Prospective flow cytometry analysis of leucocyte subsets in critically ill patients who develop sepsis: a pilot study.

PURPOSE: Sepsis in critically ill patients with injury bears a high morbidity and mortality. Extensive phenotypic monitoring of leucocyte subsets in critically ill patients at ICU admission and during sepsis development is still scarce. The main objective of this study was to identify early changes in leukocyte phenotype which would correlate with later development of sepsis. METHODS: Patients who were admitted in a tertiary ICU for organ support after severe injury (elective cardiac surgery, trauma, necessity of prolonged ventilation or stroke) were sampled on admission (T1) and 48-72 h later (T2) for phenotyping of leukocyte subsets by flow cytometry and cytokines measurements. Those who developed secondary sepsis or septic shock were sampled again on the day of sepsis diagnosis (Tx). RESULTS: Ninety-nine patients were included in the final analysis. Nineteen (19.2%) patients developed secondary sepsis or septic shock. They presented significantly higher absolute monocyte counts and CRP at T1 compared to non-septic patients (1030/µl versus 550/µl, p = 0.013 and 5.1 mg/ml versus 2.5 mg/ml, p = 0.046, respectively). They also presented elevated levels of monocytes with low expression of L-selectin (CD62Lneg monocytes) (OR[95%CI] 4.5 (1.4-14.5), p = 0.01) and higher SOFA score (p < 0.0001) at T1 and low mHLA-DR at T2 (OR[95%CI] 0.003 (0.00-0.17), p = 0.049). Stepwise logistic regression analysis showed that both monocyte markers and high SOFA score (> 8) were independently associated with nosocomial sepsis occurrence. No other leucocyte count or surface marker nor any cytokine measurement correlated with sepsis occurrence. CONCLUSION: Monocyte counts and change of phenotype are associated with secondary sepsis occurrence in critically ill patients with injury.

Platelet-rich plasma (PRP) and tendon healing: comparison between fresh and frozen-thawed PRP.

Platelet-rich plasma (PRP) is increasingly used in the treatment of musculoskeletal diseases. Its preservation by freezing it for the realization of multiple injections in clinical use has never been discussed. Calcaneal tendons of rats were surgically sectioned. Platelet concentration of the PRP was 2.5 x 106/µl with autologous plasma of rats. Frozen-thawed PRP was prepared by performing two cycles of freezing and thawing on PRP aliquots. Both platelet preparations were injected in the lesion. Biomechanical and histological evaluations were carried out after 7, 20 or 40 days post surgery. After 7 and 40 days, no significant difference was observed between the PRP and the frozen-thawed PRP group. There is however a difference 20 days after surgery: the ultimate tensile strength (UTS) was greater in the fresh PRP group. No obvious difference with histological aspect was observed between the two groups. In conclusion, fresh PRP and frozen-thawed PRP injections can lead to similar results in the healing process of section calcaneal tendons of rats. Improvements with fresh PRP are slight. PRP could thus be frozen to be preserved if multiple injections are needed (e.g. osteoarthritis).

P2X1 ion channel deficiency causes massive bleeding in inflamed intestine and increases thrombosis.

