RESUMO
In contrast to red blood cells, platelets float rather than sediment when a column of blood is placed in the gravitational field. By the analogy of erythrocyte sedimentation (ESR), it can be expressed with the platelet antisedimentation rate (PAR), which quantitates the difference in platelet count between the upper and lower halves of the blood column after 1 h of 1 g sedimentation. Venous blood samples from 21 healthy subjects were analyzed for PAR. After a 1-h sedimentation, the upper and lower fractions of blood samples were analyzed for platelet count, mean platelet volume (MPV), immature platelet fraction (IPF), and high-fluorescence IPF (H-IPF). The mechanisms behind platelet flotation were explored by further partitioning of the blood column, time-dependent measurements of platelet count and comparison with ESR. The structure and function of the platelets were assessed by electron microscopy (EM) and atomic force microscopy (AFM), and platelet aggregometry, respectively. Platelet antisedimentation is driven by density differences and facilitated by a size-exclusion mechanism caused by progressive erythrocyte sedimentation. The area under the curve (AUC) of the whole blood adenosine diphosphate (ADP) aggregation curves showed significant differences between the upper and lower samples (p < .005). AUC in the upper samples of 38% of healthy subjects exceeded the top of the normal range (53-122) suggesting that ascending platelets show an intensified ADP-induced aggregability ex vivo. H-IPF was significantly higher in the upper samples (p < .05). EM and AFM revealed that platelets in the upper samples were larger in volume and contained 1.6 times more alpha granules compared to platelets in the lower samples. Our results indicate that antisedimentation is able to differentiate platelet populations based on their structural and functional properties. Therefore, PAR may be a suitable laboratory parameter in various thromboinflammatory disorders.
It is less known that platelets do not sediment in response to gravitational force but float on the top of the blood column. This phenomenon is called antisedimentation, the rate of which, however, can be different, yet this feature has not been widely studied and used in clinical practice or diagnosis. We tested the idea that antisedimentation of platelets from venous blood samples can be a potential biomarker. We have found that platelet antisedimentation is driven by density differences and facilitated by a size-exclusion mechanism caused by progressive erythrocyte sedimentation and after 1-h upper and lower fractions develop. Interestingly, the aggregation curves showed significant differences between the upper and lower samples, suggesting that the ascending platelets show ex vivo hyperaggregability. Electron and atomic force microscopy revealed that platelets in the upper samples were larger in volume and contained more alpha granules than platelets in the lower samples. Subsequently, antisedimentation can be used to differentiate platelet populations based on their structural and functional properties; thus, it may be a promising biomarker for various thromboinflammatory disorders.
Assuntos
Plaquetas , Eritrócitos , Humanos , Contagem de Plaquetas , Volume Plaquetário Médio , Difosfato de AdenosinaRESUMO
Von Willebrand factor (VWF) is a multimer with a variable number of protomers, each of which is a head-to-head dimer of two multi-domain monomers. VWF responds to shear through the unfolding and extension of distinct domains, thereby mediating platelet adhesion and aggregation to the injured blood vessel wall. VWF's C1-6 segment uncoils and then the A2 domain unfolds and extends in a hierarchical and sequential manner. However, it is unclear whether there is any reservoir of further extensibility. Here, we explored the presence of cryptic extensibility in VWF by nanodissecting individual, pre-stretched multimers with atomic force microscopy (AFM). The AFM cantilever tip was pressed into the surface and moved in a direction perpendicular to the VWF axis. It was possible to pull out protein loops from VWF, which resulted in a mean contour length gain of 217 nm. In some cases, the loop became cleaved, and a gap was present along the contour. Frequently, small nodules appeared in the loops, indicating that parts of the nanodissected VWF segment remained folded. After analyzing the nodal structure, we conclude that the cryptic extensibility lies within the C1-6 and A1-3 regions. Cryptic extensibility may play a role in maintaining VWF's functionality in extreme shear conditions.
