XIIIa-catalyzed Cross-linking Research Articles

Citrullinated fibrinogen shows defects in FPA and FPB release and fibrin polymerization catalyzed by thrombin

Background Antibody–antigen complexes formed by IgG autoantibodies against citrullinated proteins and citrullinated forms of the α- and β-chains of fibrin in rheumatoid synovial tissue play a key role in the pathophysiology of rheumatoid arthritis. Methods Recombinant fibrinogen was citrullinated by rabbit skeletal muscle peptidylarginine deiminase so that we could analyze the function of citrullinated fibrinogen. Namely, thrombin-catalyzed fibrin polymerization and fibrinopeptide release, protection against plasmin digestion, and factor XIIIa-catalyzed cross-linking of fibrin or fibrinogen were performed. Results Strong citrullination of the Aα- and Bβ-chains and weak citrullination of the γ-chain were detected by an anti-modified citrulline detection kit. Citrullinated fibrinogen did not release FPA or FPB by thrombin catalyzation and no thrombin-stimulated conversion of fibrinogen into fibrin occurred. The citrullination of fibrinogen did not affect the 3 functions of the C-terminal γ-chain, “a-hole,” low affinity Ca binding, and γ–γ cross-linking. Conclusion Our functional analyses demonstrated that no thrombin-stimulated conversion of fibrinogen into fibrin occurred, because citrullinated fibrinogen did not release FPA or FPB after thrombin catalyzation. Our results and those of other reports suggest that citrullinated fibrin and fibrinogen are present in the synovium and might both be associated with the pathophysiology of RA.

B:b interactions are essential for polymerization of variant fibrinogens with impaired holes 'a'.

Fibrin polymerization is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b' always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes 'a': gamma364Ala, gamma364His or gamma364Val. We examined thrombin- and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of gamma364Val and gamma364His were more delayed than gamma364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs 'A'. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes 'a' and 'b', respectively. FXIIIa-catalyzed crosslinking between gamma-chains was markedly delayed for all the variants. These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes 'a'. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

Analysis of fibrinogen variants at γ387Ile shows that the side chain of γ387 and the tertiary structure of the γC-terminal tail are important not only for assembly and secretion of fibrinogen but also for lateral aggregation of protofibrils and XIIIa-catalyzed γ-γ dimer formation

AbstractTo examine the role of fibrinogen γ-chain residue 387Ile in the assembly and secretion of this multichain protein, we synthesized a series of variants with substitution at γ387 by Arg, Leu, Met, Ala, or Asp. Only the variant γ387Asp showed impaired synthesis in the cells and very low secretion into the medium. In addition, we performed thrombin-catalyzed fibrin polymerization and factor (F) XIIIa-catalyzed cross-linking of the γ-chain for 4 variants. The degree of lateral aggregation of protofibrils into fibrin fibers was slightly reduced for γ387Arg and Ala, and moderately reduced for γ387Leu and Met. Although the FXIIIa-catalyzed cross-linking for all of the variants was slower than that for γ387Ile, that of γ387Arg was much more markedly impaired than that of the others. In summary, our studies demonstrated that the specific residue at γ387 or the conformation of γ388-411 residues, but not the length of the γC tail, is critical for fibrinogen assembly and subsequent secretion. Moreover, this residue or the conformation is also important for not only the lateral aggregation of fibrin polymers but also the FXIIIa-catalyzed cross-linking of the γ-chain. Interestingly, our results clearly indicate that the conformations critical for these 2 functions are different from each other.

