Gamma Chain Of Human Fibrinogen Research Articles

Plasma fibrinogen gamma' chain content in the thrombotic microangiopathy syndrome.

Human fibrinogen gamma chain variants, termed gamma' chains, contain a unique 20-residue sequence after gamma chain residue 407 that ends at gamma'427, and is designated gamma'(427L). Full-length (FL) gamma'(427L) chains are constituents of a fibrin-dependent thrombin inhibitory system known as antithrombin I, whereas a gamma' chain processed in vivo, termed gamma'(423P), lacks the C-terminal tetrapeptide EDDL, and does not bind thrombin. Together, the gamma'(423P) and gamma'(427L) chains comprise the total plasma fibrinogen gamma' chain content. Lowered plasma gamma' chain content (i.e. gamma' chain-containing fibrinogen/total fibrinogen ratio) has been shown to correlate with susceptibility to venous thrombosis, thus prompting this study on the total and FL gamma' chain content in 45 subjects with thrombotic microangiopathy (TMA), a disorder characterized by microvascular thrombosis. We measured by enzyme-linked immunosorbent assay the total gamma' chain-containing fibrinogen/total fibrinogen (Total gamma'-fgn/Total fgn) ratio and the FL gamma' chain-containing fibrinogen/total fibrinogen (FL gamma'-fgn/Total fgn) ratio in these plasmas and in healthy subjects (n = 87). In healthy subjects, the mean Total gamma'-fgn/Total fgn ratio was 0.127, whereas the FL gamma'-fgn/Total fgn ratio was somewhat lower at 0.099 (P < 0.0001), a difference reflecting the presence of gamma'(423P) chains. In TMA plasmas, both the Total gamma'-fgn and FL gamma'-fgn/Total fgn ratios (0.099 and 0.084, respectively) were lower than those of their healthy subject counterparts (P < 0.0001). These findings in TMA suggest that reductions in the gamma' chain content indicate reduced antithrombin I activity that may contribute to microvascular thrombosis in TMA.

Structure of the fibrinogen gamma-chain integrin binding and factor XIIIa cross-linking sites obtained through carrier protein driven crystallization.

The human fibrinogen gamma-chain C-terminal segment functions as the platelet integrin binding site as well as the Factor XIIIa cross-linking substrate and thus plays an important role in blood clot formation and stabilization. The three-dimensional structure of this segment has been determined using carrier protein driven crystallization. The C-terminal segment, gamma-(398-411), was attached to a linker sequence at the C-terminus of glutathione S-transferase and the structure of this fusion protein determined at 1.8 A resolution. Functional studies of the chimeric protein demonstrate that the fibrinogen sequence in the presence of the carrier protein retains its specific functions as ligand for platelet integrin alpha(IIb)beta3 (gpIIb/IIIa) and as a cross-linking substrate for Factor XIIIa. The structure obtained for the fibrinogen gamma-chain segment is not affected by crystal packing and can provide the missing links to the recently reported model of cross-linked fibrin.

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Crystal structure of a 30 kDa C-terminal fragment from the gamma chain of human fibrinogen.

Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, (alphabetagamma)2, held together by 29 disulfide bonds. The globular C terminus of the gamma chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. The X-ray crystallographic structure of the 30 kDa globular C terminus of the gamma chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. The polymerization domain in the gamma chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.

Integrin receptor GPIIb/IIIa bound state conformation of the fibrinogen gamma-chain C-terminal peptide 400-411: NMR and transfer NOE studies.

The C-terminal dodecapeptide from human fibrinogen gamma-chain, residues 400-411, HHLG-GAKQAGDV (gamma12), is known to inhibit fibrinogen-mediated platelet cell aggregation via competitive interactions with platelet glycoprotein integrin receptor GPIIb/IIIa. NMR studies of gamma12 in the presence of purified GPIIb/IIIa (230 kDa) demonstrate that two gamma12 binding states (gamma12-I and gamma12-II) are present on the integrin receptor. The N-terminal sequence HHLG is crucial to formation of gamma12 state I since in a shorter gamma-chain octapeptide, GAKQAGDV, gamma12-I is not observed. Addition of the hexapeptide GRGDSP to the gamma12-receptor preparation effectively removes the gamma12-I population, suggesting either that gamma12 and GRGDSP share one binding site or that their binding sites are allosterically linked. Distance geometry calculations using transfer NOEs from gamma12-I (gamma12-II shows practically no NOEs) indicate the presence of helix conformation when bound to the receptor. Line broadening and chemical shift changes relative to free gamma12 suggest that gamma12 interacts with GPIIb/IIIa primarily through N-terminal residues H400 to Q407.

