RESUMO
The REG2 Anticoagulation System consists of pegnivacogin, a subcutaneously administered aptamer factor IXa inhibitor, and its intravenous active control agent, anivamersen. Its effect on thrombin generation is unknown. A prospectively designed thrombin generation study was conducted within the phase 1 ascending dose study of REG2 to assess the effect of REG2 on thrombin generation kinetics. A total of 32 healthy volunteers were recruited into four cohorts of ascending dose pegnivacogin for the phase 1 study. In this pre-specified substudy, blood samples were drawn in the presence or absence of corn trypsin inhibitor at specified times within each dosing cohort. Thrombin generation was initiated with tissue factor and thrombin generation kinetics were measured using the Calibrated Automated Thrombogram (CAT). REG2 attenuated thrombin generation in a dose-dependent manner. All parameters of the CAT assay, except for lag time, showed a dose and concentration-dependent response to pegnivacogin [time to peak thrombin generation (PTm), endogenous thrombin potential, peak thrombin generation, and velocity index (VIx)]. Reversal of the effect of pegnivacogin with anivamersen demonstrated restoration of thrombin generation without rebound effect. This first-in-human study of the effect of the REG2 Anticoagulation System on thrombin generation demonstrates concentration-dependent suppression of thrombin generation that is reversible without rebound effect, as measured by the CAT assay.
Assuntos
Anticoagulantes/administração & dosagem , Aptâmeros de Nucleotídeos/administração & dosagem , Fator IXa/antagonistas & inibidores , Trombina/metabolismo , Adulto , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Masculino , Estudos Prospectivos , Tempo de Trombina/instrumentação , Tempo de Trombina/métodosRESUMO
May-Hegglin anomaly (MHA) is an autosomal dominant macrothrombocytopenia of unclear pathogenesis characterized by thrombocytopenia, giant platelets and leukocyte inclusions. Studies have indicated that platelet structure and function are normal, suggesting a defect in megakaryocyte fragmentation. The disorder has been linked to chromosome 22q12-13. Here we screen a candidate gene in this region, encoding non-muscle myosin heavy chain A (MYH9), for mutations in ten families. In each family, we identified one of three sequence variants within either the -helical coiled coil or the tailpiece domain that co-segregated with disease status. The E1841K mutation was found in 5 families and occurs at a conserved site in the rod domain. This mutation was not found in 40 normal individuals. Four families had a nonsense mutation that resulted in truncation of most of the tailpiece. One family had a T1155I mutation present in an affected mother and daughter, but not in the mother's parents, thus representing a new mutation. Among the 30 affected individuals, 21 unaffected individuals and 13 spouses in the 10 families, there was correlation of a variant of MYH9 with the presence of MHA. The identification of MYH9 as the disease gene for MHA establishes the pathogenesis of the disorder, should provide further insight into the processes of normal platelet formation and may facilitate identification of the genetic basis of related disorders.
Assuntos
Plaquetas/patologia , Leucócitos/patologia , Proteínas Motores Moleculares , Mutação , Cadeias Pesadas de Miosina/genética , Trombocitopenia/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromossomos Humanos Par 22 , Sequência Conservada , Análise Mutacional de DNA , Éxons , Saúde da Família , Feminino , Genes Dominantes , Genótipo , Haplótipos , Humanos , Masculino , Repetições de Microssatélites , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/química , Linhagem , Polimorfismo de Fragmento de Restrição , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Patients who develop thrombocytopenia and heparin-dependent platelet factor 4 antibodies while on or shortly after receiving a heparin product are often considered for alternative anticoagulation to minimize the occurrence of life and limb-threatening events. We retrospectively reviewed the hospital records of 97 patients with heparin-dependent platelet factor 4 antibodies (at least 65 of whom were felt by the primary team to have HIT) to determine the influence of renal performance on alternative anticoagulant selection and associated clinical events. For GFR > 30, approximately 30% of patients who did not receive alternative anticoagulation had documentation of concern for HIT versus 60% of patients in the GFR < 30 group. We found that a smaller proportion of patients with severe renal insufficiency, GFRs < 30 ml/min/1.73 m(2) were treated with an alternative anticoagulant-this despite their high incidence of thromboembolic events and comparable rates of HIT. Overall, rates of hemorrhage did not differ between patients when compared to those without renal insufficiency. However, there was a higher percentage of hemorrhagic events for patients with GFR < 30 ml/min/1.73 m(2) on alternative anticoagulants. This study demonstrates that patient's with GFRs < 30 ml/min/1.73 m(2) need to be assessed for overall hemorrhagic risk at the time of starting an alternative anticoagulant and need to be monitored closely to avoid hemorrhagic events.
