Joanna Natorska

Increased thrombin generation and platelet activation are associated with deficiency in high molecular weight multimers of von Willebrand factor in patients with moderate-to-severe aortic stenosis

Background High molecular weight von Willebrand factor (vWF) multimers (HMWM) are often deficient in patients with severe aortic stenosis (AS) owing to shear stress-enhanced proteolysis of vWF. It has also been reported that AS is associated with increased activation of blood coagulation. Objective To investigate whether patients with AS with a deficiency in vWF HMWM have enhanced thrombin generation and platelet activation in vivo. Design Based on the analysis of vWF HMWM performed using immunolocalisation, 11 subjects with vWF HMWM deficiency (low %HMWM group) were identified and compared with 42 patients with AS with a normal distribution of vWF HMWM (normal %HMWM group). Plasma thrombin markers thrombin-antithrombin complexes (TAT) and prothrombin factor 1+2 (F1.2) plus platelet activation markers soluble CD40 ligand (sCD40L), β-thromboglobulin and P-selectin were also measured. Patients 48 consecutive patients with severe AS and five with moderate AS, free of angiographically-proven coronary artery disease and clinically overt bleeding, were studied. Results Patients in the low %HMWM group had 34.8% higher maximal transvalvular gradient (p=0.0003) and 44.8% higher mean gradient (p=0.0002) than those in the normal %HMWM group. Thrombin formation was enhanced in the low %HMWM group (F1.2, 284.5±63.7 vs 216.9±62.5 pmol/l, p=0.004; thrombin-antithrombin, 4.89±1.3 vs 4.06±0.9 μg/l, p=0.02) and both markers showed inverse correlations with the percentage of vWF HMWM (r=−0.59, p=0.002; r=−0.42, p=0.03, respectively). In the low %HMWM group sCD40L (279.4±60.7 vs 221.4±41.7 pmol/l, p=0.003) and β-thromboglobulin (73.1±9.2 vs 64.5±8.5 IU/ml, p=0.04), but not P-selectin, were also higher than in the remaining patients with AS. Conclusion Patients with advanced AS deficient in vWF HMWM are characterised by enhanced thrombin formation and platelet activation. This observation indicates the ambivalent impact of high shear stress in AS on haemostasis and might help explain two aspects of AS—Heyde syndrome and increased risk of thromboembolism.

Morphine attenuates pain and prevents inflammation in experimental peritonitis.

Morphine attenuates pain already at low concentrations. At high concentrations it inhibits inflammation in mice and fish but not in amphibians.

Fibrin presence within aortic valves in patients with aortic stenosis: Association with in vivo thrombin generation and fibrin clot properties

A role of coagulation in the pathogenesis of aortic stenosis (AS) is unknown. The aim of this study was to investigate the fibrin (Fn) presence and its determinants in calcified stenotic aortic valve leaflets. Twenty-one patients with dominant AS and 17 well-matched patients with dominant aortic insufficiency (AI) undergoing aortic valve replacement were studied. Immunofluorescence analysis was performed on decalcified leaflets using antibodies against human Fn and tissue factor (TF). Fn-positive (41.4%) and TF-positive (25.3%) areas were increased in AS valves compared with AI valves (7.9% and 5.9%, respectively, both p<0.001). Patients with AS had elevated plasma D-dimer (236.4 ± 28 ng/ml, p=0.002) and prothrombin fragment 1+2 (F1.2) (261.7 ± 27.1 pM, p=0.005) compared to AI subjects (142.8 ± 10 ng/ml and 131.2 ± 1.3 pM, respectively). In AS patients Fn-positive areas correlated with TF-positive areas (r=0.68, p=0.0005), D-dimer (r=0.45, p=0.018), F1.2 (r=0.64, p=0.002), the time required for plasma fibrin clot formation (r=0.44, p=0.015) and maximum absorbance of fibrin clots (r=-0.38, p<0.0001), but not with clot permeability or lysis time. Thickness of Fn layer within AS valves was associated with maximum transvalvular gradient (r =0.41, p=0.048). Patients with maximal gradient above 75 mmHg (n=11) showed significant associations between Fn-positive area and both maximal (r =0.63) and mean (r =0.67) transvalvular gradients. Large fibrin amounts, mostly co-localised with TF, are present within the valve leaflets of patients with advanced AS. In vivo thrombin generation and fibrin clot formation are associated with the extent of Fn presence within leaflets, which might contribute to the AS progression.