Assuntos
Microscopia de Força Atômica , Fator de von Willebrand , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo , Humanos , Multimerização Proteica , Domínios ProteicosRESUMO
Fibrin plays a fundamentally important role during hemostasis. To withstand the shear forces of blood flow and prevent embolisation, fibrin monomers form a three-dimensional polymer network that serves as an elastic scaffold for the blood clot. The complex spatial hierarchy of the fibrin meshwork, however, severely complicates the exploration of structural features, mechanical properties and molecular changes associated with the individual fibers of the clot. Here we developed a quasi-two-dimensional nanoscale fibrin matrix that enables the investigation of fibrin properties by topographical analysis using atomic force microscopy. The average thickness of the matrix was â¼50â¯nm, and structural features of component fibers were accessible. The matrix could be lysed with plasmin following rehydration. By following the topology of the matrix during lysis, we were able to uncover the molecular mechanisms of the process. Fibers became flexible but retained axial continuity for an extended time period, indicating that lateral interactions between protofibrils are disrupted first, but the axial interactions remain stable. Nearby fibers often fused into bundles, pointing at the presence of a cohesional force between them. Axial fiber fragmentation rapidly took place in the final step. Conceivably, the persisting axial integrity and cohesion of the fibrils assist to maintain global clot structure, to prevent microembolism, and to generate a high local plasmin concentration for the rapid, final axial fibril fragmentation. The nanoscale fibrin matrix developed and tested here provides a unique insight into the molecular mechanisms behind the structural and mechanical features of fibrin and its proteolytic degradation.
Assuntos
Produtos de Degradação da Fibrina e do Fibrinogênio/ultraestrutura , Fibrina/ultraestrutura , Fibrinolisina/química , Fibrina/química , Produtos de Degradação da Fibrina e do Fibrinogênio/química , Fibrinólise/genética , Hemostasia , Humanos , Microscopia de Força Atômica , Proteólise , Fluxo Sanguíneo RegionalRESUMO
The aim of this study was to evaluate the association between clinical signs and symptoms of dry eye disease (DED) in patients with systemic sclerosis (SSc). This cross-sectional observational study included 19 SSc patients and 19 normal subjects with no ocular symptoms or ocular surface disorders. Clinical parameters included tear film break-up time (tBUT), Schirmer I, lissamine green (LG) dye, and tear film osmolarity tests, tear production, and tear secretion flow. For assessment of the dry eye symptoms, the Ocular Surface Disease Index (OSDI) questionnaire was administered to all patients. The following mean values were found in SSc patients: OSDI 33.6 ± 19.86; osmolarity of the tear fluid 310.8 mOsmol/l ± 14.47; tBUT time 5.158 ± 2.328 s; Schirmer I test 5.395 mm/5 min; LG grading score 2.026 ± 0.8893; collected tear fluid volume 6.397 ± 2.761 µl. The calculated average tear velocity was 4.654 ± 1.963 µl/min. A significant correlation was found between the OSDI as a subjective parameter and disease duration. Early recognition of dry eye symptoms, a possible extra-intestinal manifestation of SSc, should be included in the check up of the disease to reduce ocular complications. The objective tear functional tests were strongly influenced by individual factors like age and disease duration.
Assuntos
Síndromes do Olho Seco/etiologia , Escleroderma Sistêmico/complicações , Adulto , Idoso , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Osteoprotegerin (OPG) and von Willebrand factor (VWF) form complex within endothelial cells and following secretion. The nature of blood group antigens strongly influences the levels of circulating VWF, but there is no available data concerning its ascendancy on OPG levels. We aimed to assess the relationship of AB0 blood groups with OPG, VWF levels (VWF: Ag) and collagen binding activity (VWF: CB) in peripheral arterial disease (PAD) patients. METHODS: Functional and laboratory parameters of 105 PAD patients and 109 controls were examined. Results of OPG, VWF: Ag, VWF: CB (ELISA-s) were analysed by comparative statistics, together with clinical data. RESULTS: OPG levels were higher in patients than in controls (4.64 ng/mL vs. 3.68 ng/mL, p < 0.001). Among patients elevation was marked in the presence of critical limb ischemia (5.19 ng/mL vs. 4.20 ng/mL, p = 0.011). The OPG in patients correlated positively with VWF: Ag and VWF: CB (r = 0.26, p = 0.008; r = 0.33, p = 0.001) and negatively with ankle-brachial pressure index (r = -0.22, p = 0.023). Furthermore, OPG was significantly elevated in non-0 blood groups compared to 0-groups both in patients and controls (4.95 ng/mL vs. 3.90 ng/mL, p = 0.012 and 4.09 ng/mL vs. 3.40 ng/mL, p = 0.002). CONCLUSIONS: OPG levels are associated to blood group phenotypes and higher in non-0 individuals. Increased OPG levels in PAD characterize disease severity. The significant correlation between OPG and VWF:CB might have functional importance in an atherothrombosis-prone biological environment.