Assuntos
Anticoagulantes/efeitos adversos , Heparina/efeitos adversos , Fator Plaquetário 4/imunologia , Insuficiência Renal/complicações , Trombocitopenia/complicações , Idoso , Feminino , Taxa de Filtração Glomerular , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Trombocitopenia/induzido quimicamenteRESUMO
von Willebrand disease (VWD) is a commonly encountered inherited bleeding disorder affecting both males and females, causing mucous membrane and skin bleeding symptoms, and bleeding with surgical or other haemostatic challenges. VWD may be disproportionately symptomatic in women of child-bearing age. It may also occur less frequently as an acquired disorder (acquired von Willebrand syndrome). VWD is caused by deficiency or dysfunction of von Willebrand factor (VWF), a plasma protein that mediates platelet haemostatic function and stabilizes blood coagulation factor VIII. The pathophysiology, classification, diagnosis and management of VWD are relatively complex, but understanding them is important for proper diagnosis and management of patients with VWD. These evidence-based guidelines for diagnosis and management of VWD from the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel (USA) review relevant publications, summarize current understanding of VWD pathophysiology and classification, and present consensus diagnostic and management recommendations based on analysis of the literature and expert opinion. They also suggest an approach for clinical and laboratory evaluation of individuals with bleeding symptoms, history of bleeding or conditions associated with increased bleeding risk. This document summarizes needs for further research in VWF, VWD and bleeding disorders, including clinical research to obtain more objective information about bleeding symptoms, advancements in diagnostic and therapeutic tools, and enhancement in the education and training of clinicians and scientists in bleeding and thrombotic disorders. The NHLBI Web site (http://www.nhlbi.nih.gov/guidelines/vwd) has a more detailed document, a synopsis of these recommendations, and patient education information.
Assuntos
Doenças de von Willebrand/diagnóstico , Doenças de von Willebrand/tratamento farmacológico , Antifibrinolíticos/uso terapêutico , Desamino Arginina Vasopressina/uso terapêutico , Fator VIII/análise , Feminino , Terapia Genética/métodos , Hemostáticos/uso terapêutico , Humanos , Masculino , Gravidez , Fator de von Willebrand/administração & dosagem , Fator de von Willebrand/análiseRESUMO
Coagulation Factor V is an essential component of the prothrombinase complex, which activates the zymogen prothrombin to thrombin. A patient was described who developed a Factor V inhibitor that neutralized the procoagulant activity of Factor V and resulted in a fatal hemorrhagic diathesis (Coots, M. C., A. F. Muhleman, and H. I. Glueck. 1978. Am. J. Hematol. 4:193-206). This inhibitor was shown to be an IgG antibody that bound to the light chain of Factor V. Using a series of light chain deletion mutants, we have found that this antibody binds to the second C-type domain of the light chain. Both inhibitor IgG and Fab fragments rapidly neutralized the procoagulant activity of Factor Va, implying that the neutralization resulted from specific binding to the C2 domain. We have previously demonstrated that deletion of the C2 domain results in loss of procoagulant activity, as well as loss of phosphatidylserine-specific binding. Confirming these results, both inhibitor IgG and Fab fragments interfered with phosphatidylserine-specific binding of Factor V. Conversely, preincubation of Factor Va with procoagulant phospholipids protected the cofactor from inactivation by the inhibitor. Our results suggest that this inhibitor neutralizes the procoagulant activity of Factor Va by interfering with the C2-mediated interaction with phospholipid surfaces, thereby disrupting formation of the prothrombinase complex.