Characterization of the 5′-Flanking Region of the Gene for the γChain of Human Fibrinogen

The 5'-flanking region of the gene coding for the gamma chain of human fibrinogen was characterized for its promoter activity. Reporter gene studies using chloramphenicol acetyltransferase as the indicator along with mutations in the DNA showed that a TATA-like sequence (-20 to -23 base pairs (bp)), a CAAT-like sequence (-54 to -57 bp), and an upstream stimulatory factor (USF) binding site (-66 to -77 bp) constitute a minimal promoter that mediates liver-specific expression of the gene. Electrophoretic gel mobility assays and antibody binding studies confirmed the interaction of USF with the binding site. An IL-6 responsive element with a sequence of CTGGAA located at -301 to -306 bp was shown to be a functional element in the IL-6 response. In contrast to the IL-6 responsive elements in the human alpha- and beta-fibrinogen genes, the element in the gene for the gamma chain of human fibrinogen was unaffected by the presence or elevated levels of the beta or delta isoforms of the CCAAT/enhancer binding proteins. A negative element with sequence homology to several silencer elements was also identified in the region of -348 to -390 bp of the gene for the gamma chain of human fibrinogen. A comparison of the regulatory elements in the genes coding for all three chains in fibrinogen is also presented.

Effect of pH on the Conformation and Backbone Dynamics of a 27-Residue Peptide in Trifluoroethanol: AN NMR AND CD STUDY

The C-terminal fragment, residues 385-411, from human fibrinogen gamma-chain, i.e. KIIPFNRLTIGEGQQHHLG-GAKQAGDV, shows multiple turn conformations in aqueous solution (Mayo, K. H., Burke, C., Lindon, J. N., and Kloczewiak, M. (1990) Biochemistry 29, 3277-3286). The present study investigates the effect of pH and trifluoroethanol on the conformation and backbone dynamics of this 27-residue peptide. Both circular dichroism (CD) and 1H-NMR data indicate the normally observed increased helical conformations as a function of increasing trifluoroethanol. 1H-NMR structural studies done in the presence of 40% trifluoroethanol, pH 5.3, yield a network of nuclear Overhauser effects consistent with significant populations of helix-like conformation. Distance geometry calculations based on nuclear Overhauser effect-derived distance constraints yield a family of structures with relatively well defined N- and C-terminal conformations and an ill defined mid-peptide region from Gly397 to Gly403. Similar conformational populations are observed at pH 2.5. CD studies, however, indicate an increase in average alpha-helix content on decreasing the pH from 6 to 2. This apparent conflict between CD and NMR results may be explained by a transition from multiple beta-turn character at pH 5.3 to increased alpha-helix structure at pH 2.5. 13C alpha NMR relaxation data analyzed with the Lipari-Szabo model-free approach provide order parameters that demonstrate little if any influence of pH on backbone motional restrictions within the more flexible mid-peptide domain. At low pH, however, motions become less restricted within N-terminal residues Lys385-Phe389 and more restricted within C-terminal residues Ala405-Val411.

Mechanisms Involved in Platelet Vessel Wall Interaction

The molecular mechanisms underlying formation of platelet thrombi constitute one of the central problems in vascular biology and medicine. Fibrinogen binding to its platelet integrin receptor alpha IIb beta 3 (glycoproteins IIb-IIIa complex) plays a pivotal role in formation of platelet thrombi by providing molecular bridges spanning platelets and by contributing to its receptor-mediated outside-in signaling. Conversely, fibrinogen binding to integrin alpha IIb beta 3 requires inside-out signaling mediated by intraplatelet signal transducers activated in response to external signals generated at the site of vascular injury. This tightly regulated fibrinogen-integrin alpha IIb beta 3 interaction constitutes a fundamental platelet mechanism of response to vascular injuries such as accidental and surgical wounds, rupture of atherosclerotic plaques in coronary and cerebral atherosclerosis and microvascular endothelial desquamation in septic shock. After mapping the fibrinogen site on the gamma chain responsible for recognition of platelet integrin alpha IIb beta 3 and the development of synthetic peptide inhibitors, we solved the 3D structure of the carboxy-terminal segment of the human fibrinogen gamma chain using our new technology of carrier protein-driven crystallization. Fundamental knowledge concerning a molecular 3D model of fibrinogen-integrin alpha IIb beta 3 interaction, coupled to the mechanism of its inside-out and outside-in regulation, will lead to development of a new generation of platelet-selective antithrombotic drugs.

