RESUMEN
A new robust high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS)-based screening method for angiotensin-converting enzyme (ACE)-inhibiting substances in crude samples is described. The ACE assay is carried out in a typical offline setup by incubation of the samples with ACE and angiotensin I (AI), followed by stopping the reaction with acetonitrile containing val(5)-AI serving as internal standard (I.S.). AI and the product angiotensin II (AII) are extracted from the incubation mixture by turbulent-flow chromatography (TFC) applied in backflush mode as online solid-phase extraction and are directly quantified by ESI(+)-MS. The presence of ACE inhibitors (ACEi) is detected by an increase in AI signal intensity and a corresponding decrease of AII signal, as compared to the blank assay. The overall time of analysis of the TFC/ESI-MS method was 5 min, thus making the described setup suitable for a rapid screening method. The assay was validated using a known ACE inhibitor and the IC(50) values found were in good accordance with a common HPLC/UV method and literature data. The method was successfully applied for the screening of size-exclusion chromatography fractions of the venom of the pitviper Bothrops moojeni. Three of 18 analyzed fractions inhibited ACE, due to peptides present as components of this snake venom. These compounds were extracted from the two most-active fractions by means of TFC and isolated by means of HPLC. Three peptides with ACE inhibitory activity were characterized and their structures were elucidated with ESI-MS/MS-based de novo sequencing to be ZKWPPGKVPP, ZKWPRPGPEIPP and ZNWPRPGPEIPP, respectively (Z = pyroglutamic acid).
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/análisis , Cromatografía Líquida de Alta Presión/métodos , Mezclas Complejas/química , Venenos de Crotálidos/química , Espectrometría de Masas/métodos , Angiotensina I/química , Angiotensina I/metabolismo , Angiotensina II/química , Angiotensina II/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Animales , Bothrops , Péptidos/química , Peptidil-Dipeptidasa A/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Among the proteins and peptides already characterized in Bothrops moojeni venom, two novel phospholipases A(2) (PLA(2)) have been purified and fully sequenced by ESI-MS/MS techniques. Both of them belong to the enzymatically non-active Lys49 variants of PLA(2). They consist of 122 amino acids and share a characteristic sequence in their C-terminal region composed of clusters of basic amino acids known to interact with heparin. Thus, as already established, heparin can be used as an antidote to antagonize some myotoxic PLA(2)s from venoms of Bothrops genus. The two PLA(2) variants were shown to interact in vitro with unfractionated heparin (UFH) and low molecular weight heparin (LMWH), neutralizing their anticoagulant properties. Although the influences of PLA(2)s from snake venoms on the blood coagulation system are known, their use to antagonize the anticoagulant effect of heparin in vitro or in vivo has never been proposed. These finding recommend diagnostic and therapeutic applications, which are currently investigated.
Asunto(s)
Bothrops/fisiología , Venenos de Crotálidos/química , Antagonistas de Heparina/química , Fosfolipasas A/química , Animales , Coagulación Sanguínea/efectos de los fármacos , Pruebas de Coagulación Sanguínea , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Venenos de Crotálidos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Antagonistas de Heparina/farmacología , Humanos , Lisina/química , Fosfolipasas A/farmacología , Isoformas de Proteínas/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
The objective of the present study was an investigation of the crude Bothrops moojeni venom, aiming at the identification of new compounds with platelet-activating or -inhibiting activity. The venom was separated by gel filtration chromatography into 18 fractions, which were tested by means of whole blood aggregometry for their activities affecting the aggregation of blood platelets. In order to eliminate interferences caused by prothrombin activators or thrombin like-enzymes, which are frequently present in snake venoms, a test method for screening protein mixtures was developed. To avoid clotting of the blood samples, the thrombin inhibitor hirudin and the synthetic inhibitor of fibrin polymerization Pefabloc FG were applied. In the present study, a platelet aggregation activator with an activity resembling thrombocytin from B. atrox was identified in one of the examined venom fractions. In addition, a platelet antagonist-most likely a disintegrin-with broad inhibitory activity against aggregation triggered by collagen, adenosine diphosphate and thrombin receptor activating peptide, was identified.