Assuntos
Fator V/antagonistas & inibidores , Transtornos Hemorrágicos/imunologia , Fosfatidilserinas/metabolismo , Reações Antígeno-Anticorpo , Sequência de Bases , Coagulação Sanguínea , Análise Mutacional de DNA , Epitopos , Fator V/química , Fator V/imunologia , Fator Va/antagonistas & inibidores , Fator Va/imunologia , Fator Va/metabolismo , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Testes de Precipitina , Proteínas Recombinantes/química , Deleção de SequênciaRESUMO
Essentials We evaluated antibody status, thromboembolism and survival after cardiac surgery. Positive antibody tests are common - over 50% are seropositive at 30 days. Seropositivity did not increase thromboembolism or impair survival after cardiac surgery. Results show heparin induced thrombocytopenia antibody screening after surgery is not warranted. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is a prothrombotic response to heparin therapy with platelet-activating, anti-platelet factor 4 (PF4)/heparin antibodies leading to thrombocytopenia associated with thromboembolism. Objective We tested the hypothesis that anti-PF4/heparin antibodies are associated with thromboembolism after cardiac surgery. Methods This multicenter, prospective cohort study collected laboratory and clinical data up to 30 days after surgery and longer-term clinical follow-up data. The primary outcome variable combined new arterial or venous thromboembolic complications (TECs) with all-cause death until 90 days after surgery. Laboratory analyses included platelet counts and anti-PF4/heparin antibody titers (GTI ELISA), with a confirmatory excess heparin step and serotonin release assay. Chi-square testing was used to test the relationship between our outcome and HIT antibody seropositivity. Results Initially, 1021 patients were enrolled between August 2006 and May 2009, and follow-up was completed in December 2014. Seropositivity defined by OD > 0.4 was common, being almost 20% preoperatively, > 30% by discharge, and > 60% by day 30. Death (1.7% within 30 days) or TECs (69 in total) were more likely if the partient was seronegative (OD < 0.4), but positivity defined by OD > 1.0 or including an excess heparin confirmatory step resulted in equal incidence of death or TECs, whether the patient was seronegative or seropositive. Incorporating the serotonin release assay for platelet-activating antibodies did not alter these findings. Conclusions Seropositivity for anti-PF4/heparin antibodies does not increase the risk of death or thromboembolism after cardiac surgery. Screening is not indicated, and seropositivity should only be interpreted in the context of clinical evidence for HIT. TRIAL REGISTRATION: Duke IRB Protocol #00010736.
Assuntos
Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Heparina/efeitos adversos , Fator Plaquetário 4/imunologia , Trombocitopenia/induzido quimicamente , Tromboembolia/etiologia , Idoso , Anticorpos/sangue , Anticoagulantes/efeitos adversos , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Estudos Prospectivos , Tamanho da Amostra , Tromboembolia/sangue , Tromboembolia/terapia , Resultado do TratamentoRESUMO
BACKGROUND: Closure time (CT), measured by platelet function analyzer (PFA-100) device, is now available to the clinical laboratory as a possible alternative or supplement to the bleeding time test. AIM: On behalf of the Platelet Physiology Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH-SSC), a working Group was formed to review and make recommendations on the use of the PFA-100 CT in the evaluation of platelet function within the clinical laboratory. METHODS: The Medline database was searched to review the published information on the PFA-100 CT in the evaluation of platelet disorders and platelet function. This information, and expert opinion, was used to prepare a report and generate consensus recommendations. RESULTS: Although the PFA-100 CT is abnormal in some forms of platelet disorders, the test does not have sufficient sensitivity or specificity to be used as a screening tool for platelet disorders. A role of the PFA-100 CT in therapeutic monitoring of platelet function remains to be established. CONCLUSIONS: The PFA-100 closure time should be considered optional in the evaluation of platelet disorders and function, and its use in therapeutic monitoring of platelet function is currently best restricted to research studies and prospective clinical trials.
