Fibrinogen Assembly Research Articles

Hypofibrinogenemia Associated With a Heterozygous Missense Mutation γ153Cys to Arg (Matsumoto IV): In Vitro Expression Demonstrates Defective Secretion of the Variant Fibrinogen

AbstractWe genetically analyzed a case of hypofibrinogenemia that showed no bleeding or thrombotic tendency. Direct sequencing of a polymerase chain reaction-amplified γ-chain gene segment showed a novel nucleotide substitution. This heterozygous mutation encodes both Cys (TGT) and Arg (CGT) at residue 153. To examine the basis for the fibrinogen deficiency, we prepared expression vectors containing mutant γ-chain DNAs encoding γ153R and γ153A for in vitro expression in Chinese hamster ovary (CHO) cells. Enzyme-linked immunosorbent assay and immunoblot analysis of the culture media and cell lysates showed that CHO cells transfected with γ153R or γ153A synthesized the variant γ-chain, but did not secrete variant fibrinogen into the culture medium. Metabolic pulse-chase experiments showed that fibrinogen assembly was impaired when either variant γ-chain was expressed. In cells expressing normal fibrinogen, assem- bly intermediates and intact fibrinogen were seen in cell lysates prepared after short (3 minutes) or long (1 hour) incubation with 35S-methionine. Neither intermediates nor intact fibrinogen was seen with the variant γ-chains. These data suggest that γ-chains have an important early role in fibrinogen assembly. Thus, our results support the model for fibrinogen assembly proposed by Huang et al (J Biol Chem 268:8919, 1993), in which the first step in assembly is the formation of αγ or βγ dimers, or both. This model implies that γCys153 has a critical role in the formation of these early assembly intermediates. We concluded that the γ153Cys→Arg substitution does not allow fibrinogen assembly and secretion, and this is manifest in vivo as a fibrinogen deficiency. We designated this variant as fibrinogen Matsumoto IV.

Formation of the Human Fibrinogen Subclass Fib420: Disulfide Bonds and Glycosylation in Its Unique (EChain) Domains

COS cell transfection has been used to monitor the assembly and secretion of fibrinogen molecules, both those of the subclass containing the novel E chain and those of the more abundant subclass whose  chains lack E’s globular C-terminus. That region, referred to as the EC domain, is closely related to the ends of β and γ chains of fibrinogen (βC and γC). Transfection of COS cells with E, β, and γ cDNAs alone results in secretion of the symmetrical molecule (Eβγ)2, also known as Fib420. Cotransfection with cDNA for the shorter  chain yielded secretion of both (βγ)2 and (Eβγ)2 but no mixed molecules of the structure E(βγ)2. Exploiting the COS cells’ fidelity with regard to Fib420 production, identification was made of the highly conserved Asn667 as the sole site of N-linked glycosylation in the E chain. No evidence from Cys → Ser replacements was found for interchain disulfide bridges involving the four cysteines of the EC domain. However, for fibrinogen secretion, the E, β, and γ subunits do exhibit different requirements for integrity of the two intradomain disulfide bridges located at homologous positions in their respective C-termini, indicating dissimilar structural roles in the process of fibrinogen assembly.© 1998 by The American Society of Hematology.

Formation of the Human Fibrinogen Subclass Fib420: Disulfide Bonds and Glycosylation in Its Unique (αEChain) Domains

AbstractCOS cell transfection has been used to monitor the assembly and secretion of fibrinogen molecules, both those of the subclass containing the novel αE chain and those of the more abundant subclass whose α chains lack αE's globular C-terminus. That region, referred to as the αEC domain, is closely related to the ends of β and γ chains of fibrinogen (βC and γC). Transfection of COS cells with αE, β, and γ cDNAs alone results in secretion of the symmetrical molecule (αEβγ)2, also known as Fib420. Cotransfection with cDNA for the shorter α chain yielded secretion of both (αβγ)2 and (αEβγ)2 but no mixed molecules of the structure ααE(βγ)2. Exploiting the COS cells' fidelity with regard to Fib420 production, identification was made of the highly conserved Asn667 as the sole site of N-linked glycosylation in the αE chain. No evidence from Cys → Ser replacements was found for interchain disulfide bridges involving the four cysteines of the αEC domain. However, for fibrinogen secretion, the αE, β, and γ subunits do exhibit different requirements for integrity of the two intradomain disulfide bridges located at homologous positions in their respective C-termini, indicating dissimilar structural roles in the process of fibrinogen assembly.© 1998 by The American Society of Hematology.

