaemia (ET) carry mutations in JAK2 V617F (ET-JAK2+), calreticulin (ET-CALR+) and in codon 515 (exon 10) of the thrombopoietin receptor gene (MPL W515L/K) at frequencies of 59–64%, 15–24% and 4%, respectively (Tefferi et al, 2014). ET patients and primary myelofibrosis patients (Finazzi et al, 2015), carrying CALR mutations have a lower risk of thrombotic events (Rotunno et al, 2014; Rumi et al, 2014) and longer thrombosis-free survival (Rotunno et al, 2014) in comparison with those with mutated JAK2. This has been related with a lower haemoglobin level and white blood cell (WBC) count in ET-CALR+, compared with ET-JAK2+ patients, despite higher platelet counts in the former group. Indeed, previous studies have suggested that both granulocyte and platelet activation (Arellano-Rodrigo et al, 2006), as well as increased WBC count (Passamonti et al, 2012) may be more relevant to the pathogenesis of thrombosis than the platelet count per se in this clinical condition (Rumi et al, 2014). Leucocyte and platelet activation status are known to be significantly higher in ET patients than in the general population. Among ET patients, while JAK2 mutations affect the prothrombotic phenotype by inducing increased platelet P-selectin expression and leuco-platelet aggregates (Arellano-Rodrigo et al, 2006), to the best of our knowledge, differences in leucocyte and platelet activation status between ET-CALR+ and ET-JAK2+ patients have never been addressed. With the aim of assessing markers of platelet and WBC activation in ET patients carrying mutations in JAK2 V617F versus CALR, we identified 70 consecutive patients diagnosed with ET at our Haematology and Clinical Oncology Unit, Hospital Morales-Meseguer of Murcia, Spain, between 1990 and 2006. DNA was available in 65 of them. ET was diagnosed in accordance with the criteria in use at the time of diagnosis (Vardiman et al, 2001). Molecular characterization for JAK2 V617F, MPL W515L/K and CALR exon 9 mutations were performed as previously described (Soler et al, 2015). P-selectin expression in platelets, CD14 in neutrophils, CD11b in monocytes and neutrophils, platelet-monocyte complexes (PMC) and platelet-neutrophil complexes (PNC) were quantified by flow cytometry with the appropriate monoclonal antibodies [CD42b-fluorescein isothicyanate (FITC); CD45-peridinin chlorophyll (PerCP), CD11b-FITC, CD14-FITC] on a FACScalibur platform (all from Becton Dickinson, San Jose, CA, USA), essentially as described elsewhere (Lozano et al, 2001). Platelet function status was assessed on a platelet function analyser (PFA) [PFA-100®; collagen and ADP (CADP) and collagen and epinephrine (CEPI) cartridges] (Siemens Healthcare, Barcelona, Spain) using citrated-blood samples from those patients that were free of antiplatelet therapy. Major thrombotic complications during the follow-up were recorded, including: acute coronary syndrome (ACS); vascular complications in the central nervous system [stroke, transient ischaemic attack (TIA), retinal vessel thrombosis]; intra-abdominal (Budd-Chiari syndrome, portal and splenic thrombosis); or peripheral [including peripheral arterial thrombosis; deep vein thrombosis (DVT)] and pulmonary thromboembolism. Patient's baseline characteristics are summarized in Table 1. JAK2 V617F was detected in 40 patients (61·5%). Among ET-JAK2−, 13 (20%) were CALR+ and 12 patients (18·5%) did not show mutations in CALR. Analysis of common cardiovascular risk factors displayed no differences according to the presence of CALR or JAK2 mutations. During a median follow-up of 10·4 years (interquartile range 7–14), 13 patients presented a major thrombotic event (4 stroke, 2 TIA, 1 DVT, 1 portal thrombosis, 1 subclavian thrombosis and 4 ACS). Twelve of them were JAK2+ and one patient was JAK2−CALR−. Thus, ET-JAK2+ patients had a significantly higher frequency of thrombotic events than those with CALR mutations (P = 0·023) (Table 1). 153·1 ± 41 (n = 13) 238 ± 60·4 (n = 4) 133·5 ± 10·6 (n = 2) As expected (Rotunno et al, 2014; Rumi et al, 2014), we found that CALR mutations were more frequent in younger women presenting with lower haemoglobin and WBC counts but higher platelets count than ET-JAK2+ (Table 1). In our series, these differences only reached statistical significance for haemoglobin level (P = 0·004), probably due to the limited number of patients included in each group. Regarding cell activation status, ET-CALR+ patients displayed a significantly lower percentage of PNC and CD14+ expression in neutrophils than ET-JAK2+ patients. Moreover, PFA-100 closure time with CEPI cartridge was longer in those patients who carry CALR mutations versus JAK2-mutated patients (238 s vs. 153 s; P = 0·005). No significant differences were observed in other cell activation parameters, such as platelet P-selectin expression. Thus, in accordance with previous evidence (Arellano-Rodrigo et al, 2006), our study shows an increased leucocyte activation status in ET patients carrying the JAK2 V617F mutation, a mutation that associates with higher risk or thrombotic events. Next, we tried to elucidate the role of CALR mutations per se in the lower leucocyte activation in ET patients. For that, platelet and leucocyte activation parameters were compared between ET-CALR+ and ET-CALR− patients. No significant differences were found, suggesting negligible impact of CALR mutations per se in the lower platelet/leucocyte activation in ET patients. Our data are consistent with the absence of any significant impact of these mutations in the International Prognostic Score of thrombosis for ET (IPSET) (Finazzi et al, 2014). In conclusion, we show for the first time that, in addition to decreased haemoglobin levels and WBC counts, the lower thrombotic risk of ET-CALR+ (versus ET-JAK2+) patients might be partially explained by reduced leucocyte activation. These differences seem to be related to the absence of JAK2 mutation rather than to the presence of CALR mutations. JMT analysed the data and wrote the manuscript. MJM provided clinical material. FFM provided clinical material and wrote the manuscript. AIA, MLL and JRP performed experiments. CM and VV supervised the study and reviewed the manuscript. The authors declare no conflict of interest.