Assuntos
Sistema ABO de Grupos Sanguíneos , Osteoprotegerina/sangue , Doença Arterial Periférica/sangue , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/epidemiologia , Fenótipo , Fatores de Risco , Romênia/epidemiologia , Índice de Gravidade de Doença , Ultrassonografia Doppler DuplaRESUMO
BACKGROUND: Systemic sclerosis is an autoimmune disease, characterized by widespread small vessel vasculopathy, immune dysregulation with production of autoantibodies, and progressive fibrosis. Changes in levels of proangiogenic cytokines had already been determined largely in serum. Our aim was to assess the levels of VEGF in human tears of patients with SSC. PATIENTS AND METHODS: Forty-three patients (40 female and 3 men, mean (SD) age 61 (48-74) years) with SSc and 27 healthy controls were enrolled in this study. Basal tear sample collection and tear velocity investigations were carried out followed by an ophthalmological examination. Total protein concentrations and VEGF levels were determined in tear samples. RESULTS: The average collected tear fluid volume developed 10.4 µL (1.6-31.2) in patients and 15.63 µL (3.68-34.5) in control subjects. The average total protein level was 6.9 µg/µL (1.8-12.3) in tears of patients and control tears contained an average of 4.132 µg/µL (0.1-14.1) protein. In patients with SSc the average concentration of VEGF was 4.9 pg/µL (3.5-8.1) and 6.15 pg/µL (3.84-12.3) in healthy samples. CONCLUSIONS: Total protein production was increased because of the smaller tear volume. Decreased VEGF in tear of SSc patients can be explained also by the decreased tear secretion of patients.
Assuntos
Escleroderma Sistêmico/metabolismo , Lágrimas/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fator A de Crescimento do Endotélio Vascular/análise , Adulto JovemRESUMO
Venous thromboembolism is a possible fatal complication after pelvic surgery. There is a lack of trials assessing the effect of prophylactic measures in urology. The aim of the study was to evaluate the practice of thrombosis prophylaxis in a Central European country. A questionnaire of performed radical prostatectomies, way of thrombosis prophylaxis and number of experienced thrombotic events was posted to all departments of urology in Hungary. With a response rate of 59%, 506 radical prostatectomies were reported. Low molecular weight heparin was administered by 100% of the departments. Graduated support stockings were applied by 37% of the patients. Early mobilization was the most common form of mechanic prophylaxis (57%). Thrombotic events were experienced in 1.4%, 0.2% were fatal. The thrombosis prophylaxis of patients undergoing radical prostatectomy is not unified. Due to the potential mortality of thrombotic complications it should be evaluated and prophylaxis should be recommended in urological guidelines.
Assuntos
Deambulação Precoce , Fibrinolíticos/administração & dosagem , Heparina de Baixo Peso Molecular/administração & dosagem , Padrões de Prática Médica , Prostatectomia/efeitos adversos , Meias de Compressão , Tromboembolia Venosa/prevenção & controle , Pesquisas sobre Atenção à Saúde , Humanos , Hungria , Masculino , Prostatectomia/mortalidade , Inquéritos e Questionários , Fatores de Tempo , Resultado do Tratamento , Tromboembolia Venosa/etiologia , Tromboembolia Venosa/mortalidadeRESUMO
The von Willebrand factor (VWF) is a multimeric glycoprotein composed of 80- to 120-nm-long protomeric units and plays a fundamental role in mediating platelet function at high shear. The exact nature of the shear-induced structural transitions have remained elusive; uncovering them requires the high-resolution quantitative analysis of gradually extended VWF. Here, we stretched human blood-plasma-derived VWF with molecular combing and analyzed the axial structure of the elongated multimers with atomic force microscopy. Protomers extended through structural intermediates that could be grouped into seven distinct topographical classes. Protomer extension thus progresses through the uncoiling of the C1-6 domain segment, rearrangements among the N-terminal VWF domains, and unfolding and elastic extension of the A2 domain. The least and most extended protomer conformations were localized at the ends and the middle of the multimer, respectively, revealing an apparent necking phenomenon characteristic of plastic-material behavior. The structural hierarchy uncovered here is likely to provide a spatial control mechanism to the complex functions of VWF.
