Circulating extracellular vesicles from patients with valvular heart disease induce neutrophil chemotaxis via FOXO3a and the inhibiting role of dexmedetomidine.

We previously demonstrated that circulating extracellular vesicles (EVs) from patients with valvular heart disease (VHD; vEVs) contain inflammatory components and inhibit endothelium-dependent vasodilation. Neutrophil chemotaxis plays a key role in renal dysfunction, and dexmedetomidine (DEX) can reduce renal dysfunction in cardiac surgery. However, the roles of vEVs in neutrophil chemotaxis and effects of DEX on vEVs are unknown. Here, we investigated the impact of vEVs on neutrophil chemotaxis in kidneys and the influence of DEX on vEVs. Circulating EVs were isolated from healthy subjects and patients with VHD. The effects of EVs on chemokine generation, forkhead box protein O3a (FOXO3a) pathway activation and neutrophil chemotaxis on cultured human umbilical vein endothelial cells (HUVECs) and kidneys in mice and the influence of DEX on EVs were detected. vEVs increased FOXO3a expression, decreased phosphorylation of Akt and FOXO3a, promoted FOXO3a nuclear translocation, and activated the FOXO3a signaling pathway in vitro. DEX pretreatment reduced vEV-induced CXCL4 and CCL5 expression and neutrophil chemotaxis in cultured HUVECs via the FOXO3a signaling pathway. vEVs were also found to suppress Akt phosphorylation and activate FOXO3a signaling to increase plasma levels of CXCL4 and CCL5 and neutrophil accumulation in kidney. The overall mechanism was inhibited in vivo with DEX pretreatment. Our data demonstrated that vEVs induced CXCL4-CCL5 to stimulate neutrophil infiltration in kidney, which can be inhibited by DEX via the FOXO3a signaling. Our findings reveal a unique mechanism involving vEVs in inducing neutrophils chemotaxis and may provide a novel basis for using DEX in reducing renal dysfunction in valvular heart surgery.

2-O, 3-O desulfated heparin mitigates murine chemotherapy- and radiation-induced thrombocytopenia.

Thrombocytopenia is a significant complication of chemotherapy and radiation therapy. Platelet factor 4 (PF4; CXCL4) is a negative paracrine of megakaryopoiesis. We have shown that PF4 levels are inversely related to steady-state platelet counts, and to the duration and severity of chemotherapy- and radiation-induced thrombocytopenia (CIT and RIT, respectively). Murine studies suggest that blocking the effect of PF4 improves megakaryopoiesis, raising nadir platelet counts and shortening the time to platelet count recovery. We examined the ability of 2-O, 3-O desulfated heparin (ODSH), a heparin variant with little anticoagulant effects, to neutralize PF4's effects on megakaryopoiesis. Using megakaryocyte colony assays and liquid cultures, we show that ODSH restored megakaryocyte proliferation in PF4-treated Cxcl4-/- murine and human CD34+-derived megakaryocyte cultures (17.4% megakaryocyte colonies, P < .01 compared with PF4). In murine CIT and RIT models, ODSH, started 24 hours after injury, was examined for the effect on hematopoietic recovery demonstrating higher platelet count nadirs (9% ± 5% treated vs 4% ± 4% control) and significantly improved survival in treated animals (73% treated vs 36% control survival). Treatment with ODSH was able to reduce intramedullary free PF4 concentrations by immunohistochemical analysis. In summary, ODSH mitigated CIT and RIT in mice by neutralizing the intramedullary negative paracrine PF4. ODSH, already in clinical trials in humans as an adjuvant to chemotherapy, may be an important, clinically relevant therapeutic for CIT and RIT.

Steroids Regulate CXCL4 in the Human Endometrium During Menstruation to Enable Efficient Endometrial Repair.

