Primary structure of human fibrinogen and fibrin. Structural studies on NH2-terminal part of B beta chain.

The cyanogen bromide fragment, N-DSK, containing the NH2-terminal portions of the three chains of fibrinogen, was found to exist in dimeric and polymeric forms. These different forms gave rise to identical chain fragments on reduction and alkylation. The B beta chain of N-DSK from fibrinogen and the beta chain of N-DSK from fibrin were isolated and characterized. The B beta chain fragment has a blocked NH2-terminal residue, and fibrinopeptide B is released on digestion with thrombin. The beta chain fragment has glycine as NH2-terminal residue. The molecular weight of the B beta chain fragment is 12200 as determined by ultracentrifugal analysis. Gel electrophoresis in sodium dodecyl sulphate gave the molecular weights of 14000 and 13000 for the B beta chain and beta chain fragments, respectively. The NH2-terminal B beta chain fragment consists of 118 amino acid residues and the beta chain fragment of 104 residues. The amino acid sequence of beta chain fragment is identical to B beta chain fragment except for the fibrinopeptide B portion. The isolation of a B beta-related fragment (B beta +), with a molecular weight of 30000, is also reported. The presence of B beta + was explained on the basis of incomplete cleavage at the Met-118 residue during treatment with cyanogen bromide. Some functional aspects of the B beta chain fragment are discussed.     

Polymerization site in the beta chain of fibrin: mapping of the B beta 1-55 sequence

The formation of a fibrin clot occurs through binding of putative complementary sites, called fibrin polymerization sites, located in the NH2- and COOH-terminal domains of fibrin monomer molecules. In this study, we have investigated the structure of the NH2-terminal fibrin polymerization site by using fibrinogen-derived peptides and fragments. Fibrinogen was digested with Crotalus atrox protease III, to two major molecular species: a Mr 325,000 derivative (Fg325) and a peptide of Mr 5000. The peptide and its thrombin-cleavage product were purified by ion-exchange and reverse-phase HPLC; the authenticity of the B beta 1-42 and beta 15-42 peptides, respectively, was confirmed by amino acid sequencing. Since Fg325 had decreased thrombin coagulability, we addressed the question of whether the peptide B beta 1-42 contained a fibrin polymerization site. In order to identify and map the site, the peptides B beta 1-42 and beta 15-42 were tested for their ability to inhibit fibrin monomer polymerization. In addition the following peptides prepared by chemical synthesis were also tested: beta 15-18, beta 15-26, beta 24-42, beta 40-54, beta 50-55, and alpha 17-19-Pro. While B beta 1-42 had no inhibitory activity, the peptide devoid of fibrinopeptide B, beta 15-42, was a strong inhibitor. The peptides beta 15-18, beta 15-26, and beta 15-42 decreased the rate of fibrin polymerization by 50% at a molar excess of the peptide to fibrin monomer of 500, 430, and 50, respectively. The peptides beta 24-42, beta 40-54, and beta 50-55 were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. A kallikrein-like enzyme from the venom of Crotalus ruber ruber (red rattlesnake) had been isolated and characterized by Mori and Sugihara. The enzyme was active upon the kallikrein substrates, Pro-Phe-Arg-MCA and z-Phe-Arg-MCA, and slightly hydrolyzed Boc-Val-Leu-Lys-MCA, and Boc-Phe-Ser-Arg-MCA. 2. Unlike thrombin, the newly isolated kallikrein-like enzyme did not cause formation of a fibrin clot when fibrinogen was mixed with the enzyme. 3. The B beta chain of fibrinogen was first split and A alpha chain was cleaved later. Pancreatic kallikrein hydrolyzed only the A alpha chain without affecting the B beta chain. 4. The kallikrein-like enzyme produced kallidin (Lys-bradykinin) by splitting the Met-Lys bond instead of producing bradykinin. 5. The kallikrein analog JSI-450 (Ac-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser-NH2) was also cleaved at the site of the Arg-Ser bond. 6. Its NH2-terminal amino acid sequence (Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-Arg-Pro-Phe-Leu-Val-Ala-Leu-Tyr- Asp-Ser-) is homologous to the rat pancreatic kallikrein and other snake venom proteases.     

Identification of B beta chain domains involved in human fibrinogen assembly.