Assuntos
Fator de von Willebrand , Humanos , Fator de von Willebrand/química , Subunidades ProteicasRESUMO
BACKGROUND: Bacterial infections are common cause of morbidity and mortality in patients with cirrhosis. The early diagnosis of these infections is rather difficult. AIMS: To assess the accuracy of acute phase proteins in the identification of bacterial infections. METHODS: Concentration of C-reactive protein (CRP), procalcitonin (PCT), lipopolysaccharide-binding protein (LBP), sCD14 and antimicrobial antibodies were measured in serum of 368 well-characterized patients with cirrhosis of whom 139 had documented infection. Clinical data was gathered by reviewing the patients' medical charts. RESULTS: Serum levels of CRP, PCT and LBP were significantly higher in patients with clinically overt infections. Among the markers, CRP - using a 10 mg/L cut-off value- proved to be the most accurate in identifying patients with infection (AUC: 0.93). The accuracy of CRP, however, decreased in advanced stage of the disease, most probably because of the significantly elevated CRP levels in non-infected patients. Combination of CRP and PCT increased the sensitivity and negative predictive value, compared with CRP on its own, by 10 and 5% respectively. During a 3-month follow-up period in patients without overt infections, Kaplan-Meier and proportional Cox-regression analyses showed that a CRP value of >10 mg/L (P = 0.035) was independently associated with a shorter duration to progress to clinically significant bacterial infections. There was no correlation between acute phase protein levels and antimicrobial seroreactivity. CONCLUSIONS: C-reactive protein on its own is a sensitive screening test for the presence of bacterial infections in cirrhosis and is also a useful marker to predict the likelihood of clinically significant bacterial infections in patients without overt infections.
Assuntos
Proteínas de Fase Aguda , Infecções Bacterianas/diagnóstico , Cirrose Hepática/complicações , Proteínas de Fase Aguda/análise , Adulto , Idoso , Infecções Bacterianas/complicações , Infecções Bacterianas/microbiologia , Proteína C-Reativa/análise , Calcitonina/sangue , Peptídeo Relacionado com Gene de Calcitonina , Proteínas de Transporte/sangue , Feminino , Humanos , Estimativa de Kaplan-Meier , Funções Verossimilhança , Masculino , Glicoproteínas de Membrana/sangue , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Precursores de Proteínas/sangue , Curva ROC , Análise de RegressãoRESUMO
Peripheral arterial disease (PAD) is frequently associated with atherosclerotic manifestations of the carotids and coronaries. Polyvascular involvement and low ankle−brachial index predict major cardiovascular events and high mortality. Cathepsin S (Cat S) promotes the inflammatory pathways of the arterial wall, while Cystatin C (Cys C) functions as its inhibitor; therefore, Cys C was proposed to be a biomarker of progression in PAD. In a single-center observational study, we investigated the correlations of serum Cys C and Cat S/Cys C ratio in a group of 90 PAD patients, predominantly with polyvascular involvement. Cys C and Cat S/Cys C were associated with ankle−brachial index (ABI) scores <0.4 in univariate and multiple regression models. Furthermore, both markers correlated positively with the plasma Von Willebrand Factor Antigen (VWF: Ag) and Von Willebrand Factor collagen-binding activity (VWF: CB). In addition, Cat S/Cys C was significantly decreased, whereas Cys C increased in subjects with three-bed atherosclerotic involvement. According to our results, high serum Cys C and low Cat S/Cys C ratios may indicate severe peripheral arterial disease and polyvascular atherosclerotic involvement.