Context: Repair of the endometrial surface at menstruation must be efficient to minimize blood loss and optimize reproductive function. The mechanism and regulation of endometrial repair remain undefined. Objective: To determine the presence/regulation of CXCL4 in the human endometrium as a putative repair factor at menses. Patients/Setting: Endometrial tissue was collected throughout the menstrual cycle from healthy women attending the gynecology department. Menstrual blood loss was objectively measured in a subset, and heavy menstrual bleeding (HMB) was defined as >80 mL per cycle. Monocytes were isolated from peripheral blood. Design: CXCL4 messenger RNA (mRNA) and protein were identified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The function/regulation of endometrial CXCL4 was explored by in vitro cell culture. Results: CXCL4 mRNA concentrations were significantly increased during menstruation. Intense staining for CXCL4 was detected in late secretory and menstrual tissue, localized to stromal, epithelial and endothelial cells. Colocalization identified positive staining in CD68+ macrophages. Treatment of human endometrial stromal and endothelial cells (hESCs and HEECs, respectively) with steroids revealed differential regulation of CXCL4. Progesterone withdrawal resulted in significant increases in CXCL4 mRNA and protein in hESCs, whereas cortisol significantly increased CXCL4 in HEECs. In women with HMB, CXCL4 was reduced in endothelial cells during the menstrual phase compared with women with normal menstrual bleeding. Cortisol-exposed macrophages displayed increased chemotaxis toward CXCL4 compared with macrophages incubated with estrogen or progesterone. Conclusions: These data implicate CXCL4 in endometrial repair after menses. Reduced cortisol at the time of menses may contribute to delayed endometrial repair and HMB, in part by mechanisms involving aberrant expression of CXCL4.

MKEY, a Peptide Inhibitor of CXCL4-CCL5 Heterodimer Formation, Protects Against Stroke in Mice.

BACKGROUND: MKEY, a synthetic cyclic peptide inhibitor of CXCL4-CCL5 heterodimer formation, has been shown to protect against atherosclerosis and aortic aneurysm formation by mediating inflammation, but whether it modulates neuroinflammation and brain injury has not been studied. We therefore studied the role of MKEY in stroke-induced brain injury in mice. METHODS AND RESULTS: MKEY was injected into mice after stroke with 60 minutes of middle cerebral artery occlusion. Infarct volume and neurological deficit scores were measured. Protein levels of CCL5 and its receptor CCR5 were detected by Western blot and fluorescence-activated cell sorting (FACS), respectively. Numbers of microglia-derived macrophages (MiMΦs) and monocyte-derived MΦs (MoMΦs) in the brain, and their subsets, based on the surface markers CD45, CD11b, CCR2, CX3CR1, and Ly6C, were analyzed by FACS. MΦs and neutrophil infiltration in the ischemic brain were stained with CD68 and myeloperoxidase (MPO), respectively, and assessed by immunofluorescent confocal microscopy. The results showed that expressions of CCL5 and its receptor CCR5, were increased in the ischemic brain after stroke. MKEY injection significantly reduced infarct sizes and improved neurological deficit scores measured 72 hours after stroke. In addition, MKEY injection inhibited the number of MoMΦs, but not MiMΦs, in the ischemic brain. Furthermore, MKEY inhibited protein expression levels of Ly6C,CCR2, and CX3CR1 on MoMΦs. Lastly, the confocal study also suggests that the number of CD68-positive MΦs and MPO-positive neutrophils was inhibited by MKEY injection. CONCLUSIONS: MKEY injection protects against stroke-induced brain injury, probably by inhibiting MoMΦ-mediated neuroinflammation.

Changes in plasma CXCL4 levels are associated with improvements in lung function in patients receiving immunosuppressive therapy for systemic sclerosis-related interstitial lung disease.