BACKGROUND: Intestinal inflammation is associated with bleeding and thrombosis, two processes that may involve both platelets and neutrophils. However, the mechanisms and the respective contribution of these cells to intestinal bleeding and extra-intestinal thrombosis remain largely unknown. OBJECTIVE: Our study aimed at investigating the mechanisms underlying the maintenance of vascular integrity and thrombosis in intestinal inflammation. METHODS: We used a mouse model of acute colitis induced by oral administration of dextran sodium sulfate (DSS) for 7 days. Bleeding was assessed after depletion of platelets, neutrophils, or glycoprotein VI (GPVI); treatment with aspirin or clopidogrel; or in P2X1-deficient mice. Extra-intestinal thrombosis was analyzed using a laser-induced injury model of thrombosis in cremaster muscle arterioles. RESULTS: Platelet depletion or P2X1 deficiency led to macrocytic regenerative anemia due to intestinal hemorrhage. In contrast, GPVI, P2Y12, and thromboxane A2 were dispensable. Platelet P-selectin expression and regulated on activation, normal T-cell expressed and secreted (RANTES) plasma levels were lower in DSS-treated P2X1-deficient mice as compared to wild-type mice, indicative of a platelet secretion defect. Circulating neutrophils had a more activated phenotype, and neutrophil infiltration in the colon was increased. P2X1-deficient mice also had elevated plasma granulocyte-colony stimulating factor (G-CSF) levels. Neutrophil depletion limited blood loss in these mice, whereas exogenous administration of G-CSF in colitic wild-type mice caused macrocytic anemia. Anemic colitic P2X1-deficient mice formed atypical neutrophil- and fibrin-rich, and platelet-poor thrombi upon arteriolar endothelial injury. CONCLUSIONS: Platelets and P2X1 ion channels are mandatory to preserve vascular integrity in inflamed intestine. Upon P2X1 deficiency, neutrophils contribute to bleeding and they may also be responsible for enhanced thrombosis.

Haemostatic, fibrinolytic and inflammatory profiles in West Highland white terriers with canine idiopathic pulmonary fibrosis and controls.

BACKGROUND: Canine idiopathic pulmonary fibrosis (CIPF) is a progressive interstitial lung disease mainly affecting old West Highland white terriers (WHWTs). The aetiology of CIPF is currently unknown and pathogenesis poorly understood. A genetic basis is strongly suspected based on the breed predisposition. CIPF shares clinical and pathological features with human IPF. In human IPF, coagulation disorders favouring a local and systemic pro-thrombotic state have been demonstrated in association with disease severity and outcome. The aim of this study was to compare the systemic haemostatic, fibrinolytic and inflammatory profiles of WHWTs affected with CIPF with breed-matched controls (CTRLs). Additionally, data collected in both groups were interpreted with regard to the reference intervals (when available) to assess possible pro-thrombotic features of the WHWT breed that may be related to CIPF predisposition. A total of 14 WHWTs affected with CIPF and 20 CTRLs were included. RESULTS: WHWTs affected with CIPF had prolonged activated partial thromboplastine time in comparison with CTRLs (12.2 ± 0.9 s vs. 11.5 ± 0.7 s, P = 0.028), whereas results obtained in both groups were all within reference ranges. There was no significant difference between groups for the other factors assessed including plasmatic concentrations of fibrinogen, D-dimers concentration, antithrombin III activity, protein S and protein C activities, anti-factor Xa activity, activated protein C ratio, serum C-reactive protein concentration, and rotational thromboelastometry indices. Platelet count and plasmatic fibrinogen concentration were found to be above the upper limit of the reference range in almost half of the WHWTs included, independently of the disease status. CONCLUSIONS: Results of this study provide no clear evidence of an altered systemic haemostatic, fibrinolytic or inflammatory state in WHWTs affected with CIPF compared with CTRLs. The higher platelet counts and fibrinogen concentrations found in the WHWT breed may serve as predisposing factors for CIPF or simply reflect biological variation in this breed.

IBD risk loci are enriched in multigenic regulatory modules encompassing putative causative genes.

GWAS have identified >200 risk loci for Inflammatory Bowel Disease (IBD). The majority of disease associations are known to be driven by regulatory variants. To identify the putative causative genes that are perturbed by these variants, we generate a large transcriptome data set (nine disease-relevant cell types) and identify 23,650 cis-eQTL. We show that these are determined by ∼9720 regulatory modules, of which ∼3000 operate in multiple tissues and ∼970 on multiple genes. We identify regulatory modules that drive the disease association for 63 of the 200 risk loci, and show that these are enriched in multigenic modules. Based on these analyses, we resequence 45 of the corresponding 100 candidate genes in 6600 Crohn disease (CD) cases and 5500 controls, and show with burden tests that they include likely causative genes. Our analyses indicate that ≥10-fold larger sample sizes will be required to demonstrate the causality of individual genes using this approach.