Assuntos
Transtornos Plaquetários/diagnóstico , Testes de Função Plaquetária/instrumentação , Transtornos Plaquetários/sangue , Transtornos Plaquetários/congênito , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Ponte Cardiopulmonar/efeitos adversos , Doença das Coronárias/sangue , Hemorragia/sangue , Hemorragia/terapia , Humanos , Hepatopatias/sangue , Inibidores da Agregação Plaquetária/farmacologia , Contagem de Plaquetas , Uremia/sangueRESUMO
BACKGROUND: Treatment of venous thromboembolism (VTE) in patients with cancer has a high rate of recurrence and bleeding complications. Guidelines recommend low-molecular-weight heparin (LMWH) for at least 3-6 months and possibly indefinitely for patients with active malignancy. There are, however, few data supporting treatment with LMWH beyond 6 months. The primary aim of the DALTECAN study (NCT00942968) was to determine the safety of dalteparin between 6 and 12 months in cancer-associated VTE. METHODS: Patients with active cancer and newly diagnosed VTE were enrolled in a prospective, multicenter study and received subcutaneous dalteparin for 12 months. The rates of bleeding and recurrent VTE were evaluated at months 1, 2-6 and 7-12. FINDINGS: Of 334 patients enrolled, 185 and 109 completed 6 and 12 months of therapy; 49.1% had deep vein thrombosis (DVT); 38.9% had pulmonary embolism (PE); and 12.0% had both on presentation. The overall frequency of major bleeding was 10.2% (34/334). Major bleeding occurred in 3.6% (12/334) in the first month, and 1.1% (14/1237) and 0.7% (8/1086) per patient-month during months 2-6 and 7-12, respectively. Recurrent VTE occurred in 11.1% (37/334); the incidence rate was 5.7% (19/334) for month 1, 3.4% (10/296) during months 2-6, and 4.1% (8/194) during months 7-12. One hundred and sixteen patients died, four due to recurrent VTE and two due to bleeding. CONCLUSION: Major bleeding was less frequent during dalteparin therapy beyond 6 months. The risk of developing major bleeding complications or VTE recurrence was greatest in the first month of therapy and lower over the subsequent 11 months.
Assuntos
Anticoagulantes/administração & dosagem , Dalteparina/administração & dosagem , Neoplasias/complicações , Tromboembolia Venosa/tratamento farmacológico , Idoso , Anticoagulantes/efeitos adversos , Canadá , Dalteparina/efeitos adversos , Esquema de Medicação , Europa (Continente) , Feminino , Hemorragia/induzido quimicamente , Humanos , Injeções Subcutâneas , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Neoplasias/diagnóstico , Neoplasias/mortalidade , Estudos Prospectivos , Recidiva , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Estados Unidos , Tromboembolia Venosa/sangue , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/etiologia , Tromboembolia Venosa/metabolismoRESUMO
BACKGROUND: Recommendations for management of cancer-related venous thromboembolism (VTE) in patients already receiving anticoagulant therapy are based on low-quality evidence. This international registry sought to provide more information on outcomes after a breakthrough VTE in relation to anticoagulation strategies. METHODS: Patients with cancer and VTE despite anticoagulant therapy were reported to the registry. Data on treatments, VTE events, major bleeding, residual thrombosis symptoms and death were collected for the following 3 months. Breakthrough VTE and subsequent recurrences were objectively verified. Outcomes with different treatment strategies were compared with Cox proportional hazards regression. RESULTS: We registered 212 patients with breakthrough VTE. Of those, 59% had adenocarcinoma and 73% had known metastases. At the time of the breakthrough event, 70% were on low-molecular-weight heparin (LMWH) and 27% on a vitamin K antagonist (VKA); 70% had a therapeutic or supratherapeutic dose. After breakthrough the regimen was: unchanged therapeutic dose in 33%, dose increased in 31%, switched to another drug in 24%; and other management in 11%. During the following 3 months 11% had another VTE, 8% had major bleeding and 27% died. Of the survivors, 74% had residual thrombosis symptoms. Additional VTE recurrence was less common with LMWH than with a VKA (hazard ratio [HR], 0.28; 95% confidence interval [CI], 0.11-0.70) but similar with unchanged or increased anticoagulant intensity (HR, 1.09; 95% CI, 0.45-2.63). The bleeding rate did not increase significantly with dose escalation. CONCLUSION: Morbidity and mortality are high after recurrence of cancer-related VTE despite anticoagulation. Further treatment appears to be more effective with LMWH than with a VKA.