Fibrinogen chains are assembled in a stepwise manner in the rough endoplasmic reticulum prior to secretion of the final six-chain dimeric molecule. Previous studies indicated that the synthesis of B beta may be a rate-limiting factor in the assembly of human fibrinogen. To determine the domains of B beta which interact with the other two component chains of fibrinogen, deletion mutants of B beta were transiently co-expressed, together with A alpha and gamma chains, in COS cells, and fibrinogen assembly and secretion were measured. Deletion of the COOH-terminal half of the B beta chain (amino acids 208-461) did not affect assembly and secretion. Assembly of A alpha, gamma, and B beta also occurred when the first NH2-terminal 72 amino acids of B beta were deleted, but not when 93 amino acids were deleted. This indicates that the B beta domain between amino acids 73 and 93 is necessary for the assembly of the three fibrinogen chains. This domain marks the start of the alpha-helical "coiled-coil" region of fibrinogen.     

Assembly and secretion of fibrinogen. Degradation of individual chains.

Hep G2 cells produce surplus A alpha and gamma fibrinogen chains. These excess chains, which are not secreted, exist primarily as free gamma chains and as an A alpha-gamma complex. We have determined the intracellular location and the degradative fate of these polypeptides by treatment with endoglycosidase-H and by inhibiting lysosomal enzyme activity, using NH4Cl, chloroquine, and leupeptin. Free gamma chain and the gamma component of A alpha-gamma are both cleaved by endoglycosidase-H, indicating that the gamma chains accumulate in a pre-Golgi compartment. Lysosomal enzyme inhibitors did not affect the disappearance of free gamma chains but inhibited A alpha-gamma by 50%, suggesting that A alpha-gamma is degraded in lysosomes. The degradative fate of individual chains was determined in transfected COS cells which express but do not secrete single chains. Leupeptin did not affect B beta chain degradation, had very little affect on gamma chain, but markedly inhibited A alpha chain degradation. Antibody to immunoglobulin heavy chain-binding protein (GRP 78) co-immunoprecipitated B beta but not A alpha or gamma chains. Preferential binding of heavy chain-binding protein to B beta was also noted in Hep G2 cells and in chicken hepatocytes. Taken together these studies indicate that B beta and gamma chains are degraded in the endoplasmic reticulum, but only B beta is bound to BiP. By contrast A alpha chains and the A alpha-gamma complex undergo lysosomal degradation.     

Primary structure of human fibrinogen and fibrin. Isolation and partial characterization of chains of fragment D.

Fragment D has been isolated as an apparently single molecular weight species (molecular weight about 100,000) from plasmin digests of humman fibrinogen, using a combination of affinity chromatography on insolubilized "fibrin monomer" and gel filtration. This fragment consists of three chains with molecular weights of 15,000 (Dbeta), 42,500 (Dgamma1) or 39,500 (Dgamma2), and 14,000 (Dalpha) held together by disulfide bonds. The S-carboxymethyl derivatives of the chains have been separated by gel filtration and ion exchange chromatography, and their identity has been confirmed by peptide mapping and immunological analysis. The chain with a molecular weight of 45,000 is a fragment of the Bbeta chain of fibrinogen. The chain derived from the gamma chain of fibrinogen occurred in two molecular forms having molecular weight 42,500 and 39,500. The chain derivative with molecular weight 14,000 is most likely derived from the Aalpha chain of fibrinogen. The chains were characterized by NH2-terminal sequence analysis, amino acid composition, and carbohydrate staining. The two molecular analysis, amino acid composition, and carbohydrate staining. The two molecular forms of the gamma chain appeared to be identical except for an NH2-terminal peptide extension of 23 amino acid residues in the longer chain. The latter has sequences in common with the COOH-terminal part of the gamma chain of the NH2-terminal disulfide knot (BROMBACK, B., BRONDAHL, N. J., HESSEL, B., IWANAGA, S., and WALLEN, P. (1973) J. Biol. Chem. 248, 5806-5820); its NH2-terminal residue being Ala-63 of the gamma chain of fibrinogen.     

