RESUMO
This study examines the clearance and early hydrolysis of atrial natriuretic factor (ANF) in vivo. Radiolabeled ANF was cleared from the circulation of the rat with biphasic kinetics; the majority (90%) of ANF cleared with a t1/2 of 15 s, the remaining peptide was cleared with a t1/2 of 5 min. Microsequence analysis of ANF peptides recovered from the circulation of rats revealed five major degradation products of the intact hormone. The first cleavage occurred between amino acids 12 and 13 of the hormone and would inactivate ANF. Over time, additional fragments of the hormone were generated, including fragments of 6, 7, 21, and 24 amino acids in length. Whole body radioautography of rats injected with [123I]-ANF revealed the kidney as a predominant organ involved in clearance of ANF. Subsequent amino acid sequence analyses of radiolabeled ANF exposed to the kidney in vivo indicated that this organ generated four of the five major hydrolysis products observed in circulation, namely, the 6, 7, 16, and 21 amino acid fragments of the hormone. In an attempt to stabilize ANF in vivo, a synthetic analogue of the hormone was prepared that contained the amino acid analogue, aminoisobutyric acid, substituted at position 13. This analogue completely abolished the in vivo cleavage of ANF at this site. These studies demonstrate the usefulness of a protein chemistry approach in characterizing hormone metabolism in vivo and designing analogues with enhanced in vivo stability to cleavage.
Assuntos
Fator Natriurético Atrial/metabolismo , Rim/metabolismo , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial/análogos & derivados , Autorradiografia , Cães , Feminino , Humanos , Hidrólise , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , RatosRESUMO
The N-terminal thrombin receptor peptide H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe-OH (1) fully activates the thrombin receptor with an EC50 of 10 microM. Structural features in the tetradecapeptide which are responsible for receptor activation have been elucidated. Agonist potency has been enhanced 1000-fold with the design of the shortened peptide H-Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2 (56). This analog exhibits an EC50 of 0.01 microM and is the most potent agonist for receptor activation reported to date. The monoiodinated derivative H-Ala-Phe(p-F)-Arg-Cha-HArg-Tyr(3-I)-NH2 (59) exhibits an EC50 of 0.03 microM, a level sufficient for development of a radioligand.
Assuntos
Oligopeptídeos/farmacologia , Receptores de Trombina/agonistas , Sequência de Aminoácidos , Humanos , Ligantes , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
The present work reports the discovery and characterization of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalis, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host's blood.
Assuntos
Anticoagulantes/isolamento & purificação , Inibidores do Fator Xa , Sanguessugas/análise , Proteínas e Peptídeos Salivares/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Piroglutamil-Peptidase I/metabolismo , Relação Estrutura-AtividadeRESUMO
The aggregation of platelets from a variety of animal species in response to thrombin receptor-derived activating peptides was evaluated. A series of 14-(SFLLRNPNDKYEPF), 7-(SFLLRNP-NH2), 6-(SFLLRN-HN2) or 5-(SFLLR-NH2) residue peptides, the structures of which were based on the deduced amino acid sequence of the human thrombin receptor, promoted full aggregation of platelets in plasma from humans, African Green and Rhesus monkeys, baboons and guinea pigs at 4-50 microM depending on the peptide used. Platelets in plasma from rabbit, dog, pig, and hamster underwent a shape change but failed to aggregate in response to these peptides over 3 log units of peptide up to 800 microM, despite being fully responsive to human thrombin. However, because the receptor peptides induced shape change in the platelets from these non-aggregating species, they apparently can activate some of the intracellular signaling system(s) usually initiated by thrombin in these platelets. In contrast, platelets from rats did not undergo shape change or aggregate in response to the peptides. A 7-residue receptor-derived peptide based on the deduced amino acid sequence of the clone of the hamster thrombin receptor (SFFLRNP-N2) was nearly as efficacious as the corresponding human receptor-derived 7-residue peptide to promote aggregation of human platelets. However, the hamster peptide could not promote aggregation of hamster platelets in plasma at up to 800 microM peptide, while a shape change response was elicited. Platelets from rats, rabbits and pigs also did not aggregate in response to this peptide derived from the hamster thrombin receptor, but all species except the rat underwent a shape change.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Mamíferos/sangue , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Receptores de Trombina/fisiologia , Sequência de Aminoácidos , Animais , Plaquetas/ultraestrutura , Linhagem Celular , Cricetinae , Replicação do DNA/efeitos dos fármacos , Cães/sangue , Fibroblastos/efeitos dos fármacos , Cobaias , Humanos , Masculino , Dados de Sequência Molecular , Músculo Liso Vascular/efeitos dos fármacos , Fragmentos de Peptídeos/síntese química , Primatas/sangue , Ratos , Receptores de Trombina/química , Roedores/sangue , Especificidade da Espécie , Suínos/sangueRESUMO
Optimal conditions for a method to simultaneously measure aggregation in 96 samples using a microplate reader were developed. The temperature of the assay was set at 25 degrees C, the optimal platelet concentration range was determined to be from 1-3 x 10(8) per mL, the assay volume was determined to be best at 100 microL and an agitation rate of setting #5 on the vortex was found to yield the most reliable aggregation response. After these initial assay parameters were established, EC50 values for standard platelet agonists including ADP, thrombin, collagen and thrombin receptor activating peptides were determined using the plate assay and compared to those obtained by measuring light transmittance in an aggregometer. The results were quantitatively similar, and qualitatively the shapes of the aggregations as monitored by both methods were characteristic of those expected for each agonist. The use of this assay was then extended to quantitate the inhibition of aggregation by antagonists of the fibrinogen receptor as well as by an inactive thrombin receptor peptide and by antibodies against the thrombin receptor. This method provided useful data for characterization of both platelet agonists and antagonists and should be useful for future platelet aggregation studies.