Effect of a synthetic carboxy-terminal peptide of α 2-antiplasmin on urokinase-induced fibrinolysis

α 2-Antiplasmin (α 2AP) interferes with the binding of plasminogen to fibrin because lysine residues in its carboxy-terminal region compete with those in fibrin, presumably the same way that free lysine or ε-aminocaproic acid (EACA) inhibits plasminogen binding to fibrin. While this overall process causes an inhibition of fibrinolysis, the converse was observed with a 26-residue synthetic peptide (AP26) corresponding to the carboxy-terminal region of α 2AP. The AP26 peptide, in fact, accelerated urokinase-induced lysis of (1) fully crosslinked fibrin with complete γ-dimer and α-polymer formation; (2) partially crosslinked fibrin that had undergone only γ-dimerization; and (3) noncrosslinked fibrin. The AP26 peptide also inhibited factor XIIIa-catalyzed crosslinking of fibrin α-chains, and this also accelerated lysis of fibrin. EACA had no effect. In the presence of noncrosslinked fibrin, AP26 promoted plasminogen activation by urokinase and fibrinolysis. EACA only slightly increased the rate of plasminogen activation, and as expected, it inhibited fibrinolysis. Since AP26 peptide enhanced the lysis of partially crosslinked and noncrosslinked fibrin, our results indicate that inhibition of factor XIIIa-catalyzed α-polymer formation by AP26, although associated with accelerated fibrinolysis, is not the primary mechanism. Instead, our data support the conclusion that AP26 enhances the conversion of plasminogen to plasmin ∼5-fold, probably by inducing a conformational change in plasminogen structure just as occurs with low concentrations of lysine or EACA. At higher concentrations, however, AP26 apparently does not approach the avidity or affinity of lysine or EACA for the kringle structures of plasminogen or plasmin so that their binding to fibrin is blocked. Whether AP26 alone, or as part of another molecule, could have potential for enhancing thrombolysis will require further study.

Cross-linking of fibronectin to C-terminal fragments of the fibrinogen alpha-chain by factor XIIIa.

Fibronectin binds specifically to fibrin and is covalently cross-linked to the fibrin alpha chain by activated factor XIII (XIIIa). This reaction is important for wound healing. Here we investigate XIIIa-catalyzed cross-linking of fibronectin and some of its fragments to a recombinant fragment representing the COOH-terminal 30 kDa of the fibrin alpha chain (alpha C30K:His 368-Val 610). Only fibronectin and those fragments containing an intact NH2-terminus were able to form cross-linked complexes. As many as 10 of the 17 lysines in alpha C30K can serve as amine donors in this reaction. Analysis of the rate of XIIIa-catalyzed cross-linking of fibronectin NH2-terminal peptides and fragments with alpha C30K revealed that the presence of the first type I "finger" module accelerates the cross-linking reaction; addition of fingers 2-5 had no further effect.

Studies on the Mechanism of Fibrin Fiber Formation

The arginine residue at position 275 of the fibrinogen γ-chain (γ Arg-275) has recently been shown to participate in the D-D self association, which promotes longitudinal alignment of two D domains of different fibrin moleculer bound to the thrombin-activated E domain of another molecule. Utilizing three different types of hereditary dysfibrinogens with a point mutation for γ Arg-275, i.e., Cys, His or Ser, I have compared functions of fibrinogen, which may partly be related to the D-D self association. They include fibrin monomer polymerization, factor XIIIa-catalyzed cross-linking of the fibrinogen γ-chains and facilitation of t-PA-catalyzed activation of plasminogen. Although the extent of functional derangement varied among these functions, they were all affected by a substitution of γ Arg-275 to Cys, His and Ser in this order. These functional derangements were apparently related to the degree of structural alterations, i.e., the presence of a disulfidelinked Cys molecule in the Cys-substituent and bulkiness of the side chains of His and Ser, and an electric charge for a positively charged Arg residue.

Fibrinogen Kumamoto with an Aα Arg-19 to Gly Substitution Has Reduced Affinity for Thrombin Possible Relevance to Thrombosis

