RESUMO
Proteinase 3 (PR3) is a neutral serine protease stored in neutrophil granules. It has substantial sequence homology with elastase, cathepsin G and azurocidin. PR3 is the target antigen for autoantibodies (ANCA) in Wegener's granulomatosis, a necrotizing vasculitis syndrome. ANCA have been implicated in the pathogenesis of this disease. PR3 has two potential Asn-linked glycosylation sites. This study was designed to determine the occupancy of these glycosylation sites, and to evaluate their effect on enzymatic function, intracellular processing, targeting to granules and recognition by ANCA. We found that glycosylation occurs at both sites in native neutrophil PR3 and in wild type recombinant PR3 (rPR3) expressed in HMC-1 cells. Using glycosylation deficient rPR3 mutants we found that glycosylation at Asn-147, but not at Asn-102, is critical for thermal stability, and for optimal hydrolytic activity of PR3. Efficient amino-terminal proteolytic processing of rPR3 is dependent on glycosylation at Asn-102. Targeting to granules is not dependent on glycosylation, but unglycosylated rPR3 gets secreted preferentially into media supernatants. Finally, a capture ELISA for ANCA detection, using rPR3 glycosylation variants as target antigens, reveals that in about 20% of patients, epitope recognition by ANCA is affected by the glycosylation status of PR3.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Asparagina/metabolismo , Mieloblastina/imunologia , Mieloblastina/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Estabilidade Enzimática , Glicosilação , Humanos , Mastócitos , Mieloblastina/genética , Oligopeptídeos/metabolismo , Transporte Proteico/fisiologiaRESUMO
Direct comparisons of human (h) and murine (m) neutrophil elastase (NE) and proteinase 3 (PR3) are important for the understanding and interpretation of inflammatory and PR3-related autoimmune processes investigated in wild-type-, mNE- and mPR3/mNE knockout mice. To this end, we purified recombinant mPR3 and mNE expressed in HMC1 and 293 cells and compared their biophysical properties, proteolytic activities and susceptibility to inhibitors with those of their human homologues, hPR3 and hNE. Significant species differences in physico-chemical properties, substrate specificities and enzyme kinetics towards synthetic peptide substrates, oxidized insulin B chain, and fibrinogen were detected. MeOSuc-AAPV-pNA and Suc-AAPV-pNA were hydrolyzed more efficiently by mPR3 than hPR3, but enzymatic activities of mNE and hNE were very similar. Fibrinogen was cleaved much more efficiently by mPR3 than by hPR3. All four proteases were inhibited by alpha(1)-antitrypsin and elafin. Eglin C inihibited mNE, hNE, mPR3, but not hPR3. SLPI inhibited both NEs, but neither PR3. The custom-designed hNE inhibitor, Val(15)-aprotinin, is a poor inhibitor for mNE. In conclusion, appropriate interpretation of experiments in murine models requires individual species-specific assessment of neutrophil protease function and inhibition.
Assuntos
Elastase de Leucócito/metabolismo , Serina Endopeptidases/metabolismo , Animais , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Camundongos Knockout , Mieloblastina , Proteínas Recombinantes/metabolismoRESUMO
Atherosclerosis manifests as a systemic disease with near global involvement of the named segments of the arterial tree. Acute thrombotic arterial occlusion, however, is not equally distributed. To evaluate intra-individual regional differences in arterial thrombogenicity, we compared (111)In-platelet deposition in porcine carotid and femoral arteries after a standardized crush injury. Within the unidirectional flow conditions of elastic carotid arteries, platelet deposition was more than 3-fold higher compared with predominantly muscular femoral arteries with triphasic arterial flow. To determine the influence of rheology on platelet deposition after crush injury, carotid arteries were transplanted into the femoral position and compared with the paired native carotid and femoral arteries. Similarly, femoral arteries transposed to the carotid position were compared with the paired native carotid artery. In each of these experiments, arterial transposition to a new anatomic location imparts a predilection for platelet deposition indigenous to the new location. In the controlled environment of two high-shear thrombin-independent and -dependent flow chambers, porcine carotid and femoral arterial substrates were indistinguishable from one another with respect to platelet deposition. Regional differences in arterial hemodynamics may account for substantial differences in thrombosis arising from deep arterial injury.
