RESUMEN
N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.
Asunto(s)
Proteínas de Ciclo Celular/química , Diseño de Fármacos , Pirrolidinonas/síntesis química , Factores de Transcripción/química , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Conformación Proteica , Pirrolidinonas/química , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
INTRODUCTION: Although the variability in factor VIII (FVIII):C measurement is well recognized, this has not been widely reported for post-FVIII infusion samples. AIM/METHODS: Three samples from haemophilia A patients were distributed in a UK National External Quality Assessment Scheme survey, each after treatment with either ReFacto AF, Kogenate FS or Advate. Fifty-two UK haemophilia centres performed FVIII assays using one-stage (n = 46) and chromogenic (n = 10) assays. Centres calibrated assays with the local plasma standard and with ReFacto AF laboratory standard for the ReFacto AF sample. RESULTS/CONCLUSIONS: Chromogenic assays gave significantly higher results than one-stage assays (P < 0.0001, 32% difference) in the post-Kogenate sample but not in the post-ReFacto AF (11% higher by chromogenic assay, ns) or post-Advate samples (3% lower by chromogenic, ns) when assays were calibrated with plasma standards. Twenty centres used all Instrumentation Laboratory (IL)-activated partial thromboplastin time reagents (Synthasil)/IL deficient plasma/reference plasma) in the one-stage assay and 15 used all Siemens reagents (Actin FS/Siemens deficient plasma/reference plasma); this made a significant difference to results post-ReFacto AF (41% higher by IL reagents, P < 0.0001) and Advate (39% higher by IL reagents, P < 0.0001), but not Kogenate (7% higher by IL, ns) when calibrated with plasma standards. Differences between results obtained with different one-stage assay reagents for monitoring Advate have implications for dosing patients. Furthermore, there was considerable inter-laboratory variation as indicated by CVs in the range 15-26% for chromogenic assay and 12-19% for one-stage assay results. This study suggests that external quality assessment schemes should offer participation in post-FVIII infusion schemes where haemophilic patients are monitored.
Asunto(s)
Pruebas de Coagulación Sanguínea , Coagulantes/análisis , Factor VIII/análisis , Pruebas de Coagulación Sanguínea/normas , Compuestos Cromogénicos/química , Coagulantes/normas , Coagulantes/uso terapéutico , Factor VIII/normas , Factor VIII/uso terapéutico , Hemofilia A/tratamiento farmacológico , Humanos , Tiempo de Tromboplastina Parcial , Juego de Reactivos para DiagnósticoRESUMEN
von Willebrand disease (VWD) is the most common inherited bleeding disorder, but variable severity and several classification types mean that diagnosis is often not straightforward. In many countries, the assays are not readily available and/or are not well standardized. The latest methods and the basis of VWD are discussed here, together with information from the international quality assessment programme (IEQAS). Factor XIII deficiency is a rare, but important bleeding disorder, which may be missed or diagnosed late. A discussion and update on this diagnosis is considered in the final section of our review.
Asunto(s)
Técnicas de Laboratorio Clínico/normas , Deficiencia del Factor XIII/diagnóstico , Enfermedades de von Willebrand/diagnóstico , Colágeno , Hemaglutininas , Hemofilia A/diagnóstico , Humanos , Agregación Plaquetaria , Control de Calidad , Ristocetina , Factor de von Willebrand/metabolismoRESUMEN
Congenital defects of platelets or plasma proteins involved in blood coagulation generally lead to bleeding disorders. In some of these disorders, patients with a severe phenotype are prone to spontaneous bleeds with critical consequences. This situation occurs more commonly in haemophilia A and haemophilia B and to a certain extent in severe forms (type 3) of von Willebrand disease. Defects in other plasma coagulation proteins and platelet factors are relatively rare, with an incidence of ≤ 1: 1-2 million. Molecular genetic studies of the human coagulation factors, especially factors VIII and IX, have contributed to a better understanding of the biology of these genetic disorders, the accurate detection of carriers and genetic counselling, and have also fostered new therapeutic strategies. This article reviews the evolution of genetics over the last five decades as a tool for bleeding disorder investigations, the recent advances in molecular techniques that have contributed to improved genetic diagnosis of this condition, and the development and utility of proficiency testing programmes and reference materials for genetic diagnosis of bleeding disorders.