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description.

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily. Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Soluble GPVI is elevated in injured patients: shedding is mediated by fibrin activation of GPVI.

Soluble glycoprotein VI (sGPVI) is shed from the platelet surface and is a marker of platelet activation in thrombotic conditions. We assessed sGPVI levels together with patient and clinical parameters in acute and chronic inflammatory conditions, including patients with thermal injury and inflammatory bowel disease and patients admitted to the intensive care unit (ICU) for elective cardiac surgery, trauma, acute brain injury, or prolonged ventilation. Plasma sGPVI was measured by enzyme-linked immunosorbent assay and was elevated on day 14 after thermal injury, and was higher in patients who developed sepsis. sGPVI levels were associated with sepsis, and the value for predicting sepsis was increased in combination with platelet count and Abbreviated Burn Severity Index. sGPVI levels positively correlated with levels of D-dimer (a fibrin degradation product) in ICU patients and patients with thermal injury. sGPVI levels in ICU patients at admission were significantly associated with 28- and 90-day mortality independent of platelet count. sGPVI levels in patients with thermal injury were associated with 28-day mortality at days 1, 14, and 21 when adjusting for platelet count. In both cohorts, sGPVI associations with mortality were stronger than D-dimer levels. Mechanistically, release of GPVI was triggered by exposure of platelets to polymerized fibrin, but not by engagement of G protein-coupled receptors by thrombin, adenosine 5'-diphosphate, or thromboxane mimetics. Enhanced fibrin production in these patients may therefore contribute to the observed elevated sGPVI levels. sGPVI is an important platelet-specific marker for platelet activation that predicts sepsis progression and mortality in injured patients.

Sepsis prediction in critically ill patients by platelet activation markers on ICU admission: a prospective pilot study.

BACKGROUND: Platelets have been involved in both immune surveillance and host defense against severe infection. To date, whether platelet phenotype or other hemostasis components could be associated with predisposition to sepsis in critical illness remains unknown. The aim of this work was to identify platelet markers that could predict sepsis occurrence in critically ill injured patients. METHODS: This single-center, prospective, observational, 7-month study was based on a cohort of 99 non-infected adult patients admitted to ICUs for elective cardiac surgery, trauma, acute brain injury, and post-operative prolonged ventilation and followed up during ICU stay. Clinical characteristics and severity score (SOFA) were recorded on admission. Platelet activation markers, including fibrinogen binding to platelets, platelet membrane P-selectin expression, plasma soluble CD40L, and platelet-leukocytes aggregates were assayed by flow cytometry at admission and 48 h later, and then at the time of sepsis diagnosis (Sepsis-3 criteria) and 7 days later for sepsis patients. Hospitalization data and outcomes were also recorded. METHODS: Of the 99 patients, 19 developed sepsis after a median time of 5 days. These patients had a higher SOFA score at admission; levels of fibrinogen binding to platelets (platelet-Fg) and of D-dimers were also significantly increased compared to the other patients. Levels 48 h after ICU admission no longer differed between the two patient groups. Platelet-Fg % was an independent predictor of sepsis (P = 0.0031). By ROC curve analysis, cutoff point for Platelet-Fg (AUC = 0.75) was 50%. In patients with a SOFA cutoff of 8, the risk of sepsis reached 87% when Platelet-Fg levels were above 50%. Patients with sepsis had longer ICU and hospital stays and higher death rate. CONCLUSIONS: Platelet-bound fibrinogen levels assayed by flow cytometry within 24 h of ICU admission help identifying critically ill patients at risk of developing sepsis.

Eccentric Training for Tendon Healing After Acute Lesion: A Rat Model.