Assuntos
Anticoagulantes/administração & dosagem , Heparina de Baixo Peso Molecular/administração & dosagem , Neoplasias/complicações , Tromboembolia Venosa/tratamento farmacológico , Varfarina/administração & dosagem , Idoso , Anticoagulantes/efeitos adversos , Distribuição de Qui-Quadrado , Substituição de Medicamentos , Feminino , Hemorragia/induzido quimicamente , Heparina de Baixo Peso Molecular/efeitos adversos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Neoplasias/mortalidade , Neoplasias/patologia , Modelos de Riscos Proporcionais , Estudos Prospectivos , Recidiva , Sistema de Registros , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Tromboembolia Venosa/sangue , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/etiologia , Tromboembolia Venosa/mortalidade , Vitamina K/antagonistas & inibidores , Varfarina/efeitos adversosRESUMO
BACKGROUND: Post-thrombotic syndrome (PTS) is a frequent chronic complication of deep vein thrombosis (DVT). OBJECTIVE: In the BioSOX study, we investigated whether inflammation markers predict the risk of PTS after DVT. METHODS: We measured C-reactive protein (CRP), ICAM-1, interleukin (IL)-6, and IL-10, at baseline, and 1 month and 6 months after a first proximal DVT, among 803 participants in the SOX trial. Participants were prospectively followed for 24 months for development of PTS. RESULTS: Median CRP levels at 1 month, ICAM-1 levels at baseline, 1 month and 6 months, IL-6 levels at 1 month and 6 months and IL-10 levels at 6 months were higher in patients who developed PTS than in those who did not. Multivariable regression with the median as a cutoff showed risk ratios (RRs) for PTS of 1.23 (95% confidence interval [CI] 1.05-1.45) and 1.25 (95% CI 1.05-1.48) for ICAM-1 at 1 month and 6 months, respectively, and 1.27 (95% CI 1.07-1.51) for IL-10 at 6 months. Quartile-based analysis demonstrated a dose-response association between ICAM-1 and PTS. ICAM-1 and IL-10 were also associated with PTS severity. Analysis of biomarker trajectories after DVT demonstrated an association between the highest-trajectory group of ICAM-1 and PTS. CONCLUSIONS: In this prospective study, ICAM-1 over time was most consistently associated with the risk of PTS. Further study is required to confirm these findings and assess their potential clinical relevance.
Assuntos
Mediadores da Inflamação/sangue , Molécula 1 de Adesão Intercelular/sangue , Síndrome Pós-Trombótica/etiologia , Trombose Venosa/sangue , Adulto , Idoso , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Canadá , Distribuição de Qui-Quadrado , Feminino , Seguimentos , Humanos , Interleucina-10/sangue , Interleucina-6/sangue , Modelos Lineares , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Síndrome Pós-Trombótica/diagnóstico , Síndrome Pós-Trombótica/prevenção & controle , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Meias de Compressão , Fatores de Tempo , Resultado do Tratamento , Estados Unidos , Trombose Venosa/complicações , Trombose Venosa/diagnóstico , Trombose Venosa/terapiaRESUMO
A rapidly acting anticoagulant that can either inhibit thrombin generation or inhibit thrombin itself is the optimum therapy for acute thrombosis associated with heparin-induced thrombocytopenia (HIT). In this review, the newer treatment approaches that fulfill this requirement are discussed. These newer treatments include hirudin and argatroban, direct thrombin inhibitors, and danaparoid, which inhibits thrombin generation. Preliminary outcome results from the extensive compassionate-use program for danaparoid in HIT and from a recently completed randomized clinical trial that compared danaparoid with dextran in patients with HIT are provided. Based on these data, danaparoid appears to be a useful and safe replacement for heparin in patients who develop HIT.