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.

Purification and Functional Characterization of Homodimeric γB-γB Fibrinogen From Rat Plasma

Heterogeneity in the human and rat plasma fibrinogen (FBG) gamma chains is due to differential splicing of the primary gamma chain mRNA transcript. The subunit composition of FBG containing the predominant form of the gamma chain is homodimeric (human: A alpha 2, B beta 2, gamma 50-gamma 50; rat: A alpha 2, B beta 2, gamma A-gamma A). The subunit composition of FBG containing the longer, minor form of the gamma chain is heterodimeric (human: A alpha 2, B beta 2, gamma 50-gamma 57.5; rat: A alpha 2, B beta 2, gamma A-gamma B). The variant gamma chain-FBGs comprise about 10% of the total plasma FBG in the human and 30% in the rat. Although the presence of homodimeric gamma B-gamma B FBG has been speculated, proof of its existence has been difficult to obtain experimentally. We now show that 5% to 6% of rat plasma FBG is found as homodimeric FBG with a subunit composition of A alpha 2, B beta 2, gamma B-gamma B. Commercially purified rat FBG was further purified by diethylaminoethyl-Sephacel chromatography with a combined pH and ionic strength gradient. The enriched gamma B-gamma B FBG fraction eluted at the lowest pH (6.3) and highest ionic strength (4.5 mmho) due to its increased net negative charge. To further purify gamma B-gamma B FBG, a Mono Q column with an NaCl gradient was used. The subunit composition of the purified gamma B-gamma B FBG was confirmed by its electrophoretic mobility under reducing and denaturing conditions both as FBG, and clotted and cross-linked fibrin. Homodimeric gamma B-gamma B FBG was unable to support adenosine diphosphate (ADP)-induced platelet aggregation, whereas heterodimeric gamma A-gamma B FBG was able to support ADP-induced platelet aggregation at 40% of that achieved with homodimeric gamma A-gamma A FBG, similar to previous observations with human FBGs. Taken together, these results support the hypothesis that the A alpha-RGD sites alone are not sufficient for dimeric FBG support of platelet aggregation. Furthermore, the data suggest that at least one intact platelet recognition site at the carboxyterminus of the gamma A or gamma 50 chain is required.

Purification and functional characterization of homodimeric gamma B- gamma B fibrinogen from rat plasma

Heterogeneity in the human and rat plasma fibrinogen (FBG) gamma chains is due to differential splicing of the primary gamma chain mRNA transcript. The subunit composition of FBG containing the predominant form of the gamma chain is homodimeric (human: A alpha 2, B beta 2, gamma 50-gamma 50; rat: A alpha 2, B beta 2, gamma A-gamma A). The subunit composition of FBG containing the longer, minor form of the gamma chain is heterodimeric (human: A alpha 2, B beta 2, gamma 50- gamma 57.5; rat: A alpha 2, B beta 2, gamma A-gamma B). The variant gamma chain-FBGs comprise about 10% of the total plasma FBG in the human and 30% in the rat. Although the presence of homodimeric gamma B- gamma B FBG has been speculated, proof of its existence has been difficult to obtain experimentally. We now show that 5% to 6% of rat plasma FBG is found as homodimeric FBG with a subunit composition of A alpha 2, B beta 2, gamma B-gamma B. Commercially purified rat FBG was further purified by diethylaminoethyl-Sephacel chromatography with a combined pH and ionic strength gradient. The enriched gamma B-gamma B FBG fraction eluted at the lowest pH (6.3) and highest ionic strength (4.5 mmho) due to its increased net negative charge. To further purify gamma B-gamma B FBG, a Mono Q column with an NaCl gradient was used. The subunit composition of the purified gamma B-gamma B FBG was confirmed by its electrophoretic mobility under reducing and denaturing conditions both as FBG, and clotted and cross-linked fibrin. Homodimeric gamma B-gamma B FBG was unable to support adenosine diphosphate (ADP)-induced platelet aggregation, whereas heterodimeric gamma A-gamma B FBG was able to support ADP-induced platelet aggregation at 40% of that achieved with homodimeric gamma A-gamma A FBG, similar to previous observations with human FBGs. Taken together, these results support the hypothesis that the A alpha-RGD sites alone are not sufficient for dimeric FBG support of platelet aggregation. Furthermore, the data suggest that at least one intact platelet recognition site at the carboxyterminus of the gamma A or gamma 50 chain is required.

