Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains

Integrin alpha v beta 3, a widely distributed fibrinogen receptor, recognizes the RGD572-574 motif in the alpha chain of human fibrinogen. However, this motif is not conserved in other species, nor is it required for alpha v beta 3-mediated fibrin clot retraction, suggesting that fibrinogen may have other alpha v beta 3 binding sites. Fibrinogen has conserved C-terminal domains in its alpha (E variant), beta, and gamma chains (designated alpha EC, beta C, and gamma C, respectively), but their function in cell adhesion is not known, except that alpha IIb beta 3, a platelet fibrinogen receptor, binds to the gamma C HHLGGAKQAGDV400-411 sequence. Here we used mammalian cells expressing recombinant alpha v beta 3 to show that recombinant alpha EC and gamma C domains expressed in bacteria specifically bind to alpha v beta 3. Interaction between alpha v beta 3 and gamma C or alpha EC is blocked by LM609, a function-blocking anti-alpha v beta 3 mAb, and by RGD peptides. alpha v beta 3 does not require the HHLGGAKQAGDV400-411 sequence of gamma C for binding, and alpha EC does not have such a sequence, indicating that the alpha v beta 3 binding sites are distinct from those of alpha IIb beta 3. A small fragment of gamma C (residues 148-226) supports alpha v beta 3 adhesion, suggesting that an alpha v beta 3 binding site is located within the gamma chain 148-226 region. We have reported that the CYDMKTTC sequence of beta 3 is responsible for the ligand specificity of alpha v beta 3. gamma C and alpha EC do not bind to wild-type alpha v beta 1, but do bind to the alpha v beta 1 mutant (alpha v beta 1-3-1), in which the CYDMKTTC sequence of beta 3 is substituted for the corresponding beta 1 sequence CTSEQNC. This suggests that gamma C and alpha EC contain determinants for fibrinogen's specificity to alpha v beta 3. These results suggest that fibrinogen has potentially significant novel alpha v beta 3 binding sites in gamma C and alpha EC.     

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.     

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.     

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.     

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.     

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.     

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.     

Impaired platelet aggregation and sustained bleeding in mice lacking the fibrinogen motif bound by integrin alpha IIb beta 3.

Blood loss at sites of vascular rupture is controlled by the adhesion and aggregation of platelets and the formation of an insoluble fibrin matrix. Fibrinogen is considered to be critical in these processes by both providing an abundant dimeric ligand for alpha IIb beta 3-mediated platelet aggregation, and serving as the fundamental building block of the fibrin polymer. To establish an in vivo model system to examine in detail the importance of alpha IIb beta 3-fibrinogen interactions in platelet function, hemostasis, response to injury and vasoocclusive disease, and to test the prevailing hypothesis that the C-terminal segment of the fibrinogen gamma chain is essential for alpha IIb beta 3 binding, we have used gene-targeting technology in mice to eliminate the last five residues (QAGDV) from the gamma chain. Mice homozygous for the modified gamma chain gene (gamma delta 5/gamma delta 5) displayed a generally normal hematological profile, including normal platelet count, plasma fibrinogen level, clotting time and fibrin crosslinking. However, both gamma delta 5-fibrinogen binding to alpha IIb beta 3 and platelet aggregation were highly defective. Remarkably, another alpha IIb beta 3-dependent process, clot retraction, was unaffected by the gamma delta 5 mutation. Despite the preservation of clotting function, gamma delta 5/gamma delta 5 mice were unable to control blood loss following a surgical challenge and occasionally developed fatal neonatal bleeding events.     

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.     

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.     

Conservation of human fibrinogen conformation after cleavage of the B beta chain NH2 terminus

Human fibrinogen exposed to protease III from Crotalus atrox venom is cleaved near the NH2 terminus of the B beta chain yielding a species of Mr 325,000 (Fg325) with impaired thrombin clottability. The derivative was compared with intact fibrinogen in a number of ways to determine whether the functional defect resulted from a conformational change or from the loss of a polymerization site. NH2-terminal amino acid sequencing of isolated A alpha, B beta, and gamma chains showed that Fg325 contained intact A alpha and gamma chains, but differed from fibrinogen by the absence of the first 42 residues of the B beta chain. Fibrinopeptide A was present and was cleaved at the same rate in both fibrinogen and Fg325. The rate and extent of A alpha and gamma cross-linking by factor XIIIa was also indistinguishable. In contrast, the thrombin-catalyzed coagulation of Fg325 was 46% less in extent and 180-fold slower than observed for intact fibrinogen. A conformational comparison of Fg325 and fibrinogen was made using immunochemical and spectroscopic approaches. Antisera specific for different regions of the fibrinogen molecule were used to characterize the epitopes in Fg325. The only significant differences were found in the NH2-terminal region of the B beta chain, probed with antiserum to B beta 1-118. The conformational similarity of Fg325 and fibrinogen was confirmed by the identity of both near and far UV CD spectra of the two proteins. Structural, functional, and immunochemical results imply that cleavage of 42 NH2-terminal residues from the B beta chain is not accompanied by a measurable conformational change. The residues of this B beta chain segment, which are evidently located on the surface of the molecule, in conjunction with the NH2-terminal part of the A alpha chain appear to play an important role in the expression of a fibrin polymerization site.     