BACKGROUND: The tendon is a dynamic entity that remodels permanently. Platelet-rich plasma (PRP) injection has been shown to have a beneficial effect on tendon healing after lesion in rats. Furthermore, eccentric exercise seems to improve the mechanical quality of the tendon. HYPOTHESIS: A combination of PRP injection and eccentric training might be more effective than either treatment alone. STUDY DESIGN: Controlled laboratory study. METHODS: Adult male rats were anesthetized, an incision was performed in the middle of their left patellar tendon and an injection of physiological fluid (PF) or homologous PRP was randomly made at the lesion level. The rats were then divided into 2 groups: the eccentric group, undergoing eccentric training 3 times a week, and the untrained group, without any training. Thus, 4 groups were compared. After 5 weeks, the tendons were removed and their ultimate tensile strength and energy were measured. Tendons were frozen for proteomic analyses when all biomechanical tests were completed. Statistical analysis was performed with linear mixed effect models. RESULTS: No significant difference was found between the treatments using PF injection or PRP injection alone. However, the value of the ultimate tensile force at rupture was increased by 4.5 N (108% of control, P = .006) when eccentric training was performed. An intragroup analysis revealed that eccentric training significantly improved the ultimate force values for the PRP group. Proteomic analysis revealed that eccentric training led to an increase in abundance of several cytoskeletal proteins in the PF group, while a decrease in abundance of enzymes of the glycolytic pathway occurred in the PRP-treated groups, indicating that this treatment might redirect the exercise-driven metabolic plasticity of the tendon. CONCLUSION: Eccentric training altered the metabolic plasticity of tendon and led to an improvement of injured tendon resistance regardless of the treatment injected (PF or PRP). CLINICAL RELEVANCE: This study demonstrates the necessity of eccentric rehabilitation and training in cases of tendon lesion regardless of the treatment carried out.

Efficacy of the RemoweLL cardiotomy reservoir for fat and leucocyte removal from shed mediastinal blood: a randomized controlled trial.

INTRODUCTION: Re-transfusion of lipid particles and activated leucocytes with shed mediastinal blood (SMB) can aggravate cardiopulmonary bypass-associated inflammation and increase the embolic load. This study evaluated the fat and leucocyte removal capacity of the RemoweLL cardiotomy reservoir. METHODS: Forty-five patients undergoing elective on-pump cardiac surgery were randomly allocated to filtration of SMB using the RemoweLL or the Admiral cardiotomy reservoir. The primary outcome was a drop in leucocytes and lipid particles obtained with the two filters. The effect of the filters on other blood cells and inflammatory mediators, such as myeloperoxidase (MPO), was also assessed. RESULTS: The RemoweLL cardiotomy filter removed 16.5% of the leucocytes (p<0.001) while no significant removal of leucocytes was observed with the Admiral (p=0.48). The percentage reductions in lipid particles were similar in the two groups (26% vs 23%, p=0.2). Both filters similarly affected the level of MPO (p=0.71). CONCLUSION: The RemoweLL filter more effectively removed leucocytes from SMB than the Admiral. It offered no advantage in terms of lipid particle clearance.

[Is there a place for thrombin generation assay in routine clinical laboratory?]. / Quelle place pour le test de génération de thrombine au sein du laboratoire de biologie clinique ?

Laboratory diagnosis of coagulopathies primarily relies on assays selectively exploring either the extrinsic (PT), the intrinsic (aPTT) or the common (TT) pathway of the coagulation system. Although these tests are very useful to rapidly identify severe coagulation disorders or to monitor anticoagulant therapy, they only poorly correlate with the clinical manifestations. Global assays that evaluate the whole coagulation process could potentially more accurately reflect the hemorrhagic or thrombotic phenotype of an individual. Thrombin generation assay (TGA), first described in the 1950's, has been developed and automated in the 1990's. This technique is widely used in fundamental research but has yet failed to integrate clinical laboratories. In this article, we describe TGA and review its clinical applications. Laboratory aspects and technical issues will also be discussed.

Reflections about the optimisation of the treatment of tendinopathies with PRP.

