Fibrinogen Variants Research Articles

Molecular weight fibrinogen variants determine angiogenesis rate in a fibrin matrix in vitro and in vivo.

During wound repair, fibrin acts both as a barrier to prevent blood loss and as a temporary matrix for the invasion and ingrowth of endothelial and tissue cells. A well-controlled angiogenesis process in the fibrinous exudate matrix is crucial for optimal wound healing. The composition and structure of the fibrin matrix are important determinants of the invasion of endothelial cells and capillary-tube formation into the matrix. Fibrinogen circulates in a high and low molecular weight form (HMW and LMW, respectively) and the purpose of this study was to investigate how fibrin matrices from these naturally occurring fibrinogen variants influence angiogenesis. Angiogenesis was studied using an in vitro model in which human microvascular endothelial cells (hMVEC) were cultured on three-dimensional fibrin matrices from different fibrinogen forms, and using two in vivo mouse models. The in vitro angiogenesis in an HMW-fibrin matrix shows increased cell and tubular structure ingrowth compared with unfractionated fibrin matrix (median increase 58%, range 46-234%). The ingrowth of tubular structures in an LMW-fibrin matrices is decreased when compared with unfractionated fibrin (median decrease 70%, range 67-100%). Similar results were observed for in vivo angiogenesis. The naturally occurring fibrinogen variants HMW- and LMW-fibrin modulate the angiogenic capacity of endothelial cells in fibrin matrices. The different effects of the molecular weight fibrinogen variants provide further insight in the matrix characteristics in angiogenesis and could possibly be applied in the context of tissue engineering and wound healing.

Mutant Fibrinogen Cleared from the Endoplasmic Reticulum via Endoplasmic Reticulum-Associated Protein Degradation and Autophagy: An Explanation for Liver Disease

The endoplasmic reticulum (ER) quality control processes recognize and remove aberrant proteins from the secretory pathway. Several variants of the plasma protein fibrinogen are recognized as aberrant and degraded by ER-associated protein degradation (ERAD), thus leading to hypofibrinogenemia. A subset of patients with hypofibrinogenemia exhibit hepatic ER accumulation of the variant fibrinogens and develop liver cirrhosis. One such variant named Aguadilla has a substitution of Arg375 to Trp in the gamma-chain. To understand the cellular mechanisms behind clearance of the aberrant Aguadilla gamma-chain, we expressed the mutant gammaD domain in yeast and found that it was cleared from the ER via ERAD. In addition, we discovered that when ERAD was saturated, aggregated Aguadilla gammaD accumulated within the ER while a soluble form of the polypeptide transited the secretory pathway to the trans-Golgi network where it was targeted to the vacuole for degradation. Examination of Aguadilla gammaD in an autophagy-deficient yeast strain showed stabilization of the aggregated ER form, indicating that these aggregates are normally cleared from the ER via the autophagic pathway. These findings have clinical relevance in the understanding of and treatment for ER storage diseases.

In vitro fibrin clot formation and fibrinolysis using heterozygous plasma fibrinogen from γAsn319, Asp320 deletion dysfibrinogen, Otsu I

Introduction We have reported a heterozygous dysfibrinogenemia, fibrinogen Otsu I, caused by the deletion of γAsn319 and γAsp320, which was originally identified in the dysfibrinogen Vlissingen/Frankfurt IV (V/FIV) associated with thrombosis. Unlike the V/FIV family, the Otsu propositus showed no thrombotic tendencies. To analyze the relationship between thrombosis and the heterozygous plasma variant fibrinogen, we used purified plasma fibrinogen from the Otsu patient and compared it with a normal control. Materials and methods Thrombin-induced fibrin clot formation and clot structure were observed by fibrin polymerization and scanning electron microscopy, respectively. For in vitro observation of fibrinolysis, plasmin generation and clot lysis assays were performed by the addition of tissue type plasminogen activation (tPA) and plasminogen. Results and conclusions Polymerization of Otsu was markedly impaired, while fibrin fibers were much thicker and the density of the bundles of fibrin fibers was less and porous compared with normal. Lysis of the Otsu clot was not significantly different from normal when a tPA and plasminogen mixture was overlaid onto the clots. For Otsu, the penetration of the tPA/plasminogen mixture into the clot was much faster than normal and the protection against plasmin cleavage was impaired; however, tPA-induced plasmin activation of the Otsu fibrin was slower than that of normal fibrin, resulting in a clot lysis of Otsu similar to normal.