Maintenance of patency after vascular trauma by topical irrigation with a peptide homologous to the carboxy-terminus of the fibrinogen gamma chain

Synthetic peptides with amino acid sequences homologous to the carboxy-terminal sequence of the gamma chain of human fibrinogen were evaluated for their capacity to inhibit thrombus development. A 21-residue peptide effectively inhibited surface-mediated fibrin clot propagation in vitro. Since this effect was localized to the surface, we proceeded with in vivo evaluation, using site-specific topical application only. In a rat model of arterial trauma and microvascular repair, topical application of the 21-residue peptide significantly reduced the rate of thrombosis to 17%, as compared with a control rate of 83%; in comparison, a 12-residue homologue reduced the thrombosis rate only modestly to 61%, which was not significant compared with the control rate. These results indicate the feasibility of antithrombotic therapy with topically applied agents, an approach that may obviate the use of systemic anticoagulation in extremity-replantation surgery.

Fibrinogen γ-Chain mRNA Is Not Detected In Human Megakaryocytes

Human megakaryocytes and platelets contain counterparts of several plasma proteins. The origin of most of these alpha-granule proteins is unclear. Fibrinogen represents one of those molecules, being essential in hemostasis, thrombosis, and platelet aggregation. To study whether fibrinogen is endocytosed by megakaryocytes and packaged into alpha-granules or newly synthesized by these cells, we established a highly sensitive nested primer polymerase chain reaction for the detection of human fibrinogen gamma-chain mRNA. In enriched megakaryocyte fractions, as well as fluorescence-activated cell sorter-purified megakaryocytes from bone marrow samples of healthy volunteers, no fibrinogen gamma-chain mRNA could be detected, despite the presence of the corresponding fibrinogen gamma-chain DNA. We conclude that fibrinogen gamma-chain mRNA, as detectable by our amplification system, is missing in megakaryocytes. This finding suggests that fibrinogen might be acquired from plasma by endocytosis and sequestered in alpha-granules before reentering the circulation after platelet activation.

Fibrinogen gamma-chain mRNA is not detected in human megakaryocytes

Human megakaryocytes and platelets contain counterparts of several plasma proteins. The origin of most of these alpha-granule proteins is unclear. Fibrinogen represents one of those molecules, being essential in hemostasis, thrombosis, and platelet aggregation. To study whether fibrinogen is endocytosed by megakaryocytes and packaged into alpha- granules or newly synthesized by these cells, we established a highly sensitive nested primer polymerase chain reaction for the detection of human fibrinogen gamma-chain mRNA. In enriched megakaryocyte fractions, as well as fluorescence-activated cell sorter-purified megakaryocytes from bone marrow samples of healthy volunteers, no fibrinogen gamma- chain mRNA could be detected, despite the presence of the corresponding fibrinogen gamma-chain DNA. We conclude that fibrinogen gamma-chain mRNA, as detectable by our amplification system, is missing in megakaryocytes. This finding suggests that fibrinogen might be acquired from plasma by endocytosis and sequestered in alpha-granules before reentering the circulation after platelet activation.

Polymorphism of the human gamma chain fibrinogen gene

Nucleotide sequence comparisons of the published cDNAs and genomic sequences coding for the gamma-chain of human fibrinogen revealed several differences. We isolated two independent human cDNA clones, coding for part of this protein and compared their sequences, which are identical with the published relevant data. Our sequence allowed us to solve a conflict for aminoacid 88. All the remaining differences resulting from this comparison occurred in the third position of a codon and did not change codon properties or restriction sites. The level of polymorphism of this gene is discussed, taking into account also the nucleotide differences among all the published relevant data.

Proton 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.