Assuntos
Hematologia/métodos , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/fisiologia , Difosfato de Adenosina/farmacologia , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Colágeno/farmacologia , Humanos , Técnicas In Vitro , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/efeitos dos fármacos , Receptores de Trombina/efeitos dos fármacos , Trombina/farmacologiaRESUMO
A protein that blocks collagen-stimulated platelet aggregation has been identified and isolated from the soluble fraction of salivary glands from Haementeria officinalis leeches. We have named this protein leech antiplatelet protein (LAPP). LAPP was isolated from soluble crude salivary gland extract by heparin-agarose, size exclusion, and C18 reverse phase high-performance chromatography. Its molecular weight is approximately 16,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reduced and nonreduced conditions. The sequences of peptides generated by V8 digestion of LAPP as well as its amino acid composition suggested no homology to other known proteins. The IC50 for LAPP to inhibit platelet aggregation was approximately 60 nM. This inhibitory activity is specific for collagen-induced aggregation. Platelet aggregation in response to ADP, arachidonic acid, U46619, thrombin, and ionophore A23187 was not inhibited by LAPP at a concentration that blocked platelet aggregation to collagen by 100%. In contrast, crude salivary gland-soluble extract contained activity(ies) which inhibited aggregation to all these agonists except thrombin at 1 unit/ml and 2 microM A23187. Thus, the H. officinalis leech has evolved multiple mechanisms to prevent hemostasis, including an inhibitor of collagen-stimulated platelet aggregation. The identification and isolation of LAPP demonstrates the existence of a new type of platelet inhibitor that should be useful to better understand the mechanism of collagen stimulation of platelets.
Assuntos
Colágeno/antagonistas & inibidores , Sanguessugas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/metabolismo , Glândulas Salivares/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Aminoácidos/análise , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Colágeno/farmacologia , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Humanos , Hidrólise , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/isolamento & purificação , Proteínas e Peptídeos Salivares/isolamento & purificação , Proteínas e Peptídeos Salivares/farmacologiaRESUMO
The phosphoprotein (NS) gene from the Indiana serotype of vesicular stomatitis virus (VSV; Mudd-Summers strain) was cloned and sequenced. The NS gene encodes a protein of 265 amino acids which was expressed from a simian virus 40 vector in COS cells. The post-translational modification characteristic of viral NS, the extensive phosphorylation of a cluster of serine and threonine residues, was also evident in recombinant NS protein. The NS gene displays a property common to the phosphoprotein genes of negative-strand RNA viruses: the phosphoprotein mRNA has a second open reading frame (ORF) which could encode a small (7500 mol. wt.) protein. Both measles virus and Sendai virus employ the second ORF of their phosphoprotein gene, and the resultant proteins have an amino acid composition similar to that predicted for the VSV ORF. Comparison of phosphoproteins from different VSV strains revealed two conserved domains that we propose are critical for the function of NS in transcription and replication.