A heterozygous dysfibrinogen, fibrinogen Kumamoto, derived from a 29-year-old woman with episodes of recurrent blurred vision accompanied by weakness of the ipsilateral lower extremity was found to have an Arg-19 to Gly substitution. Both the thrombin and ancrod times were prolonged, and fibrin monomer polymerization profiles were abnormal with a slightly prolonged lag phase and a moderately reduced amplitude. When analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis, thrombin-catalyzed cleavage of both fibrinopeptides A and B was only minimally delayed and factor XIIIa-catalyzed cross-linking of the fibrin γ-chains was obviously altered. Nevertheless, that of the fibrinogen γ-chains was identical with the normal control in the early stage of the reaction, but was substantially delayed in the later stage. These results may imply that the initial contact between two adjacent D domains of different fibrinogen molecules took place normally, but that the molecular realignment of partially cross-linked single-stranded fibrinogen oligomers to create double-strands was perturbed. In addition to these dysfunctions, decreased adsorption of thrombin to fibrin gels was observed. This finding suggests that immobilization of thrombin onto fibrin clots is incomplete, and thus thrombin action on fibrinogen and platelets in blood circulation is seemingly potentiated, accounting for the transient ischemic attacks in the patient. This type of amino acid substitution has been shown in two dysfibrinogens, Aarhus and Mannheim I, and a transient ischemia was observed in fibrinogen Aarhus I as well.

A γ Gly-268 to Glu Substitution Is Responsible for Impaired Fibrin Assembly in a Homozygous Dysfibrinogen Kurashiki I

A new type of gamma Gly-268 (GGA) to Glu (GAA) substitution has been identified in a homozygous dysfibrinogen by analyses of the affected polypeptide and its encoding gene derived from a 58 year-old man manifesting no major bleeding or thrombosis. The functional abnormality was characterized by impaired fibrin assembly most likely due to failure to construct properly aligned double-stranded fibrin protofibrils. This presumption was deduced from the following findings: (1) Factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains progressed in a normal fashion, indicating that the contact between the central E domain of one fibrin monomer and the D domain of another took place normally; (2) Nevertheless, factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chains was obviously delayed, suggesting that longitudinal association of D domains of different fibrin monomers, ie, D:D association was perturbed; (3) Plasminogen activation catalyzed by tissue-type plasminogen activator was not as efficiently facilitated by polymerizing fibrin monomer derived from the patient as by the normal counterpart. Therefore, gamma Gly-268 would not be involved in the 'a' site residing in the D domain, which functions as a complementary binding site with the thrombin-activated 'A' site in the central E domain, but would be rather involved in the D:D self association sites recently proposed for human fibrinogen. Thus, the gamma Glu-268 substitution newly identified in this homozygous dysfibrinogen seems to impair proper alignment of adjacent D domains of neighboring fibrin molecules in the double-stranded fibrin protofibril, resulting in delayed fibrin gel formation.

A monoclonal Antibody that Inhibits fibrin Polymerization and Cross-Linking of the Fibrin γ-Chains

IEF-13 is an anti-human fibrinogen monoclonal antibody that belongs to the subclass of IgGi with κ-type light chains. IEF-13 recognizes the carboxy-terminal segment of the fibrinogen γ-chain containing the factor XIIIa-catalyzed cross-linking sites, but it is unable to react with the preformed γ-dimer, indicating that the local conformation of the carboxyterminal γ-chain segment contributes to the integrity of epitope for this antibody. As anticipated, IEF-13 inhibited factor XIIIa-catalyzed crosslinking of the fibrin γ-chains. Of interest was a finding that the antibody inhibited fibrin monomer polymerization at a molar ratio to fibrin monomer ≥1 even in the absence of factor XIIIa and Ca2+. Thus it appears that the antiparallel alignment of two γ-chains from different molecules is closely linked with the interaction of fibrin monomers in the formation of double-stranded fibrin protofibrils. In an in vivo situation, crosslinking of the γ-chains readily progresses when factor XIIIa and Ca2+ are present in fibrin clots.

Three-dimensional structure of the platelet integrin recognition segment of the fibrinogen gamma chain obtained by carrier protein-driven crystallization.