Asunto(s)
Trastornos de la Coagulación Sanguínea/genética , Factores de Coagulación Sanguínea/genética , Hemostasis/genética , Biología Molecular/métodos , Trastornos de la Coagulación Sanguínea/diagnóstico , Humanos , Análisis de Secuencia de ADNRESUMEN
N-Methylpyrrolidone is one of several chemotypes that have been described as a mimetic of acetyl-lysine in the development of bromodomain inhibitors. In this paper, we describe the synthesis of a 4-phenyl substituted analogue - 1-methyl-4-phenylpyrrolidin-2-one - and the use of aryl substitution reactions as a divergent route for derivatives. Ultimately, this has led to structurally complex, chiral compounds with progressively improved affinity as inhibitors of bromodomain-containing protein 4.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Diseño de Fármacos , Pirrolidinonas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Transferencia Resonante de Energía de Fluorescencia , Humanos , Modelos Moleculares , Estructura Molecular , Pirrolidinonas/química , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
External quality assessment (EQA) has been shown to improve laboratory performance and diagnosis in haemostasis. We report here findings from the World Federation of Haemophilia (WFH) EQA programme during the period 2004-2007. Samples for PT, APTT, FVIII:C, FIX:C and VWF assays were distributed to centres in both established and emerging countries, and results were compared with results obtained by United Kingdom National External Quality Assessment Scheme (UK NEQAS) participants on the same samples. In general, good agreement was seen throughout between WFH and UK NEQAS for screening tests, and it was possible to identify an improvement in WFH centre agreement for results for VWF assays during the period of study. Agreement between emerging and established WFH centres was comparable for screening tests, possibly indicative of the relative simplicity of these tests and the degree of automation now employed in almost all haemostasis laboratories. However, CVs and performance compared with UK NEQAS participant results for factor assays amongst established centres was better than between emerging centres. Distribution of a questionnaire revealed different application of methodology for these assays, which may contribute to the observed difference in performance. Several centres participated in supplementary exercises, with comparable results obtained by emerging and established centres performing FVIII and fibrinogen measurement on cryoprecipitate, and all centres performing FVIII inhibitor assays correctly identifying the presence of an inhibitor. Participation in EQA programmes should continue to encourage improvement in laboratory performance and therefore improvements in the diagnosis and care of patients with haemophilia.
Asunto(s)
Técnicas de Laboratorio Clínico/normas , Trastornos Hemorrágicos/diagnóstico , Hemostasis , Garantía de la Calidad de Atención de Salud/normas , Factor IX/análisis , Factor VIII/análisis , Humanos , Tiempo de Protrombina , Encuestas y Cuestionarios , Factor de von Willebrand/análisisRESUMEN
INTRODUCTION: Mutations in fibrinogen (Fgn) genes, causing dysfibrinogenaemia, can result in either a bleeding or thrombophilic diathesis. Dysfibrinogenaemia is infrequently encountered in hospital laboratories, and the utility of different assays in the diagnosis of dysfibrinogenaemia has not previously been explored in a multicentre study. We describe here an exercise in which PRO-RBDD project (prospective data collection on patients with fibrinogen and Factor XIII deficiencies) centres, and UK NEQAS centres, performed investigations for dysfibrinogenaemia. METHODS: Samples from donors with dysfibrinogenaemia (sample 1: gamma p.Arg301Cys, sample 2: Bbeta166Arg3Cys-Fgn Longmont, sample 3: Aalpha p.Arg35His) and a normal donor were sent to laboratories for investigation for possible dysfibrinogenaemia. Median, coefficient of variation and range were determined for each assay method. RESULTS: Results were returned from 62 UK NEQAS and 24 PRO-RBDD centres. PT, APTT, Clauss fibrinogen and thrombin times were performed by >90% of centres, with 51% performing reptilase times, and 31% fibrinogen antigen. All centres identified samples 1 and 3 as abnormal. However, 39% of centres reported a normal or raised fibrinogen for the Fgn Longmont sample, and marked differences in Clauss fibrinogen results with different reagents were noted for this sample (median 1.01 g/L vs 5.10 g/L for the two mostly widely used reagents). CONCLUSION: In-house studies suggest that the method of detection of fibrin clot formation may result in different Clauss fibrinogen measurements with FgnLongmont plasma. It is possible that some widely used methodologies, both using optical and mechanical end-point detection systems, will fail to detect this rare fibrinogen variant.