A unique proteolytic fragment of human fibrinogen containing the A alpha COOH-terminal domain of the native molecule

The COOH-terminal portion of the A alpha chain of human fibrinogen is highly susceptible to proteolytic degradation. This property has prevented isolation of the COOH-terminal domain of fibrinogen for the direct investigation of its functional characteristics. Human fibrinogen was degraded with hementin, a fibrinogen-olytic protease from the posterior salivary glands of the leech, Haementeria ghilianii. Two initial fragments, Yhem1 and Dhem1, produced by cleavage through the three polypeptide chains in the connector region, were characterized and shown to retain the entire A alpha COOH-terminal domain. Late cleavages by hementin occurred in the A alpha chain COOH-terminal region to produce fragments Yhem and Dhem with shorter A alpha chain remnants. Fragments Dhem were isolated from an intermediate hementin digest of fibrinogen using anion-exchange chromatography. Fragment Dhem1 was separated further from Dhem fragments with shorter alpha chain remnants by affinity chromatography on immobilized plasma fibronectin. Fragment Dhem1 represents a unique proteolytic fragment of fibrinogen containing an intact A alpha chain COOH-terminal region. NH2-terminal sequence analysis of isolated chains from fragment Dhem1 located hementin cleavage sites in the connector region to A alpha Asn102-Asn103, B beta Lys130-Gln131, and gamma Pro76-Asn77. The specific interaction of fragment Dhem1 with immobilized fibronectin indicated that the binding site probably was located within the COOH-terminal 111 amino acids of the A alpha chain. The overall pattern of fibrinogen cleavage by hementin is similar to that of plasmin, yet hementin cleaves preferably in the coiled-coil connector, sparing the A alpha COOH-terminal domain.     

Localization of a fibrin polymerization site

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.     

Characterization of laminin 5B and NH2-terminal proteolytic fragment of its alpha3B chain: promotion of cellular adhesion, migration, and proliferation

Various laminin isoforms have specific biological functions depending on their structures. Laminin 5A, which consists of the three truncated chains alpha3A, beta3, and gamma2, is known to have strong activity to promote cell adhesion and migration, whereas a laminin 5 variant consisting of a full-sized alpha3 chain (alpha3Beta) and the beta3 and gamma2 chains, laminin 5B, has not been characterized yet. In the present study, we for the first time cloned a full-length human laminin alpha3B cDNA and isolated the human laminin 5B protein. The molecular size of the mature alpha3B chain (335 kDa) was approximately twice as large as the mature alpha3A chain in laminin 5A. Laminin 5B had significantly higher cell adhesion and cell migration activities than laminin 5A. In addition, laminin 5B potently stimulated cell proliferation when added into the culture medium directly. Furthermore, we found that the alpha3B chain undergoes proteolytic cleavage releasing a 190-kDa NH(2)-terminal fragment. The 190-kDa fragment had activities to promote cellular adhesion, migration, and proliferation through its interaction with integrin alpha(3)beta(1). These activities of the NH(2)-terminal structure of the alpha3B chain seem to contribute to the prominent biological activities and the physiological functions of laminin 5B.     

Anticoagulant proteases from western diamondback rattlesnake (Crotalus atrox) venom

Crotalus atrox venom contains agents that render human fibrinogen and plasma incoagulable by thrombin. To elucidate the mechanism of alteration of fibrinogen clotting function by the venom, four immunochemically different proteases, I, II, III, and IV, were purified from the venom by anion-exchange chromatography and column gel filtration. All four proteases had anticoagulant activity rendering purified fibrinogen incoagulable. Proteases I and IV do not affect fibrinogen in plasma but in purified fibrinogen cleave the A alpha chain first and then the B beta and gamma chains. Both enzymes are metalloproteases containing a single polypeptide chain with 1 mol of zinc, are inhibited by (ethylenedinitrilo)tetraacetate and human alpha 2-macroglobulin, and have an optimal temperature of 37 degrees C and an optimal pH of 7. Protease I has a molecular weight (Mr) of 20 000 and is the most cationic. Protease IV has an Mr of 46 000 and is the most anionic glycoprotein with one free sulfhydryl group. Proteases II and III degrade both purified fibrinogen and fibrinogen in plasma, cleaving only the B beta chain and leaving the A alpha and gamma chains intact. Both enzymes are alkaline serine proteases, cleave chromogenic substrates at the COOH terminal of arginine or lysine, are inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride, and have an optimal temperature of 50-65 degrees C. Protease II is a single polypeptide chain glycoprotein with an Mr of 31 000. Protease III is a two polypeptide chain protein with an Mr of 24 000, each of the two chains having an Mr of 13 000; its activity is not affected by major protease inhibitors of human plasma. Proteases II and III are enzymes with unique and limited substrate specificity by cleaving only the B beta chain, releasing a peptide of Mr 5000 and generating a fibrinogen derivative of Mr 325 000, with intact A alpha and gamma chains and poor coagulability. Since the two enzymes are active in human plasma and serum, it is postulated that proteases II and III can mediate anticoagulant effects in vivo after envenomation.     