Analysis of rodent platelet glycoprotein IIb: evidence for evolutionarily conserved domains and alternative proteolytic processing

Recently, the full-length primary amino acid sequence for human glycoproteins (GP) IIb and IIIa have been derived from their respective cDNAs. Potential functional domains within these proteins have been proposed based primarily on homology with similar domains in other proteins having known biologic function. To further understand the relationship between structure and function of the platelet fibrinogen receptor, we have begun comparative studies of the human GPIIb/IIIa receptor with the corresponding rodent receptor. The rodent rGPIIb/IIIa receptor differs from the human receptor, having low affinity for R.G.D- containing oligopeptides and not binding at all to the C-terminus of the gamma chain of human fibrinogen. We describe the structure of rodent platelet GPIIb derived from a combination of peptide sequencing, and cDNA and partial genomic DNA sequence analysis. The initial transcript is 1037 amino acid residues, having 78% amino acid identity with its 1039 residue human analog. Both heavy chains have the N- terminal sequence L.N.L.D, agreeing with the consensus derived from other integrin family alpha heavy chains. All 18 cysteine residues occur at positions conserved in human GPIIb and the vitronectin receptor alpha subunit VNR alpha. The putative calcium-binding domains of the GPIIbs have a high level of amino acid identity (92%), supporting the supposition that these regions have a critical biologic role. The final 48 C-terminal amino acid residues of the heavy chain of rodent GPIIb share only 56% identity with its human counterpart, and the proposed cleavage site of human GPIIb into its heavy and light chains is not present in the rodent sequence. Although we demonstrate that rodent GPIIb is split into two subunits during its maturation, this process either involves a different recognition sequence in the rodent or occurs at a different site. Finally, partial genomic DNA sequence analysis indicates that there are at least two rodent GPIIb genes: a normal gene, containing introns in positions similar to those in the human gene, and a processed pseudogene. The human haploid genome contains only a single GPIIb gene.

Analysis of Rodent Platelet Glycoprotein IIb: Evidence for Evolutionarily Conserved Domains and Alternative Proteolytic Processing

Recently, the full-length primary amino acid sequence for human glycoproteins (GP) IIb and IIIa have been derived from their respective cDNAs. Potential functional domains within these proteins have been proposed based primarily on homology with similar domains in other proteins having known biologic function. To further understand the relationship between structure and function of the platelet fibrinogen receptor, we have begun comparative studies of the human GPIIb/IIIa receptor with the corresponding rodent receptor. The rodent rGPIIb/IIIa receptor differs from the human receptor, having low affinity for R.G.D-containing oligopeptides and not binding at all to the C-terminus of the gamma chain of human fibrinogen. We describe the structure of rodent platelet GPIIb derived from a combination of peptide sequencing, and cDNA and partial genomic DNA sequence analysis. The initial transcript is 1037 amino acid residues, having 78% amino acid identity with its 1039 residue human analog. Both heavy chains have the N-terminal sequence L.N.L.D, agreeing with the consensus derived from other integrin family alpha heavy chains. All 18 cysteine residues occur at positions conserved in human GPIIb and the vitronectin receptor alpha subunit VNR alpha. The putative calcium-binding domains of the GPIIbs have a high level of amino acid identity (92%), supporting the supposition that these regions have a critical biologic role. The final 48 C-terminal amino acid residues of the heavy chain of rodent GPIIb share only 56% identity with its human counterpart, and the proposed cleavage site of human GPIIb into its heavy and light chains is not present in the rodent sequence. Although we demonstrate that rodent GPIIb is split into two subunits during its maturation, this process either involves a different recognition sequence in the rodent or occurs at a different site. Finally, partial genomic DNA sequence analysis indicates that there are at least two rodent GPIIb genes: a normal gene, containing introns in positions similar to those in the human gene, and a processed pseudogene. The human haploid genome contains only a single GPIIb gene.