Asunto(s)
Afibrinogenemia/diagnóstico , Afibrinogenemia/genética , Errores Diagnósticos , Fibrinógeno , Pruebas de Coagulación Sanguínea/métodos , Femenino , Fibrinógeno/genética , Fibrinógeno/metabolismo , Humanos , Masculino , Mutación , Reino UnidoRESUMEN
INTRODUCTION: FXIII deficiency is a rare bleeding disorders, and specific FXIII assays are recommended to detect this deficiency. We investigated the performance and accuracy of FXIII investigations in two exercises, comparing centres enrolled in the PRO-RBDD project (prospective data collection on patients with fibrinogen and Factor XIII deficiencies), and UK NEQAS BC centres. METHODS: Samples from a FXIII deficient subject and a normal donor were sent to participating centres, to investigate for FXIII deficiency, and interpret their results. Median, coefficient of variation and range were determined. RESULTS: Results were returned from 98 UK NEQAS BC and 28 PRO-RBDD centres. Up to 40% of UK NEQAS BC and 52% of PRO-RBDD centres reported clot solubility results - with diagnostic errors by two NEQAS BC centres (false negatives for the FXIII deficient sample) and one PRO-RBDD centre (false positive for the normal sample). Over 70% of UK NEQAS BC centres and PRO-RBDD centres performed FXIII assays. Median results were similar between the two groups, with the exception of sample 3 in survey 2 (5.5 vs. 14.0 µ/dl for UK NEQAS BC and PRO-RBDD centres respectively, P < 0.001). Diagnostic errors were made by 2 UK NEQAS BC centres. CONCLUSION: Approximately 70% of centres now employ FXIII assays, complying with international recommendations. However, solubility tests continue to be used. Our data show this can be successful, depending on the sensitivity of the method in use. Diagnostic errors are made by centres using both solubility screens and FXIII assays, and laboratories should ensure good quality assurance procedures to improve diagnostic accuracy.
Asunto(s)
Deficiencia del Factor XIII/diagnóstico , Coagulación Sanguínea , Pruebas de Coagulación Sanguínea/métodos , Pruebas de Coagulación Sanguínea/normas , Servicios de Laboratorio Clínico/normas , Factor XIII , Deficiencia del Factor XIII/sangre , Femenino , Encuestas de Atención de la Salud , Humanos , Laboratorios , Masculino , Garantía de la Calidad de Atención de Salud , Reproducibilidad de los Resultados , Reino UnidoRESUMEN
The recently determined crystal structure of the PR65/A subunit of protein phosphatase 2A reveals the architecture of proteins containing HEAT repeats. The structural properties of this solenoid protein explain many functional characteristics and account for the involvement of solenoids as scaffold, anchoring and adaptor proteins.
Asunto(s)
Fosfoproteínas Fosfatasas/química , Conformación Proteica , Proteína Fosfatasa 2RESUMEN
Phenylalanine hydroxylase (PAH) exists as an equilibrium of dimers and tetramers. However, there is little information concerning the inter- or intra-molecular interactions required for enzyme quaternary structure. It is predicted that the formation of a PAH tetramer will require at least two points of contact per enzyme subunit. Sequence analysis has suggested the existence of a C-terminal domain with characteristics of a leucine zipper or a variant of this called a coiled-coil. By deletion of 24 amino acids from the C-terminus or conversion of leucine 448 to an alanine residue, we have shown that this putative leucine zipper/coiled-coil domain is involved in the assembly of an active enzyme tetramer from dimers. The removal of this C-terminal domain of PAH reduces enzyme activity but does not abolish it. Furthermore, we report that an alanine 447 to aspartate mutation associated with phenylketonuria may affect subunit assembly which suggests the formation of enzyme tetramers is physiologically relevant. Our analysis of subunit interactions in vivo, show that in the absence of the C-terminal coiled-coil domain, dimers can form and this is only possible when the N-terminal domain is present. This provides the first evidence that N-terminal domain is required for multimerisation. We propose that the N-terminal regulatory domain in conjunction with the C-terminal coiled-coil domain, mediates the formation of fully active enzyme tetramers.
Asunto(s)
Fenilalanina Hidroxilasa/metabolismo , Conformación Proteica , Secuencia de Aminoácidos , Animales , Dimerización , Humanos , Cinética , Leucina Zippers/genética , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Fenilcetonurias/genética , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Eliminación de Secuencia/genética , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
INTRODUCTION: External quality assessment (EQA) is an important component of quality assurance for laboratory tests of haemostasis. Lyophilization of plasma confers stability of labile clotting factors, allowing valid comparison of results between participating centres. However, elevated ambient temperatures in some geographical areas could affect the stability of lyophilized samples in transit. METHODS: The effect on lyophilized plasma samples of consistent elevated temperature with respect to haemostasis tests was determined in a single centre. The temperature to which packages were exposed during transit was also monitored. RESULTS: Survey packages were exposed to average temperatures up to 31.9 °C and maximum temperatures up to 39.7 °C over delivery periods between 1 and 8 weeks. In-house studies revealed samples to be stable over a 6-week period at a constant 30 °C, and only small changes were observed for samples exposed to 37 °C for 4 weeks. 6-week storage at 37 °C was associated with average changes of up to 15% in factor assay activity. CONCLUSION: Lyophilized EQA material employed in UK NEQAS surveys is stable under conditions encountered for the majority of participants, but in cases of delayed delivery of samples, the effect of temperature on sample integrity must be considered when assessing laboratory performance.