RESUMO
Autologous hematopoietic stem cell transplantation (HSCT) is often complicated by hemostatic and thrombotic events associated with endothelial cell injury. Thrombotic complications are affected by a disturbed balance between platelets, circulating von Willebrand factor (VWF), and its specific protease, ADAMTS13. HSCT-associated endothelial dysfunction, impaired hemostasis, and inflammation are interrelated processes, and research on the complex interplay of conditioning regimens from engraftment to bone marrow regeneration remains intensive. This prospective observational study comparing lymphoma and multiple myeloma (MM) patients who underwent autologous HSCT explored how platelet count, VWF level, ADAMTS13 activity, and C-reactive protein (CRP) level as potential markers (1) vary in response to therapy, (2) differ between the 2 groups, and (3) correlate with the remission state at 100 days after HSCT. We correlated the quantitative changes in platelet count and levels of VWF, ADAMTS13, and CRP with one another during HSCT and in the remission state in 45 patients with lymphoma and 59 patients with MM who underwent autologous HSCT between 2010 and 2013 at the University of Debrecen. Samples were collected at the start of conditioning chemotherapy, on the day of stem cell transplantation, and at 5, 11, and 100 days following HSCT. CRP levels peaked when platelet counts dropped to a minimum, and these changes were much more pronounced in the lymphoma group. VWF level was the highest, with lower ADAMTS13 activity, at platelet engraftment in both patient groups equally. Diagnostic evidence indicative of thrombotic complications was not found. In the lymphoma group, VWF level prior to conditioning had statistically significant correlations with platelet count, CRP level, and hemoglobin concentration at the time of bone marrow regeneration (P < .001) and during the remission state (P = .034). In the MM group, platelet count before conditioning was correlated with platelet count (P < .001) and white blood cell count (P = .012) at the time of bone marrow regeneration. The statistically significant correlation of the markers at the time of bone marrow regeneration with the preconditioning VWF levels in lymphoma and with the preconditioning platelet counts in MM might indicate the clinical significance of the bone marrow niches of arterioles and megakaryocytes, respectively, where the stem cells are located and regulated. Because preconditioning VWF levels are associated with remission after HSCT in lymphoma patients, VWF should be screened before conditioning, along with the markers used in HSCT protocols, to optimize personalized treatment and reduce therapeutic risks.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Trombose , Humanos , Fator de von Willebrand/metabolismo , Medula Óssea/metabolismo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante Autólogo , Condicionamento Pré-Transplante/métodos , Trombose/patologia , BiomarcadoresRESUMO
BACKGROUND & AIMS: Mannose-binding lectin (MBL) is a serum lectin synthesized by the liver and involved in innate host defense. MBL deficiency increases the risk of various infectious diseases mostly in immune-deficient conditions. Bacterial infections are a significant cause of morbidity and mortality in liver cirrhosis due to the relative immuncompromised state. METHODS: Sera of 929 patients with various chronic liver diseases [autoimmune liver diseases (ALD), 406; viral hepatitis C (HCV), 185; and liver cirrhosis (LC) with various etiologies, 338] and 296 healthy controls (HC) were assayed for MBL concentration. Furthermore, a follow-up, observational study was conducted to assess MBL level as a risk factor for clinically significant bacterial infections in cirrhotic patients. RESULTS: MBL level and the prevalence of absolute MBL deficiency (<100 ng/ml) was not significantly different between patients and controls (ALD: 14.5%, HCV: 11.9%, LC: 10.7%, HC: 15.6%). In cirrhotic patients, the risk for infection was significantly higher among MBL deficient subjects as compared to non-deficient ones (50.0% vs. 31.8%, p=0.039). In a logistic regression analysis, MBL deficiency was an independent risk factor for infections (OR: 2.14 95% CI: 1.03-4.45, p=0.04) after adjusting for Child-Pugh score, co-morbidities, gender, and age. In a Kaplan-Meier analysis, MBL deficiency was associated with a shorter time to develop the first infectious complication (median days: 579 vs. 944, pBreslow=0.016, pLogRank=0.027) and was identified as an independent predictor in a multivariate Cox-regression analysis (p=0.003, OR: 2.33, 95% CI: 1.34-4.03). CONCLUSIONS: MBL deficiency is associated with a higher probability and shorter time of developing infections in liver cirrhosis, further supporting the impact of the MBL molecule on the host defense.