43. Fibrinogen assembly in extracellular matrix is heparan sulfate proteoglycan-dependent

Departments of 1Medicine-Vascular, 2Microbiology and Immunology and 3Pathology, University of Rochester, Rochester, New York, USA

36. The pattern of fibrinogen biosynthesis and assembly in lung and liver epithelial cells during inflammation

36. The pattern of fibrinogen biosynthesis and assembly in lung and liver epithelial cells during inflammation

Current Progress in the Production of Recombinant Human Fibrinogen in the Milk of Transgenic Animals

The mammary gland of transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein. While the mammary gland appears to be able to secrete fully assembled recombinant human fibrinogen (rhfib) at 0.1 to 5 g/l levels, some unassembled rhfib chains are also secreted. Presently, the relationship between unassembled rhfib and the coordinated translation of each nascent rhfib polypeptide in the mammary epithelia is unknown. The secretion of fully and partially assembled rhfib is widely variable among mammalian cell lines and where previously no cell line has been shown to secrete beta chain alone. We have observed that mammary epithelia can secrete B beta chain into milk as well as immature forms of other recombinant proteins, suggesting it likely uses a different secretory pathway than does the liver. This difference in secretory behavior is possibly due to the natural design of milk, where the precise regulation of post translational modifications and intracellular pools of nascent polypeptides needed to achieve fibrinogen assembly may be less important to fulfill the nutritional function of most milk proteins.

The Role of βγ and αγ Complexes in the Assembly of Human Fibrinogen

The role of alphagamma and betagamma dimers as intermediates in the assembly of fibrinogen was examined in cell fusion experiments using stably transfected baby hamster kidney cell lines expressing one or combinations of fibrinogen chains. Fibrinogen was readily formed and secreted into the culture media when cells co-expressing beta and gamma chains and generating betagamma complexes were fused with cells expressing only the alpha chain. Likewise, when cells co-expressing alpha and gamma chains and generating alphagamma complexes were fused with cells expressing only the beta chain, fibrinogen was also formed and secreted. The relative amounts of alphagamma or betagamma intermediates observed during fibrinogen biosynthesis were determined by the levels of the component chains; i.e. when the beta chain was limiting, the alphagamma dimer was the predominant intermediate; likewise, when the alpha chain was limiting, the betagamma complex was the predominant intermediate. The incorporation of preformed alphagamma and betagamma complexes into secreted fibrinogen did not require concurrent protein synthesis, as shown by experiments employing cycloheximide. These data strongly support the role of alphagamma and betagamma complexes as functional intermediates in the assembly of fibrinogen.

Fibrinogen Mediates Leukocyte-Endothelium Bridging In Vivo at Low Shear Forces

In addition to preserving hemostasis, fibrinogen assembly on leukocytes mediates inflammatory responses and may aberrantly contribute to vascular injury. In this study, we used real-time intravital video microscopy in exposed rabbit mesentery to investigate the potential role of fibrinogen on leukocyte adherence mechanisms, in vivo. At physiologic concentrations of 0.15 to 0.5 mg/mL, human fibrinogen dose-dependently enhanced by threefold to fivefold the adhesion of chemoattractant-stimulated monocytic HL-60 cells to rabbit mesenteric endothelium, by acting as a bridging molecule between the two types. Fibrinogen-dependent intercellular bridging occurred in venules, but not in arterioles or capillaries (1), was optimal at reduced flow shear forces (range: 0.77 to 2.79 dyne/cm2) (2), and produced a firm attachment of monocytic cells to endothelium, rather than transient rolling (3). Consistent with this model, rabbit fibrinogen failed to support human leukocyte adhesion, while human fibrinogen enhanced monocytic cell attachment to rabbit endothelial cells in vitro, in a reaction indistinguishable from that observed with human endothelium. Antagonists of the recently described association of fibrinogen with intercellular adhesion molecule-1 (ICAM-1), including monoclonal antibodies (MoAbs) LB-2 or 2D5, or the fibrinogen gamma 3 peptide gamma Asn117-Ala133, blocked fibrinogen-dependent leukocyte-endothelium interaction in vitro or in vivo, respectively, while a control nonbinding antibody or the fibrinogen L10 peptide gamma Leu402-Val411 were ineffective. These data suggest that simultaneous assembly of fibrinogen on leukocytes and endothelial ICAM-1 provides a pathway of intercellular adhesion which may act in concert with beta 2 integrins to stabilize firm leukocyte attachment to endothelium, in vivo. Given the recognized role of fibrinogen as a major risk factor for atherosclerosis, this mechanism may directly contribute to thrombus formation and endothelial cell damage in vascular diseases.