BACKGROUND: Increased circulatory levels of the chemokine CXCL4 have been associated with the presence of interstitial lung disease (ILD) in an observational study of patients with systemic sclerosis (SSc). The purpose of the present study was to evaluate the relationship between baseline CXCL4 level and extent of ILD in the context of a randomized controlled trial and to determine whether changes in CXCL4 levels in response to immunosuppression are associated with future progression of SSc-ILD. METHODS: A total of 142 SSc-ILD patients from Scleroderma Lung Study (SLS) II were randomized in a double-blind, parallel-arm trial, to receive mycophenolate (MMF) for 2 years or oral cyclophosphamide (CYC) for 1 year followed by 1 year of placebo. Plasma CXCL4 levels were measured at baseline, 12 months, and 24 months in SLS II participants (N = 136) and at a single time point in healthy controls (N = 67). A mixed-effects model evaluated the relationship between change in CXCL4 levels and SSc-ILD progression. The primary outcome was the course of the forced vital capacity. RESULTS: Baseline CXCL4 levels were significantly higher in SSc-ILD patients compared with healthy controls (2699 ± 1489 ng/ml vs 2233 ± 1351 ng/ml (mean ± SD); P = 0.019). However, no significant correlations were identified between CXCL4 levels and extent of ILD at baseline, as measured by the forced vital capacity, diffusing capacity of carbon monoxide, or radiographic extent of ILD. Plasma CXCL4 decreased significantly from baseline to 12 months in all patients (CYC: P < 0.001; MMF: P = 0.006) with no between-treatment differences (CYC vs MMF). Patients with the largest decline in CXCL4 levels during the first 12 months had an improved course of forced vital capacity %-predicted from 12 to 24 months (P = 0.040), even after adjusting for baseline disease severity and treatment arm assignment. CONCLUSIONS: Levels of CXCL4 were higher in patients with SSc-ILD compared with controls and decreased in all patients treated with immunosuppressive therapy. While CXCL4 levels were not correlated with extent of ILD at baseline, changes in CXCL4 at 12 months predicted future progression of SSc-ILD from 12 to 24 months. These findings suggest that intermediate-term changes in CXCL4 may have predictive significance for long-term progression of SSc-ILD in patients receiving immunosuppressive therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00883129 . Registered 16 April 2009.

Clopidogrel enhances periodontal repair in rats through decreased inflammation.

AIM: We hypothesized that platelet inactivation induced by drugs might interfere with periodontal repair in experimental periodontitis by suppressing the release of biological mediators from platelets at the site of injury. MATERIAL AND METHODS: Sixty rats were randomly assigned to six groups (n = 10) and ligatures were placed around lower first molars of three groups. The other three groups were used as negative controls. Ligatures were removed after 10 days of periodontitis induction and all groups were submitted to treatment with aspirin (Asp) (30 mg/kg), clopidogrel (Clop) (75 mg/kg) or NaCl 0.9% intra-gastrically once daily for 3 days. Periodontal tissue was assessed by the measurement of CXCL12, CXCL4, CCL5 and platelet-derived growth factor (PDGF) by enzyme-linked immunosorbent assay; histomorphometrical analysis of polymorphonuclear (PMN) infiltration, attachment loss, bone loss and osteoclast numbers and quantification of blood vessels by imunnohistochemistry. RESULTS: During periodontal repair and treatment with NaCl 0.9%, CCL5 was decreased and CXCL12 increased when compared with negative control groups. Asp and Clop did not affect CCL5 expression, decreased CXCL12 but only Clop decreased CXCL4 and PDGF content compared with saline-treated animals. Clop increased blood vessel number, reduced PMN count and decreased attachment and bone loss, also decreased osteoclast number in animals submitted or not to periodontal repair. CONCLUSION: Systemic administration of Clop for 3 days improved the repair process associated with experimental periodontal disease, suggesting that it may have therapeutic value under situations where tissues undergo a transition from inflammation to repair.

Propofol lowers serum PF4 level and partially corrects hypercoagulopathy in endotoxemic rats.

Propofol, an anesthetic drug, has been shown to exhibit antioxidant and anti-inflammatory properties in vitro and in vivo. Hypercoagulopathy is a common clinical feature of sepsis, but the effects of propofol on the coagulation system in septic conditions are unclear. Using the gel-based comparative proteomic approach, together with Western blot analysis, ELISA, antithrombin III activity assay, and blood coagulation test, the effect of propofol on serum proteomic profiles in endotoxemic rats was examined. We identified that serum platelet factor-4 (PF4), an endogenous pro-coagulant, was up-regulated in LPS-challenged rats (p<0.001). Endotoxemia also resulted in hypercoagulopathy as evidenced by the shortening of thromboplastin time and thrombin time. Administration of propofol attenuated LPS-stimulated PF4 release and partially reversed the effect of LPS on thromboplastin time (p=0.0012) and thrombin time (p=0.0072). We demonstrated that propofol reduces serum levels of PF4 and partially corrects the hypercoagulopathy associated with endotoxemia in rats.

Heparin-induced thrombocytopenia (HIT II) - a drug-associated autoimmune disease.