The C-terminal sequences of the gamma 57.5 chain of human fibrinogen constitute a plasmin sensitive epitope that is exposed in crosslinked fibrin

The gamma chain of human plasma fibrinogen is heterogeneous with three forms differing in length at the C-terminus. Alternative RNA splicing produces two gamma chain mRNAs encoding gamma 50 and gamma 57.5 polypeptides, while fibrinogen gamma 55 is produced by post- translational modification of the gamma 57.5 chain. The composition of purified variant gamma chain fibrinogens, which comprise 10% to 13% total plasma fibrinogen, is predominantly heterodimeric (A alpha, B beta, gamma 50/gamma 55 or A alpha, B beta, gamma 50/gamma 57.5), whereas the composition of purified fibrinogen with the major form of the gamma chain is homodimeric (A alpha, B beta, gamma 50/gamma 50). These gamma chain variations interrupt sequences that mediate platelet- fibrinogen interactions. Therefore, the structure and function of gamma 57.5 C-terminal sequences were investigated using synthetic peptides and a specific monoclonal antibody (MoAb), L2B. The L2B epitope was localized and included gamma 57.5 chain residues 409–412 (Arg-Pro-Glu- His), as determined by differential enzyme-linked immunosorbent assay (ELISA) reactivity with a His-412 deleted synthetic peptide and by Western blot analysis of plasmin cleaved fibrinogen gamma 57.5. L2B had no effect on adenosine diphosphate (ADP)-induced platelet aggregation supported by either fibrinogen gamma 50 or gamma 57.5. High concentrations (0.5 to 1 mmol/L) of synthetic peptide gamma 57.5 405– 416 only weakly inhibited ADP-induced platelet aggregation supported by either fibrinogen gamma 50 or gamma 57.5. Binding of fibrinogen gamma 50 (IC50 = 780 mumol/L) or gamma 57.5 (IC50 = 650 mumol/L) to ADP- stimulated platelets was weakly inhibited, and MoAb L2B failed to inhibit fibrinogen gamma 57.5 binding. Peptide gamma 57.5 408–416 failed to dissociate platelet-bound fibrinogens. These data indicate that the gamma 408–416 sequence of fibrinogen gamma 55 or gamma 57.5 alone is unlikely to bind to the platelet fibrinogen receptor, glycoprotein llb-llla (GPllb-llla), in support of platelet aggregation under physiologic conditions. The sequence recognized by L2B does not resemble known GPllb-llla binding site peptide sequences [Arg-Gly-Asp- Ser (RGDS) or gamma 50 400–411] as determined by competitive inhibition ELISA comparing these binding site synthetic peptides with gamma 57.5 408–416. This epitope is available for binding MoAb L2B in gamma 55 or gamma 57.5 chain dimers and binds to all gamma 57.5 408–416 epitopes equally in non-crosslinked and factor Xllla crosslinked fibrin clots.(ABSTRACT TRUNCATED AT 400 WORDS).

Megakaryocyte and Hepatocyte Origins of Human Fibrinogen Biosynthesis Exhibit Hepatocyte-Specific Expression of y Chain-Variant Polypeptides

Abstract The gamma chain of human fibrinogen is heterogeneous in length at the C- terminus due to differential RNA processing of the gamma chain-gene primary transcript. We have produced two specific monoclonal antibodies (MoAbs) against the gamma-chain epitopes generated by this alternative processing event: anti-gamma 57.5(408–416) (L2B), which reacts with gamma 57.5 and gamma 55 chains, and anti-gamma 50(337–411) (H9B7), which reacts preferentially with gamma 50 chains. Using these MoAbs we have studied the expression of gamma-chain polypeptides by immunofluorescence microscopy in the tissues of fibrinogen biosynthesis and have determined that gamma 57.5 polypeptide is expressed in hepatocytes but is absent or present in significantly reduced amounts in megakaryocytes. Therefore the gamma 50 chain is found in plasma, platelet, and megakaryocyte fibrinogens, but the gamma 57.5 chain is found only in plasma fibrinogen. The C-terminal amino acid sequence of gamma 55 includes the L2B epitope 57.5(408–416). Using MoAb L2B we have determined that gamma 55, which is a post-translationally modified gamma 57.5 chain, is found only in plasma fibrinogen and is absent or present in markedly reduced amounts in platelet or megakaryocyte fibrinogen. In addition, the conformation of the L2B epitope is preserved in gamma 55, as determined by Western blot analysis. The hepatocyte-specific expression of the gamma 57.5-chain polypeptide and the post-translational modification to gamma 55 result in a compartmentalization of gamma-chain polypeptide expression. This is suggestive of different mechanisms regulating human fibrinogen gamma- chain gene expression in hepatocytes v megakaryocytes that may operate in a tissue-specific manner at the level of 3′ RNA processing events.