Interaction of fibrin(ogen) with leukocyte receptor alpha M beta 2 (Mac-1): further characterization and identification of a novel binding region within the central domain of the fibrinogen gamma-module

The fibrinogen gamma-module sequences, gamma190-202 or P1, and gamma377-395 or P2, were implicated in interaction with the alpha(M)I-domain of the leukocyte receptor alpha(M)beta(2). P1 is an integral part of the gamma-module central domain, while P2 is inserted into this domain forming an antiparallel beta-strand with P1. We hypothesized earlier that separation of P2 from P1 may regulate interaction of fibrin(ogen) with leukocytes during the inflammatory response. To test the relative contributions of these sequences to the interaction and the effect of their separation, we prepared the recombinant gamma-module (gamma148-411) and its halves, gamma148-286 and gamma287-411 fragments containing P1 and P2, respectively, and evaluated their affinities for the recombinant alpha(M)I-domain. In a solid-phase binding assay, the immobilized gamma-module exhibited high affinity for alpha(M)I (K(d) = 22 nM), while the affinities of the isolated gamma148-286 and gamma287-411 halves were much lower (K(d)'s = 521 and 194 nM, respectively), indicating that both halves contribute to the interaction in a synergistic manner. This is consistent with the above hypothesis. Further, we prepared the recombinant gamma148-191 and gamma192-286 fragments corresponding to the NH(2)-terminal and central domains, respectively, as well as gamma148-226 containing P1, and tested their interaction with alpha(M)I. The immobilized gamma192-286 fragment bound to alpha(M)I with K(d) = 559 nM, while both gamma148-191 and gamma148-226 failed to bind suggesting that P1 does not contribute substantially to the binding and that the binding occurs mainly through the gamma227-286 region. To further localize a putative binding sequence, we cleaved gamma192-286 and analyzed the resulting peptides. The only alpha(M)I-binding activity was associated with the gamma228-253 peptide, indicating that this region of the central domain contains a novel alpha(M)beta(2)-binding sequence.     

Factor XIIIa incorporates thymosin beta4 preferentially into the fibrin(ogen) alphaC-domains

It was shown recently that tissue transglutaminase and presumably plasma transglutaminase, factor XIIIa, can covalently incorporate into fibrin(ogen) a physiologically active peptide, thymosin beta(4) [(Huff et al. (2002) FASEB J. 16, 691-696]. To clarify the mechanism of this incorporation, we studied the interaction of thymosin beta(4) with fibrinogen, fibrin, and their recombinant fragments, the gamma-module (gamma-chain residues 148-411), and the alphaC-domain (Aalpha-chain residues 221-610) and its truncated variants by immunoblot and ELISA. No significant noncovalent interaction between them was detected in the absence of activated factor XIII, while in its presence thymosin beta(4) was effectively incorporated into fibrin and to a lesser extent into fibrinogen. The incorporation at physiological concentrations of fibrin(ogen) and factor XIII was significant with molar incorporation ratios of thymosin beta(4) to fibrinogen and fibrin of 0.2 and 0.4, respectively. Further experiments revealed that although activated factor XIII incorporates thymosin beta(4) into the isolated gamma-module and alphaC-domain, in fibrin the latter serves as the major incorporation site. This site was further localized to the COOH-terminal portion of the alphaC-domain including residues 392-610.     

Evidence that three adhesive proteins interact with a common recognition site on activated platelets

Synthetic peptides corresponding to the extreme COOH terminus of the gamma chain of fibrinogen gamma 400-411, (400)HHLGGAKQAGDV(411), have been used to analyze recognition specificities of the platelet-binding sites for fibrinogen, fibronectin, and von Willebrand factor. gamma 403-411 did not inhibit 125I-fibrinogen binding to platelets. In contrast, gamma 402-411 produced dose-dependent inhibition of fibrinogen binding to ADP and thrombin-stimulated living or fixed cells and was a competitive antagonist. Inhibitory activity was not modified by addition of one (gamma 401-411) or two (gamma 400-411) histidinyl residues to the NH2 terminus, but peptides with a trifluoroacetyl group on the epsilon-amino group of lysine 406 were inactive. 125I-Fibronectin and 125I-von Willebrand factor binding to thrombin-stimulated living or fixed cells was inhibited in the same dose range by the same set of peptides which inhibited fibrinogen binding and not by gamma 403-411 or trifluoroacetate-blocked peptides. The capacity of the peptides to inhibit binding to cells with an expressed receptor, i.e. fixed cells, excludes an effect on receptor induction. Thus, these results suggest that the three adhesive glycoproteins share a common site on thrombin-activated platelets, and a 10-amino acid peptide, corresponding to gamma 402-411, defines the recognition specificity of this site.     

Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.     

1H NMR sequential assignments and secondary structure analysis of human fibrinogen gamma-chain C-terminal residues 385-411

The human fibrinogen gamma-chain, C-terminal fragment, residues 385-411, i.e., KIIPFNRLTIGEGQQHHLGGAKQAGDV, contains two biologically important functional domains: (1) fibrinogen gamma-chain polymerization center and (2) platelet receptor recognition domain. This peptide was isolated from cyanogen bromide degraded human fibrinogen and was investigated by 1H NMR (500 MHz) spectroscopy. Sequence-specific assignments of NMR resonances were obtained for backbone and side-chain protons via analysis of 2D NMR COSY, double quantum filtered COSY, HOHAHA, and NOESY spectra. The N-terminal segment from residues 385-403 seems to adopt a relatively fixed solution conformation. Strong sequential alpha CH-NH NOESY connectivities and a continuous run of NH-NH NOESY connectivities and several long-lived backbone NH protons strongly suggest the presence of multiple-turn or helix-like structure for residues 390 to about 402. The conformation of residues 403-411 seems to be much less constrained as evidenced by the presence of weaker and sequential alpha CH-NH NOEs, the absence of sequential NH-NH NOEs, and the lack of longer lived amides. Chemical shifts of resonances from backbone and side-chain protons of the C-terminal dodecapeptide, residues 400-411, differ significantly from those of the parent chain, suggesting that some preferred C-terminal conformation does exist.     

Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIbIIIa

Platelet membrane GPIIbIIIa is a member of the family receptors named integrins that recognize RGD sequences in their ligands. GPIIbIIIa interacts with at least three different adhesive ligands: fibrinogen, fibronectin, and von Willebrand factor. These interactions are inhibited by RGD-containing peptides and by peptides corresponding to a sequence unique to fibrinogen in the COOH-terminal domain of its gamma chain (HLGGAKQAGDV). Two RGD sequences are present in fibrinogen A alpha chain: an RGDS sequence at A alpha 572-575, and an RGDF sequence at A alpha 95-98. Polyclonal antibodies raised against the RGDF sequence and the gamma COOH-terminal domain both reacted specifically with fibrinogen in solid phase enzyme-linked immunosorbent assays and immunoprecipitated the protein in solution. The Fab fragments prepared from these antibodies inhibited fibrinogen-platelet interaction and aggregation. These results demonstrate that these two sequences are both accessible within the fibrinogen molecule and are both implicated in ligand binding and cell-cell interaction. In addition, by further examining the interaction of the gamma chain peptide with platelets, it was found that RGDF and the gamma peptide produced a similar dose-dependent inhibition of the binding of the labeled gamma peptide to ADP-stimulated platelets. These results provide evidence that the RGDF sequence present at the A alpha 95-98 constitutes with the gamma 401-411 sequence two recognition sites interacting with the same site or with mutually exclusive sites on GPIIbIIIa.     

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.     

Localization of segments essential for polymerization and for calcium binding in the gamma-chain of human fibrinogen