Autoimmune thrombocytopenia (ITP) is an acquired autoimmune disease characterised by isolated persistent thrombocytopenia and normal megakaryopoiesis. This definition also applies to heparin-induced thrombocytopenia (HIT II), a frequent side effect of heparin treatment. In HIT II, the immunogen is a coagulation active complex of heparin and platelet factor 4 (PF4). By now, diagnostics of HIT II is often material and time consuming. Three groups of patients were investigated for HIT II antibodies (HIT II-AB): 54 hospitalised stroke patients, 87 hospitalised cardiac patients, and 71 patients on chronic haemodialysis, all treated with heparin. Furthermore, 100 healthy volunteers were investigated. For detection of HIT II-AB the innovative whole blood test PADA-HIT (PADA: platelet adhesion assay) was used. PADA-HIT quantifies the interaction of IgG antibodies with FcgammaIIA receptors by comparing the activation state of platelets in citrated and heparinised whole blood. The occurrence of HIT II-AB in blood was very high with 44 % of stroke patients, 69% of cardiac patients and 38% of haemodialysis patients compared to only 15% of healthy volunteers. This demonstrates a high incidence and a rapid onset of HIT II-AB in patients being acutely treated with heparin. HIT II is one of the most frequent and severe autoimmune diseases bearing a great thrombosis risk. PADA-HIT represents an innovative diagnostic method for detection of autoimmune antibodies of IgG type that are directed against platelet factor 4 (PF4)-heparin-complex. By early and fast diagnostics and appropriate treatment severe complications of HIT II can be prevented.

Heparin-induced thrombocytopenia in myeloproliferative disorders: a rare or under-diagnosed complication?

Patients with myeloproliferative disorders (MPD) are prone to develop thrombotic complications and thus frequently receive heparin. Surprisingly heparin-induced thrombocytopenia (HIT) has been rarely reported in MPD and is potentially under-diagnosed due to the relatively high platelet count. We report three patients with MPD who developed HIT; all presented with a relative fall of platelet counts (although without an absolute thrombocytopenia), thrombosis or skin necrosis and a positive test for HIT antibodies (particle gel immunoassay). Risk factors for developing HIT in our patients were exposure to unfractionated heparin, a recent surgical procedure and female gender. We review the literature on HIT in MPD and discuss the diagnosis of HIT in the absence of an absolute thrombocytopenia.

High prevalence of antibodies to platelet factor 4 heparin in patients with antiphospholipid antibodies in absence of heparin-induced thrombocytopenia.

Seventy-two patients with antiphospholipid antibodies (aPL), with or without antiphospholipid syndrome (APS), were studied for detection of heparin-PF4-induced antibodies (HPIA) using a commercial kit (Asserachrom HPIA) PF4-dependant enzyme-linked immunoassay (ELISA) test. None of the patients had a medical history of heparin induced thrombocytopenia (HIT). Eleven percent of patients were positive for HPIA. Plasma from 40 of the 72 patients (seven positive and 33 negative), was also tested with the other available HPIA ELISA (GTI) kit. Five patients were positive with both ELISA kits, two were highly positive only with Asserachrom HPIA and four only with GTI. None of the positive patients had severe thrombocytopenia. Two patients have never received heparin treatment. No relationship was found between HPIA presence and patients' age, sex, aPL levels or presence of lupus anticoagulant. No significant difference in HPIA presence was observed in patients with primary APS, secondary APS or aPL without APS. We found a poor correlation between the two commercial ELISA showing that, on the same blood sample, a patient could be highly positive with one technique and negative with the other. The PF4-dependant enzyme-linked immunoassay, which is often the first test used for the diagnosis of HIT, should be interpreted cautiously in patients with aPL since there is a danger of overdiagnosis and overtreatment.

Heparin-platelet factor 4 antibodies in patients presenting to the ED with thrombosis.