We have developed a method for crystallizing small functional protein segments so that their three-dimensional structure can be determined by x-ray diffraction analysis. This method consists of linking a small protein segment of unknown tertiary structure to either the amino or carboxyl terminus of a larger carrier protein of known tertiary structure. Crystallization of the small segment is then driven by crystallization of the carrier protein. Using this approach, we have obtained crystals of the human fibrinogen gamma-chain carboxyl-terminal segment linked to the carboxyl terminus of chicken egg white lysozyme. The three-dimensional structure of the carboxyl-terminal segment of the fibrinogen gamma chain was determined by x-ray diffraction analysis at a resolution of 2.4 A. This segment encompasses the recognition site for the integrin alpha IIb beta 3 receptor on activated platelets and for the clumping receptor on pathogenic staphylococci and also bears donor and acceptor sites for factor XIIIa-catalyzed crosslinking of fibrin. Therefore, the structural information derived from our analysis will provide a rational basis for the design of inhibitors of these important functions of fibrinogen. Moreover, carrier protein-driven crystallization will facilitate the determination of the three-dimensional structure of functional segments of other proteins that are, like fibrinogen, difficult to crystallize in toto.

Factor XIII cross-linking of fibronectin at cellular matrix assembly sites.

We describe the effect of activated Factor XIII (Factor XIIIa, plasma transglutaminase) on the incorporation of plasma fibronectin into extracellular matrix by cultured human fibroblasts. In the absence of added Factor XIIIa, fibronectin binds to cultured fibroblast cell layers and is assembled into disulfide-bonded multimers of the extracellular matrix. When Factor XIIIa was included in the binding medium of skin fibroblasts, accumulation of 125I-fibronectin in the deoxycholate-insoluble matrix was increased. Fibronectin accumulating in the cell layer was cross-linked into nonreducible high molecular weight aggregates. The 70-kDa amino-terminal fragment of fibronectin inhibited the binding and cross-linking of 125I-fibronectin to cell layers, whereas fibrinogen had little effect. When 125I-fibronectin was incubated with isolated matrices or with cell layers pretreated with cytochalasin B, it did not bind and could not be cross-linked by Factor XIIIa into the matrix. HT-1080 human fibrosarcoma cells bound exogenous fibronectin following treatment with dexamethasone; Factor XIIIa cross-linked the bound fibronectin and caused its efficient transfer to the deoxycholate-insoluble matrix. These results indicate that exogenous fibronectin is susceptible to Factor XIIIa-catalyzed cross-linking at cellular sites of matrix assembly. Thus, Factor XIIIa-mediated fibronectin cross-linking complements disulfide-bonded multimer formation in the stabilization of assembling fibronectin molecules and thus enhances the formation of extracellular matrix.

Immunochemical Characterization of Crosslinked Derivatives Isolated from Alpha Chain Oligomers Formed During Early Stages of Fibrin Crosslinking

Early crosslinked alpha chain oligomers (E alpha XL) that formed within 1/2 h of in vitro clot formation were isolated from preparations of reduced, carboxymethylated fibrin by gel filtration on Sepharose CL-4B. Cyanogen bromide (CNBr) digestion of E alpha XL released a mixture of fragments which could be partially separated by Sephadex G-150 chromatography. Crosslinked and non-crosslinked forms of the A alpha chain peptides, CNBr VIII (A alpha #241-476) and CNBr X (A alpha #518-584) were identified in the column effluent both by radioimmunoassay (RIA) and by immunoblotting using polyclonal antisera (anti-CNBr VIII, anti-CNBr X) and monoclonal antibodies (F-103, binds to A alpha #259-276; F-102, binds to A alpha #540-554), respectively. Immunoaffinity chromatography of the crosslinked material, on F-103, followed by F-102 Sepharose, resulted in the separation of two types of fragments, each of which contained an early alpha chain crosslink. One of these (35-37K) exhibited coincident CNBr VIII and CNBr X immunoreactivities, while the other (54-59K) exhibited only CNBr VIII immunoreactivity, based on immunoblotting and RIA findings. NH2-terminal sequencing and cyanoethylation data provided biochemical evidence for the occurrence of covalent CNBr VIII-X as well as covalent CNBr VIII-VIII interactions during early alpha chain crosslinking. These findings indicate, for the first time, that both glutamine acceptor and lysine donor activities may be localized within CNBr VIII. This information should facilitate the chemical identification of alpha chain residues that partner during factor XIIIa-catalyzed cross-linking and thus enable development of reagents for the generation of fibrin-specific antibodies.