We have isolated an intermediate plasmic degradation product, D2, of fibrinogen that does not inhibit the polymerization of fibrin monomer but does bind Ca2+. Fibrinogen was digested to a limited extent with plasmin in the presence of Ca2+, and a "large" fragment D (fragment D1A) was isolated with a gamma-chain remnant consisting of residues 63-411. Fragment D1A was digested further in the presence of Ca2+, yielding fragment D1 (with its gamma-chain containing residues 86-411). The digestion of fragment D1 [in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to complex Ca2+] led to a gradual shortening of the carboxyl-terminal portion of the gamma-chain. Fragment D2 (with its gamma-chain containing residues 86-335/356) was isolated from an intermediate digest in the presence of EGTA. The Lys-338-Cys-339 peptide bond of the gamma-chain is intact in this preparation of D2, even though it is split in the isolated peptide gamma303-355 (with an intact disulfide bond at Cys-326-Cys-339). Fragment D2 does not interfere with the polymerization of fibrin monomer, whereas fragment D1 is a potent inhibitor of this polymerization. We conclude that the gamma-chain segment 356/357-411, present in fragment D1 but absent from fragment D2, is essential for maintenance of a polymerization site located in the outer (D) nodule of fibrinogen. This segment (356/357-411) is longer than two shorter ones reported earlier [Olexa, S.A., & Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3549; Horwitz, B.H., Váradi, A., & Scheraga, H.A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5980-5984]; the data for the earlier reports are reinterpreted here. Finally, fragment D2 possesses a single Ca2+ binding site, as revealed by equilibrium dialysis binding studies. Since fragment D3 (with its gamma-chain containing residues 86-302) fails to bind Ca2+, we conclude that segment gamma 303-355/356 plays a crucial role in Ca2+ binding.     

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.     

Identification of a site in fibrin(ogen) which is involved in the acceleration of plasminogen activation by tissue-type plasminogen activator

The rate of activation of plasminogen by tissue-type plasminogen activator is greatly increased by fibrin, but not by fibrinogen. A possible explanation for this phenomenon could be that conformational changes take place during the transformation of fibrinogen to fibrin which lead to exposure of sites involved in the accelerated plasmin formation. This is also supported by our recent observation that some enzymatically prepared fragments of fibrinogen and fibrin (D EGTA, D-dimer, Y) and also CNBr fragment 2 from fibrinogen have this property. CNBr fragment 2 consists of amino acid residues A alpha (148-207), B beta (191-224) + (225-242) + (243-305) and gamma 95-265, kept together by disulphide bonds. In order to study the localization of a stimulating site within this structure we purified the chain remnants of CNBr fragment 2 after reduction and carboxymethylation, and found that only A alpha 148-207 was stimulating. This was further confirmed by digesting pure A alpha-chains with CNBr and purifying the resulting A alpha-chain fragments. CNBr digests of B beta- and gamma-chains were not stimulatory. The A alpha-chain remnant (residues 111-197) in D EGTA and D-dimer also comprise the major part (residues A alpha 148-197) of the CNBr A alpha-chain fragment. We conclude that a site capable of accelerating the plasminogen activation by tissue-type plasminogen activator preexists in fibrinogen, that this site becomes exposed upon fibrin formation or disruption of fibrinogen by plasmin or CNBr and that this site is within the stretch A alpha 148-197, which is retained in the A alpha-chain remnants of fibrinogen degradation products.     

Regulated unmasking of the cryptic binding site for integrin alpha M beta 2 in the gamma C-domain of fibrinogen

Fibrinogen is a ligand for leukocyte integrin alpha(M)beta2 (CD11b/CD18, Mac-1) and mediates adhesion and migration of leukocytes during the immune-inflammatory responses. The binding site for alpha(M)beta2 resides in gammaC, a constituent subdomain in the D-domain of fibrinogen. The sequence gamma383-395 (P2-C) in gammaC was implicated as the major binding site for alpha(M)beta2. It is unknown why alpha(M)beta2 on leukocytes can bind to immobilized fibrinogen in the presence of high concentrations of soluble fibrinogen in plasma. In this study, we have investigated the accessibility of the binding site in fibrinogen for alpha(M)beta2. We found that the alpha(M)beta2-binding site in gammaC is cryptic and identified the mechanism that regulates its unmasking. Proteolytic removal of the small COOH-terminal segment(s) of gammaC, gamma397/405-411, converted the D100 fragment of fibrinogen, which contains intact gammaC and is not able to inhibit adhesion of the alpha(M)beta2-expressing cells, into the fragment D98, which effectively inhibited cell adhesion. D98, but not D100, bound to the recombinant alpha(M)I-domain, and the alpha(M)I-domain recognition peptide, alpha(M)(Glu253-Arg261). Exposure of the P2-C sequence in fibrinogen, D100, and D98 was probed with a site-specific mAb. P2-C is not accessible in soluble fibrinogen and D100 but becomes exposed in D98. P2-C is also unmasked by immobilization of fibrinogen onto a plastic and by deposition of fibrinogen in the extracellular matrix. Thus, exposure of P2-C by immobilization and by proteolysis correlates with unmasking of the alpha(M)beta2-binding site in the D-domain. These results demonstrate that conformational alterations regulate the alpha(M)beta2-binding site in gammaC and suggest that processes relevant to tissue injury and inflammation are likely to be involved in the activation of the alpha(M)beta2-binding site in fibrinogen.     