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.     

Effects of starvation and different culture conditions on the phospholipid content of isolated pancreatic islets

Three forms of the normal human plasma fibrinogen gamma-chain which differ in molecular weight have been purified. Plasma fibrinogen was separated by ion exchange chromatography on DEAE-Sephacel into three populations of molecules, each with a unique gamma-chain composition. Following reduction and S-carboxymethylation, the fibrinogen polypeptide chains in each chromatographic peak were separated by ion exchange chromatography on DEAE-Sephacel and identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The A alpha, B beta and smallest gamma-chain (gamma 50) eluted at progressively higher ionic strengths, but the elution positions of A alpha, B beta and gamma 50 chains were identical for fibrinogen from each of the three different chromatographic fractions. The unique gamma chain of fibrinogen in the second chromatographic peak (gamma 55) eluted at an ionic strength higher than that of the gamma 50 chain, while the largest gamma-chain (gamma 57.5), which was contained only in the third chromatographic peak of fibrinogen, eluted at the highest ionic strength. The higher ionic strengths needed to elute fibrinogen in the second and third peaks was paralleled by the higher ionic strengths needed to elute the gamma-chains unique to them, suggesting that the gamma-chain composition of the three fibrinogen fractions accounted for their differential binding to the ion exchange resin. Following desialation with neuraminidase, the differences in electrophoretic mobilities between the three gamma-chain forms was maintained, indicating that differential migration on SDS-polyacrylamide gel electrophoresis was not due to variation in sialic acid content.     

The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins

Laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed alpha, beta, and gamma. The putative binding site for integrins has been mapped to the G domain of the alpha chain, although trimerization with beta and gamma chains is necessary for the G domain to exert its integrin binding activity. The mechanism underlying the requirement of beta and gamma chains in integrin binding by laminins remains poorly understood. Here, we show that the C-terminal region of the gamma chain is involved in modulation of the integrin binding activity of laminins. We found that deletion of the C-terminal three but not two amino acids within the gamma1 chain completely abrogated the integrin binding activity of laminin-511. Furthermore, substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the gamma1 chain, also abolished the integrin binding activity, underscoring the role of Glu-1607 in integrin binding by the laminin. We also found that the conserved Glu residue of the gamma2 chain is necessary for integrin binding by laminin-332, suggesting that the same mechanism operates in the modulation of the integrin binding activity of laminins containing either gamma1 or gamma2 chains. However, the peptide segment modeled after the C-terminal region of gamma1 chain was incapable of either binding to integrin or inhibiting integrin binding by laminin-511, making it unlikely that the Glu residue is directly recognized by integrin. These results, together, indicate a novel mechanism operating in ligand recognition by laminin binding integrins.     

Evolution and structure of the fibrinogen genes: Random insertion of introns or selective loss?

Chromosomal linkage as well as sequence homologies provide unequivocal evidence that the genes for the alpha, beta and gamma chains of fibrinogen arose by successive duplication of a single ancestral gene. Yet, when the three fibrinogen chains are aligned by amino acid homology, the positions of intervening sequences coincide at only two positions for all three chains. While one additional intron occurs at a homologous site in the beta and gamma chains, none of the positions of the remaining 11 introns in the three genes is shared. This arrangement of introns in the three fibrinogen genes suggests that either introns were selectively lost, implying that there is essential information in the retained introns, or the common introns were present in the ancestral fibrinogen gene and introns have been randomly inserted since the triplication of the original gene. The more likely possibility of selective loss of introns implies that the ancestral gene, as it existed about one billion years ago, must have been composed of numerous small exons.     

Noncoordinate synthesis of the fibrinogen subunits in hepatocytes cultured under hormone-deficient conditions

Differential detergent gel electrophoresis conditions are described which enable the accurate quantitation of radiolabel incorporated into each of the closely migrating, constituent polypeptides of chicken fibrinogen: glycosylated and nonglycosylated A alpha, B beta, gamma', and gamma. These methods were applied to analysis of fibrinogen synthesis by monolayer cultures of chick embryo hepatocytes to determine whether the cells coordinate biosynthesis of the fibrinogen subunits under nonstimulated or basal conditions (i.e. in the absence of hormones) and in the presence of serum, which is a potent stimulator of fibrinogen production. Since secretion of the subunits apparently depends on their oligomeric assembly into the general structure (A alpha, B beta, gamma)2, it was thought that their synthesis might be stoichiometric. Incorporation of [35S]methionine into the subunit chains was determined for both cellular and secreted fibrinogen, immunoprecipitated from pulse-labeled and continuously labeled cultures. Molar ratios of subunit synthesis and the degree of serum-induced stimulation for each subunit were calculated. Specific subunit mRNA levels were also evaluated with a cell-free translation assay as well as microinjection of RNA into Xenopus oocytes. The results indicate, to the contrary, that in hormone-deprived hepatocytes there is a deficiency in A alpha chain synthesis, correlating with reduced A alpha-specific mRNA levels, which leads to hepatocellular degradation of surplus B beta and gamma chains. Addition of serum to the cellular environment, while increasing rates of subunit synthesis, also corrects the deficiency in A alpha chain synthesis, thereby restoring a measure of balance and preventing much of the degradation. The outcome of this serum-induced enhancement and coordination of fibrinogen subunit gene expression is a dramatic (more than 20-fold) stimulation of fibrinogen secretion.     