In Vitro Assembly of the Component Chains of Fibrinogen Requires Endoplasmic Reticulum Factors

Human fibrinogen (340 kDa) is a dimer, with each identical half-molecule composed of three different polypeptides (Aalpha, 66 kDa; Bbeta, 55 kDa; and gamma, 48 kDa). To understand the mechanisms of chain assembly, a coupled in vitro transcription translation system capable of assembling fibrinogen chains was developed. Fibrinogen chain assembly was assayed in an expression system coupled to rabbit reticulocyte lysate in the presence or absence of dog pancreas microsomal membranes. Fibrinogen chain assembly required microsomal membranes and oxidized glutathione. Co-expression of two of the chains, Bbeta and gamma or Aalpha and gamma, yielded free chains and two-chain complexes. Unlike combinations of Aalpha with gamma and Bbeta with gamma, co-expression of Aalpha and Bbeta did not form a single two-chain complex but produced a mixture of two-chain complexes. Co-expression of all three chains yielded free chains, two-chain complexes, and higher molecular weight complexes that corresponded to a half-molecule and to fully formed fibrinogen. Upon treatment of this mixture with thrombin and factor XIIIa, a gamma.gamma dimer, similar to that obtained from cross-linked human fibrin, was produced, indicating that properly folded fibrinogen was formed in vitro. Molecular chaperones may participate in fibrinogen assembly, since antibodies to resident proteins of the endoplasmic reticulum (BiP, Hsp90, protein disulfide isomerase, and calnexin) co-precipitated the chaperones together with nascent fibrinogen chains and complexes.

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

Intermolecular end-to-middle domain pairing between a thrombin-exposed 'A' polymerization site in the central 'E' domain of fibrin, and a constitutive complementary 'a' site in each outer 'D' domain ('D:E'), is necessary but not alone sufficient for normal fibrin assembly, as judged from previous studies of a congenital dysfibrinogen, Tokyo II (gamma 275 arg-->cys), which showed defective fibrin clot assembly and a normal D:E interaction (Matsuda, M., M. Baba, K. Morimoto, and C. Nakamikawa, 1983. J. Clin. Invest. 72:1034-1041). In addition to the 'a' polymerization site, two other constitutive intermolecular association sites on fibrinogen D domains have been defined: between gamma chain regions containing the carboxy-terminal factor XIIIa crosslinking site ('gamma XL:gamma XL'); and between sites located at the outer ends of each molecule ('D:D') (Mosesson, M. W., K. R. Siebenlist, J. F. Hainfeld, and J. S. Wall, manuscript submitted for publication). We evaluated the function of these sites in Tokyo II fibrinogen, and confirmed that there was a normal fibrin D:E interaction, as determined from a normal fibrin crosslinking rate in the presence of factor XIIIa. We also found a normal gamma XL: gamma XL interaction, as assessed by a normal fibrinogen crosslinking rate. Judging from electron microscopic images, factor XIIIa-crosslinked Tokyo II fibrinogen failed to form elongated double-stranded fibrils like normal fibrinogen. Instead, it formed aggregated disordered collections of molecules, with occasional short fibrillar segments. In addition, Tokyo II fibrin formed an abnormal, extensively branched clot network containing many tapered terminating fibers. These findings indicate that the Tokyo II fibrinogen defect results in a functionally abnormal D:D self-association site, and that a normal D:D site interaction is required, in addition to D:E, for normal fibrin or fibrinogen assembly.