Diurnal mechanisms are central to regulating host responses. Recent studies uncovered a novel family of mediators termed as specialized proresolving mediators that terminate inflammation without interfering with the immune response. Herein, we investigated the diurnal regulation of specialized proresolving mediators in humans and their role in controlling peripheral blood leukocyte and platelet activation. Using lipid mediator profiling and healthy volunteers, we found that plasma concentrations of n-3 docosapentaenoic acid-derived D-series resolvins (RvDn-3 DPA) were regulated in a diurnal manner. The production and regulation of these mediators was markedly altered in patients at risk of myocardial infarct. These changes were associated with decreased 5-lipoxygenase expression and activity, as well as increased systemic adenosine concentrations. We also found a significant negative correlation between plasma RvDn-3 DPA and markers of platelet, monocyte, and neutrophil activation, including CD63 and CD11b. Incubation of RvDn-3 DPA with peripheral blood from healthy volunteers and patients with cardiovascular disease significantly and dose-dependently decreased platelet and leukocyte activation. Furthermore, administration of RvD5n-3 DPA to ApoE-/- (apolipoprotein E deficient) mice significantly reduced platelet-leukocyte aggregates, vascular thromboxane B2 concentrations, and aortic lesions. These results demonstrate that peripheral blood RvDn-3 DPA are diurnally regulated in humans, and dysregulation in the production of these mediators may lead to cardiovascular disease.

Platelets possess two receptors for ADP, P2Y(1) and P2Y(12). ADP is released from platelet dense granules upon platelet activation by numerous agonists and thereby amplifies platelet responses regardless of the initial stimulus. The P2Y(1) receptor is one of many platelet receptors coupled to Gq and initiates ADP-induced activation. The P2Y(12) receptor on the other hand is linked to Gi and plays a special role in the amplification of platelet activation initiated by numerous other pathways. Platelet activation leads to a range of responses that play a critical role in arterial thrombosis and the inflammatory responses associated with this, including platelet aggregation, dense and alpha granule secretion and procoagulant activity. P2Y(12) receptor activation yields powerful amplification of these processes such that P2Y(12) receptor antagonists may have dramatic inhibitory effects on platelet function regardless of the activating stimuli. This phenomenon, coupled with the restricted distribution of the P2Y(12) receptor in humans, makes the receptor an ideal target for pharmaceutical therapy. This has already been established by the therapeutic success of clopidogrel, which acts, via an active metabolite, on this receptor. However, current therapeutic regimens of clopidogrel yield variable and incomplete P2Y(12) receptor blockade and more effective strategies to block P2Y(12) receptor activation offer the potential of greater clinical efficacy.

Platelet Activation Promotes Formation of Platelet-Leukocyte Conjugates (PLC) in Non-Valvular Atrial Fibrillation (AF).

Mutations That Alter Use of Hepatitis C Virus Cell Entry Factors Mediate Escape From Neutralizing Antibodies

Endothelial Microparticles, Platelet and Leukocyte Activation and Cellular Interactions in Patients with Non-Valvular Atrial Fibrillation.

Leukocytes and the kidney contribute to interstitial inflammation in lupus nephritis

Cardiovascular disease, a progressive disorder characterized by the accumulation of lipids in the artery wall, is a leading cause of death in Western societies. One of the initial events in atherogenesis involves the recruitment of inflammatory cells from the circulation into the developing lesion. Studies during the past decade have underscored the role of inflammatory mediators in disease initiation and progression. Critical progress has been made in our understanding of the complex mechanisms by which monocytes, macrophages, and T-cells accumulate in atherosclerotic plaques. Experimental research has identified several candidate adhesion proteins and chemokines that are critically involved in the recruitment process, and encouraging data provide a mechanistic framework for new therapeutic targets. This review provides an overview of our current understanding of the mechanisms that direct the recruitment of monocytes to, and their retention in, atherosclerotic lesions.