Assuntos
RNA Viral/genética , Vírus da Estomatite Vesicular Indiana/genética , Proteínas Virais/genética , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Genes Virais , Teste de Complementação Genética , Haplorrinos , Mutação , Fosforilação , RNA Mensageiro/genética , Moldes Genéticos , Transfecção , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas não Estruturais Virais , Proteínas Virais/metabolismo , Proteínas Virais/fisiologiaRESUMO
In vitro transcripts of vesicular stomatitis virus (VSV) were either 5'-terminally labeled by incorporation of [beta-(32)P]GTP or were selected on Hg-agarose after incorporation of gamma-thio-GTP. Capped RNAs ranged in size from 23 nucleotides, the shortest capped RNA detected, to full-length message size. The 5'-terminal sequences corresponded to those of N message and to a small amount of NS message. Approximately 14% of the capped N gene transcripts were terminated at positions 86 to 90 of the VSV genome, giving rise to specific, 36 to 40-nucleotide-long, capped RNA species. The GTP-initiated RNAs were short with a predominant 28-nucleotide-long RNA species. A minor portion was as large as mRNAs. Nucleotide sequence analyses of the short RNA revealed that it was specifically initiated at positon 91 of the VSV genome, 41 nucleotides within the N cistron. This corresponds exactly to the site where transcription of the 40-nucleotide-long, capped RNA terminated. Initiation with GTP at position 91 occurred at approximately the same frequency as termination of the capped RNA at position 90, suggesting that intracistronic initiation at position 91 may depend upon termination of transcription of the 5'-proximal region and therefore may be sequential. This unique RNA represents the first transcript of VSV which was initiated at an intracistronic site with GTP, and may also represent the first example of a transcript derived from a stop/start mechanism of VSV transcription in vitro. Although initiation occurred frequently at the beginning of the N cistron yielding 11 to 14-nucleotide-long, [beta-(32)P]ATP-labeled transcripts (D. F. Pinney and S. U. Emerson, J. Virol. 42:889-896, 1982), capping of these short RNAs was not detected. This suggests that transcripts may have to be 15 to 23 nucleotides long to be accepted as substrates by the guanyltransferase.
Assuntos
Genes Virais , Genes , Guanosina Trifosfato/metabolismo , Transcrição Gênica , Vírus da Estomatite Vesicular Indiana/genética , Sequência de Bases , Capuzes de RNA , RNA Mensageiro/genética , RNA Viral/genéticaRESUMO
Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP), that specifically blocks collagen-mediated platelet aggregation (Connolly, T. M., Jacobs, J. W., and Condra, C. (1992) J. Biol. Chem. 267, 6893-6898). Degenerate oligonucleotides whose sequences were derived from two short peptides from V8 digests of the native LAPP were used as primers to generate a polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides. Using this PCR product as a hybridization probe, phage containing cDNA clones were isolated containing the entire deduced amino acid sequence for LAPP. Computer analysis of the amino acid sequence predicts a peptidase cleavage site between a 21-residue pre-peptide and a mature protein of 126 amino acids. A DNA insert to express the predicted mature LAPP protein was generated by PCR amplification using phage-derived cDNA clones as a substrate. This insert encoded a fusion protein with the leader sequence of the yeast alpha mating factor and the mature LAPP cDNA. These PCR products were cloned into the yeast expression vector pKH4 alpha 2. A KEX 2 Lys-Arg endopeptidase cleavage site was placed NH2-terminal to the predicted mature protein. This vector transfected into the yeast Saccharomyces cerevisiae directs expression of a secreted mature protein at levels up to 200 mg of LAPP/liter of culture medium. The recombinant protein was comparable to native LAPP in its electrophoretic mobility, its reactivity with anti-LAPP antisera, and its biological activity including inhibition of collagen-stimulated platelet aggregation and the adhesion of platelets to collagen. Availability of significant quantities of recombinant LAPP opens the way to further biochemical structure/function studies and to studies on the effects of an inhibitor of collagen-stimulated platelet aggregation in vivo.