Assuntos
Artérias/fisiopatologia , Hemodinâmica , Ativação Plaquetária , Trombose/diagnóstico por imagem , Trombose/fisiopatologia , Animais , Artérias/diagnóstico por imagem , Artérias/lesões , Artérias Carótidas/transplante , Complacência (Medida de Distensibilidade) , Feminino , Artéria Femoral/lesões , Artéria Femoral/transplante , Fluxo Sanguíneo Regional , Suínos , Trombose/etiologia , Ultrassonografia , Ferimentos não Penetrantes/complicações , Ferimentos não Penetrantes/fisiopatologia , Ferimentos não Penetrantes/cirurgiaRESUMO
The media layer of the arterial cryo-cross sections, is defective for vWf-dependent platelet adhesion. Exposure of the same layer by stripping off the most inner portions of the vessel wall results in a highly thrombogenic surface. Stripping or balloon dilation was applied to porcine arteries prior to functional assays. Cryosections of treated or untreated arteries were perfused with porcine blood at 3,350 s(-1) and platelet deposition was detected by indirect immunofluorescence. Following balloon dilation, vWf-dependent platelet deposition increased; covering 9.08 +/- 1.36% of the total media surface area, this value for untreated vessels was 0.88 +/- 0.14%. A 10-fold increase was also found in the binding of vWf-coated fluorescent beads to the media. In addition to mechanical procedures, treatment by serine-proteases like trypsin, chymotrypsin and proteinase 3, or by chondroitinase ABC, but not by heparitinase also resulted in a 7-10-fold increase in platelet coverage over the media. Collagen in the media may be complexed with another vessel wall component shielding the vWf-binding sites. Mechanical or biochemical processes unmask these sites, and increase the thrombogenicity of the vessel wall.
Assuntos
Trombose/etiologia , Túnica Média/patologia , Animais , Artérias/patologia , Sítios de Ligação , Cateterismo/efeitos adversos , Microscopia de Fluorescência , Adesividade Plaquetária , Serina Endopeptidases/metabolismo , Suínos , Trombose/patologia , Túnica Média/fisiologia , Fator de von Willebrand/metabolismoRESUMO
Fibrillar collagen serves as a thrombogenic surface for platelet adhesion mediated by von Willebrand factor (vWf) at high shear. Although abundant throughout the arterial wall, vWf-dependent platelet deposition to artery cross-sections from perfused citrated blood is localized to the adventitia of the vessel wall. Here we describe a similarly skewed distribution of vWf-binding sites in artery cross-sections. Binding of vWf-coated fluorescent beads, as well as detection of plasma vWf bound to artery cross-section at 3350 s(-1) shear rate with indirect particle-immunofluorescence or immunoelectron microscopy demonstrate vWf binding sites in the adventitia, but not in the media. A monoclonal anti-vWf antibody that interferes with vWf-binding to collagen in a microplate ELISA inhibits vWf-binding to both the adventitia and sections of collagen fibrils. Our data suggest that the media, despite its fibrillar collagen content, evidenced by electron microscopy, is defective for vWf-binding, which may explain its thromboresistant nature at high shear rates.