Asunto(s)
Bioensayo/métodos , Plaquetas/efectos de los fármacos , Bothrops , Venenos de Crotálidos , Animales , Plaquetas/fisiología , Venenos de Crotálidos/química , Venenos de Crotálidos/farmacología , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/fisiología , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacologíaRESUMEN
The prothrombinase-induced clotting time assay (PiCT, Pentapharm, Basel, Switzerland) is a clotting assay sensitive to factor Xa and factor IIa inhibitors. It is based on the addition of factor Xa and snake venom RVV-V (Russell viper venom factor V activator) specifically activating factor V and phospholipids to platelet-poor plasma. Following an incubation time, the mixture is recalcified and the clotting time is determined. An almost linear dose-response and high sensitivity of the assay for unfractionated heparin (UFH), low-molecular-weight heparins (LMWHs), r-hirudin, and argatroban was found. Fondaparinux showed a nonlinear dose-response. By using ex vivo samples, the following Pearson correlation coefficients were found: r=0.85 between amidolytic anti-Xa and PiCT for 120 LMWH and 24 control samples; r=0.86 between amidolytic anti-Xa activity and PiCT for 68 UFH and 24 control samples; and r=0.94 between ECT and PiCT for 38 hirudin samples. Thus, PiCT is a promising assay for the monitoring of anticoagulants inhibiting factor Xa and/or factor IIa.
Asunto(s)
Pruebas de Coagulación Sanguínea/métodos , Fibrinolíticos/uso terapéutico , Heparina de Bajo-Peso-Molecular/uso terapéutico , Heparina/uso terapéutico , Polisacáridos/uso terapéutico , Tromboplastina/fisiología , Fondaparinux , Humanos , Sensibilidad y EspecificidadRESUMEN
Snake venoms are known to be an extensive source of bioactive peptides. Bradykinin-potentiating peptides (BPPs) are inhibitors of the angiotensin-converting enzyme that have already been identified in the venom of many snake, scorpion, spider and batrachian species. Their most characteristic structural features are an invariable N-terminal pyroglutamate residue (pGlu or Z) and two consecutive proline residues at the C-terminus. Fragmentation of BPPs by collision-induced dissociation during electrospray tandem mass spectrometry analysis (ESI-MS/MS) generates a predominant signal at m/z 213.1 corresponding to the y-ion of the terminal Pro-Pro fragment. In addition, signals at m/z 226.1 and 240.1 that correspond to the b ions of the N-terminus pGlu-Asn and pGlu-Lys, respectively, can often be observed. Based on these structural determinants, the present work describes an original methodology for the discovery of BPPs in natural extracts using liquid chromatography coupled to ESI-MS/MS operated in precursor ion-scan mode. The venom of the Bothrops moojeni snake was used as a model and the methodology was applied for subsequent structural analysis of the identified precursors by tandem mass spectrometry on quadrupole-time-of-flight (Q-TOF) and matrix-assisted laser-desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) instruments. More than 40 peptides below 2500 Da could be detected, among them 20 were shown to belong to the BPP-like family including the related tripeptides pGlu-Lys-Trp and pGlu-Asn-Trp. A total of 15 new sequences have been identified using this approach.