BACKGROUND: platelet-rich plasma (PRP) infiltration represents a recent therapy for chronic tendinopathies. However, in the literature, this treatment remains controversial. PURPOSE: we suggest some ideas for improving this treatment. METHODS: these suggestions were based on a review of published studies and our clinical experience. CONCLUSION: optimizing the technique for PRP collection is paramount. Different risk factors must be corrected before infiltration and chronic tendinopathies must be carefully selected. Finally, post-infiltration rehabilitation remains absolutely critical. Standardization of the use of PRP remains necessary in order to optimize the results.

Purinergic control of inflammation and thrombosis: Role of P2X1 receptors.

Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Upon tissue damage or infection, a sudden increase of extracellular ATP occurs, that might contribute to the crosstalk between inflammation and thrombosis. On platelets, P2X1 receptors act to amplify platelet activation and aggregation induced by other platelet agonists. These receptors critically contribute to thrombus stability in small arteries. Besides platelets, studies by our group indicate that these receptors are expressed by neutrophils. They promote neutrophil chemotaxis, both in vitro and in vivo. In a laser-induced injury mouse model of thrombosis, it appears that neutrophils are required to initiate thrombus formation and coagulation activation on inflamed arteriolar endothelia. In this model, by using P2X1-/ - mice, we recently showed that P2X1 receptors, expressed on platelets and neutrophils, play a key role in thrombus growth and fibrin generation. Intriguingly, in a model of endotoxemia, P2X1-/ - mice exhibited aggravated oxidative tissue damage, along with exacerbated thrombocytopenia and increased activation of coagulation, which translated into higher susceptibility to septic shock. Thus, besides its ability to recruit neutrophils and platelets on inflamed endothelia, the P2X1 receptor also contributes to limit the activation of circulating neutrophils under systemic inflammatory conditions. Taken together, these data suggest that P2X1 receptors are involved in the interplay between platelets, neutrophils and thrombosis. We propose that activation of these receptors by ATP on neutrophils and platelets represents a new mechanism that regulates thrombo-inflammation.

Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

BACKGROUND: A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. METHODS AND RESULTS: This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. CONCLUSIONS: DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug.

P2X1 expressed on polymorphonuclear neutrophils and platelets is required for thrombosis in mice.

Adenosine triphosphate (ATP) and its metabolite, adenosine, are key regulators of polymorphonuclear neutrophil (PMN) functions. PMNs have recently been implicated in the initiation of thrombosis. We investigated the role of ATP and adenosine in PMN activation and recruitment at the site of endothelial injury. Following binding to the injured vessel wall, PMNs are activated and release elastase. The recruitment of PMNs and the subsequent fibrin generation and thrombus formation are strongly affected in mice deficient in the P2X1-ATP receptor and in wild-type (WT) mice treated with CGS 21680, an agonist of the A2A adenosine receptor or NF449, a P2X1 antagonist. Infusion of WT PMNs into P2X1-deficient mice increases fibrin generation but not thrombus formation. Restoration of thrombosis requires infusion of both platelets and PMNs from WT mice. In vitro, ATP activates PMNs, whereas CGS 21680 prevents their binding to activated endothelial cells. These data indicate that adenosine triphosphate (ATP) contributes to polymorphonuclear neutrophil (PMN) activation leading to their adhesion at the site of laser-induced endothelial injury, a necessary step leading to the generation of fibrin, and subsequent platelet-dependent thrombus formation. Altogether, our study identifies previously unknown mechanisms by which ATP and adenosine are key molecules involved in thrombosis by regulating the activation state of PMNs.

Connection between cardiac vascular permeability, myocardial edema, and inflammation during sepsis: role of the α1AMP-activated protein kinase isoform.

