Elena M. Egorina

Infusion of methylene blue in human septic shock: A pilot, randomized, controlled study

ObjectiveTo evaluate the effects of continuous infusion of methylene blue (MB), an inhibitor of the nitric oxide pathway, on hemodynamics and organ functions in human septic shock. DesignProspective, randomized, controlled, open-label, pilot study. SettingMultidisciplinary intensive care unit of a university hospital. PatientsTwenty patients with septic shock diagnosed <24 hrs before randomization. InterventionsPatients were randomized 1:1 to receive either MB (MB group, n = 10) or isotonic saline (control group, n = 10), adjunctive to conventional treatment. MB was administered as an intravenous bolus injection (2 mg/kg), followed 2 hrs later by infusion at stepwise increasing rates of 0.25, 0.5, 1, and 2 mg/kg/hr that were maintained for 1 hr each. During infusion, mean arterial pressure was maintained between 70 and 90 mm Hg, while attempting to reduce concurrent adrenergic support. Measurements and Main Results Hemodynamics and organ function variables were assessed over a 24-hr period, and the survival rate at day 28 was noted. Infusion of MB prevented the stroke volume and the left-ventricular stroke work indexes from falling and increased mean arterial pressure. Compared with the control group, MB reduced the requirement for norepinephrine, epinephrine, and dopamine by as much as 87%, 81%, and 40%, respectively. Oxygen delivery remained unchanged in the MB group and decreased in the control group. MB also reduced the body temperature and the plasma concentration of nitrates/nitrites. Leukocytes and organ function variables such as bilirubin, alanine aminotransferase, urea, and creatinine were not significantly affected. Platelet count decreased in both groups. Five patients treated with MB survived vs. three patients receiving conventional treatment. ConclusionsIn human septic shock, continuously infused MB counteracts myocardial depression, maintains oxygen transport, and reduces concurrent adrenergic support. Infusion of MB appears to have no significant adverse effects on the selected organ function variables.

Intracellular and Surface Distribution of Monocyte Tissue Factor: Application to Intersubject Variability

Objective—The high and low responder phenomenon describes individual differences in lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) activity. We characterized patterns of intracellular accumulation, externalization, and shedding of TF in response to LPS in mononuclear cells (MNCs) from high responders (HRs) and low responders (LRs). Methods and Results—After 2 hours of LPS stimulation of whole blood, flow cytometry analyses revealed a larger population of TF-positive monocytes in HRs (32.0±3.5%) versus LRs (11.2±1.2%; P≤0.05), along with a stronger mean fluorescence intensity of TF signal in HRs (7.1±0.5 arbitrary units [AU]) compared with LRs (5.4±0.4 AU; P≤0.05). The LPS-treated blood of the HR group contained 2-fold more TF-positive microparticles than LRs. In-cell Western assay demonstrated higher intracellular accumulation of TF in mononuclear cells (MNCs) from LRs because LPS induced a 3.7-fold increase of total TF levels in LRs versus a 1.5-fold increase in HRs. In contrast, in response to LPS stimulation, MNCs from HRs exhibited a 4-fold induction of surface TF, whereas MNCs from LRs only had a minor increase in surface TF levels. Conclusions—The higher availability of surface TF antigen on MNCs from HRs and TF-containing microparticles might make these individuals more susceptible to hypercoagulation.

Endothelial dysfunction enhances vasoconstriction due to scavenging of nitric oxide by a hemoglobin-based oxygen carrier

Background:To date, there is no safe and effective hemoglobin-based oxygen carrier (HBOC) to substitute for erythrocyte transfusion. It is uncertain whether a deficiency of endothelial nitric oxide bioavailability (endothelial dysfunction) prevents or augments HBOC-induced vasoconstriction. Methods:Hemodynamic effects of infusion of PolyHeme (1.08 g hemoglobin/kg; Northfield Laboratories, Evanston, IL) or murine tetrameric hemoglobin (0.48 g hemoglobin/kg) were determined in awake healthy lambs, awake mice, and anesthetized mice. In vitro, a cumulative dose-tension response was obtained by sequential addition of PolyHeme or tetrameric hemoglobin to phenylephrine-precontracted murine aortic rings. Results:Infusion of PolyHeme did not cause systemic hypertension in awake lambs but produced acute systemic and pulmonary vasoconstriction. Infusion of PolyHeme did not cause systemic hypertension in healthy wild-type mice but induced severe systemic vasoconstriction in mice with endothelial dysfunction (either db/db mice or high-fat fed wild-type mice for 4–6 weeks). The db/db mice were more sensitive to systemic vasoconstriction than wild-type mice after the infusion of either tetrameric hemoglobin or PolyHeme. Murine aortic ring studies confirmed that db/db mice have an impaired response to an endothelial-dependent vasodilator and an enhanced vasoconstrictor response to HBOC. Conclusions:Reduction in low molecular weight hemoglobin concentrations to less than 1% is insufficient to abrogate the vasoconstrictor effects of HBOC infusion in healthy awake sheep or in mice with reduced vascular nitric oxide levels associated with endothelial dysfunction. These findings suggest that testing HBOCs in animals with endothelial dysfunction can provide a more sensitive indication of their potential vasoconstrictor effects.