A beta-turn is present in the 392-411 segment of the human fibrinogen gamma-chain. Effects of structural changes in this segment on affinity to antibody 4A5.

The interaction between fibrinogen gamma-peptide 392-411, LTIGEGQQHHLGGAKQAGDV, and monoclonal antibody 4A5, an antibody with a high affinity for both for the peptide and native fibrinogen, is being studied as a model for peptide-antibody interaction. Two-dimensional NMR studies of the free peptide at pH 5.2 indicated the presence of a significant population, about 60%, of type II beta-turn, spanning residues Gln407-Asp410. At pH 2.7, little, if any, turn structure is present. The D-Ala409 analog, which, for steric reasons, would be expected to preserve the beta-turn, and the L-Ala409 analog, which would not be expected to have this conformational feature, were synthesized, and NMR studies confirmed the respective structural predictions. The affinity of the D-Ala analog for antibody 4A5 is even greater than that displayed by native gamma 392-411, while the affinity of the L-Ala analog is less than one-tenth that of the native peptide. Both conformational and steric effects involving residues 407-410 may be important in recognition by antibody 4A5. Since gamma 392-411 includes a platelet receptor binding locus of fibrinogen, and this and related peptides are inhibitors of platelet aggregation, the D-Ala409 and L-Ala409 analogs were tested for platelet binding. Neither of the analogs displays any measurable platelet binding, indicating that the recognition requirements for the platelet receptor differ considerably from those for antibody 4A5.     

Sequence gamma 377-395(P2), but not gamma 190-202(P1), is the binding site for the alpha MI-domain of integrin alpha M beta 2 in the gamma C-domain of fibrinogen

The interaction between the leukocyte integrin alpha(M)beta(2) (CD11b/CD18, Mac-1, CR3) and fibrinogen mediates the recruitment of phagocytes during the inflammatory response. Previous studies demonstrated that peptides P2 and P1, duplicating gamma 377-395 and gamma 190-202 sequences in the gamma C domain of fibrinogen, respectively, blocked the fibrinogen-binding function of alpha(M)beta(2), implicating these sequences as possible binding sites for alpha(M)beta(2). To determine the role of these sequences in integrin binding, recombinant wild-type and mutant gamma C domains were prepared, and their interactions with the alpha(M)I-domain, a ligand recognition domain within alpha(M)beta(2), were tested. Deletion of gamma 383-411 (P2-C) and gamma 377-411 produced gamma C mutants which were defective in binding to the alpha(M)I-domain. In contrast, alanine mutations of several residues in P1 did not affect alpha(M)I-domain binding, and simultaneous mutations in P1 and deletion of P2 did not decrease the binding function of gamma C further. Verifying the significance of P2, inserting P2-C and the entire P2 into the homologous position of the beta C-domain of fibrinogen imparted the higher alpha(M)I-domain binding ability to the chimeric proteins. To further define the molecular requirements for the P2-C activity, synthetic peptides derived from P2-C and a peptide array covering P2-C have been analyzed, and a minimal recognition motif was localized to gamma(390)NRLTIG(395). Confirming a critical role of this sequence, the cyclic peptide NRLTIG retained full activity inherent to P2-C, with Arg and Leu being important residues. Thus, these data demonstrate the essential role of the P2, but not P1, sequence for binding of gamma C by the alpha(M)I-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.     