Isolation of two hemorrhagic toxins from Crotalus basiliscus basiliscus (Mexican west coast rattlesnake) venom and their effect on blood clotting and complement

1. Two hemorrhagic toxins of mol. wt 27,000 (B1) and 27,500 (B2) and pI 9.8 and 5.2 respectively were isolated from Crotalus basiliscus venom. 2. The two proteinases did not cross-react antigenically. 3. Both toxins caused hemorrhage in mice and each was capable of hydrolyzing hide power azure, casein, collagen and fibrin. 4. B1 hydrolyzed the A alpha, B beta and gamma chains of fibrinogen. B2 hydrolyzed the A alpha and B beta chains of fibrinogen, but not the gamma chain. 5. Both proteinases inactivated guinea pig complement.     

Recombinant human fibrinogen and sulfation of the gamma' chain

Human fibrinogen and the homodimeric gamma'-chain-containing variant have been expressed in BHK cells using cDNAs coding for the alpha, beta, and gamma (or gamma') chains. The fibrinogens were secreted at levels greater than 4 micrograms (mg of total cell protein)-1 day-1 and were biologically active in clotting assays. Recombinant fibrinogen containing the gamma' chain incorporated 35SO4 into its chains during biosynthesis, while no incorporation occurred in the protein containing the gamma chain. The identity of the sulfated gamma' chain was verified by its ability to form dimers during clotting. In addition, carboxypeptidase Y digestion of the recombinant fibrinogen containing the gamma' chain released 96% of the 35S label from the sulfated chain, and the radioactive material was identified as tyrosine O-sulfate. These results clarify previous findings of the sulfation of tyrosine in human fibrinogen.     

Identification of amino acid sequences in fibrinogen gamma -chain and tenascin C C-terminal domains critical for binding to integrin alpha vbeta 3

Integrin alpha(v)beta(3) recognizes fibrinogen gamma and alpha(E) chain C-terminal domains (gammaC and alpha(E)C) but does not require the gammaC dodecapeptide sequence HHLGGAKQAGDV(400-411) for binding to gammaC. We have localized the alpha(v)beta(3) binding sites in gammaC using gammaC-derived synthetic peptides. We found that two peptides GWTVFQKRLDGSV(190-202) and GVYYQGGTYSKAS(346-358) block the alpha(v)beta(3) binding to gammaC or alpha(E)C, block the alpha(v)beta(3)-mediated clot retraction, and induce the ligand-induced binding site 2 (LIBS2) epitope in alpha(v)beta(3). Neither peptide affects fibrinogen binding to alpha(IIb)beta(3). Scrambled or inverted peptides were not effective. These results suggest that the two gammaC-derived peptides directly interact with alpha(v)beta(3) and specifically block alpha(v)beta(3)-gammaC or alpha(E)C interaction. The two sequences are located next to each other in the gammaC crystal structure, although they are separate in the primary structure. Asp-199, Ser-201, Gln-350, Thr-353, Lys-356, Ala-357, and Ser-358 residues are exposed to the surface. This suggests that the two sequences are part of alpha(v)beta(3) binding sites in fibrinogen gammaC domain. We also found that tenascin C C-terminal fibrinogen-like domain specifically binds to alpha(v)beta(3). Notably, a peptide WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG from this domain that includes the sequence corresponding to gammaC GVYYQGGTYSKAS(346-358) specifically binds to alpha(v)beta(3), suggesting that fibrinogen and tenascin C C-terminal domains interact with alpha(v)beta(3) in a similar manner.     

Comparison of structures of various human fibrinogens and a derivative thereof by a study of the kinetics of release of fibrinopeptides

The kinetics of the thrombin-induced release of fibrinopeptides from several variants of human fibrinogen, and from the plasmin digestion fragment E thereof, have been studied by using an HPLC technique to separate the reaction products. The data were analyzed in terms of a Michaelis-Menten mechanism in which the A alpha and B beta chains compete for thrombin. Phosphorylation of Ser-3 of the A alpha chain appears to increase the rate of release of the corresponding phosphorylated peptide A from fibrinogen, due to enhanced binding of thrombin (lower value of the Michaelis-Menten constant KM). However, phosphorylation does not affect the rate of release of the unphosphorylated A or B peptides. Increase in the length of the gamma chain (at the C-terminus) does not affect the rate of release of any of the fibrinopeptides. The rate of release of the A peptide from fragment E (which is devoid of the B peptide) is similar to that for the complete fibrinogen molecule. These results are in agreement with an earlier conclusion [Martinelli, R. A., & Scheraga, H. A. (1980) Biochemistry 19, 2343] that the A alpha and B beta chains behave independently in their competition for thrombin; i.e., the hydrolyzable Arg-Gly bonds of the A alpha and B beta chains are both accessible to thrombin.