RESUMO
The von Willebrand factor cleaving protease (VWFCP) modulates the von Willebrand factor (VWF) multimeric size in normal plasma. VWFCP activity levels are decreased in different physiological and pathologic situations. Different techniques have been developed to unfold the purified VWF (perfusion at high shear rate, dialysis against urea in nitrocellulose filters), to detect the VWFCP activity on it (multimeric analysis of VWF, collagen binding to VWF assay) and to use the patient plasma both as the source of the enzyme and substrate. In this paper we compared the above mentioned methods with new ones: normal plasma dialyzed on membranes instead of purified VWF, dialysis of the samples against urea in tubing instead of nitrocellulose filters, and sonicated plasma to remove the endogenous VWF. The perfusion assay and detection by multimeric analysis showed a limit of detection (25%) of VWFCP activity. Dialysis against urea in both supports and detection by multimeric analysis, showed a better limit of detection (3%), but the recovery of the samples was not as efficient in nitrocellulose filters as it was in tubing. The detection by collagen binding to VWF has more advantages because it allows to analyze more samples than the multimeric analysis does in the same assay. The dialysis of plasma by membranes to obtain the source of exogenous VWF requires no complex equipment. The method, which uses patient plasma as the source of the enzyme and substrate, was inapplicable in our experience because the values could not be interpolated in the reference curve (AU)
Assuntos
Humanos , Fator de von Willebrand , Metaloendopeptidases/metabolismo , Púrpura Trombocitopênica Trombótica/fisiopatologia , Metaloendopeptidases/sangue , Púrpura Trombocitopênica Trombótica/metabolismo , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/fisiopatologia , Diálise , Fator de von Willebrand/isolamento & purificação , Fator de von Willebrand/metabolismo , Plasma/enzimologia , Colágeno/metabolismoRESUMO
This work is an investigation on the microvasculature of the cutaneous infiltrates of leprosy with the immunohistochemical staining of endothelial cells in cutaneous biopsies. Anti-Factor VIII-related antigen antibody (anti-FVIII-ra) and Ulex Europaeus-1 lectin (UEA-1) binding were utilized as endothelial cell markers. Thirty-nine patients grouped according to the Ridley-Jopling classification (14 borderline tuberculoid, 18 borderline lepromatous, 6 lepromatous, and 1 indeterminate leprosy) were selected for this study. Two microvascular architectural patterns could be clearly distinguished: lepromatous lesions presented a dense and tortuous mesh of microvessels among the Mycobacterium leprae-glutted macrophages; whereas the microvessels in the tuberculoid lesions were restricted to the periphery of the granulomas and were not seen among the central epithelioid cells. We were able to distinguish three basic morphological kinds of infiltrate distribution related to the microvessels: micronodules, cords and macronodules. Intensifications of the FVIII-ra immunoreactivity and UEA-1 binding capacity were observed in the endothelial cells of microvessels involved by the inflammatory infiltrate. A distinct cytokine expression profile at the leprosy poles and the role of mast cells in angiogenesis were speculated as factors contributing to these distinct patterns. Growth of the lesion and systemic dissemination of M. leprae in the bipolar spectrum of leprosy may hypothetically be influenced by the vascular-infiltrate relationship. The detection of angiogenesis in the cutaneous lesions of leprosy may bring about alternate and/or additional strategies for leprosy treatment.
Assuntos
Hanseníase Virchowiana/patologia , Hanseníase Tuberculoide/patologia , Pele/irrigação sanguínea , Biópsia , Endotélio Vascular/patologia , Granuloma , Humanos , Imuno-Histoquímica , Pele/patologia , Fator de von Willebrand/isolamento & purificaçãoRESUMO
Patients and methods: Five hundred eighty nine patients whose main symptom was the presence of mucocutaneous hemorrhages were studied. Bleeding time, platelet count, coagulant activity of factor VIII (FVIII:C), FvW: Ag and FvW:CoRis and ABO blood group were measured in all patients in a first stage. According to the results of these tests, further studies were decided. Results: In patients younger than 13 years old, males predominated and, in older patients, females consulted with higher frequency. There was a higher proportion of individuals with O blood type than in the normal population. Bleeding time was abnormal in 330 patients (56 percent). One hundred ten patients (19 percent) had von Willebrand disease and, among them, one third had a normal bleeding time. Isolated reduction of factor VIII activity was found in 66 patients (11 percent, 51 males) and 32 of these had normal bleeding time. Eighty one patients (14 percent) were considered to have an hereditary platelet function defect. A precise diagnosis was not achieved in 332 patients (56 percent). Conclusions: Among patients consulting for mucocutaneous hemorrhages, 19 percent had von Willebrand disease, 11 had an isolated reduction of factor VIII activity, 14 percent had platelet function defects and in 56 percent, a precise diagnosis was not reached
Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto , Transtornos Hemorrágicos/epidemiologia , Mucosa/fisiopatologia , Doenças de von Willebrand/epidemiologia , Fator de von Willebrand/isolamento & purificaçãoRESUMO
The perfusion of serum, citrated whole blood and citrated plasma, through a simple tube system resulted in a significant loss of large von Willebrand factor (vWf) multimers, without decrease in antigen levels. Maximum loss of large multimers was observed at a shear rate of 15,000 s-1 for 15 min. Heparin, aprotinin, soybean trypsin inhibitor, phenylmethylsulphonylfluoride, N-ethylmaleimide, leupeptin or calpain inhibitor peptide could not prevent the loss of large vWf multimers in citrated plasma. The addition of EDTA calcium salt partially prevented it, and it was totally prevented by EDTA without calcium. Perfusion of purified vWf did not induce the loss of large multimers, but this did happen after the addition of either whole serum or a plasma fraction. The activity of this plasma fraction disappeared at pH < 6.8. Besides, we have found that the binding to subendothelium of purified vWf diluted in dialyzed serum was lower at pH 7.2 than at pH 6.0. Chromatographic studies demonstrated that the loss of large vWf multimers, induced by high shear rates, involves a plasma substance(s) of molecular weight larger than 200 kD; calpain and granulocyte or cysteine proteases do not seem to be this plasma substance(s).
