Beta-chain Of Human Fibrinogen Research Articles

A novel fragment derived from the beta chain of human fibrinogen, beta43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo.

Background:Angiogenesis and haemostasis are closely linked within tumours with many haemostatic proteins regulating tumour angiogenesis. Indeed we previously identified a fragment of human fibrinogen, fibrinogen E-fragment (FgnE) with potent anti-angiogenic properties in vitro and cytotoxic effects on tumour vessels in vivo. We therefore investigated which region of FgnE was mediating vessel cytotoxicity.Methods:Human dermal microvascular endothelial cells (ECs) were used to test the efficacy of peptides derived from FgnE on proliferation, migration, differentiation, apoptosis and adhesion before testing the efficacy of an active peptide on tumour vasculature in vivo.Results:We identified a 20-amino-acid peptide derived from the β chain of FgnE, β43–63, which had no effect on EC proliferation or migration but markedly inhibited the ability of activated ECs to form tubules or to adhere to various constituents of the extracellular matrix – collagen IV, fibronectin and vitronectin. Furthermore, our data show that β43–63 interacts with ECs, in part, by binding to αvβ3, so soluble αvβ3 abrogated β43–63 inhibition of tubule formation by activated ECs. Finally, when injected into mice bearing tumour xenografts, β43–63 inhibited tumour vascularisation and induced formation of significant tumour necrosis.Conclusions:Taken together, these data suggest that β43–63 is a novel anti-tumour peptide whose anti-angiogenic effects are mediated by αvβ3.

Twofold symmetry of human fibrinogen proved at the beta chain distal domains by monoclonal-immunoelectron microscopy and image analysis.

Using a murine antibody (F7) specific for the C-terminal domain of the beta chain of human fibrinogen combined with electron microscopy and image analysis, we show unequivocally that the epitopes for F7 are at the distal nodules of fibrinogen, equidistant from the center of the molecule and arranged not colinearly with the long axis of the molecule but at opposite sides of it, i.e., following twofold symmetry. Thus, given the monoclonality of the immunochemical probe used and the dimeric nature of the fibrinogen molecule, we can conclude that the distal domains of the two beta chains are arranged in the same manner as these epitopes and, therefore, that the fibrinogen molecule has twofold symmetry. This symmetry pattern found here for F7 is the same as that found recently for the platelet fibrinogen receptor binding sites [Weisel, J. W., Nagaswami, C., Vilaire, G., & Bennett, J. S. (1992) J. Biol. Chem. 23, 16637-16643], located almost certainly at the C-terminal end of the gamma chains, and gives further support to the most accurate model of fibrinogen available so far. We discuss the consequences of this symmetry pattern and of the molecular rigidity of fibrinogen in the actual models of fibrin polymerization and platelet aggregation and adhesion.

Isoforms of a highly specific B??-chain fibrinogenase from the venom of Crotalus atrox

Three variants of a Crotalus atrox venom enzyme that cleaves primarily the Arg42 - Ala bond of the B beta-chain of human fibrinogen were separated by high-performance liquid chromatography and were shown by N-terminal sequence analysis to be amino acid isoforms. All are single-chain glycoproteins whose approximate molecular weights lie between 26,000 and 28,000. The isoenzymes also cleaved the A alpha-chain at Arg491 - His, though much more weakly, with strengths for both bonds that varied among the isoforms.

Characterization of complementary deoxyribonucleic acid and genomic deoxyribonucleic acid for the beta chain of human fibrinogen.

A total of 148 cDNAs coding for the beta chain of human fibrinogen have been identified from a human liver cDNA library employing a bovine cDNA as a probe. The largest cDNA insert contained 1932 base pairs cloned into the PstI site of plasmid pBR322. This cDNA insert contained 66 base pairs coding for a portion or all of a signal sequence, 1383 base pairs coding for 461 amino acids in the mature protein, a stop codon of TAG, a noncoding region of 431 base pairs, and a poly(A) tail of 19 base pairs. Most of the cDNA inserts coding for the beta chain were found to have a noncoding region of 98 or 167 base pairs rather than 431 base pairs at the 3'-end. The bovine cDNA for the beta chain was also employed as a probe for screening a lambda phage library containing human genomic DNA. Seven positive phage were identified. One of the phage, which contained the entire gene for the beta chain of fibrinogen, was examined by electron microscopy, and portions of its DNA sequence are presented. Seven intervening sequences were identified in the gene for the beta chain of human fibrinogen. The largest intervening sequence (approximately 1.3 kilobases) was found at the 5'-end of the gene and was located between amino acid residues 8 and 9, which are present in fibrinopeptide B. A sequence analysis of the 5'-end of the gene also indicated that the B chain of human fibrinogen contained a signal sequence of either 16, 27, or 30 amino acid residues.