Assuntos
Artérias Carótidas/metabolismo , Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Hemorreologia , Artéria Torácica Interna/metabolismo , Adesividade Plaquetária , Artéria Renal/metabolismo , Fator de von Willebrand/metabolismo , Animais , Sítios de Ligação , Artérias Carótidas/ultraestrutura , Ensaio de Imunoadsorção Enzimática , Hemostasia , Humanos , Imuno-Histoquímica , Artéria Torácica Interna/ultraestrutura , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Ligação Proteica , Artéria Renal/ultraestrutura , Estresse Mecânico , SuínosRESUMO
OBJECTIVES: Infants and children undergoing cardiopulmonary bypass for repair of congenital heart defects are at substantial risk for excessive bleeding, contributing greatly to morbidity and mortality. Aprotinin significantly reduces bleeding and transfusion requirements in adults but is of indeterminate value for pediatric patients. The aim of this study was to determine plasma aprotinin concentrations in these patients with a functional aprotinin assay. METHODS: Thirty patients less than 16 years of age scheduled for cardiac surgery with aprotinin were enrolled. Aprotinin was administered as a 25,000 KIU/kg bolus, 35,000 KIU/kg cardiopulmonary bypass prime, and 12,500 KIU.kg(-1).h(-1) continuous infusion. Blood samples for aprotinin concentrations (kallikrein-inhibiting units/milliliter) were obtained before aprotinin; 5 minutes post-bolus; 5 minutes after cardiopulmonary bypass initiation; 30 and 60 minutes on cardiopulmonary bypass; on discontinuation of aprotinin; 1 hour after aprotinin discontinuation; and 4 hours after permanent separation from cardiopulmonary bypass. For analysis, patients were grouped according to weight (<10 kg, 10-20 kg, >20 kg). Differences between weight groups were assessed using an exact test for categoric variables and 1-way analysis of variance for continuous variables. RESULTS: Aprotinin concentrations differed significantly across weight groups. Five minutes after aprotinin bolus and initiation of cardiopulmonary bypass, there was significant correlation between weight and aprotinin concentration (r =.57, P =.003; r =.69, P =.001, respectively). CONCLUSION: A functional assay reveals significant variability in aprotinin concentration for pediatric patients using current weight-based aprotinin dosing. Additional investigation is necessary to determine target aprotinin concentration dosing regimens to provide better efficacy.
Assuntos
Aprotinina/administração & dosagem , Aprotinina/farmacocinética , Perda Sanguínea Cirúrgica/prevenção & controle , Ponte Cardiopulmonar/métodos , Cardiopatias Congênitas/cirurgia , Adolescente , Peso Corporal , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Seguimentos , Cardiopatias Congênitas/diagnóstico , Humanos , Infusões Intravenosas , Masculino , Monitorização Intraoperatória/métodos , Cuidados Pré-Operatórios/métodos , Probabilidade , Estudos Prospectivos , Medição de Risco , Índice de Gravidade de Doença , Estatísticas não Paramétricas , Resultado do TratamentoRESUMO
INTRODUCTION: The quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure, the quality and properties of reagents used and concentrations of other plasma proteins, including von Willebrand factor (VWF). OBJECTIVE: To compare FVIII concentrations measured by activity-based assays with those obtained by an immunoassay and to establish the influence of plasma dilution on the FVIII clotting activity (FVIIIc). METHODS: The APTT, a chromogenic assay (Coatest) and two in-house immunoassays were used. Albumin-free recombinant FVIII was used as the calibrator in all assays. RESULTS: For a group of 44 healthy individuals (HI), the mean value observed for FVIII antigen (FVIIIag; 1.22+/-0.56 nM; S.D.) is substantially higher than that for FVIIIc (0.65+/-0.29 nM) and the chromogenic assay (FVIIIch; 0.50+/-0.23 nM). A positive correlation between FVIIIag and VWFag with R(2)=0.20 was observed. Since plasma VWF has an inhibitory effect on FVIIIc, we evaluated the influence of plasma dilutions on FVIIIc in HI (n=105). At a 4-fold dilution, estimates of FVIIIc by clotting assay were much lower than FVIIIag (0.77+/-0.31 vs. 1.14+/-0.48 nM). At 10- and 25-fold dilutions, the estimated FVIIIc increased to 0.87+/-0.36 and 0.94+/-0.44 nM, respectively. CONCLUSIONS: 1) In plasma, FVIIIag is higher than FVIIIc and FVIIIch; and 2) Real FVIII concentrations in plasma can be estimated by measuring FVIIIag.