Fibrinogen assembly: Insights from chicken hepatocytes

In all vertebrate species studied, the complex, disulfide-linked structure of fibrinogen is essentially the same: a hexamer assembled from three different subunits (A alpha, B beta, gamma)2. This study utilized species differences in fibrinogen subunit monomer pools to address the question of how these surplus subunit pools may affect the assembly process. We used a chicken model system in which B beta and gamma-subunits are present in excess, in contrast to the A alpha and gamma-subunit surplus found in human model systems. Analysis was based on pulse-chase experiments with electrophoretic separation of intracellular forms and secreted fibrinogen on reducing and nonreducing gels. The chicken liver-derived cells employed for this purpose, primary hepatocytes and a hepatoma cell line with a fortuitous defect in fibrinogen synthesis, together offer advantages over human systems for resolving the complexes formed in the early stages of assembly. The results demonstrate that in chicken hepatocytes there is an initial binding of gamma to A alpha subunits rather than to B beta subunits, as occurs in human hepatoma cells. Nevertheless, the presence of similar intracellular fibrinogen-related forms in both chicken- and human-derived cells, in the context of their differing subunit monomer pools, suggests an assembly pathway common to both species, with the versatility to be regulated by limitation of A alpha or B beta subunit production.

Role of interchain disulfide bonds on the assembly and secretion of human fibrinogen.

The cysteines involved in joining the 2 half-molecules of fibrinogen and also those located on either side of the alpha-helical coiled-coil region, were substituted, by site-directed mutagenesis, with serine. Fibrinogen assembly and secretion were determined in transiently transfected COS cells. Our studies indicate that in order to assemble the 2 half-molecules into a dimer, it is not sufficient to only have the disulfide linkages which keep the 2 half-molecules intact. The disulfide rings which flank the coil-coiled region also play important roles in dimer assembly. Intact interchain disulfide linkages at the NH2-terminal end of the coiled-coil region are essential for assembly of the 2 half-molecules. Disruption of these disulfide rings leads to the formation and secretion of half-molecules. Disruption of the interchain disulfide rings at the COOH-terminal end of the coiled-coil region allows dimer formation, but the 6-chain molecule which is assembled is not secreted. Disruption of both disulfide rings at either end of the coiled-coil region disallows assembly of half-molecules and of dimeric fibrinogen.

Symmetrical disulfide bonds are not necessary for assembly and secretion of human fibrinogen

Human fibrinogen is a dimer, and each half-molecule is composed of three different polypeptides (A alpha, B beta, and gamma). The two half-molecules are joined together at the amino-terminal ends by three symmetrical disulfide bonds between adjacent A alpha chains, at position 28, and between adjacent gamma chains at gamma 8 and 9. To determine the role of these disulfide bonds in fibrinogen assembly and secretion, site-directed mutagenesis was used to change cysteines of A alpha 28 and gamma 8 and 9 to serine, the mutant chains were coexpressed with normal fibrinogen chains in COS cells, and assembly and secretion of fibrinogen were measured. Elimination of the symmetrical disulfide bonds did not affect assembly of the chains and dimeric fibrinogen was secreted. Analysis by plasmin digestion indicated that the secreted mutant fibrinogens have a similar structure to normal fibrinogen. Our results indicate that other domains of fibrinogen participate in dimer formation and that the three symmetrical disulfide bonds are not crucial for assembly and secretion of fibrinogen.