In-cell Western assay: a new approach to visualize tissue factor in human monocytes.

Summary.  Background: Tissue factor (TF) is an integral membrane protein essential for the initiation of the extrinsic pathway of hemostasis. A precise understanding of the TF regulation is still limited and dependent on the availability of methodological tools. Here, we describe a new approach for assessing TF amounts in human mononuclear cells (MNCs) by using the whole blood experimental conditions. Aim: In order to study TF antigen levels in human MNCs, we applied a quantitative immunostaining technique – in‐cell Western (ICW) assay using an Odyssey Infrared Imaging System. Methods and results: The ICW assay of TF in resting or lipopolysaccharide (LPS)‐stimulated human MNCs was performed. Several sample preparation conditions were tested, namely the plating of MNCs prior to immunostaining, paraformaldehyde fixation, and an adequate cell number was used in the assay. By the use of recombinant human TF standards, it was possible, for the first time, to measure TF amounts in LPS‐stimulated MNCs as 0.09 ± 0.02 ng and 0.43 ± 0.15 ng 10−6 cells of surface and total TF, respectively. The concentrations of TF in resting MNCs, however, were below the detection limit. Conclusions: A novel TF ICW assay is a reproducible, time‐ and cost‐saving method, which could become useful for studies in the fields of physiology and pathophysiology of human hemostasis.

No evidence for the presence of tissue factor in high‐purity preparations of immunologically isolated eosinophils

Summary.  Background: To date, there is no unequivocal opinion on whether human eosinophils express tissue factor (TF). Therefore, we studied the expression of TF protein and activity in resting or stimulated immunologically purified human eosinophils. Methods and results: By use of immunologic isolation, we achieved over 96% purity of eosinophil preparations, and contamination by CD14‐positive cells was below 0.3%. Flow cytometric [fluorescence‐activated cell sorting (FACS)] analysis of eosinophils revealed no surface expression of TF antigen in resting or stimulated eosinophils. Immunoblotting of eosinophil lysates did not show any TF protein under resting or stimulated conditions. The lysates of resting or stimulated eosinophils contained no detectable levels of TF procoagulant activity. In contrast, monocytes, stimulated in plasma or medium, possessed readily detectable TF levels on the cell surface and in cell lysates as detected by FACS and immunoblotting. This was active TF antigen, as confirmed by TF activity assay (19.2 ± 4.2 and 28.6 ± 3.1 mU per 106 cells, stimulated in medium or plasma, respectively). We found no detectable TF mRNA levels in resting or stimulated eosinophils by real‐time polymerase chain reaction (PCR), whereas in monocytes TF mRNA levels were significantly increased after stimulation. Conclusions: Our data indicate that there is no evidence for TF expression in high‐purity preparations of immunologically isolated eosinophils.

Regulation of tissue factor procoagulant activity by post-translational modifications

Post-translational modification of amino acid residues is a common way to regulate localization, stability and ultimately the function of the protein. Tissue factor (TF), the major initiator of blood coagulation cascade, receives several post-translational modifications, like glycosylation, phosphorylation, palmitoylation and nitrosylation. Recent studies have demonstrated that these processes play important roles in modulating biological functions of TF. The present review highlights the mechanisms of several common protein post-translational modifications of TF with the special reference on the recent knowledge about their roles in regulation of trafficking, stability as well as procoagulant and signaling functions of TF.

The role of tissue factor in systemic inflammatory response syndrome.

Tissue factor (TF) is a major initiator of extrinsic pathway of blood coagulation. A dual role of TF in the extensive crosstalk between blood coagulation and inflammation has recently become apparent. The majority of the cases of systemic inflammatory response syndrome, disseminated intravascular coagulation, and sepsis are accompanied by hyperactivation of TF in circulating monocytes and damaged tissue. Systemic Gram-negative infection induces expression of TF by vascular cells. In addition to extrinsic coagulation pathway, TF induces proinflammatory signaling cascade originating from activation of protease-activated receptors. Because TF-activated proteolytic cascade is placed in a nexus between coagulation and inflammation, early modulation of TF activity presently becomes a tempting experimental therapeutic strategy in systemic inflammatory response syndrome patients.