Assuntos
Fator VIII/metabolismo , Imunoensaio , Tempo de Tromboplastina Parcial , Adulto , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Hemofilia A/sangue , Humanos , Imunoensaio/métodos , Masculino , Tempo de Tromboplastina Parcial/métodos , Plasma/metabolismo , Adulto Jovem , Fator de von Willebrand/metabolismoRESUMO
OBJECTIVE: The purpose of this study was to determine if substitution of a heparin-coated oxygenator and salvaged autologous blood for cardiotomy suction would improve platelet function. DESIGN: A prospective, randomized trial. SETTING: A large academic medical center. PARTICIPANTS: Sixty adult patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass (CPB). INTERVENTIONS: Patients were randomized into 1 of 4 groups in a 2 x 2 factorial design by oxygenator (heparinized v nonheparinized) and blood salvage during CPB (cardiotomy suction v salvaged autologous blood). MEASUREMENTS AND MAIN RESULTS: Primary outcome measures were platelet function, glass-bead retention, platelet dense-body adenosine triphosphate secretion, platelet-rich plasma (PRP) aggregometry, Plateletworks platelet-function analyzer (Helena Laboratories Corp, Allen Park, MI), and platelet count. Secondary outcome measures were chest-tube drainage and allogeneic blood transfusion requirements. All platelet-function tests except thrombin-receptor activator peptide-induced PRP aggregometry showed a reduction in platelet function during and immediately after CPB (all p < 0.05). The only statistically significant difference in platelet-function tests between the groups was the glass-bead assay at 5 minutes before discontinuation of CPB (p < 0.05). This difference resolved 10 minutes after protamine administration. There were no differences between the groups in the amount of blood transfused, chest-tube drainage, and routine laboratory test results. CONCLUSIONS: The authors concluded that the effects of these changes to the CPB circuit were small and inconsequential in this cohort of patients.
Assuntos
Perda Sanguínea Cirúrgica , Transfusão de Sangue Autóloga , Ponte Cardiopulmonar , Ponte de Artéria Coronária , Heparina , Oxigenadores de Membrana , Testes de Função Plaquetária , Sucção , Adulto , Transfusão de Sangue , Tubos Torácicos , Materiais Revestidos Biocompatíveis , Feminino , Humanos , Masculino , Agregação Plaquetária , Contagem de PlaquetasRESUMO
Microvascular thrombosis is a prominent feature in cardiac delayed xenograft rejection (DXR). We investigated the impact of warfarin or low-molecular-weight heparin (LMWH) anti-coagulation on xenograft function using a heterotopic pig-to-primate model. Donor hearts were from CD46 transgenic pigs and baboon immunosuppression included tacrolimus, sirolimus, anti-CD20 and TPC, an alpha-galactosyl-polyethylene glycol conjugate. Three groups of animals were studied. Group 1 (n = 9) was treated with warfarin, Group 2 (n = 13) with LMWH and Group 3, received no anti-coagulant drugs. The median duration of xenograft function was 20 days (range 3-62 days), 18 days (range 5-109 days) and 15 days (range 4-53 days) in Groups 1 to 3 respectively. Anti-coagulation achieved the targeted international normalized prothrombin ratio (INR) and anti-factor Xa levels consistent with effective in vivo therapy yet, no significant impact on median xenograft function was observed. At rejection, a similar histology of thrombosis and ischemia was apparent in each group and the levels of fibrin deposition and platelet thrombi in rejected tissue was the same. Anti-coagulation with warfarin or LMWH did not have a significant impact on the onset of DXR and microvascular thrombosis. However, a role for specific anti-coagulant strategies to achieve long-term xenograft function cannot be excluded.
Assuntos
Transplante de Coração/métodos , Heparina de Baixo Peso Molecular/uso terapêutico , Transplante Heterólogo/métodos , Varfarina/uso terapêutico , Animais , Animais Geneticamente Modificados , Anticoagulantes/farmacologia , Antígenos CD/biossíntese , Fator Xa/química , Imunoglobulina G/química , Imunoglobulina M/química , Imunossupressores/farmacologia , Coeficiente Internacional Normatizado , Isquemia , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/biossíntese , Microcirculação , Miocárdio/metabolismo , Papio , Primatas , Protrombina/metabolismo , Sirolimo/farmacologia , Suínos , Tacrolimo/farmacologia , Trombose/metabolismo , Fatores de Tempo , Resultado do Tratamento , Vitamina K/metabolismoRESUMO
