fibrinopeptide-a and arginyl-glycyl-aspartic-acid

The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin. Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets. We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids. The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3' fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography. The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin. The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Escherichia coli; Fibrinolytic Agents; Fibrinopeptide A; Genes, Synthetic; Hirudins; Humans; Metalloendopeptidases; Molecular Sequence Data; Oligopeptides; Recombinant Fusion Proteins

Several degradation products of fibrinogen have been shown to possess regulatory functions. Using peptide extracts from human blood filtrate, a large number of fibrinogen A alpha fragments was identified. These fragments are generated at known plasmin attack sites and at several novel cleavage sites especially at hydrophobic and basic amino acid residues. One fragment containing the cell attachment site (RGD sequence) of fibrinogen A alpha efficiently inhibits fibrinogen binding and platelet aggregation (IC50:20-50 microM) in vitro. We conclude that in vivo degradation of fibrinogen A alpha results in generation of endogenous antithrombotic peptides with local importance in fibrinolysis and platelet aggregation.

Topics: Adenosine Diphosphate; Amino Acid Sequence; Binding Sites; Blood Platelets; Fibrinogen; Fibrinolysin; Fibrinopeptide A; Humans; Molecular Sequence Data; Oligopeptides; Platelet Aggregation; Platelet Aggregation Inhibitors

Haemorrhagic metalloproteinases from Bothrops jararaca and other venoms degrade vessel-wall and plasma proteins involved in platelet plug and fibrin clot formation. These enzymes also cause proteolytic digestion of fibrinogen which has been suggested to cause defective platelet function. Fibrinogen degradation by jararhagin, a metalloproteinase from B. jararaca, and the effect of jararhagin fibrinogenolysis on both platelet aggregation and fibrin clot formation were investigated. Jararhagin was found to cleave human fibrinogen in the C-terminal region of the A alpha-chain giving rise to a 285-290 kDa fibrinogen molecule lacking the A alpha-chain RGD 572-574 platelet-binding site. Platelet binding and aggregation of ADP-activated platelets is unaffected by this modification. This indicates that the lost site is not essential for platelet aggregation, and that the remaining platelet binding sites located in the N-terminal portion of A alpha chains (RGD 95-97) and the C-terminal of gamma chains (dodecapeptide 400-411) are unaffected by jararhagin-digestion of fibrinogen. Fibrin clot formation with thrombin of this remnant fibrinogen molecule was defective, with poor polymerization of fibrin monomers but normal release of FPA. The abnormal polymerization could be explained by the loss of one of the two complementary polymerization sites required for side-by-side association of fibrin protofibrils. Jararhagin-induced inhibition of platelet function, an important cause of haemorrhage in envenomed patients, is not caused by proteolysis of fibrinogen, as had been thought, and the mechanism remains to be elucidated.

Topics: Adenosine Diphosphate; Amino Acid Sequence; Animals; Bothrops; Bothrops jararaca Venom; Crotalid Venoms; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinopeptide A; Hemostasis; Humans; Metalloendopeptidases; Molecular Sequence Data; Oligopeptides; Platelet Aggregation

The partial amino acid sequences of fibrinogen A alpha-chains from five mammalian species have been inferred by means of the polymerase chain reaction (PCR). From the genomic DNA of the rhesus monkey, pig, dog, mouse and Syrian hamster, the DNA fragments coding for alpha-C domains in the A alpha-chains were amplified and sequenced. In all species examined, four cysteine residues were always conserved at the homologous positions. The carboxy- and amino-terminal portions of the alpha-C domains showed a considerable homology among the species. However, the sizes of the middle portions, which corresponded to the internal repeat structures, showed an apparent variability because of several insertions and/or deletions. In the rhesus monkey, pig, mouse and Syrian hamster, 13 amino acid tandem repeats fundamentally similar to those in humans and the rat were identified. In the dog, however, tandem repeats were found to consist of 18 amino acids, suggesting an independent multiplication of the canine repeats. The sites of the alpha-chain cross-linking acceptor and alpha 2-plasmin inhibitor cross-linking donor were not always evolutionally conserved. The arginyl-glycyl-aspartic acid (RGD) sequence was not found in the amplified region of either the rhesus monkey or the pig. In the canine alpha-C domain, two RGD sequences were identified at the homologous positions to both rat and human RGDS. In the Syrian hamster, a single RGD sequence was found at the same position to that of the rat. Triplication of the RGD sequences was seen in the murine fibrinogen alpha-C domain around the homologous site to the rat RGDS sequence. These findings are of some interest from the point of view of structure-function and evolutionary relationships in the mammalian fibrinogen A alpha-chains.

Topics: Amino Acid Sequence; Animals; Base Sequence; Consensus Sequence; Cricetinae; Dogs; Fibrinopeptide A; Macaca mulatta; Mammals; Mesocricetus; Mice; Molecular Sequence Data; Oligopeptides; Phylogeny; Polymerase Chain Reaction; Protein Structure, Tertiary; Rats; Repetitive Sequences, Nucleic Acid; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Swine