Assuntos
Perfusão/métodos , Plasma/química , Fator de von Willebrand/análise , Fator de von Willebrand/isolamento & purificação , Técnicas In VitroRESUMO
The perfusion of serum, citrated whole blood and citrated plasma, through a simple tube system resulted in a significant loss of large von Willebrand factor (vWf) multimers, without decrease in antigen levels. Maximum loss of large multimers was observed at a shear rate of 15,000 s(-1) for 15 min. Heparin, aprotinin, soybean trypsin inhibitor, phenylmethylsulphonylfluoride, N-ethylmaleimide, leupeptin or calpain inhibitor peptide could not prevent the loss of large vWf multimers in citrated plasma. The addition of EDTA calcium salt partially prevented it, and it was totally prevented by EDTA without calcium. Perfusion of purified vWf did not induce the loss of large multimers, but this did happen after the addition of either whole serum or a plasma fraction. The activity of this plasma fraction disappeared at pH < 6.8. Besides, we have found that the binding to subendothelium of purified vWf diluted in dialized serum was lower at pH 7.2 than at pH 6.0. Chromatographic studies demonstrated that the loss of large vWf multimers, induced by high shear rates, involves a plasma substance(s) of molecular weight larger than 200 kD; calpain and granulocyte or cysteine proteases do not seem to be this plasma substance(s).
Assuntos
Técnicas In Vitro , Perfusão/métodos , Plasma/química , Fator de von Willebrand/análise , Fator de von Willebrand/isolamento & purificaçãoRESUMO
The perfusion of serum, citrated whole blood and citrated plasma, through a simple tube system resulted in a significant loss of large von Willebrand factor (vWf) multimers, without decrease in antigen levels. Maximum loss of large multimers was observed at a shear rate of 15,000 s(-1) for 15 min. Heparin, aprotinin, soybean trypsin inhibitor, phenylmethylsulphonylfluoride, N-ethylmaleimide, leupeptin or calpain inhibitor peptide could not prevent the loss of large vWf multimers in citrated plasma. The addition of EDTA calcium salt partially prevented it, and it was totally prevented by EDTA without calcium. Perfusion of purified vWf did not induce the loss of large multimers, but this did happen after the addition of either whole serum or a plasma fraction. The activity of this plasma fraction disappeared at pH < 6.8. Besides, we have found that the binding to subendothelium of purified vWf diluted in dialized serum was lower at pH 7.2 than at pH 6.0. Chromatographic studies demonstrated that the loss of large vWf multimers, induced by high shear rates, involves a plasma substance(s) of molecular weight larger than 200 kD; calpain and granulocyte or cysteine proteases do not seem to be this plasma substance(s). (AU)
Assuntos
Técnicas In Vitro , RESEARCH SUPPORT, NON-U.S. GOVT , Plasma/química , Perfusão/métodos , Fator de von Willebrand/análise , Fator de von Willebrand/isolamento & purificaçãoRESUMO
Ajoene, (E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide, is a potent antiplatelet compound isolated from alcoholic extracts of garlic (Allium sativum). Ajoene reversibly inhibits in vitro platelet aggregation as well as release reaction induced by all known agonists. We used a well characterized cylindrical perfusion chamber to study the effect of ajoene on platelet deposition onto physiological substrates such as pig aortic subendothelium and tunica media as a model of mildly and severely damaged vessel wall respectively. Experiments were performed under flow conditions of high and low shear rate that mimic laminar blood flow in small and medium size arteries (1690 sec-1 and 212 sec-1). Our results indicate that ajoene prevents thrombus formation both at low and high shear rate in citrated whole blood. The inhibitory effect of ajoene on platelet-thrombus formation seems to be dependent on its inhibition of fibrinogen binding, since significantly higher concentrations of ajoene are needed to affect von Willebrand factor binding to GPIIb/IIIa receptors. Further, ajoene does not impair Ristocetin-induced platelet agglutination, mediated by GPIb. Our results suggest that ajoene may be useful for the acute prevention of thrombus formation induced by vascular damage.
Assuntos
Dissulfetos/farmacologia , Alho , Extratos Vegetais/farmacologia , Plantas Medicinais , Inibidores da Agregação Plaquetária/farmacologia , Reologia , Trombose/prevenção & controle , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Endotélio Vascular/metabolismo , Radioisótopos de Índio , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ligação Proteica , Estresse Mecânico , Sulfóxidos , Suínos , Fator de von Willebrand/isolamento & purificação , Fator de von Willebrand/metabolismoRESUMO
Se realizó la purificación del FVIII/FvW a partir de un concentrado comercial que había pasado la fecha de vencimiento para su uso terapéutico y se obtuvo un inmunógeno el cual se administró a un conejo con el objetivo de obtener un antisuero contra esa proteína. El antisuero obtenido se purificó posteriormente mediante una cromatografía de afinidad antigenoanticuerpo. Con la utilización de estos métodos hemos logrado obtener un antisuero el cual se puede utilizar para la determinación cuantitativa de FVIII/FvW sin necesidad de imponer este reactivo