Thrombin binding to the A alpha-, B beta-, and gamma-chains of fibrinogen and to their remnants contained in fragment E

In order to study thrombin interaction with fibrinogen, thrombin binding to fragments D and E (prepared by plasmin digestion of fibrinogen) and to intact S-carboxymethylated chains of fibrinogen (A alpha, B beta, and gamma) was analyzed by autoradiography, immunoblotting, and affinity chromatography. Complex formation was observed between late fragment E and thrombin but not with fragment D. The three reduced chain remnants of fragment E all formed complexes with thrombin. Also, thrombin bound to the intact, separated A alpha, B beta, and gamma chains of fibrinogen as well as to the alpha and beta chains of fibrin. In these experiments the extended substrate-binding site, but not the catalytic-binding site, was being examined because fragment E had as its amino-terminal amino acids Val20 in the alpha chain, Lys54 in the beta chain, and Tyr1 in the gamma chain. Also, thrombin inhibited in its active center by D-phenyl-alanyl-L-prolyl-L-arginine-chloromethyl ketone bound to fragment E and to the separated chains in the same manner as unmodified thrombin. A lysine residue to thrombin was essential for its binding to fibrinogen. Thrombin attached to CNBr-activated Sepharose through its amino groups did not bind to fragment E, but when thrombin was attached through its carboxyl groups, it bound fragment E.     

Regulation of fibrinogen assembly. Transfection of Hep G2 cells with B beta cDNA specifically enhances synthesis of the three component chains of fibrinogen

Previous studies indicated that synthesis of B beta chain may be a rate-limiting factor in the production of human fibrinogen since Hep G2 cells contain surplus pools of A alpha and gamma but not of B beta chains, and fibrinogen assembly commences by the addition of preformed A alpha and gamma chains to nascent B beta chains attached to polysomes. To test whether B beta chain synthesis is rate limiting Hep G2 cells were transfected with B beta cDNA, and its effect on fibrinogen synthesis and secretion was measured. Two sets of stable B beta cDNA-transfected Hep G2 cells were prepared, and both cell lines synthesized 3-fold more B beta chains than control cells. The B beta-transfected cells also synthesized and secreted increased amounts of fibrinogen. Transfection with B beta cDNA not only increased the synthesis of B beta chain but also increased the rate of synthesis of the other two component chains of fibrinogen and maintained surplus intracellular pools of A alpha and gamma chains. Transfection with B beta cDNA did not affect the synthesis of albumin, transferrin, or anti-chymotrypsin and had a small inhibitory effect on the synthesis of C-reactive protein. Taken together these studies demonstrate that increased B beta chain synthesis specifically causes increased production of the other two component chains of fibrinogen and that unequal and surplus amounts of A alpha and gamma chains are maintained intracellularly.     