Gly-Pro-Arg-Pro modifies the glutamine residues in the α- and γ-chains of fibrinogen: inhibition of transglutaminase cross-linking

During blood clotting Factor XIIIa, a transglutaminase, catalyzes the formation of covalent bonds between the ϵ-amino group of lysine and the γ-carboxamide group of peptide-bound glutamine residues between fibrin molecules. We report that glycyl- l-prolyl- l-arginyl- l-proline (GPRP), a terapeptide that binds to the fibrin polymerization sites (D-domain) in fibrin(ogen), inhibits transglutaminase cross-linking by modifying the glutamine residues in the α- and γ-chains of fibrinogen. Purified platelet Factor XIIIa, and tissue transglutaminase from adult bovine aortic endothelial cells were used for the cross-linking studies. Gly-Pro (GP) and Gly-Pro-Gly-Gly (GPGG), peptides which do not bind to fibrinogen, had no effect on transglutaminase cross-linking. GPRP inhibited platelet Factor XIIIa-catalyzed cross-linking between the γ-chains of the following fibrin(ogen) derivatives: fibrin monomers, fibrinogen and polyermized fibrin fibers. GPRP functioned as a reversible, noncompetitive inhibitor of Factor XIIIa-catalyzed incorporation of [ 3H]putrescine and [ 14C]methylamine into fibrinogen and Fragment D1. GPRP did not inhibit 125I-Factor XIIIa binding to polymerized fibrin, demonstrating that the Factor XIIIa binding sites on fibrin were not modified. GPRP also had no effect on Factor XIIIa cross-linking of [ 3H]putrescine to casein. This demonstrates that GPRP specifically modified the glutamine cross-linking sites in fibrinogen, and had no effect on either Factor XIIIa or the lysine residues in fibrinogen. GPRP also inhibited [ 14C]putrescine incorporation into the α- and γ-chains of fibrinogen without inhibiting β-chain incorporation, suggesting that the intermolecular cross-linking sites were selectively affected. Furthermore, GPRP inhibited tissue transglutaminase-catalyzed incorporation of [ 3H]putrescine into both fibrinogen and Fragment D1, without modifying [ 3H]putrescine incorporation into casein. GPRP also inhibited intermolecular α-α-chain cross-linking catalyzed by tissue transglutaminase. This demonstrates that the glutamine residues in the α-chains involved in intermolecular cross-linking are modified by GPRP. This is the first demonstration that a molecule binding to the fibrin polyermization sites on the D-domain of fibrinogen modifies the glutamine cross-linking sites on the α- and γ-chains of fibrinogen.

Fibrinogen petoskey: Identification of a new dysfibrinogenemia characterized by altered release of fibrinopeptide A

A new congenital dysfibrinogenemia, designated fibrinogen Petoskey, which was traced through four generations of a Michigan family, was found to exhibit an abnormally slow rate of release of fibrino-peptide A upon treatment with thrombin and batroxobin. Batroxobin only partially hydrolyzed and polymerized the fibrinogen from affected individuals, suggesting that these patients had both normal and abnormal fibrinogen in their circulation and that batroxobin was not capable of releasing fibrinopeptide A from the abnormal fibrinogen. Polymerization of fibrin monomers from fibrinogen Petoskey and plasmin mediated digestion of fibrinogen Petoskey were normal. The Factor XIIIa-catalyzed cross-linking of fibrinogen Petoskey was slightly delayed at low (but not at high) concentrations of thrombin. This delayed cross-linking appeared to be a secondary effect of the lower rate of release of fibrinopeptide A.

Human plasma fibrinogen heterogeneity: evidence for an extended carboxyl-terminal sequence in a normal gamma chain variant (gamma').