Determination of the carboxyl termini of the alpha and beta subunits of yeast K1 killer toxin. Requirement of a carboxypeptidase B-like activity for maturation

The carboxyl-terminal sequences of the two polypeptide chains of the Saccharomyces cerevisiae K1 killer toxin were determined by protein sequencing and amino acid analysis of peptide fragments generated from the mature, secreted toxin. The COOH-terminal amino acid of the beta chain is histidine 316, the final residue encoded by the precursor gene. The COOH terminus of the alpha chain is at alanine 147 of the preprotoxin. Amino acid composition data for the purified toxin are consistent with that predicted from the gene sequence of the preprotoxin where the alpha and beta subunits consist of amino acid residues 45-147 and 234-316, respectively. The molecular weight of the mature alpha beta dimer is about 20,658. The COOH-terminal sequence determination completes the location of the toxin subunits in the precursor, and its configuration may be represented as prepropeptide-Pro-Arg-alpha-Arg-Arg-gamma-Lys-Arg-beta, where gamma represents the interstitial glycosylated peptide. The COOH terminal side of the paired basic residues (Arg-148 Arg-149 and Lys-232 Arg-233 of preprotoxin) are endoproteolytic processing sites for the product of the KEX2 gene (Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984) Cell 37, 1075-1089), and thus maturation of the alpha subunit of killer toxin apparently requires a carboxypeptidase B-like activity. A possible candidate for this activity is the product of the KEX1 gene (Dmochowska, A., Dignard, D., Henning, D., Thomas, D.Y., and Bussey, H. (1987) Cell, in press).     

Analysis of engineered fibrinogen variants suggests that an additional site mediates platelet aggregation and that "B-b" interactions have a role in protofibril formation

The C-terminal domain of the fibrinogen gamma-chain includes multiple functional sites that have been defined in high-resolution structures and biochemical assays. Calcium binds to this domain through the side chains of gammaD318 and gammaD320 and the backbone carbonyls of gammaF322 and gammaG324. We have examined variant fibrinogens with alanine at position gamma318 and/or gamma320 and found that calcium binding, fibrin polymerization, and fibrinogen-mediated platelet aggregation, but not FXIIIa-catalyzed cross-linking, were abnormal. When measured by turbidity, thrombin-catalyzed polymerization was severely reduced, and batroxobin-catalyzed polymerization was completely obliterated. Moreover, thrombin-catalyzed polymerization was abolished by the peptide GHRP, which binds to the polymerization site in the beta-chain but does not inhibit polymerization of normal fibrinogen. ADP-induced platelet aggregation was also severely impaired. In contrast, as measured by SDS-PAGE, FXIIIa introduced cross-links between gamma-chains for all three variants, as expected if the gamma-chain C-terminal sites were normal. In addition, binding of the monoclonal antibody 4A5, which recognizes the C-terminal residues, was not different from normal. These data suggest two specific conclusions: (1) a site in the gamma-module other than the C-terminus is critical for platelet aggregation and (2) "B-b" interactions have a role in protofibril formation.     

Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence

Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.     

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 re-examination of the cleavage of fibrinogen and fibrin by plasmin.

Three Fragment D species (D1, D2, D3) were isolated with time from a plasmin digest of fibrinogen and had molecular weights of 92,999, 86,000 and 82,000 by summation of subunit molecular weights from sodium dodecyl sulfate polyacrylamide gel electrophoresis. Their molecular weights by sedimentation equilibrium ultracentrifugation were 94,000 t87,000, 88,000 to 82, 000, and 76,000 to 70,000 depending on the values calculated for the partial specific volumes. Each of the Fragment D species contained three disulfide-linked subunits derived from the Aalpha, Bbeta, and gamma chains of fibrinogen and differed only in the extent of COOH-terminal degradation of their gamma chain derivatives. Plasmin cleaved Fragment D1 to release the cross-link sites from its gamma' subunit of 38,000 molecular weight; however, the beta' subunit of 42,000 molecular weight and the alpha' subunit of 12,000 molecular weight were resistant to further digestion by plasmin. Fragment D isolated from highly cross-linked fibrin had a dimeric structure due to cross-link formation between the gamma' subunits of two fibrinogen Fragment D species. The molecular weight of fibrin Fragment D was 184,000 by summation of subunit molecular weights and 190,000 to 175,000 by sedimentation equilibrium. Cross-linking the gamma chain, as well as incorporating the site-specific fluorescent label monodansyl cadaverine into the gamma chain cross-link acceptor site, prevented its COOH-terminal degradation by plasmin. Therefore, only one species of fibrin Fragment D, as well as only one species of monodansyl cadaverine-labeled fibrin Fragment D monomer, was generated during plasmin digestion. These results show unequivocally that each fibrinogen Fragment D contains only three subunit chains and therefore the digestion of fibrinogen by plasmin must result in the production of two Fragment D molecules from each fibrinogen molecule. The recently proposed model of fibrinogen cleavage that postulates the generation of a single Fragment D with three pairs of subunit chains from each fibrinogen molecule is incorrect. Incorporation of monodansyl cadaverine into the cross-link acceptor sites of the alpha chain did not alter its cleavage by plasmin detectably. A series of monodansyl cadaverine-labeled peptides, which ranged in molecular weight from 40,000 to 23,000, were cleaved from the alpha chain of monodansyl cadaverine-labeled fibrin monomer during the early stages of plasmin digestion. These peptides were degraded progressively to a brightly fluorescent plasmin-resistant peptide of 21,000 molecular weight and a weakly fluorescent peptide of 2,500 molecular weight. Thus both alpha chain cross-link acceptor sites are contained within a peptide segment of 23,000 molecular weight.     