Assuntos
Heparina/efeitos adversos , Trombocitopenia/induzido quimicamente , Trombocitopenia/tratamento farmacológico , Anticoagulantes/uso terapêutico , Arginina/análogos & derivados , Sulfatos de Condroitina/uso terapêutico , Dermatan Sulfato/uso terapêutico , Combinação de Medicamentos , Heparitina Sulfato/uso terapêutico , Terapia com Hirudina , Humanos , Ácidos Pipecólicos/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , SulfonamidasRESUMO
Acute lung injury (ALI) is characterized by fibrin deposition in the tissue and vascular spaces. Coagulation is activated after exposure to endotoxin or bacteria, and a procoagulant environment rapidly develops in the vascular, interstitial, and alveolar spaces of the lung. These changes are tissue factor (TF)-dependent and associated with increases in inflammatory cytokines. Procoagulant changes also occur in the lungs of patients with the acute respiratory distress syndrome (ARDS), suggesting that epithelial inflammation activates the extrinsic pathway. Many inflammatory mediators have specific effects on coagulation; however, the role of TF in regulation of pulmonary inflammatory responses is less clear. Here we report initial data on blockade of TF-initiated coagulation in baboons with Escherichia coli sepsis-induced ALI, using active site-inactivated FVIIa (FVIIai ASIS). Treatment with FVIIai prevented plasma fibrinogen depletion and attenuated fibrin deposition in the tissues. The drug also decreased systemic cytokine responses and inflammatory changes in the lung, including neutrophil infiltration, and decreased edema. Coagulation blockade with FVIIai improved lung function by preserving gas exchange and compliance, decreased pulmonary hypertension, and enhanced renal function. These results show that TF-FVIIa complex is an important regulatory site for the pathologic response of the lung to sepsis.
Assuntos
Síndrome do Desconforto Respiratório/metabolismo , Tromboplastina/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Papio , Síndrome do Desconforto Respiratório/microbiologia , Síndrome do Desconforto Respiratório/fisiopatologia , Tromboplastina/fisiologiaRESUMO
Four factor VIII light chain constructs containing hemophilia A mutations at R2304 and R2307 were prepared and expressed in mammalian cells. These mutations are located in a putative phosphatidylserine binding site identified by peptide studies (spanning amino acids 2303-2332). The levels of all four mutants in conditioned medium were significantly less than wild type by immunoprecipitation and ELISA. R2304H and wild type factor VIII light chains were concentrated by cation exchange chromatography from medium. R2304H and wild type factor VIII light chains bound immobilized phosphatidylserine similarly. The reconstituted cofactor activity of R2304H factor VIII light chain was slightly greater than wild type factor VIII light chain. These results are consistent with the recently reported crystal structure of factor VIII C2 domain that suggests R2304H is not directly involved in phospholipid binding. The observed clinical phenotype is probably due to decreased circulating levels of a functional protein.
Assuntos
Cisteína Endopeptidases/metabolismo , Fator VIII/genética , Fator VIII/metabolismo , Proteínas de Neoplasias , Fosfatidilserinas/metabolismo , Substituição de Aminoácidos , Animais , Células COS , Linhagem Celular , Hemofilia A/genética , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica , TransfecçãoRESUMO
Factor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIII/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIII A1 domain dramatically reduced secretion of factor V approximately 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII approximately 3-fold, whereas substitution of the factor V A2 domain actually reduced secretion approximately 4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.
Assuntos
Fator VIII/metabolismo , Fator V/química , Fragmentos de Peptídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Mapeamento de Epitopos , Fator VIII/imunologia , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismoRESUMO
We evaluated the utility of the PFA-100 platelet function analyzer in identifying disorders in platelet function and/or von Willebrand factor (vWF) in patients with various systemic disorders being followed at a tertiary care center. Closure times were determined with collagen/ ADP (CADP) and collagen/epinephrine (CEPI) cartridges for 305 patients, and abnormal results were further evaluated with platelet aggregometry and vWF analysis. Prolonged CADP and/or CEPI closure times were identified in 114 patients (37.3%), but most were isolated prolonged CEPI closure times predominantly due to aspirin therapy (79 patients). Prolonged CADP closure times were most frequently due to qualitative platelet defects and/or decreased vWF levels. Prolonged CADP closure times were encountered most frequently in patients with sickle cell disease and were associated with a decreased hematocrit. This study demonstrated that the PFA-100 analyzer can accurately assess vWF-dependent platelet function and detect other platelet defects under high shear stress in complex patient populations.