Two types of normal human plasma fibrinogen--peak 1 and peak 2--are distinquishable by DEAE-cellulose gradient elution chromatography. The elution characteristics of peak 2 fibrinogen, which amounts to about 15% of the total, are attributable to the presence of a gamma chain variant, gamma', which is more negatively charged than gamma chains and makes up about half of all such chains in that peak [Mosesson M. W., Finlayson, J. S. & Umfleet, R. A. (1972), J. Biol. Chem. 247, 5223-5227]. Analyses of reduced S-carboxymethylated fibrin that had first been incubated in the presence of Factor XIIIa plus the fluorescent amine donor dansylcadaverine (DNScad) showed that the same amount of this compound could be incorporated covalently into either type of gamma chain. Furthermore, the DNScad-labeled COOH-terminal CNBr fragment (CNBr e) derived from the S-carboxymethylated gamma chain was smaller than the DNScad-labeled fragment (CNBr e') from the gamma' chain (Mr, 3200 and 4900) by about the same amount as the difference in size between the respective parent chains (Mr, 49,400 and 51,500). DNScad-CNBr e or DNScad-cNBR e' could be further cleaved by trypsin to yield a smaller fluorescent fragment corresponding to the penultimate tryptic gamma chain peptide containing the DNScad-glutamine acceptor and lysine donor crosslinking functions. The COOH-terminal amino acids of gamma and gamma' chains were valine and leucine, respectively. The rates of Factor XIIIa-catalyzed crosslinking of peak 1 and peak 2 fibrin were the same, but peak 1 fibrin gamma chains formed only one species of crosslinked dimer (gamma gamma) whereas peak 2 fibrin gamma chains yielded three (gamma gamma, gamma gamma', gamma'gamma'). We conclude that gamma' chains are functionally normal but have an extended COOH-terminal sequence accounting for their more negative charge and larger size relative to gamma chains.

Factor XIIIa-catalyzed cross-linking of platelet and muscle actin regulation by nucleotides

Actin from human blood platelets or rabbit skeletal muscle can serve as substrate for factor XIIIa. The latter catalyzes the incorporation of 1.5–2 mol monodansylcadaverine/mol rabbit actin and 0.5 mol/mol platelet actin. Highly cross-linked platelet and muscle actin polymers form in the absence of added amines, indicating the presence of both acceptor and donor sites. As expected, the cross-link was found to be a γ-glutamyl-ϵ-lysine bond, with an average of 0.3–0.4 mol dipeptide/mol platelet actin. Both cross-linking and amine incorporation are prevented by ATP, ADP, GTP, CTP, but not by AMP and cyclic AMP. These nucleotides may have important regulatory role in muscle and non-muscle systems.

Studies on the structural abnormality of fibrinogen Paris I.

The structural properties of an inherited fibrinogen abnormality designated fibrinogen Paris I were investigated. Dodecyl sulfate gel electrophoresis of unreduced samples revealed no discernible differences in molecular weight from normal; this implied that in fibrinogen Paris I, the normal fibrinogen architecture of six covalently linked chains per molecule is preserved. Examination of dithiothreitol reduced samples before and after treatment with Reptilase or thrombin revealed that the Aalpha- and Bbeta-chains could release the A and B peptides, respectively. A mutant chain (mol wt 52,500, termed gammaParis I) which replaces a large proportion of gamma-chains (mol wt 49,400) was shown, like normal gamma-chains, to lack thrombin- and Reptilase-sensitive sites. The gamma-chains and alpha-chains of Paris I fibrin underwent Factor XIIIa-catalyzed cross-linking slowly; this behavior was not attributable to an intrinsic abnormality of these chains themselves but rather to the inhibitory effect of the mutant gammaParis I chains on this process. Results of DEAE-cellulose gradient elution chromatography of Paris I fibrinogen preparations revealed the presence of small amounts of normal fibrinogen molecules and also indicated that the gammaParis I chains possessed structural overlap with gamma-chains. Unlike gamma-chains however, the gammaParis I chains did not incorporate dansylcadaverine in the prescence of Factor XIIIa, nor, as previously reported, did they undergo cross-linking. The observations indicate that the amine acceptor site found in the COOH-terminal region of the gamma-chain is either not present on the gammaParis I chain or is unavailable for cross-linking. Further support for localization of the abnormality in the COOH-terminal region of the molecule was obtained from the observation that during plasmic hydrolysis of Paris I fibrinogen, at least one unique form of core Fragment D (DParis I) was evolved, whereas Fragment E did not differ from normal.