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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The simultaneous emergence in evolution of a ligand and its receptor might have entailed their active sites being drawn from the pool of common oligopeptides. This was tested on the principal components of cell-matrix interaction: the RGD (Arg-Gly-Asp) site of matrix proteins and the EKKD (Gly-Lys-Lys-Asp) site of integrin cell-surface receptor. In the 32 diverse proteins scrutinized, which totalled 14,806 residues, there were 104 Arg-Gly dipeptides. Most common of the tripeptides beginning with Arg-Gly were Arg-Gly-Leu, Arg-Gly-Gly, and Arg-Gly-Asp; each was found in ten copies. RGD tripeptide was one of the commonest; the fortuitous presence of an RGD site was noted in two enzymes, fibrinogen, a pituitary hormone precursor, and a viral structural protein. The 32 proteins also contained 121 Lys-Lys dipeptides. Of the tetrapeptides centered by Lys-Lys, the commonest was Lys-Lys-Lys-Lys, in four copies. Second most common were Gly-Lys-Lys-Lys, Val-Lys-Lys-Leu, and Glu-Lys-Lys-Asp; each occurred in three copies. The fortuitous presence of an EKKD site was noted in three proteins--an intracellular transport protein, a pituitary hormone precursor and a protein of the cerebrospinal fluid. In most instances, protein-protein interaction between the fortuitously present active sites appears to bring about deleterious consequences. Occasionally, however, the fortuitous active site appears to confer a new function to a protein bearing it.
J Mol Evol 1995 Jan
PMID:Active sites of ligands and their receptors are made of common peptides that are also found elsewhere. 771 8

In the present study, the roles of Ca2+ and fibrinogen receptor occupancy in the regulation of phospholipase C by G protein-coupled and tyrosine kinase-linked receptor pathways in human platelets have been investigated. Agonist stimulation of phospholipase C was not altered significantly in the absence of stirring or in the presence of the fibrinogen receptor antagonist arginine-glycine-aspartate-serine, conditions that prevent platelet aggregation. Similarly, elevation of intracellular Ca2+ levels by the ionophores A23187 or ionomycin did not induce formation of inositol phosphates. In contrast, chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) reduced formation of inositol phosphates by G protein receptor (thrombin)- and tyrosine kinase (Fc receptor and peroxovanadate)-regulated pathways. Similarly, short term exposure to Ni2+ ions, which also prevent Ca2+ entry, inhibited thrombin-stimulated formation of inositol phosphates. Loading of platelets with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) markedly suppressed elevation of intracellular Ca2+ and formation of inositol phosphates in platelets stimulated by G protein receptor- and tyrosine kinase-regulated pathways. The greater inhibition of phospholipase C by BAPTA, relative to that induced by EGTA, is consistent with the more pronounced inhibition of intracellular Ca2+ elevation. The tyrphostin tyrosine kinase inhibitor ST271 also reduced intracellular Ca2+ levels and inhibited activation of phospholipase C. The degree of inhibition of phospholipase C by ST271 was slightly greater than that induced by EGTA but was not additive with the effect of EGTA, suggesting a common mode of action. It is concluded that elevation of intracellular Ca2+ regulates agonist-induced activation of phospholipase C and that this contributes to the inhibition of thrombin-induced formation of inositol phosphates by the tyrphostin ST271.
Mol Pharmacol 1995 Apr
PMID:Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and the tyrphostin ST271 inhibit phospholipase C in human platelets by preventing Ca2+ entry. 772 44