Structure of fragment E species from human cross-linked fibrin

Fragments E1, E2, and E3 are plasmic derivatives of fibrin encompassing the NH2-terminal region of the molecule. The first two species, but not the third, can bind to fragment DD, forming a (DD)E complex, and therefore probably contain binding sites involved in the polymerization of fibrin. For localization of these sites the structure of the fragments was determined by establishing the NH2- and COOH-terminal boundaries of the molecules and using the published amino acid sequence of fibrinogen. Fragment E1 encompasses Gly-alpha 17 to Lys-alpha 78, Gly-beta 15 to Lys-beta 122, and Tyr-gamma 1 to Lys-gamma 62, this representing the intact NH2-terminal region of fibrin. Fragment E2 is an asymmetric molecule which is lacking the sequence of Gly-beta 15 to Lys-beta 53 in one beta-chain remnant. This fragment E2 also lost Lys-beta 122 from the COOH terminal of the beta chain as compared with fragment E1. These cleavages did not affect the ability of fragment E2 to bind to fragment DD. Fragment E3 was heterogeneous, the main species encompassing Val-alpha 20 to Lys-alpha 78, Lys-beta 54 to Leu-beta 120, and Tyr-gamma 1 to Lys-gamma 53. Thus, the loss of the binding function involved in the formation of fibrin clot was associated with the removal of small fragments from all three polypeptide chains: alpha 17-19 (Gly-Pro-Arg), beta 15-53 from the remaining half of the molecule, beta 121 (Leu), and gamma 54-58 (Thr-Ser-Glu-Val-Lys).     

Studies on the assembly and secretion of fibrinogen.

cDNAs of fibrinogen A alpha and gamma chains were individually subcloned into a eukaryotic expression vector by using the polymerase chain reaction. Triple cotransfection into COS cells of the two plasmids together with a B beta chain expression plasmid, constructed as described previously (Danishefsky, K.J., Hartwig, R., Banerjee, D., and Redman, C. (1990) Biochim. Biophys. Acta 1048, 202-208), resulted in the secretion of complete fibrinogen into the media and the formation of four additional intracellular complexes which we also showed to be present in the hepatocyte cell line Hep 3B. The complexes, which have Mr = 232, 150, 135, and 128 (x 10(-3) conform with the Mr expected for A alpha B beta gamma 2, B beta gamma 2 and gamma 3, respectively. A A mechanism of assembly is proposed based on the assumption that all these complexes are precursors of complete fibrinogen. Each of the expressed fibrinogen chains in transfected COS cells interacts noncovalently with binding protein (BiP, GRP 78), but not to the same extent; gamma chain binds less BiP than the A alpha and B beta chains. Assembly of fibrinogen is not absolutely required for its secretion. In addition to complete fibrinogen, the conditioned media of hepatocytes and of transfected COS cells contained free A alpha, free gamma, and two of the above-mentioned complexes, A alpha gamma 2 and A alpha B beta gamma 2.     

The NH2-terminal alpha subunit attenuator domain confers regulation of G protein activation by beta gamma complexes.

Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the alpha s or alpha i subunit. The beta gamma subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and alpha subunit activation by GTP. Multiple beta and gamma polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The beta gamma complex from retinal rod outer segments (beta gamma t) has been shown to discriminate between alpha i and alpha s subunits (Helman et al: Eur J Biochem 169:431-439, 1987). beta gamma t efficiently interacts with alpha i-like G protein subunits, but poorly recognizes the alpha s subunit. beta gamma t was, therefore, used to define regions of the alpha i subunit polypeptide that conferred selective regulation compared to the alpha s polypeptide. A series of alpha subunit chimeras having NH2-terminal alpha i and COOH-terminal alpha s sequences were characterized for their regulation by beta gamma t, measured by the kinetics of GTP gamma S activation of adenylyl cyclase. A 122 amino acid NH2-terminal region of the alpha i polypeptide encoded within an alpha i/alpha s chimera was sufficient for beta gamma t to discriminate the chimera from alpha s. A shorter 54 amino acid alpha i sequence substituted for the corresponding NH2-terminal region of alpha s was insufficient to support the alpha i-like interaction with beta gamma t. The findings are consistent with our previous observation (Osawa et al: Cell 63:697-706, 1990) that a region in the NH2-terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the alpha subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by beta gamma complexes.     

Fibrinogens Kawaguchi and Osaka: an amino acid substitution of A alpha arginine-16 to cysteine which forms an extra interchain disulfide bridge between the two A alpha chains

Structural analyses of fibrinogens from patients with congenital dysfibrinogenemia, designated as fibrinogens Kawaguchi and Osaka, have been performed to identify the difference responsible for the lack of fibrinopeptide A release. For the structural studies, a new strategy was employed. Amino acid sequence analysis of one of the lysyl endopeptidase-peptides isolated from the abnormal fibrinogens indicated that in both fibrinogens, arginine-16 of the A alpha chain had been replaced by cysteine. To characterize the chemical nature of the sulfhydryl group of cysteine-16, a tryptic peptide containing cysteine-16 of the A alpha chain was prepared from intact fibrinogen Kawaguchi. The amino acid composition and the molecular weight determination of this aberrant peptide revealed that it was a dimeric NH2-terminal peptide corresponding to residues 1-19 derived from the abnormal A alpha chain. These results indicate that the half-cystine at position 16 in the abnormal A alpha chain forms an intramolecular disulfide bridge with the same residue in the other abnormal A alpha chain and that fibrinogen Kawaguchi is a homo dimer composed of two identical abnormal halves.     