Asunto(s)
Pruebas de Coagulación Sanguínea/normas , Proteínas Sanguíneas/química , Plasma/química , Liofilización , Humanos , Estabilidad Proteica , Control de Calidad , TemperaturaRESUMEN
Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.
Asunto(s)
Fenilalanina Hidroxilasa/química , Fosfoproteínas/química , Animales , Baculoviridae/genética , Cristalografía por Rayos X , Dimerización , Activación Enzimática , Humanos , Espectrometría de Masas , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fenilalanina Hidroxilasa/genética , Fenilalanina Hidroxilasa/metabolismo , Fenilcetonurias/etiología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Conformación Proteica , Ratas , Proteínas Recombinantes/química , Eliminación de Secuencia , Spodoptera/citologíaRESUMEN
Phenylalanine hydroxylase (PAH) is the enzyme that converts phenylalanine to tyrosine as a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. Over 300 mutations have been identified in the gene encoding PAH that result in a deficient enzyme activity and lead to the disorders hyperphenylalaninaemia and phenylketonuria. The determination of the crystal structure of PAH now allows the determination of the structural basis of mutations resulting in PAH deficiency. We present an analysis of the structural basis of 120 mutations with a 'classified' biochemical phenotype and/or available in vitro expression data. We find that the mutations can be grouped into five structural categories, based on the distinct expected structural and functional effects of the mutations in each category. Missense mutations and small amino acid deletions are found in three categories: 'active site mutations', 'dimer interface mutations', and 'domain structure mutations'. Nonsense mutations and splicing mutations form the category of 'proteins with truncations and large deletions'. The final category, 'fusion proteins', is caused by frameshift mutations. We show that the structural information helps formulate some rules that will help predict the likely effects of unclassified and newly discovered mutations: proteins with truncations and large deletions, fusion proteins and active site mutations generally cause severe phenotypes; domain structure mutations and dimer interface mutations spread over a range of phenotypes, but domain structure mutations in the catalytic domain are more likely to be severe than domain structure mutations in the regulatory domain or dimer interface mutations.
Asunto(s)
Mutación , Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/enzimología , Fenilcetonurias/genética , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Cristalografía por Rayos X , Dimerización , Mutación del Sistema de Lectura , Genotipo , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional/genética , Fenotipo , Fenilalanina Hidroxilasa/biosíntesis , Estructura Terciaria de Proteína/genética , Ratas , Eliminación de Secuencia/genéticaRESUMEN
Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine and inhibited by its cofactor tetrahydrobiopterin (BH(4)). The crystal structure of PAH revealed that the N-terminal sequence of the enzyme (residues 19-29) partially covered the enzyme active site, and suggested its involvement in regulation. We show that the protein lacking this N-terminal sequence does not require activation by phenylalanine, shows an altered structural response to phenylalanine, and is not inhibited by BH(4). Our data support the model where the N-terminal sequence of PAH acts as an intrasteric autoregulatory sequence, responsible for transmitting the effect of phenylalanine activation to the active site.
Asunto(s)
Biopterinas/análogos & derivados , Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/metabolismo , Sitios de Unión , Biopterinas/metabolismo , Biopterinas/farmacología , Quimotripsina/metabolismo , Activación Enzimática/efectos de los fármacos , Modelos Moleculares , Fenilalanina/antagonistas & inhibidores , Fenilalanina/metabolismo , Fenilalanina/farmacología , Fenilalanina Hidroxilasa/antagonistas & inhibidores , Fenilalanina Hidroxilasa/genética , Fosforilación/efectos de los fármacos , Conformación Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia/genéticaRESUMEN
Familial (F)XIII deficiency is an extremely rare bleeding disorder. In most laboratories the diagnosis is initially established through a clot-solubility screening test. We report here results from a series of UK NEQAS (Blood Coagulation). Proficiency Testing investigations, in which laboratories were provided with samples from normal individuals and from various subjects with FXIII deficiency with a request to perform their usual test for this disorder and to provide an interpretation of their results. Over 95% of centers were able to diagnose severe familial FXIII deficiency in previously untreated patients and to identify samples from normal subjects. However, both quantitative and qualitative methods produced widely variable results on samples obtained from previously treated individuals with FXIII deficiency but having measurable levels of FXIII. Data generated by UK NEQAS investigations suggested that solubility tests employing thrombin show greater sensitivity to FXIII deficiency, and this was confirmed in a subsequent single-center study. Our results lead us to recommend the use of thrombin and acetic acid in the clot-solubility screening test. Use of sensitive screening tests, and improvement in the accuracy and precision of quantitative FXIII assays will aid study of the clinical importance of moderate FXIII deficiency.