Assuntos
Infecções Bacterianas/sangue , Infecções Bacterianas/etiologia , Cirrose Hepática/sangue , Cirrose Hepática/complicações , Lectina de Ligação a Manose/deficiência , Adulto , Idoso , Infecções Bacterianas/imunologia , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Hungria , Imunidade Inata , Cirrose Hepática/imunologia , Hepatopatias/sangue , Hepatopatias/complicações , Hepatopatias/imunologia , Masculino , Lectina de Ligação a Manose/sangue , Lectina de Ligação a Manose/imunologia , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Fatores de Risco , Fatores de TempoRESUMO
OBJECTIVES: Thromboembolic complications are present in 0.8%-16.8% of the cases after radical prostatectomy (RP). Association between elevated plasma von Willebrand factor (VWF) levels-as an endothelial activation marker-and increased risk of thrombotic events has been evidenced. We aimed to elicit new data on the VWF after RP in prostate cancer patients and explore the role of it as a thrombotic risk factor. Upon perioperative plasma VWF levels (VWF:Ag) its collagen-binding (CB) activity (VWF:CB), multimerization, and cleaving enzyme (ADAMTS13 [a disintegrin and metalloprotease with thrombospondin type repeats, motif 1, type 13]) of the VWF multimers were quantitated along with Factor VIII and routine laboratory parameters in this observational pilot study. METHODS: Plasma samples of 24 prostate cancer patients were collected before (-1 day; D-1) and after RP (1 hour, 6 days, 1 month, and 10 months; H1, D6, M1, and M10). VWF:Ag, VWF:CB, ADAMTS13:Ag were measured by ELISA, and the multimer distribution by electrophoresis and quantitative densitometry. Factor VIII, fibrinogen, D-dimer, and other routine laboratory parameters were determined as well. Preoperative values served as baselines which were compared to controls (24 healthy individuals). RESULTS: VWF:Ag and CB elevated by 122% and 143% respectively at H1 after RP then plateaued at D6 compared to baseline values. ADAMTS13/VWF:Ag ratio reduced by 41% at H1, and by 46% at D6, meanwhile the ratio of high molecular weight multimers increased as well. Values returned to baseline at M1 and further reduced to the levels of the controls at M10. All of the 24 patients at H1 and D6 and 14 at M10 were in potential prothombotic state according ROC analysis of the VWF parameters as indicators. CONCLUSIONS: Prostate malignancy and then surgical stress, and inflammatory reactions induced release of VWF from the endothelial cells, along with an increasing amount of large multimers and relative reduction of ADAMTS13 level. Because these changes mark a prothrombotic state even at M1 after RP, more than 1 month follow-up and prophylactic targeting through the thrombotic and inflammatory activity of the VWF is proposed. Evaluation of VWF parameters provides new information about the long-term disturbances of primary hemostasis after radical pelvic oncologic surgery like RP and might improve the understanding the physiological and pathological recovery.
Assuntos
Plasma/química , Prostatectomia/métodos , Neoplasias da Próstata/sangue , Fator de von Willebrand/metabolismo , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/cirurgiaRESUMO
Von Willebrand factor (VWF) is built up from a varying number of subunits, of which the larger molecules have higher haemostatic activity. Von Willebrand disease (VWD) and thrombotic thrombocytopenic purpura are the best known disorders with pathognomonic changes of the highly multimerised VWF forms. There is an established method to calculate the relative amount of large oligomers. Our aim is to quantify the degree of VWF multimerisation as well, to complete the densitometric analysis of VWF electrophoresis. After optimisation, we have defined this new parameter (M(MW)) as the molecular weight corresponding to the lower boundary of the largest 25% of VWF protein. M(MW) was significantly different (p < .0001) in platelet lysate, normal samples and VWD type 2 samples (10.4, 6.3, 2.1, respectively). There was strong correlation between the M(MW) and the amount of large multimers in normal samples (r(2) = 0.98) and in platelet lysate. However M(MW) was higher in platelet lysate, in which VWF is not cleaved by ADAMTS-13, than in healthy samples with the same amount of large multimers. Comparison of the new parameter and the collagen binding and ristocetin cofactor activity of VWF, showed that the functional tests are at least partially determined by the multimerisation; however, about 15% of VWD samples had normal activity to antigen ratios. The quantification of multimerisation aids the classification in these cases, especially at low antigen concentrations, and also might help in the detection of thrombotic conditions.