Processing of the carboxyl 15-amino acid extension in the alpha-chain of fibrinogen

The cDNA for the alpha-chain of human fibrinogen codes for 15 amino acids at the carboxyl terminus that are not found in the native protein circulating in plasma. In the present studies, experiments were designed to test whether the carboxyl extension was required for fibrinogen assembly or was simply the result of limited proteolysis and maturation of the protein during or following secretion. Baby hamster kidney cells were transfected with cDNAs coding for the alpha-, beta-, and gamma-chains, and the recombinant fibrinogen secreted into the cell culture medium was identified either with an antibody against the mature molecule or with an antibody directed toward the carboxyl extension of the alpha-chain. The secreted protein contained primarily two species of alpha-chains, including one with the carboxyl extension and one that was the same as that in plasma fibrinogen. A mutant fibrinogen, in which Arg-611 at the putative cleavage site in the alpha-chain was converted to Gly, was also readily assembled, and, in this case, the protein was secreted with the alpha-chain carboxyl extension. Similarly, a mutant fibrinogen that completely lacked the carboxyl extension of the alpha-chain was assembled and secreted from the transfected baby hamster kidney cells. These two mutants with modified alpha-chains were readily clotted by thrombin and cross-linked by factor XIIIa. These results indicate that the alpha-chain carboxyl extension in fibrinogen is not required for assembly or secretion of the molecule. Accordingly, it was concluded that the cleavage of the alpha-chain removing the carboxyl-terminal 15 amino acids is a normal and specific processing event occurring during the maturation of the protein.

Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly.

Stable transfected baby hamster kidney (BHK) cells expressing human alpha, beta, and gamma fibrinogen chains together, in various combinations of any two, or individually, were established. Several types of subunit interactions were observed in the intracellular extracts of the transfected BHK cell lines as well as in Hep G2 cells. These included: 1) formation of alpha gamma dimers linked by a disulfide bond(s), 2) formation of beta gamma dimers linked by a disulfide bond(s), 3) formation of alpha beta gamma half-molecules linked by disulfide bonds, and 4) formation of mature fibrinogen, which was also secreted into the cell culture medium. Analysis of the chain composition confirmed the stoichiometry of the alpha gamma, beta gamma, and alpha beta gamma complexes. These data are consistent with the proposal that the alpha gamma and beta gamma dimers as well as the alpha beta gamma half-molecules are intermediates in the assembly and biosynthesis of fibrinogen. In contrast, disulfide-linked alpha beta complexes were not found in transfected BHK cells or in Hep G2 cells, suggesting that the formation of disulfide bonds between these two chains most likely occurs when alpha beta gamma half-molecules are formed from alpha gamma and/or beta gamma complexes and when alpha beta gamma half-molecules dimerize to generate the mature molecule. Dimers, trimers, and larger oligomers of each individual chain linked by disulfide bonds were also identified when each chain was expressed in the absence of the other two chains. Preferential formation of alpha gamma and beta gamma complexes, rather than oligomers of individual chains, suggested that the oligomers were less likely to be intermediates in the assembly of fibrinogen. A model for fibrinogen assembly is presented based on these results.

The Role of Amino-Terminal Disulfide Bonds in the Structure and Assembly of Human Fibrinogen

Human fibrinogen contains two half-molecules, each composed of an α, β, and γ chain linked by disulfide bonds. The two half-molecules (αβγ) are held together in the native protein by additional disulfide bonds located in the amino terminus of each chain. Site-directed mutagenesis, in which the amino-terminal Cys residues (α-Cys28 and 36, β-Cys65, and γ-Cys8 and 9) were converted to Ser, was carried out in order to study the role of the amino-terminal disulfide bonds in the structure and assembly of fibrinogen. An analysis of the fibrinogen synthesized in transfected baby hamster kidney (BHK) cells employing various combinations of these mutations revealed that α-Cys36 and β-Cys65 form disulfide bonds between two αβγ half-molecules, rather than within the same half-molecule; furthermore, these two disulfide bonds are sufficient to hold the two αβγ half-molecules together as intact fibrinogen. Disulfide bonds formed by γ-Cys8 and 9 were also sufficient to hold the two fibrinogen αβγ half-molecules together, while the disulfide bond between the two α-Cys28 residues failed to form in the absence of the disulfide bonds linking the α and β chains and the two γ chains.

Assembly and secretion of fibrinogen. Degradation of individual chains

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

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

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

Studies on the assembly and secretion of fibrinogen.

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

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

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