OBJECTIVE: As adenosine monophosphate (AMP)-activated protein kinase both controls cytoskeleton organization in endothelial cells and exerts anti-inflammatory effects, we here postulated that it could influence vascular permeability and inflammation, thereby counteracting cardiac wall edema during sepsis. DESIGN: Controlled animal study. SETTINGS: University research laboratory. SUBJECTS: C57BL/6J, α1AMPK, and α1AMPK mice. INTERVENTION: Sepsis was triggered in vivo using a sublethal injection of lipopolysaccharide (O55B5, 10 mg/kg), inducing systolic left ventricular dysfunction. Left ventricular function, edema, vascular permeability, and inflammation were assessed in vivo in both wild-type mice (α1AMPK) and α1AMP-activated protein kinase-deficient mice (α1AMPK). The 5-aminoimidazole-4-carboxamide riboside served to study the impact of AMP-activated protein kinase activation on vascular permeability in vivo. The integrity of endothelial cell monolayers was also examined in vitro after lipopolysaccharide challenge in the presence of aminoimidazole-4-carboxamide riboside and/or after α1AMP-activated protein kinase silencing. MEASUREMENTS AND MAIN RESULTS: α1AMP-activated protein kinase deficiency dramatically impaired tolerance to lipopolysaccharide challenge. Indeed, α1AMPK exhibited heightened cardiac vascular permeability after lipopolysaccharide challenge compared with α1AMPK. Consequently, an increase in left ventricular mass corresponding to exaggerated wall edema occurred in α1AMPK, without any further decrease in systolic function. Mechanistically, the lipopolysaccharide-induced α1AMPK cardiac phenotype could not be attributed to major changes in the systemic inflammatory response but was due to an increased disruption of interendothelial tight junctions. Accordingly, AMP-activated protein kinase activation by aminoimidazole-4-carboxamide riboside counteracted lipopolysaccharide-induced hyperpermeability in wild-type mice in vivo as well as in endothelial cells in vitro. This effect was associated with a potent protection of zonula occludens-1 linear border pattern in endothelial cells. CONCLUSIONS: Our results demonstrate for the first time the involvement of a signaling pathway in the control of left ventricular wall edema during sepsis. AMP-activated protein kinase exerts a protective action through the preservation of interendothelial tight junctions. Interestingly, exaggerated left ventricular wall edema was not coupled with aggravated systolic dysfunction. However, it could contribute to diastolic dysfunction in patients with sepsis.

Overexpression of CD39 in mouse airways promotes bacteria-induced inflammation.

In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of inflammation because accelerated ATP metabolism occurs in chronic inflammatory lung diseases. We sought to determine whether constant elevated CD39 activity in lung epithelia is sufficient to cause inflammation and whether this affects the response to acute LPS or Pseudomonas aeruginosa exposure. We generated transgenic mice overexpressing human CD39 under the control of the airway-specific Clara cell 10-kDa protein gene promoter. Transgenic mice did not develop any spontaneous lung inflammation. However, intratracheal instillation of LPS resulted in accelerated recruitment of neutrophils to the airways of transgenic mice. Macrophage clearance was delayed, and the amounts of CD8(+) T and B cells were augmented. Increased levels of keratinocyte chemoattractant, IL-6, and RANTES were produced in transgenic lungs. Similarly, higher numbers of neutrophils and macrophages were found in the lungs of transgenic mice infected with P. aeruginosa, which correlated with improved bacteria clearance. The transgenic phenotype was partially and differentially restored by coinstillation of P2X(1) or P2X(7) receptor antagonists or of caffeine with LPS. Thus, a chronic increase of epithelial CD39 expression and activity promotes airway inflammation in response to bacterial challenge by enhancing P1 and P2 receptor activation.

P2X1 ion channels promote neutrophil chemotaxis through Rho kinase activation.