Soluble guanylate cyclase agonists inhibit expression and procoagulant activity of tissue factor.

Objective—Tissue factor (TF), a major initiator of blood coagulation, contributes to inflammation, atherosclerosis, angiogenesis, and vascular remodeling. Pharmacological agonists of soluble guanylate cyclase (sGC) attenuate systemic and pulmonary hypertension, vascular remodeling, and platelet aggregation. However, the influence of these novel pharmacophores on TF is unknown. Methods and Results—We evaluated effects of BAY 41-2272 and BAY 58-2667 on expression and activity of TF in human monocytes and umbilical vein endothelial cells (HUVECs). Both compounds reduced expression of active TF protein in monocytes stimulated with lipopolysaccharide, as demonstrated by immunoblotting and a TF procoagulant activity assay. In-cell Western assay revealed that this effect was associated with a marked reduction of total and surface TF presentation. Furthermore, BAY 41-2272 and BAY 58-2667 decreased TF protein expression and the TF-dependent procoagulant activity in HUVECs stimulated with TNF-&agr;. The sGC agonists also suppressed transcriptional activity of NF-&kgr;B. A siRNA-mediated knockdown of the &agr;1-subunit of sGC in monocytes and HUVECs confirmed that the inhibitory effect of BAY 41-2272 and BAY 58-2667 on TF expression is mediated through the sGC-dependent mechanisms. Conclusions—Inhibition of TF expression and activity by sGC agonists might provide therapeutic benefits in cardiovascular diseases associated with enhanced procoagulant and inflammatory response.

Bone morphogenetic protein -7 increases thrombogenicity of lipid-rich atherosclerotic plaques via activation of tissue factor

Thrombogenicity of atherosclerotic plaques largely depends on plaque morphology. Tissue factor (TF) expression is higher in lipid-rich than in calcified lesions. Although bone morphogenetic protein (BMP) -7 is a known inhibitor of vascular calcification, the role of BMP-7 in the development of plaque thrombogenicity is uncertain. We hypothesized that increased thrombogenic potential of lipid-rich plaques is attributed to activation of TF by BMP-7. We measured levels of BMP-7 and TF proteins in lipid-rich and calcified carotid plaques, and tested the effects of BMP-7 on TF expression in human monocytes in vitro. Quantitative immunohistochemical analysis of endarterectomy specimens for TF and BMP-7 revealed that lipid-rich plaques contained more TF antigen than calcified ones (158.6±25.3 vs 37.4±8.8 AU, p<0.008). Lipid-rich plaques also expressed higher levels of BMP-7 (60.7±5.2 AU) than calcified lesions (31.8±8.6 AU, p<0.021). In vitro treatment of whole blood with BMP-7 markedly increased the population of TF-positive monocytes from 1.5±0.6 % to 31.0±7.6 % (p<0.001). Stimulation of blood with BMP-7 was accompanied by elevated surface presentation of TF antigen in monocytes as TF-dependent fluorescence intensity increased from 5.0±2.6 AU in unstimulated conditions to 15.8±1.9 AU after incubation with BMP-7 (p<0.002). Our data suggest that BMP-7 contributes to increased thrombogenicity of lipid-rich plaques via enhancement of TF expression.

Evidence for direct transfer of tissue factor from monocytes to platelets in whole blood.

Varying specificity of anti-tissue factor (anti-TF) antibodies gives rise to erroneous conclusions on TF positivity of platelets. Although monocytes are a well established source of TF in whole blood, there is no consensus whether platelets express or acquire TF from external sources. To test whether platelets can acquire TF expressed in monocytes, we studied a transfer of TF-yellow fluorescent protein (TF-YFP) from monocytes nucleofected with TF-YFP to platelets in a whole blood model. Platelets isolated from whole blood were found positive for TF when immunostained with anti-TF antibody from one supplier, whereas no platelet TF antigen was found in whole blood immunostained with anti-TF antibody from another supplier. Both antibodies recognized TF in monocytes. Platelets isolated from whole blood reconstituted with monocytes expressing TF-YFP fusion protein were found positive for TF-YFP only after stimulation with lipopolysaccharide (LPS). Taken together, TF protein could be transferred from monocytes upon stimulation with LPS.