Assuntos
Transtornos Plaquetários/diagnóstico , Hospitais Universitários , Ambulatório Hospitalar , Testes de Função Plaquetária/instrumentação , Doenças de von Willebrand/diagnóstico , Difosfato de Adenosina/farmacologia , Adulto , Idoso , Anemia/sangue , Anemia Falciforme/sangue , Transtornos Plaquetários/sangue , Colágeno/farmacologia , Epinefrina/farmacologia , Feminino , Seguimentos , Hematócrito , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , North Carolina/epidemiologia , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária , Gravidez , Estresse Mecânico , Fatores de Tempo , Doenças de von Willebrand/sangue , Doenças de von Willebrand/epidemiologiaRESUMO
Many of the autoantibodies in antiphospholipid syndrome (APS) are directed against beta2-glycoprotein I (beta2-GPI). Recent studies from our laboratories have indicated that the immunodominant binding epitope(s) for high titer, affinity purified antibodies from 11 APS patients are localized to the amino terminal domain (domain 1) of beta2-GPI. The present study employed surface plasmon resonance to localize the immunodominant domain in serum samples from a large cohort of patients with GPL values ranging from 21 to 230 units (n = 106 patients). Eighty-eight percent of patients showed > or = threefold selectivity for beta2-GPI containing domain 1 relative to the domain deletion mutant that lacked domain 1. The domain 1 binding activity in patient serum was abolished by removing the IgG fraction from the serum and the binding activity could be fully reconstituted with the IgG fraction. Thus, analysis of serum samples from a large cohort of APS patients indicates that the immunodominant binding epitope(s) for anti-beta2 antibodies are localized to the amino terminal domain of beta2-GPI.
Assuntos
Anticorpos Anticardiolipina/imunologia , Glicoproteínas/imunologia , Adulto , Anticorpos Anticardiolipina/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Síndrome Antifosfolipídica/imunologia , Autoanticorpos/análise , Autoanticorpos/imunologia , Estudos de Casos e Controles , Estudos de Coortes , Epitopos/análise , Epitopos/química , Epitopos/metabolismo , Feminino , Glicoproteínas/metabolismo , Humanos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , beta 2-Glicoproteína IRESUMO
Progressive thrombocytopenia may develop in as many as 5% of patients receiving heparin anticoagulation. In these patients, the risk of thromboembolic complications as well as continued thrombocytopenia necessitates discontinuation of heparin and initiation of an alternative anticoagulant when indicated. The heparinoid Lomoparan (Org 10172) is a mixture of several non-heparin low molecular weight glycosaminoglycans with proven anticoagulant efficacy that is generally non-reactive with platelets in the presence of plasma from patients with heparin induced thrombocytopenia, whereas standard heparin will induce platelet aggregation. We evaluated the role of heparinoid as a potential alternative anticoagulant in patients with heparin induced thrombocytopenia. During a 6 month period, we identified six patients with heparin induced thrombocytopenia who required an alternative parenteral anticoagulant, four as primary treatment for specific medical problem, and two as anticoagulation during a necessary surgical procedure. Heparinoid was used successfully in both medical and surgical patients requiring parenteral anticoagulation. In no case was there an exacerbation of the thrombocytopenia nor thromboembolic complications while on heparinoid therapy. Three of our patients sustained hemorrhagic complications, predominantly in the post-surgical setting in association with elevated anti-factor Xa levels and additional anticoagulant agents. We feel that these results confirm the utility of heparinoid anticoagulation in a select subset of patients with heparin induced thrombocytopenia who require continued parenteral anticoagulation.
Assuntos
Anticoagulantes/uso terapêutico , Sulfatos de Condroitina , Dermatan Sulfato , Glicosaminoglicanos/uso terapêutico , Heparina/efeitos adversos , Heparinoides/farmacologia , Heparitina Sulfato , Trombocitopenia/induzido quimicamente , Trombose/induzido quimicamente , Anticoagulantes/efeitos adversos , Feminino , Glicosaminoglicanos/efeitos adversos , Heparinoides/efeitos adversos , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos , Trombocitopenia/sangue , Trombocitopenia/tratamento farmacológico , Trombose/sangue , Trombose/tratamento farmacológicoRESUMO
Hemorrhagic factor V inhibitors frequently bind to the second C-type (C2) domain of factor V and interfere with phospholipid binding. To define specific residues recognized by inhibitors from four patients (one bovine thrombin-induced and three spontaneous antibodies), epitope mapping was performed using recombinant human factor V lacking most of the B-type domain (FV des B) and alanine-substituted mutants within the C2 domain (FV des B C2 mutants). FV des B C2 mutants located in the region between Lys2060 and Glu2069 were resistant to inhibition by three IgG preparations including the bovine thrombin-induced antibody in both prothrombinase and phospholipid-binding assays. In contrast, mutations at Lys2087 and Lys2092/Glu2096 were significantly resistant to inhibition by the fourth IgG preparation in both prothrombinase and phospholipid-binding assays. These results confirm interference of phospholipid binding by hemorrhagic factor V inhibitors and support the role(s) of these residues in phospholipid binding.