Asunto(s)
Deficiencia del Factor XIII/diagnóstico , Ácido Acético , Pruebas de Coagulación Sanguínea/normas , Técnicas de Laboratorio Clínico/normas , Salud de la Familia , Humanos , Tamizaje Masivo/métodos , Tamizaje Masivo/normas , Garantía de la Calidad de Atención de Salud , Juego de Reactivos para Diagnóstico , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Trombina , Reino UnidoRESUMEN
We report here results from a United Kingdom National Quality Assessment Scheme (UK NEQAS) exercise in which both plasma spiked with monoclonal antibodies and plasma from a patient known to have lupus anticoagulant (LA) were distributed to 245 hemostasis laboratories with a request for them to test for possible LA using their routine screening procedure. In general, good agreement was seen in the diagnosis of samples spiked with monoclonal antibodies against beta2-glycoprotein 1 (beta2GP1) and prothrombin, the LA-positive patient sample, and a normal pooled plasma; over 87% of centers correctly identified each sample. However, methods employing platelet neutralizing procedures were associated with a higher proportion of false-negative responses with the antiprothrombin-spiked sample, and it is important to recognize that sensitivity and responsiveness of different methods may vary between artificial plasmas and different LA-positive patient plasmas.
Asunto(s)
Anticuerpos Monoclonales/química , Pruebas de Coagulación Sanguínea/métodos , Laboratorios/normas , Inhibidor de Coagulación del Lupus/análisis , Inhibidor de Coagulación del Lupus/sangre , Algoritmos , Glicoproteínas/química , Humanos , Tiempo de Tromboplastina Parcial , Protrombina/química , Control de Calidad , Reproducibilidad de los Resultados , Tromboplastina/biosíntesis , Factores de Tiempo , Reino Unido , Tiempo de Coagulación de la Sangre Total , beta 2 Glicoproteína IRESUMEN
To assess the practicality of the recent Scientific and Standardization committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) recommendations in respect of the classification of hemophilia we distributed samples from three untreated subjects with hemophilia A to 91 UK hemophilia centers (HCs), comprising 20 comprehensive care centers (CCCs) and 71 HCs. Laboratories were requested to perform their routine factor (F)VIII:C assays and to classify the severity of hemophilia. Median values of < 1 U dL-1 were obtained on two samples. However, for each of the two, approximately 30% of laboratories obtained results in the range 1-29 U dL-1 and 1-33 U dL-1 respectively. For one of these samples 17 laboratories diagnosed severe hemophilia despite obtaining FVIII:C levels in the range 1-5 U dL-1. The median FVIII:C for the third sample was 5.8 U dL-1 with a range of 1.5-36 U dL-1. For this sample eight centers diagnosed severe hemophilia. Fifty-four laboratories obtained a result > 5 U dL-1; 21 of these diagnosed mild hemophilia, 31 moderate hemophilia and two severe hemophilia. Results from CCCs were more accurate and more precise than those from HCs. Our results indicate a need for improved standardization of FVIII assays. In the UK there remains a lack of consensus in respect of the laboratory diagnostic criteria for the classification of hemophilia A.
Asunto(s)
Factor VIII/análisis , Hemofilia A/clasificación , Técnicas de Laboratorio Clínico/normas , Hemofilia A/diagnóstico , Humanos , Estándares de Referencia , Reino UnidoRESUMEN
Acute cellular rejection and hepatic vessel thrombosis are significant postoperative complications of liver transplantation. The study investigated changes in endothelial cell-related hemostatic proteins in the peripheral circulation of patients after liver transplantation, and assays for hemostatic parameters were compared with data from routine hematologic and biochemical investigations, together with clinical information. Of the 12 patients, 8 underwent acute rejection episodes. No significant differences in any hemostatic parameter measured were seen between rejection and nonrejection groups, with the exception of the platelet count, which increased after treatment of the rejection episode. Two of the 12 patients suffered fatal hepatic vessel thrombosis during the study. A number of significant differences were found between these patients and those with no thrombotic complications, most notably and increase in the von Willebrand factor antigen to ristocetin cofactor ratio and thrombin-antithrombin complex generation. These changes occurred before clinical detection of thrombosis. Thus, measurement of these parameters may be of predictive value in the diagnosis and monitoring of post-transplant thrombosis.