Impaired fibrinolysis is associated with the severity of aortic stenosis in humans

A role of fibrinolysis in the pathogenesis of aortic valve stenosis (AS) is unknown, although fibrinolytic proteins have been detected in aortic stenotic valves.

Increased bleeding risk in patients with aortic valvular stenosis: From new mechanisms to new therapies

Aortic stenosis (AS), the most prevalent acquired valvular disease in the adults that requires invasive treatment, coexists with coagulopathy, resulting in bleeding in approximately 20% of patients. In the current review, we summarize the available knowledge on the mechanisms underlying the bleeding tendency observed in AS, and discuss potential compensatory mechanisms preventing most patients with severe AS from experiencing bleeding. We offer an update on Heydes syndrome and other types of bleeding, and study extensively their pathobiology, providing insights into the new emerging concepts on coagulation regulation in AS. The focus is given to the impact of valvular interventions on coagulation abnormalities in AS. Both surgical valve replacement and transcatheter aortic valve implantation are discussed. Finally, we discuss current treatment recommendations in AS related bleeding.

Strain-specific differences in modulatory effects of morphine on peritoneal inflammation in mice.

It has been previously shown that local administration of a high dose of morphine together with a proinflammatory agent has anti-inflammatory effects in several (but not all) strains of mice. In the present paper, behavioural and cellular consequences of morphine co-injection are compared between several strains of mice with zymosan-induced peritonitis. Males of C57C3H, Swiss, Balb/c, C57BL/6, and CBA strains were ip injected with Zymosan (40 mg/kg) and/or morphine at 0, 5, 10, 20 mg/kg without or with naltrexone pretreatment. Early stages of Zymosan-induced peritonitis were connected with intraperitoneal accumulation of leukocytes (mainly PMNs), pain symptoms (body writhes of strain-specific frequency: C57C3H>CBA>Swiss>>Balb/c-C57BL/6), and sedation (significant in Swiss and Balb/c). Morphine co-injection abolished pain symptoms at all doses in every investigated strain, and restored locomotor activity or induced hyperlocomotion in a dose- and strain-specific manner. The highest dose of morphine inhibited intraperitoneal accumulation of peritoneal leukocytes (PTLs) including polymorphonuclear cells (PMNs) in all but the CBA strain of mice. Anti-inflammatory and anti-nociceptive effects of morphine were reversed in naltrexone pre-treated animals. The pre-incubation of Swiss but not CBA leukocytes with morphine inhibited cell chemotaxis towards zymosan-activated serum. In conclusion, morphine exerts various strain-specific effects on peritonitis.

Factor XIII expression within aortic valves and its plasma activity in patients with aortic stenosis: association with severity of disease

Aortic valve stenosis (AS) shares several similarities with atherosclerosis. Factor XIII (FXIII) has been detected within atherosclerotic plaques and may contribute to the development of atherosclerosis via multiple mechanisms. In the current study, we sought to investigate FXIII expression within human stenotic aortic valves and its association with severity of the disease. We prospectively enrolled 91 consecutive patients with AS scheduled for isolated valve replacement. Valvular FXIII subunit A (FXIII-A), fibrin and macrophages expression was evaluated by immunostaining. FXIII-A subunit transcripts and FXIII-A Val34Leu polymorphism was determined by real-time PCR. Plasma FXIII (pFXIII) activity was measured. We demonstrated that the valvular FXIII-A was predominantly expressed on the aortic side of leaflets, colocalized with alternatively activated macrophages (AAM). Areas stained for FXIII-A showed positive correlations with valvular fibrin presence, degree of calcification, pFXIII activity and the severity of AS, reflected by mean and maximum transvalvular gradients (all, p<0.001). The FXIII-A mRNA in the stenotic leaflets was significantly elevated compared to control leaflets. Interestingly, pFXIII activity was also positively correlated with mean (p<0.001) and maximum (p=0.001) transvalvular gradient. The FXIII-A Val34Leu polymorphism did not affect FXIII-A and fibrin expression in AS valves. In conclusion, the study is the first to show abundant expression of FXIII-A at the mRNA and protein levels within human stenotic aortic valves, which is associated with the severity of AS. Our findings might suggest that FXIII in the stenotic valves is presented in AAM and may be involved in the AS progression.