Assuntos
Densitometria/métodos , Fator de von Willebrand/química , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Soluções Tampão , Estudos de Casos e Controles , Colágeno/metabolismo , Densitometria/estatística & dados numéricos , Humanos , Técnicas In Vitro , Peso Molecular , Complexos Multiproteicos/química , Ligação Proteica , Estrutura Quaternária de Proteína , Doenças de von Willebrand/sangue , Fator de von Willebrand/análise , Fator de von Willebrand/metabolismoRESUMO
The activity of ADAMTS13, the von Willebrand factor (VWF) cleaving protease is low in several conditions, including HELLP (haemolysis, elevated liver enzymes, and low platelet count) syndrome. As HELLP syndrome develops in most cases on the basis of preeclampsia, our aim was to determine whether plasma ADAMTS13 activity is decreased in preeclampsia. Sixty-seven preeclamptic patients, 70 healthy pregnant women and 59 healthy non-pregnant women were involved in this case-control study. Plasma ADAMTS13 activity was determined with the FRETS-VWF73 assay, while VWF antigen (VWF:Ag) levels with an enzyme-linked immunosorbent assay. The multimeric pattern of VWF was analyzed by SDS-agarose gel electrophoresis. There was no significant difference in plasma ADAMTS13 activity between the preeclamptic and the healthy pregnant and non-pregnant groups (median [25-75 percentile]: 98.8 [76.5-112.8] %, 96.3 [85.6-116.2] % and 91.6 [78.5-104.4] %, respectively; p > 0.05). However, plasma VWF:Ag levels were significantly higher in preeclamptic patients than in healthy pregnant and non-pregnant women (187.1 [145.6-243.1] % versus 129.3 [105.1-182.8] % and 70.0 [60.2-87.3] %, respectively; p < 0.001). The multimeric pattern of VWF was normal in each group. Primiparas had lower plasma ADAMTS13 activity than multi-paras (92.6 [75.8-110.6] % versus 104.2 [92.1-120.8] %; p = 0.011). No other relationship was found between clinical characteristics, laboratory parameters and plasma ADAMTS13 activity in either study group. In conclusion, plasma ADAMTS13 activity is normal in preeclampsia despite the increased VWF:Ag levels. However, further studies are needed to determine whether a decrease in plasma ADAMTS13 activity could predispose preeclamptic patients to develop HELLP syndrome.
Assuntos
Proteínas ADAM/sangue , Síndrome HELLP/etiologia , Pré-Eclâmpsia/sangue , Fator de von Willebrand/análise , Proteína ADAMTS13 , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Síndrome HELLP/sangue , Síndrome HELLP/enzimologia , Humanos , Paridade , Pré-Eclâmpsia/enzimologia , Gravidez , Multimerização Proteica , Regulação para Cima , Adulto JovemRESUMO
Decreased activity of ADAMTS13, the von Willebrand factor (VWF) cleaving protease, was recently reported in cardiovascular diseases and in hepatic failure. Chronic heart failure (CHF) is characterised by abnormalities of left ventricular function accompanied by the failure of the liver and dysregulation of endothelial activation. Therefore, the aim of our study was to measure ADAMTS13 activity in CHF, and determine the prognostic value of VWF and ADAMTS13 on major clinical events in CHF. ADAMTS13 activity (measured by FRETS-VWF73 substrate) was decreased in CHF (n = 152, left ventricular ejection fraction <45%), and it correlated negatively with B-type natriuretic peptide (BNP) NYHA (New York Heart Association) classes, markers of synthetic capacity of the liver and endothelial dysfunction (all p < 0.005). Both, high VWF:Ag levels (hazard ratio [HR] 1.52, 95% confidence interval [CI] 1.189-1.943), and low ADAMTS13/VWF:Ag ratios (HR 0.70, 95% CI 0.58-0.84) independently and significantly predicted short-term (1 year follow-up) clinical adverse events in heart failure (HF). Decreased activity of ADAMTS13 with concomitant high VWF:Ag levels is a significant independent predictor of clinical events in CHF. The levels of the two molecules may integrate the impaired synthetic capacity of the liver and the disturbed endothelial regulation and can therefore be a useful tool to predict clinical events in CHF.
Assuntos
Proteínas ADAM/biossíntese , Insuficiência Cardíaca/sangue , Fator de von Willebrand/biossíntese , Proteína ADAMTS13 , Idoso , Biomarcadores/metabolismo , Doença Crônica , Estudos de Coortes , Endotélio Vascular/patologia , Feminino , Insuficiência Cardíaca/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Risco , Resultado do TratamentoRESUMO
The present study describes severe multiplex cerebral ischaemic laesions in a male patient being diagnosed with polycythaemia vera (PV). In contrast to previous publications, unique platelet receptor pattern with normal platelet count was identified. Glycoprotein Ib receptor number on the surface of resting platelets was increased two-fold and almost three-fold in case of activated platelets compared to the controls. More over, in an in vitro study when whole blood was circulated both at venous and arterial shear conditions and shear rate was adjusted according to the blood viscosity, platelet aggregate/thrombus formation was characteristic on surfaces covered with purified von Willebrand factor while in case of controls the surface was covered with single platelets or platelet monolayer. Similar results with pathological findings have not been published in PV until now. Our result undersigns the necessity of antiplatelet therapy of PV patients, even at normal platelet count.