Characterization of a complementary deoxyribonucleic acid coding for the gamma chain of human fibrinogen.

A number of cDNAs coding for the gamma chain of human fibrinogen have been isolated from a liver cDNA library by employing a synthetic nucleotide mixture as a probe. One of the positive clones was then employed to screen the entire cDNA library of 18000 recombinants, yielding 320 positive clones for the gamma chain. The largest cDNA was 1638 base pairs in length and contained 10 base pairs of poly(G) at the 5'-end followed by 71 base pairs of noncoding nucleotides. The next 78 base pairs coded for a leader sequence that was 26 amino acids in length and included a methionine start signal and a typical hydrophobic core. The following 1233 base pairs coded for 411 amino acids that are present in the mature protein followed by a stop codon of TAA, 207 base pairs of noncoding nucleotides, a poly(A) track of 15 base pairs, and 22 base pairs of poly(C). Specific regions of the cDNA of the gamma chain were then compared with the cDNAs for the alpha and beta chains of human fibrinogen.

Cloning of fibrinogen genes and their cDNA.

Cross-species hybridizations have enabled us to isolate and clone the gene for the beta chain of human fibrinogen. Highlights of the gene for the beta chain revealed by nucleotide sequence analyses, particularly in areas that have a direct bearing on defining the overall organization of the gene, have been presented. Nucleotide sequence determination has confirmed the presence of seven intervening sequences. The positions where several of these intervening sequences interrupt the coding region appear to be related to the functional domains of the polypeptide. A putative signal peptide has been identified. Studies on the cDNA for the human alpha chain indicate that the alpha chain polypeptide may be synthesized in a precursor form with a COOH-terminal extension of 15 amino acids as compared to the alpha chain present in the mature molecule found in plasma. We are in the process of isolating the genes for the alpha and gamma chains by a similar approach. We are hopeful that these studies will provide information as to how they are regulated and how they have undergone changes in the course of evolution.

Amino acid sequence of the .beta. chain of human fibrinogen

The beta chain of human fibrinogen contains 461 amino acid residues, 15 of which are methionines. The calculated molecular weight, independent of a single carbohydrate cluster, is 52 230. In this regard, we have isolated and characterized all 16 cyanogen bromide fragments. In one case (CNI), we have concentrated on a disputed portion of a previously reported fragment. The arrangement of the cyanogen bromide peptides was deduced by the use of overlap fragments obtained from the tryptic digestion of modified and unmodified beta-chains and from digestions with staphylococcal protease, as well as by considerations involving the plasmic digestion products of fibrinogen. In one case two adjacent fragments were aligned by homology with the corresponding segments of the gamma chain. The homology of the beta chain with the gamma chain is especially strong over the course of the carboxy-terminal two-thirds of the sequence. Neither of these chains appears to be homologous with the alpha chain in these regions. With a few minor exceptions, the sequence reported in this article is in agreement with data reported by other groups in Stockholm and Munich.

Amino acid sequence of the beta chain of human fibrinogen: homology with the gamma chain.

The beta chain of human fibrinogen is composed of 452 +/- 5 amino acid residues, 14 of which are methionines. Consistent with these findings we have isolated and characterized 15 fragments after cyanogen bromide digestion of carboxymethylated beta chains. The arrangement of several of these peptides was deduced on the basis of overlapping peptides isolated from the fragments D and E produced by the plasmic digestion of fibrinogen and/or from a tryptic digest of citraconylated beta chains. Most of the other cyanogen bromide fragments can be aligned by homology with the alpha and/or gamma chains from human fibrinogen, although the positioning of a few of the smallest peptides is still ambiguous. The homology of the beta chain with the gamma chain is especially strong in certain regions of the domain that includes fragment D.