Structurally guided design approaches to low-molecular-weight platelet aggregation antagonists addressing the platelet-associated heterodimeric cell surface receptor gpIIb/IIIa rely on comparative studies of an ensemble of conformationally and biologically characterized compounds, since no high-resolution structure of the receptor system is available. We report a classical indirect and comparative pharmacophore refinement approach based on a series of small cyclic Arg-Gly-Asp (RGD) peptides as gpIIb/IIIa-fibrinogen interaction antagonists. These peptides have previously been investigated as potent and selective tumor cell adhesion inhibitors. The definition of geometrical descriptors classifying the RGD peptide conformations and their subsequent analysis over selected RGD- and RXD-containing protein structures allows for a correlation of distinct structural features for platelet aggregation inhibition. An almost parallel alignment of the Arg and Asp side chains was identified by a vector analysis as being present in all active cyclic hexa- and pentapeptides. This orientation is induced mainly by the constraint of backbone cyclization and is not of any covalent tripeptide-inherent origin, which was rationalized by a 500 ps high-energy MD simulation of a sequentially related linear model peptide. The incorporation of the recognition tripeptide Arg-Gly-Asp into the cyclic peptide templates acted as a filter mechanism, restricting the otherwise free torsional relation of both side chains to a parallel orientation. Based on the derived results, several detailed features of the receptor binding site could be deduced in terms of receptor complementarity. These findings should govern the design of next-generation compounds with enhanced activities. Furthermore, the complementary stereochemical characteristics of the substrate can be used as boundary conditions for pseudoreceptor modelling studies that are capable of reconstructing a hypothetical binding pocket, qualitatively resembling the steric and electronic demands of gpIIb/IIIa. It is interesting to note that these features provide clear differentiation to requirements for inhibition of alpha v beta 3 substrate binding. This can account for the extremely high selectivity and activity of some of our constrained peptides for either the alpha IIb beta 3 or the alpha v beta 3 receptor.
J Comput Aided Mol Des 1994 Dec
PMID:Pharmacophore refinement of gpIIb/IIIa antagonists based on comparative studies of antiadhesive cyclic and acyclic RGD peptides. 773 6

Substrate specificity of two collagenolytic proteases from the king crab Paralithodes camtschatica has been studied. Both proteases are shown to hydrolyze effectively type I and III collagens, gelatin and fibrinogen. The variety of products formed during the enzymatic hydrolysis of the proteins appeared to be different for crab proteases A and C. Studies on peptide hydrolysis demonstrated that protease A cleaves preferably peptide bonds with Arg and Lys as carbonyl components, while protease C prefers hydrophobic amino acids. Kinetic constants of hydrolysis for low molecular weight substrates in the presence of crab proteases have been determined. This allowed us to characterize collagenolytic protease A as a trypsin-like protease. By contrast, collagenolytic protease C was classified as chymotrypsin-like protease although this protease and bovine chymotrypsin are not completely similar. Collagenase substrates Pz-Pro-Leu-Gly-Pro-D-Arg and Z-Gly-Pro-Ala-Gly-Pro-Ala were found to be resistant to both crab proteases.
Comp Biochem Physiol Biochem Mol Biol 1994 Mar
PMID:Substrate specificity of collagenolytic proteases from the king crab Paralithodes camtschatica. 774 10

The conversion by thrombin of soluble plasma fibrinogen to an insoluble fibrin matrix is central to hemostasis and subsequent wound healing. Fibroblasts adhere to and rapidly grow into fibrin clots, resulting in collagen deposition and, ultimately, scar formation. Although a number of soluble mediators have been implicated in this process, a role for fibrin(ogen) itself has not been described. The present study further investigated the nature of mitogenic activity remaining in solution after in vitro fibrin clot formation. Liquid expressed from a fibrin clot (clot supernatant) elicited a mitogenic response of up to 83 +/- 4.7% above media control. Upon addition of a polyclonal fibrinogen antibody, this activity was reduced by 50%. The remaining activity was attributed to the presence of thrombin and was neutralized by the addition of a specific thrombin inhibitor. Fibrinogen cleavage products were separated by molecular sieve chromatography and the mitogenic potential of each fraction assessed. A peak of activity was observed in fractions containing proteins with apparent molecular weights of 200 to 300 kD. Enhanced chemiluminescence Western blotting of these fractions established the presence of several fibrin(ogen)-derived protein bands. It is therefore proposed that thrombin cleavage of fibrinogen, in addition to producing fibrin, generates high-molecular-weight soluble cleavage products that may play an important role during normal wound healing and in the pathogenesis of disease states associated with vascular leakage and fibrosis.
Am J Respir Cell Mol Biol 1995 Jun
PMID:Partially degraded fibrin(ogen) stimulates fibroblast proliferation in vitro. 776 31

Infections, trauma and inflammatory processes induce a host response with increases in a large group of structurally and functionally diverse plasma proteins. Parental administration of foreign proteins also induce an increase in plasma fibrinogen. Interleukin-6 (IL-6) is a monocyte-derived mediator and has regulatory effects on acute phase protein genes which result in the induction of fibrinogen synthesis in primary hepatocytes, while the addition of interleukin-1 (IL-1) exerts a negative modulating influence on the IL-6-stimulated fibrinogen. In order to understand the mechanisms by which IL-1 inhibits IL-6-stimulated fibrinogen transcription and translation, and since IL-1 is believed to act through PGE2 stimulation, we have studied the influence of PGE2 in IL-6 or IL-1, alone and in combination, on Fg mRNA expression (by Northern blot analysis) and the influence of PGE2, indomethacin, and arachidonic acid on Fg secretion. Moreover, since human recombinant interleukin-1 receptor antagonist (hrIL-1ra) is a strong inhibitor of IL-1 induced IL-1 transcription and translation and has an inhibitory effect on PGE2, we have studied the effects of IL-1ra on the down-regulation of IL-6 stimulated fibrinogen by IL-1, using an Fg ELISA method.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1995 Jan 26
PMID:The down-regulation of IL-6-stimulated fibrinogen steady state mRNA and protein levels by human recombinant IL-1 is not PGE2-dependent: effects of IL-1 receptor antagonist (IL-1RA). 777 69