BACKGROUND: Autoantibodies directed against neutrophil proteinase 3 (PR3-ANCA) from patients with Wegener's granulomatosis and microscopic polyangiitis recognize conformational epitopes of PR3. During maturation of neutrophils, PR3 undergoes amino-terminal and carboxy-terminal processing. In contrast to amino-terminal processing, the effects of carboxy-terminal processing on recognition of PR3 by PR3-ANCA remain unknown. Carboxy-terminally modified or tagged recombinant PR3 (rPR3) molecules may be useful for the refinement of diagnostic assays and for the study of biological processes. METHODS: This study was designed to determine whether 293 cells can be used to express specifically designed carboxy-terminal variants of rPR3, and to evaluate the effects of different carboxy-terminal modifications on the recognition by PR3-ANCA in the capture ELISA. RESULTS: The rPR3-variants secreted into the media supernatants of transfected 293 cells escaped proteolytic processing. Furthermore, in contrast to the effects of amino-terminal pro-peptide deletion on PR3-ANCA binding, carboxy-terminal modifications (deletion and additions) did not significantly affect recognition by PR3-ANCA. CONCLUSIONS: This expression system is ideally suited for the expression of custom-designed carboxy-terminal rPR3 variants, and major conformational effects of carboxy-terminal modifications seem unlikely.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Serina Endopeptidases/química , Serina Endopeptidases/imunologia , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Linhagem Celular , Ensaio de Imunoadsorção Enzimática/métodos , Variação Genética , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Mieloblastina , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
UNLABELLED: Aprotinin is effective during cardiac surgery for reducing blood loss and transfusion requirements, but it is expensive. Aprotinin is usually administered to adults according to a fixed protocol regardless of the patient's weight. We previously developed a weight-based dosing protocol for aprotinin. The purpose of this prospective observational study was to determine aprotinin levels in four patient groups (n = 10 each) using the new weight-based aprotinin dosing schedule that should achieve concentrations over 100, 150, 200, and 250 kallikrein inhibitory units/mL compared with full-dose aprotinin regimen (n = 10) by a simple functional aprotinin assay. There was no difference in patient demographic or surgical variables among groups. There was less within patient variation in plasma aprotinin concentrations over time in the new weight-based aprotinin dosing schedule groups compared with the full-dose aprotinin regimen group (P < 0.02 for all comparisons). The mean plasma aprotinin concentration achieved with the new weight-based aprotinin dosing schedule was similar to the desired concentrations, but we were unable to reduce between-patient variability in aprotinin concentrations. IMPLICATIONS: The current dosing schedule for aprotinin results in a large variation in aprotinin plasma concentrations between patients and a large variation within each patient over time. A new weight-based dosing schedule reduced variation of aprotinin concentration over time, but was unable to reduce between-patient variability in aprotinin concentration.
Assuntos
Aprotinina/administração & dosagem , Peso Corporal , Procedimentos Cirúrgicos Cardíacos , Hemostáticos/administração & dosagem , Idoso , Aprotinina/farmacocinética , Perda Sanguínea Cirúrgica , Transfusão de Sangue , Ponte Cardiopulmonar , Feminino , Hemostáticos/farmacocinética , Humanos , Masculino , Estudos ProspectivosRESUMO
BACKGROUND: Microvascular thrombosis is a prominent characteristic of delayed xenograft rejection, therefore the effects of antiplatelet therapy with aspirin and clopidogrel on long-term cardiac xenograft function was investigated in a heterotopic pig-to-baboon cardiac transplant model. METHODS: Donor hearts from human CD46 transgenic pigs were transplanted heterotopically to baboons. The recipients received immunosuppression that included tacrolimus, sirolimus, corticosteroids, anti-CD20 monoclonal antibody and TPC, an alpha-galactosyl-polyethylene glycol conjugate. In group 1 (n = 9) in addition to immunosuppression, the recipients received combination therapy consisting of aspirin (80 mg/day) and clopidogrel (75 mg/day) beginning 2 days after transplant and continuing until cessation of graft function. Antiaggregatory efficacy was evaluated by platelet aggregation assay. In group 2 (n = 9) antiplatelet drugs were not given. RESULTS: Functional assays confirmed inhibition of platelet aggregation in group 1 suggesting sufficient systemic effects of the treatment. However, anticoagulant therapy did not result in significant prolongation of xenograft function (group 1: median survival 22 days, range 15 to 30 days; group 2: median survival 15 days, range 4 to 53 days). Histologic analysis at rejection revealed no difference in the level of platelet containing thrombi between the groups. CONCLUSIONS: Inhibition of platelet aggregation by a combination of aspirin and clopidogrel did not have a significant impact on the length of xenograft survival or on the development of microvascular thrombosis in this pig-to-primate model.