Assuntos
Anticorpos/imunologia , Mapeamento de Epitopos/métodos , Fator V/imunologia , Mutação/imunologia , Idoso , Anticorpos/farmacologia , Sítios de Ligação , Testes de Coagulação Sanguínea , Fator V/genética , Fator V/metabolismo , Hemorragia/etiologia , Humanos , Masculino , Membranas Artificiais , Fosfolipídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , TromboplastinaRESUMO
Bovine thrombin is often used topically to promote hemostasis during vascular surgery, including dialysis-access placement. Patients frequently develop antibodies to bovine thrombin preparations, and some may develop antiphospholipid antibodies. We evaluated 88 hemodialysis patients for the presence of antibodies to topical bovine thrombin to determine if elevated antibody levels correlated with vascular access thrombosis. Twenty-seven patients (30.7%) had elevated antibody levels to topical bovine thrombin. More patients with elevated antibody levels had prior vascular access thrombosis than patients with normal antibody levels (13 of 27 versus 5 of 61 patients; P < 0.001). This difference was almost entirely the result of greater levels of thrombosis in patients with polytetrafluoroethylene (PTFE) grafts and elevated antibody levels. In these patients, 11 of 13 patients (84.6%) with elevated antibody levels had a previous thrombosis compared with 2 of 15 patients (13. 3%) with normal antibody levels (P < 0.001). Patients with elevated antibody levels and PTFE grafts also had more prior thromboses (1.92 +/- 1.60 versus 0.133 +/- 0.35 thromboses; P < 0.01) and a greater thrombosis rate (66.89 +/- 63.71 versus 4.65 +/- 12.05 thromboses/100 patient-years; P < 0.01) than patients with normal antibody levels. There were no differences in the frequency of myocardial infarction, coronary artery bypass, access age, presence of diabetes mellitus, platelet counts, anticardiolipin antibody, albumin, lactate dehydrogenase, or C-reactive protein levels. In conclusion, patients with PTFE grafts and elevated antibody levels to topical bovine thrombin had significantly more vascular access thrombosis.
Assuntos
Anticorpos/sangue , Cateteres de Demora/efeitos adversos , Diálise Renal , Trombina/imunologia , Trombose/imunologia , Idoso , Animais , Bovinos , Feminino , Humanos , MasculinoRESUMO
PURPOSE: The purpose of this study was to determine the frequency and types of abnormalities at arteriography in patients with antiphospholipid antibodies (APA) and ischemic cerebrovascular events. METHODS: Twenty-three patients with APA and ischemic cerebrovascular events who underwent arteriography were identified. Patients over the age of 65 years were excluded. No patients met diagnostic criteria for systemic lupus erythematosus. All angiograms were reviewed by two neuroradiologists. RESULTS: Seventeen patients (74%) between the ages of 28 and 64 years (average age, 40 years) had abnormal angiograms. Sixteen patients had arterial abnormalities and one had dural sinus thrombosis. Ten had solely intracranial abnormalities (nine arterial and one venous), six had solely extracranial arterial abnormalities, and one had both intracranial and extracranial arterial abnormalities. Intracranial arterial abnormalities included stem or branch occlusions of the cerebral or basilar arteries, which were generally solitary (six patients), and findings suggestive of vasculitis (four patients). Four patients had stenoses of the origins of two or more great vessels. Two patients had extracranial internal carotid artery stenoses or occlusions that were not typical of atheromatous disease, considered to be embolic in one patient. In another patient, a stenosis of the origin of the internal carotid artery was present that appeared typical of atheromatous disease. Infarctions were seen on CT or MR studies in 13 of 17 patients with abnormal angiograms. CONCLUSION: In our group of patients, typical atheromatous lesions at the common carotid artery bifurcation were rare. Some lesions that are infrequent in the general stroke population (eg, vasculitis-like findings and stenoses at the origin of great vessels) were common. Patients with APA and cerebrovascular events appear to differ from the general stroke population with regard to types of arterial abnormalities seen at arteriography.