Relations between circulating microRNAs (miR-21, miR-26, miR-29, miR-30 and miR-133a), extracellular matrix fibrosis and serum markers of fibrosis in dilated cardiomyopathy

Relations between circulating microRNAs (miR-21, miR-26, miR-29, miR-30 and miR-133a), extracellular matrix fibrosis and serum markers of fibrosis in dilated cardiomyopathy Paweł Rubiś ⁎, Justyna Totoń-Żurańska , Sylwia Wiśniowska-Śmiałek , Katarzyna Holcman , Maria Kołton-Wróż , Paweł Wołkow , Ewa Wypasek , Joanna Natorska , Lucyna Rudnicka-Sosin , Agnieszka Pawlak , Artur Kozanecki , Piotr Podolec a,g,1

Presence of B cells within aortic valves in patients with aortic stenosis: Relation to severity of the disease.

BACKGROUND Aortic stenosis (AS) shares several similarities with atherosclerosis. Recent reports showed that B cells are implicated in atherosclerosis progression through macrophage-B cells bidirectional interaction. We aimed to study the in loco presence of B cells within aortic valves and to determine its modulators. METHODS Thirty-seven patients with severe AS were studied. Immunohistochemistry was performed on valve leaflets using antibodies against CD20, B cell-activating factor of the tumor necrosis factor family receptor (BAFF-R) and CD68. Plasma inflammatory markers were also determined. RESULTS The B cells were detected within aortic leaflets from 5 to 31/mm(2) (17.9±11.6/mm(2)). Double-staining showed that 27±13.5% of B cells express BAFF-R. There were positive correlations between the number of B cells and macrophages (r=0.45, p=0.018), and between macrophages and B cell-associated BAFF-R expression (r=0.66, p=0.002). The number of B cells was associated with the valve calcification (r=0.41, p=0.039), and with the maximum transvalvular gradient (r=0.63, p=0.02). The BAFF-R expression was positively correlated with maximum transvalvular gradient (r=0.39, p=0.031) and negatively with aortic valve area (r=-0.41, p=0.048). There were no correlations between the number of B cells and plasma markers. CONCLUSIONS It might be hypothesized that, like in atherosclerosis, increasing number of B cells within aortic valves may accelerate inflammation and thus potentiate the progression of AS.

Toll-like receptors expression and NF-κB activation in peritoneal leukocytes in morphine-mediated impairment of zymosan-induced peritonitis in swiss mice.

Zymosan-induced peritonitis represents a well-described model of acute inflammation. The binding of zymosan with its specific Toll-like receptors (TLR2 and TLR6) on leukocytes initiates activation and phosphorylation of nuclear factor (NF)-κB, which leads to accumulation of NF-κB p65 subunits in the nucleus and subsequently up-regulation of the proinflammatory cytokine genes expression. Intraperitoneal co-administration of zymosan and morphine significantly inhibits peritonitis in several strains of mice by decreasing the influx of exudatory cells; however, mechanisms of this action still remain unclear. We aimed to verify the effects of morphine on NF-κB and TLRs expression at messenger RNA and protein levels during the early stages of zymosan-induced peritonitis. Peritonitis was induced by a single injection of zymosan A or zymosan supplemented with morphine in Swiss mice. At selected time points, after stimulation, peritoneal leukocytes were harvested. The TLRs and NF-κB expression was assessed by real-time PCR and flow cytometry. In comparison with the mice injected with zymosan only, morphine co-injection significantly decreased the expression of phospho-NF-κB and TLR2 in all investigated immunocompetent cells as well as up-regulated the levels of nitric oxide (NO) in peritoneal fluid. Moreover, supplementation of zymosan with morphine altered the TLR, NF-κB and some proinflammatory cytokines (keratinocyte-derived chemokine, tumor necrosis factor-α) gene expression during ongoing inflammation. We may postulate that after morphine stimulation peritoneal leukocytes recognize less effectively zymosan antigens because of impaired TLRs expression. The lower TLR expression attenuates TLR-mediated signal transduction, which prevents NF-κB activation. Additionally, during zymosan-induced peritonitis, morphine may modulate the NF-κB expression, at least partially, by an up-regulated release of NO, as suggested by others.