Amino-Terminal Sequence of the Tryptophan Synthetase α Chain of Bacillus subtilis

The sequence of the 46 NH(2)-terminal residues of the tryptophan synthetase alpha chain of Bacillus subtilis was determined and compared with the corresponding sequences of Escherichia coli, Shigella dysenteriae, Salmonella typhimurium, Aerobacter aerogenes, Serratia marcescens, and Pseudomonas putida. A deletion of six residues was found at the NH(2)-terminal end of the alpha chain of B. subtilis.     

Structure-functional analysis of peptide P3 identical to gamma370-383 binding sites with integrin alphaIIbbeta3 in gammaC-domain of fibrinogen

The interactions between platelet integrin alpha IIb beta 3 and fibrinogen (Fg) mediate a range of adhesive reactions, which are necessary for platelet aggregation and fibrin clot retraction. The binding site for alpha IIb beta 3 resides in the gamma C domain of Fg. In our previous work we have identified a novel binding site in the gamma C domain, gamma 370-383 (P3), for integrin alpha IIb beta 3 and have demonstrated that the P3 sequence together with the C-terminal gamma C sequence 408AGDV411 accounts for the full binding of alpha IIb beta 3. In our present study, in order to define the amino acid residues in P3 involved in the interaction with alpha IIb beta 3, we have used SPOT-synthesis analyses. Libraries consisting of peptides covering P3 were created and probed with radiolabeled alpha IIb beta 3. Screening of the libraries showed that several positively charged residues may be critical for interaction of P3 with integrin alpha IIb beta 3.     

Carboxyl-terminal residues of mammalian fibrinogen and fibrin

The carboxyl-terminal residues of mammalian fibrinogens of six different species and the chain peptides, alpha(A), beta(B) and gamma, isolated from these fibrinogens were determined by hydrazinolysis, digestion with carboxypeptidases and selective tritium labelling. The C-terminal ends of bovine fibrinogen and fibrin were identified as proline and valine, in the molar ratio of approximately 1:2. Proline was identified as the C-terminus of the alpha(A)-chain, and C-terminal valine was found on both the beta(B)- and gamma-chains. On hydrazinolysis after selective tritium labelling of fibrinogen, radioactive C-terminal valine was also identified. The same C-terminal ends as those of bovine fibrinogen were found on the corresponding chain peptides isolated from sheep fibrinogen. The C-terminal residues of all the chain peptides of human and horse fibrinogens, however, were valine. In hog and dog fibrinogens, proline was identified at the C-termini of the alpha(A)-chains, and C-terminal valine and isoleucine were found on the beta(B)- and gamma-chains, respectively. Thus, the C-terminal amino acid residues of the fibrinogens of all mammalian species tested were very similar. It should be noted that hydrophobic amino acids, like isoleucine, valine and proline, are mainly located in the C-terminal ends of all three chain peptides in the fibrinogen molecule.     

Analysis of fibrinogen genes in patients with congenital afibrinogenemia

Several cDNA clones coding for A alpha, B beta and gamma chains of fibrinogen have been isolated from a human liver cDNA library. They were selected by differential hybridization with probes raised against fractionated liver mRNA (positive probes) and muscle and albumin mRNA (negative probes), then firmly identified by positive hybridization selection. Three of these clones, encoding A alpha, B beta and gamma fibrinogen chain sequences, were further characterized by restriction mapping and used as probes to characterize fibrinogen mRNAs from adult and fetal liver and fibrinogen genes in normal individuals and two afibrinogenemic patients. The results indicate that there is a single copy of the fibrinogen genes which are present and grossly intact in afibrinogenemic DNA.