Assuntos
Colágeno/antagonistas & inibidores , DNA/genética , Expressão Gênica , Sanguessugas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Glândulas Salivares/metabolismo , Proteínas e Peptídeos Salivares/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Adesão Celular/efeitos dos fármacos , Clonagem Molecular , Colágeno/farmacologia , Vetores Genéticos , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Saccharomyces cerevisiae/metabolismo , Proteínas e Peptídeos Salivares/farmacologiaRESUMO
Moubatin, a new type of specific inhibitor of collagen-induced platelet aggregation, has been isolated from the soft tick Ornithodoros moubata (Waxman, L., and Connolly, T. M. (1993) J. Biol. Chem. 268, 5445-5449). A polymerase chain reaction-generated hybridization probe, produced using primers based on moubatin protein sequence, identified phage containing the entire cDNA sequence of moubatin. Analysis of the predicted amino acid sequence yielded a mature protein of 156 amino acids with a putative prepeptide of 15 amino acids. Comparison of the sequence of moubatin to that of other proteins in the Swiss PROT data base revealed no significant homology. The cDNA sequence was cloned into the yeast expression vector pKH4 alpha 2, producing a biologically active protein which inhibited collagen-stimulated aggregation of washed human platelets with an IC50 of about 100 nM, which is similar to the potency of native tick moubatin. A concentration of recombinant moubatin that fully inhibited collagen-stimulated aggregation did not inhibit aggregation induced by a variety of other platelet agonists, again demonstrating comparable properties of the recombinant and native proteins. Moubatin did not inhibit platelet adhesion to collagen even at a concentration up to 16 times its IC50 for the inhibition of aggregation. This specificity for inhibiting collagen-stimulated aggregation and not adhesion to collagen indicates that moubatin is unique among the natural product inhibitors of collagen stimulation of platelets. Further examination of the mechanism of moubatin-mediated inhibition of collagen-stimulated aggregation revealed that 1-6 microM moubatin diminished the second phase of aggregation induced by ADP, inhibited aggregation in response to submaximal concentrations of the thromboxane A2 mimetic U46619, and competed for the binding of a thromboxane A2 receptor antagonist to platelet membranes. Therefore, at higher concentrations, moubatin may affect more than one aspect of platelet signal transduction including the thromboxane A2 receptor. The availability of recombinant moubatin will allow further investigation of its unique activities in vitro and in vivo.
Assuntos
Inibidores da Agregação Plaquetária/química , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Colágeno/farmacologia , DNA , Humanos , Dados de Sequência Molecular , Plasmídeos , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/isolamento & purificação , Proteínas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae , Carrapatos/químicaRESUMO
BACKGROUND: Thrombin inhibitors have been shown to be efficacious in animal models of thrombosis and in initial human clinical trials. It is unknown if their efficacy is due to their prevention of thrombin-mediated fibrin formation or to an inhibitory effect on thrombin-stimulated platelet activation. Appropriate tools to address this question have not been available. Therefore, to evaluate the role of the platelet thrombin receptor in intravascular thrombus formation, a polyclonal antibody was raised against a peptide derived from the thrombin-binding exosite region of the cloned human thrombin receptor. This antibody serves as a selective inhibitor of the thrombin receptor for in vivo evaluation. METHODS AND RESULTS: The immune IgG (IgG 9600) inhibited thrombin-stimulated aggregation and secretion of human platelets. In contrast, it had no effect on platelet activation induced by other agonists including ADP, collagen, or the thrombin receptor-derived peptide SFLLR-NH2. IgG 9600 also inhibited thrombin-induced aggregation of African Green monkey (AGM) platelets. By Western blot analysis, the IgG identified a protein of approximately 64 kD in homogenates of both human and AGM platelets. The effect of thrombin receptor blockade by this antibody on arterial thrombosis was evaluated in an in vivo model of platelet-dependent cyclic flow reductions (CFRs) in the carotid artery of the AGM. The intravenous administration of IgG 9600 (10 mg/kg) abolished CFRs in three monkeys and reduced CFR frequency by 50% in a fourth monkey. Ex vivo platelet aggregation in response to up to 100 nmol/L thrombin was completely inhibited during the 120-minute postbolus observation period in all four animals. There was a twofold increase in bleeding time, which was not statistically different from baseline, and ex vivo clotting time (APTT) was not changed. The glycoprotein IIb/IIIa receptor antagonist MK-0852 and the thrombin inhibitor recombinant hirudin also demonstrated inhibitory effects on CFRs at doses that did not significantly prolong template bleeding time. Control IgG had no effect on CFRs, ex vivo platelet aggregation, bleeding time, or APTT. CONCLUSIONS: These results demonstrate that blockade of the platelet thrombin receptor can prevent arterial thrombosis in this animal model without significantly altering hemostatic parameters and suggest that the thrombin receptor is an attractive antithrombotic target.
Assuntos
Anticorpos/uso terapêutico , Receptores de Trombina/imunologia , Trombose/tratamento farmacológico , Animais , Formação de Anticorpos , Chlorocebus aethiops , Agregação Plaquetária/efeitos dos fármacos , Receptores de Trombina/química , Receptores de Trombina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Thrombin is the most potent agonist of platelet activation, and its effects are predominantly mediated by platelet thrombin receptors. Therefore, antagonists of the thrombin receptor have potential utility for the treatment of thrombotic disorders. Screening of combinatorial libraries revealed 2 to be a potent antagonist of the thrombin receptor. Modifications of this structure produced 11k, which inhibits thrombin receptor stimulated secretion and aggregation of platelets.