An experimental strategy based on solution viscosity perturbation allowed us to study the energetics of amide-substrates, p-aminobenzamidine (p-ABZ) and proflavin binding to the catalytic site of two proteolyzed forms of alpha-thrombin, i.e. zeta- and gamma T-thrombin. These thrombin derivatives are cleaved at the Leu144-Gly150 loop and at the fibrinogen recognition exosite (FRS), respectively. A phenomenological analysis of thermodynamic data showed that the amide substrates and p-ABZ interactions with zeta-thrombin were respectively, associated with a chemical compensation (i.e. the linear relationship between entropy and enthalpy of binding) and a hydrophobic phenomenon (i.e. a change in the standard heat capacity). The latter was slightly lower than that previously observed for a alpha-thrombin (0.78 +/- 0.25 versus 1.01 +/- 0.17 kcal/mol K). Both phenomenon were absent in gamma T-thrombin. The interaction of a alpha-, zeta- and gamma T-thrombin with macromolecular substrates that "bridge-bind" to both the catalytic site (CS) and fibrinogen recognition exosite (FRS), such as fibrinogen and the cleavable platelet receptor (CPR), was also evaluated. These interactions were studied by following fibrinopeptide A (FpA) release and by measuring intraplatelet Ca2+ changes induced by thrombin-CPR interaction. It was found that the free energy of activation (RT ln Kcat/Km) for both fibrinogen and CPR hydrolysis followed the same hierarchy, i.e. alpha > zeta > gamma. Moreover, the values of delta Cp for alpha-, zeta- and gamma T-thrombin interaction with p-ABZ were found to be linearly correlated to the free energy of activation for both fibrinogen and CPR cleavage. In conclusion, these data demonstrate that: (1) the Leu144-Gly150 loop and the FRS are both involved in the conformational transition linked to the binding of p-aminobenzamidine to the thrombin active site; (2) the extent of thrombin's capacity to undergo conformational transitions in alpha-, zeta- and gamma T forms is positively correlated to the free energy of activation for hydrolysis of macromolecular substrates interacting with both the catalytic domain and the FRS.
J Mol Biol 1995 Jan 27
PMID:Conformational transitions linked to active site ligation in human thrombin: effect on the interaction with fibrinogen and the cleavable platelet receptor. 783 75

A semi-micro method (BR BLUE) is presented using Coomassie Brilliant Blue G-250 color reagent for the determination of fibrinogen on potentially turbid specimens. It is compared with the reference Clauss method using the automated MLA Electra 1000C Automatic Coagulation Timer and the semi-automated Mechrolab Clot Timer. Correlation is excellent (r = 0.97). The BR BLUE method is also seen to be reasonably precise and linear.
Res Commun Mol Pathol Pharmacol 1994 Oct
PMID:A semi-micro method for fibrinogen determination essentially unaffected by turbidity. 785 Feb 50

The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.
J Mol Biol 1995 Feb 10
PMID:Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin. 785 94

Staphylococcus aureus has been shown to interact specifically with fibrinogen. Three different extracellular fibrinogen-binding proteins, two of which have coagulase activity, are produced by S. aureus strain Newman. The role of these fibrinogen-binding proteins during staphylococcal colonization and infection has not yet been fully elucidated. Here we describe the cloning, sequencing and expression of a gene for a 19 kDa fibrinogen-binding protein. This gene, called fib, encodes a 165-amino-acid polypeptide, including a 29-amino-acid signal sequence. The recombinant protein, which has an estimated molecular mass of 15.9 kDa, bound fibrinogen and was recognized by a polyclonal antiserum against the native Fib protein. Homologies between the Fib protein and the fibrinogen-binding domain of coagulase suggest that amino acids within this domain are involved in the binding to fibrinogen.
Mol Microbiol 1994 May
PMID:Cloning and characterization of a gene for a 19 kDa fibrinogen-binding protein from Staphylococcus aureus. 793 83


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