Platelet receptor recognition domain on the gamma chain of human fibrinogen and its synthetic peptide analogues

We have shown previously that the domain recognizing receptors on activated human platelets is located on the human fibrinogen gamma chain between residues 400 and 411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767]. To study the correlation between the structure of this segment of the gamma chain and its reactivity toward receptors on ADP-activated human platelets, we designed a series of analogues containing replacements at 9 out of 12 positions. A double substitution of the normal His400-His401 sequence by Ala-Ala reduced the inhibitory potency of the dodecapeptide 3-fold. When Lys406 was replaced by Arg, the inhibitory potency of the dodecapeptide decreased 15 times. On the other hand, substitution of Ala408 with Arg increased the inhibitory potency of the dodecapeptide 6-fold. A drastic decrease in the reactivity of the dodecapeptide toward platelet receptors was observed when Val411 was replaced by leucine or cysteine or tyrosine. A 3-fold decrease in reactivity was noted when Val411 was substituted with phenylalanine. Amidation of the carboxy-terminal Val411 also produced a significant decrease in dodecapeptide reactivity. With seven residues (His400, His401, Leu402, Lys406, Gln407, Asp410, and Val411) preserved, substitution of the intervening five amino acids with nonpolar leucine or polar serine, increasing or decreasing the hydrophobicity of the dodecapeptide, reduced more than 16-fold its inhibitory potency. Rabbit antibody Fab fragments directed against the human fibrinogen gamma-chain peptide encompassing residues 385-411 inhibited 50% of 125I-fibrinogen binding at a 2:1 stoichiometry with regard to 125I-fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of primary polymerization sites in the D domain of human fibrinogen depends on intact conformation

Fragments D1 and DD, plasmic degradation products of human fibrinogen and cross-linked fibrin, respectively, originate from the COOH-terminal domain of the parent molecule. Since a specific binding site for fibrin resides in the COOH-terminal region of the gamma chain, the primary structure of the two fragments was compared and their affinity for fibrin monomer measured. Fragments D1 and DD contained the same segments of the three fibrinogen chains, corresponding to the sequences alpha 105-206, beta 134-461, and gamma 63-411. Fragment DD had a double set of the same chain remnants. Fragments D1 and DD inhibited polymerization of fibrin monomer in a dose-dependent manner; 50% inhibition occurred at a molar ratio of fragment to monomer of 1:1 and 0.5:1, respectively. To prevent fibrin monomer polymerization and render it suitable for binding studies in the liquid phase, fibrinogen was decorated with Fab fragments isolated from rabbit antibodies to human fragment D1. Fibrinogen molecules decorated with 6 molecules of this Fab fragment did not clot after incubation with thrombin, and the decorated fibrin monomer could be used to measure binding of fragments D1 and DD in a homogeneous liquid phase. The data analyzed according to the Scatchard equation and a double-reciprocal plot gave a dissociation constant of 12 nM for fragment D1 and 38 nM for fragment DD. There were two binding sites/fibrin monomer molecule for each fragment. After denaturation in 5 M guanidine HCl, the inhibitory function on fibrin polymerization was irreversibly destroyed. Denatured fragments also lost binding affinity for immobilized fibrin monomer. The preservation of the native tertiary structure in both fragments was essential for the expression of polymerization sites in the structural D domain.