Assuntos
Isquemia Encefálica/sangue , Adesividade Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/análise , Policitemia Vera/complicações , Adulto , Células Cultivadas , Humanos , Masculino , Inibidores da Agregação Plaquetária/uso terapêutico , Contagem de Plaquetas , Testes de Função Plaquetária , Policitemia Vera/tratamento farmacológicoRESUMO
BACKGROUND: There is an unstable balance between pro- and anti-haemostatic processes in patients with cirrhosis. We hypothesized, that in patients with acute decompensation (AD) the major alterations of von Willebrand factor (VWF) could contribute to the pro-thrombotic situation as compared to patients with stable (ST) cirrhosis. PATIENTS AND METHODS: We analysed different parameters of VWF, including detailed multimer distribution by densitometry and platelet adhesion, together with adisintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) activity and antigen and C-reactive protein (CRP) levels in patients with ST cirrhosis (n = 99), with AD (n = 54) and controls (n = 92). RESULTS: VWF antigen, ristocetin co-factor as well as collagen-binding activities were elevated in both cirrhotic groups in a stepwise manner. There was a decrease in high and an increase in low molecular weight multimer ratios in the majority of ST cirrhosis. However, in 24 out of 54 AD patients, ultra-large VWF multimers (ultra-large molecular weight multimers [ULMWM]) were found. ADAMTS13 activity in ST and AD patients without ULMWM was similar to controls (median [interquartile range; IQR]%: 98 [67-132] and 91 [60-110] vs. 106 [88-117], respectively). The presence of ULMWM in AD patients was associated with low ADAMTS13 activity [33 (24-49)%] and high CRP level [23 (7.1-83.6) mg/L]. Adhesion of normal platelets showed a stepwise increase in the presence of cirrhotic plasmas, reaching the highest level in AD patients with ULMWM. CONCLUSION: Characteristic changes of VWF parameters are seen in ST cirrhosis. In AD patients, highly increased VWF and reduced ADAMTS13 activity could be found, along with the presence of ULMWM, which are possible markers and contributors of the disease progression.
Assuntos
Coagulação Sanguínea , Cirrose Hepática/sangue , Falência Hepática Aguda/sangue , Trombose/sangue , Fator de von Willebrand/metabolismo , Proteína ADAMTS13/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Progressão da Doença , Feminino , Humanos , Mediadores da Inflamação/sangue , Cirrose Hepática/complicações , Cirrose Hepática/diagnóstico , Falência Hepática Aguda/diagnóstico , Falência Hepática Aguda/etiologia , Masculino , Pessoa de Meia-Idade , Peso Molecular , Trombose/diagnóstico , Trombose/etiologia , Adulto JovemRESUMO
Many polymorphisms in vonWillebrand factor (VWF) have been reported and their association with VWF plasma levels or cardiovascular diseases has been investigated. The aim of this study was to examine whether the amino acid polymorphism A/T1381 in the VWF A1-domain would affect VWF binding to platelet GPIbalpha. Sixty-one normal individuals were genotyped at the A/T1381 locus. Twenty-one A/A1381 homozygotes, 30 A/T1381 heterozygotes and 10 T/T1381 homozygotes were identified. Remarkably, when compared to VWF of A/T1381 and A/A1381 individuals, VWF of individuals carrying the T/T1381 variant showed an increased affinity for its platelet receptor GPIbalpha under static conditions, as reflected by an increased sensitivity to low concentrations of ristocetin or botrocetin. In addition, also the rVWF-T1381 demonstrated a higher affinity for GPIbalpha than rVWF-A1381. Interestingly, this enhanced affinity of the T/T variant over the A/T and A/A variant was, however, too subtle to affect platelet adhesion under physiological flow conditions, which fully corroborates the normal haemostatic phenotype of all individuals. We demonstrate that the VWF A/T1381 polymorphism plays an important role in inter-individual variability of the affinity of VWF for GPIbalpha, with T/T variants having a higher affinity than A/A and A/T variants, at least under static conditions in vitro. Further genetic linkage and association studies are necessary to establish whether the A/T1381 polymorphism could correlate with an increased risk of thrombotic events.