BACKGROUND: Patients with heparin-platelet factor 4 (PF4) antibodies, particularly platelet-activating ones, are at risk for heparin-induced thrombocytopenia if administered heparin. We determined the heparin-PF4 antibody prevalence in emergency department (ED) patients presenting with chest pain or symptoms of thrombosis. METHODS: Admission samples from 324 ED patients with chest pain or symptoms of thrombosis were tested for heparin-PF4 antibodies and, if positive, platelet-activating antibodies. RESULTS: Twenty-four (7.4%; 95% confidence interval, 4.8%-10.8%) patients had heparin-PF4 antibodies. Seropositivity occurred in 18 (9.2%) of 196 patients recently (< or =6 months) hospitalized vs 6 (4.7%) of 128 not recently hospitalized (P = .19), and in 16/231 (6.9%) patients with chest pain vs 8/93 (8.6%) with other thrombosis (P = .64). Of 22 seropositive patients retested, 8 (7 recently hospitalized) had platelet-activating antibodies. CONCLUSION: Heparin-PF4 antibody prevalence is 7.4% in ED patients with chest pain or thrombosis, with approximately 1 in 3 seropositive patients having platelet-activating antibodies. Alternative, nonheparin anticoagulation would be prudent in these at-risk patients.

Bivalirudin anticoagulation for a patient with hypercoagulable immune syndromes undergoing mitral valve surgery.

Unfractionated heparin has been a near universal anticoagulant for cardiac surgery; however it is contraindicated in heparin-induced thrombocytopenia type II. Alternative anticoagulants such as bivalirudin (a direct thrombin inhibitor) are being utilized. Bivalirudin was successfully used in an immunologically complex patient (diagnoses of heparin-induced thrombocytopenia type II, systemic lupus erythematosus, antiphospholipid syndrome, and dialysis-dependent renal failure) requiring cardiopulmonary bypass. Thrombotic events are common in antiphospholipid syndrome patients undergoing cardiac surgery utilizing high-dose heparin. This may represent unrecognized heparin-induced thrombocytopenia type II. Our patient did not experience perioperative thrombotic or bleeding complications. The possible cross-reactivity between heparin induced thrombocytopenia type II and antiphospholipid syndrome has not been investigated.

Mechanistic basis of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) occurs in 1% to 5% of patients treated with unfractionated high-molecular weight heparin and often results in limb- and/or life-threatening thrombotic complications. This disorder involves the formation of antibodies to a complex of Platelet Factor 4 (PF4) with the heparin. We believe that the pathogenesis of this disease begins with having an excess of platelet PF4 release. The freed PF4 is bound to platelet and other vascular membrane surfaces and has three critical roles in the initiation of HIT. (1) Infused heparin neutralizes a portion of excess surface PF4, directly enhancing local thrombosis. (2) The excess PF4 is mobilized into PF4/heparin complexes that stimulates HIT antibody production. (3) The remaining PF4 complexed to heparanoids and heparin on the vascular surfaces now bind to these HIT antibodies and through surface Fc gammaRII receptors leads to more platelet activation and removal, thrombus formation, and inflammation. These events, in turn, further stimulate PF4 release, perpetuating repetitive cycle that results in the clinical manifestations of this disorder.

[From heparin to synthetic antithrombotic oligosaccharides]. / De l'héparine aux oligosaccharides antithrombotiques de synthèse.

In the early eighties, following breakthroughs in oligosaccharide chemistry, we achieved the total chemical synthesis of pentasaccharides related to the antithrombin-binding domain in heparin. Selective inhibitors of coagulation factor Xa, thus obtained, represent a new class of efficient antithrombotic drugs. In a further step, we have designed and synthesised oligosaccharides (pentadeca--to eicosasaccharides), comprising an antithrombin binding domain prolonged by a thrombin binding domain. These compounds inhibit both factor Xa and thrombin, in the presence of antithrombin, while they are devoid of undesirable non-specific interactions, particularly with platelet factor 4 (PF4). The efficacy of these new synthetic antithrombotics, with a unique biological profile, will be evaluated in clinical trials.

A selective effect of Mpl ligand on mRNA stabilization during megakaryocyte differentiation.

Megakaryocytes, the platelet precursors, are induced to differentiate in response to Mpl ligand. Here we report that stability of the megakaryocyte-specific platelet factor 4 (PF4) mRNA is substantially augmented in the presence of Mpl ligand. This stabilization requires protein synthesis, but the 3'-untranslated region of PF4 mRNA is not sufficient for granting the effect. This cytokine also significantly or mildly stabilizes Mpl receptor or GAPDH mRNAs, respectively, in contrast to a previously reported lack of effect on P2Y(1) receptor mRNA. Our study is the first to suggest that Mpl ligand-induced lineage specification is also determined by message stabilization.