ATP, released at the leading edge of migrating neutrophils, amplifies chemotactic signals. The aim of our study was to investigate whether neutrophils express ATP-gated P2X(1) ion channels and whether these channels could play a role in chemotaxis. Whole-cell patch clamp experiments showed rapidly desensitizing currents in both human and mouse neutrophils stimulated with P2X(1) agonists, alphabeta-methylene ATP (alphabetaMeATP) and betagammaMeATP. These currents were strongly impaired or absent in neutrophils from P2X(1)(-/-) mice. In Boyden chamber assays, alphabetaMeATP provoked chemokinesis and enhanced formylated peptide- and IL-8-induced chemotaxis of human neutrophils. This agonist similarly increased W-peptide-induced chemotaxis of wild-type mouse neutrophils, whereas it had no effect on P2X(1)(-/-) neutrophils. In human as in mouse neutrophils, alphabetaMeATP selectively activated the small RhoGTPase RhoA that caused reversible myosin L chain phosphorylation. Moreover, the alphabetaMeATP-elicited neutrophil movements were prevented by the two Rho kinase inhibitors, Y27632 and H1152. In a gradient of W-peptide, P2X(1)(-/-) neutrophils migrated with reduced speed and displayed impaired trailing edge retraction. Finally, neutrophil recruitment in mouse peritoneum upon Escherichia coli injection was enhanced in wild-type mice treated with alphabetaMeATP, whereas it was significantly impaired in the P2X(1)(-/-) mice. Thus, activation of P2X(1) ion channels by ATP promotes neutrophil chemotaxis, a process involving Rho kinase-dependent actomyosin-mediated contraction at the cell rear. These ion channels may therefore play a significant role in host defense and inflammation.

Fibrillar type I collagens enhance platelet-dependent thrombin generation via glycoprotein VI with direct support of alpha2beta1 but not alphaIIbbeta3 integrin.

The role of collagens and collagen receptors was investigated in stimulating platelet-dependent thrombin generation. Fibrillar type-I collagens, including collagen from human heart, were most potent in enhancing thrombin generation, in a way dependent on exposure of phosphatidylserine (PS) at the platelet surface. Soluble, non-fibrillar type-I collagen required pre-activation of integrin alpha2beta1 with Mn2+ for enhancement of thrombin generation. With all preparations, blocking of glycoprotein VI (GPVI) with 9O12 antibody abrogated the collagen-enhanced thrombin generation, regardless of the alpha2beta 1 activation state. Blockade of alpha2beta1 alone or antagonism of autocrine thromboxane A2 and ADP were less effective. Blockade of alphaIIbbeta3 with abciximab suppressed thrombin generation in platelet-rich plasma, but this did not abolish the enhancing effect of collagens. The high activity of type-I fibrillar collagens in stimulating GPVI-dependent procoagulant activity was confirmed in whole-blood flow studies, showing that these collagens induced relatively high expression of PS. Together, these results indicate that: i) fibrillar type-I collagen greatly enhances thrombin generation, ii) GPVI-induced platelet activation is principally responsible for the procoagulant activity of fibrillar and non-fibrillar collagens, iii) alpha2beta1 and signaling via autocrine mediators facilitate and amplify this GPVI activity, and iv) alphaIIbbeta3 is not directly involved in the collagen effect.

Contribution of platelet glycoprotein VI to the thrombogenic effect of collagens in fibrous atherosclerotic lesions.

Collagens (types I and III) are among the strongest thrombus-forming components of the vascular subendothelium. We compared the thrombogenic effects of four collagen-containing advanced atherosclerotic lesions with those of purified types I and III collagen fibers. Cell-free homogenates from the human plaques effectively promoted platelet adhesion and aggregate formation under high-shear flow conditions, as well as exposure of procoagulant phosphatidylserine (PS) on platelets. With all plaques, blocking of the glycoprotein VI (GPVI) receptor for collagen abolished aggregation and PS exposure. Blocking of platelet ADP receptors resulted in similar, but less complete inhibitory effects. Type I collagen was more potent than type III collagen in inducing aggregation and PS exposure under flow, via stimulation of GPVI and ADP receptors. Type I collagen also more strongly enhanced thrombin generation with platelets and tissue factor, again via GPVI activation and PS exposure. The plaque material enhanced thrombin generation, partly due to the presence of tissue factor and partly via GPVI and ADP receptors. Together, these results indicate that in advanced plaques collagen type I is a major trigger of thrombus formation and PS exposure, acting via GPVI and ADP release, while tissue factor directly enhances coagulation.