Asunto(s)
Bothrops/metabolismo , Bradiquinina/química , Venenos de Crotálidos/química , Espectrometría de Masas/métodos , Péptidos/química , Animales , Fragmentos de Péptidos/química , Mapeo Peptídico , Ácido Pirrolidona Carboxílico/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en TándemRESUMEN
A new clotting assay, Pefakit APC-R Factor V Leiden (Pentapharm Ltd., Switzerland), for the detection of an increased resistance of coagulation factor V against degradation by activated protein C, caused mainly by the factor V Leiden mutation, was evaluated in clinical studies at two University Centers in Europe and the US. The performance was compared with the performance of the routinely used predicate device COATEST APC Resistance V (Chromogenix IL, USA). Both tests were run in parallel on a STA-R analyzer (Diagnostica Stago, France). Samples from subjects undergoing routine laboratory thrombophilia screening were examined, 187 at the Institute of Medical and Chemical Laboratory Diagnostics (IMCLD), University of Vienna, Austria, and 236 at the Duke University Medical Center (DUMC), Durham/Raleigh NC, USA. All samples were analyzed for factor V Leiden mutation and prothrombin 20210 G/A mutation using standard PCR methods. The data show that the Pefakit APC-R Factor V Leiden assay discriminates very well between healthy controls and carriers of the factor V Leiden mutation, even in patients with lupus anticoagulant or with deficiency in Protein C or Protein S. Furthermore, this new test is able to discriminate well between heterozygous and homozygous carriers of the factor V Leiden mutation. In both studies the Pefakit assay showed 100% sensitivity and 100% specificity for detection of the factor V Leiden mutation, compared to 93.1% sensitivity and 93.0% specificity for the COATEST APC Resistance V in the IMCLD study and 93.9% sensitivity and 95.6% specificity in the DUMC study. The new test has PCR-like discrimination power which will help to decrease costs by reducing the need for PCR verification of borderline cases.
Asunto(s)
Resistencia a la Proteína C Activada/diagnóstico , Química Clínica/métodos , Factor V/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Europa (Continente) , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Protrombina/genética , Sensibilidad y Especificidad , Trombofilia/etiología , Trombofilia/genética , Estados UnidosAsunto(s)
Carboxipeptidasa B2/genética , Carboxipeptidasa B2/fisiología , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Bioensayo , Plaquetas/metabolismo , Carboxipeptidasa B2/química , Estudios de Casos y Controles , Enfermedad de la Arteria Coronaria/patología , Ensayo de Inmunoadsorción Enzimática , Humanos , Modelos Estadísticos , Infarto del Miocardio , Polimorfismo Genético , Factores de RiesgoRESUMEN
A multiplexed mass spectrometry based assay scheme for the simultaneous determination of five different substrate/product pairs was developed as a tool for screening of proteolytic activities in snake venom fractions from Bothrops moojeni. The assay scheme was employed in the functional characterization of eight model proteases. Time-resolved reaction profiles were generated and the relative reaction progress at each time point was determined. These were used to semi-quantitatively sort the catalytic activities of each enzyme towards the respective substrates into six classes. The resulting activity pattern served as an activity fingerprint for each enzyme. The multiplex assay scheme was then applied to a screening for proteolytic activities in fractions of the pre-separated venom from B. moojeni. Activity patterns of each fraction were generated and used to sort the fractions into three different categories of activity. By comparison of the fingerprint activity patterns of the venom fractions and the model enzymes, a compound with proteolytic properties similar to activated protein C was detected.
Asunto(s)
Venenos de Crotálidos/análisis , Venenos de Crotálidos/química , Ensayo de Inmunoadsorción Enzimática/métodos , Péptido Hidrolasas/análisis , Péptido Hidrolasas/química , Mapeo Peptídico/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Bothrops , Bovinos , Activación Enzimática , Humanos , Especificidad por SustratoRESUMEN
A new prothrombin-based activated protein C resistance (APC-R) test is described. In this method, the patient sample is prediluted in a plasma depleted of factor V (FV). A reagent containing APC and a specific activator of FV is added. After an incubation period, clotting is initiated by the addition of the FV-dependent prothrombin activator Noscarin. We analyzed 703 samples from patients undergoing thrombophilia screening. By using a predefined cutoff ratio of 2.5, 100% sensitivity and specificity for the detection of a factor V Leiden (FVL) mutation was found. With a cutoff ratio of 1.2, a complete but narrow distinction of FVL heterozygous (n = 192) and FVL homozygous samples (n = 27) was determined. No interference by the international normalized ratio, activated partial thromboplastin time (aPTT), protein S activity, fibrinogen and factor VIII (FVIII) levels, or lupus anticoagulant ratio was detected. The new prothrombin-based APC-R assay provides improved distinction of FV wild-type and FVL carriers compared with the aPTT-based method. By the use of an FV-dependent prothrombin activator, the assay is not influenced by FVIII concentration or lupus anticoagulants.