A novel variant fibrinogen, deletion of Bβ111Ser in coiled-coil region, affecting fibrin lateral aggregation

Background Functional fibrinogen concentration of a male infant showed < 0.50 g/l and we speculated this patient as a dysfibrinogenemia or hypofibrinogenemia. Methods We analyzed propositus and his parent by DNA sequencing and by thrombin-catalyzed fibrin polymerization for purified plasma fibrinogen. Results Although functional fibrinogen determinations based on Clauss method showed the marked discrepancy of values among 3 sets of reagent and analyzer, we found a novel heterozygous variant fibrinogen, Kyoto IV, caused by 3-bp deletion in Bβ-chain gene corresponding to the deletion of 111Ser located in coiled-coil region. We suggested that the discrepancy of fibrinogen values among 3 assays was caused by the difference in NaCl concentration in reagents for determination and analyzed the polymerization under the conditions of various NaCl concentrations. Although under normal physiological conditions Kyoto IV fibrinogen augmented the polymerization as compared with normal control, in 0.21 mol/l NaCl Kyoto IV fibrinogen showed abruptly impaired polymerization curve compared with normal control. Conclusion Variant fibrinogen, BβΔ111Ser, showed augmented lateral aggregation under normal physiological conditions and the residue located in coiled-coil region, Bβ111Ser, plays an important role in the lateral aggregation.

Novel monoclonal antibody that recognizes new neoantigenic determinant of D-dimer

Our novel monoclonal antibody (mAb) B4 reacted with only D-dimer but not intact fibrinogen, or fibrinogen degradation products (FgDP) such as D-monomer, E fragment on ELISA. B4 didn't react with denatured D-dimer, while it reacted well with denatured D-monomer rather than the native form, indicating that B4 recognizes some neoconformational epitope in D-dimer. In our epitope study, B4 recognized the N-terminal (Bβ134-142) of D-dimer, which corresponds to the most flexible segment of coiled coil backbone. It was confirmed by inhibition assay of B4 binding to D-dimer using the synthesized peptides with this sequence. As the other evidence, B4 didn't bind to some D-dimer species produced from a particular fibrinogen variant. This fibrinogen variant is mutated BβLys133 residue to Gln133 thus it doesn't produce the particular N-terminal epitope of D134∼ by plasmin. Finally, our mAb was useful for clinical application. ELISA using our mAbs was well correlated with other commercial D-dimer ELISAs and in some clinical samples it was preferable to them. These results suggest that the epitope for B4 is another neoantigenic determinant in native D-dimer as distinct from native D-monomer.

Does Elevated Plasma Fibrinogen Increase the Risk of Coronary Heart Disease?

The purpose of this study was to assess whether a genetic variant associated with higher fibrinogen levels is associated with increased coronary heart disease (CHD) risk, as a test of the causal influence of fibrinogen on CHD. We performed a meta-analysis of case-control and prospective studies of the G-455-->A and C-148-->T beta-fibrinogen promoter region variants, in relation to CHD risk. The 19 studies found included 12,393 cases and 21,649 controls. Fibrinogen levels were robustly related to the genetic variants (mean increase per allele, 0.117 g/L; 95% CI, 0.091-0.142 g/L). However, the genetic variants were unrelated to CHD risk (odds ratio per allele, 0.976; 95% CI, 0.916-1.040). The predicted causal odds ratio for a 1 g/L higher plasma fibrinogen level, given the genetic variant-fibrinogen and genetic variant-CHD associations, was 0.81 (95% CI, 0.46-1.40). Although imprecise, the predicted causal effect of fibrinogen on CHD is clearly different from the odds ratio of 1.8 (95% CI, 1.6-2.0) for an increase of 1 g/L derived from a meta-analysis of observational studies. This evidence suggests that lowering the fibrinogen level may not, in itself, reduce CHD risk.

In vitro expression demonstrates impaired secretion of the γAsn319, Asp320 deletion variant fibrinogen

The hypodysfibrinogenemia Otsu is caused by the two-residue deletion, gammaAsn319 and gammaAsp320. Analysis of plasma or purified fibrinogen from the heterozygous propositus revealed that the amount of variant gamma-chain was lower than that of normal gamma-chain. In order to examine the basis for this difference, we transfected Chinese hamster ovary cells and established stable cell lines that expressed both chains, gammaDelta/gammaN, only the normal chain, gammaN, and only the variant chain, gammaDelta. We measured fibrinogen concentration of confluent cultures by ELISA. We found the ratios of the concentrations in the media to the concentrations in the cell lysates of gammaDelta, gammaDelta/gammaN, and gammaN-cells were 0.42, 0.60, and 1.00, respectively. We measured the concentrations of the gammaDelta and gammaN chains by densitometric analysis of samples following separation by SDS-PAGE and found the fraction of gammaDelta-chains in cell lysates was always greater than the fraction in the respective culture media. We examined the kinetics of fibrinogen synthesis, assembly and secretion in pulse-chase experiments, and found that the gammaDelta-chain was assembled into intact fibrinogen at a rate similar to assembly of the gammaN-chain into normal fibrinogen, but was secreted into the medium at a slightly slower rate than normal fibrinogen. Considered together, these experiments indicate secretion of the variant fibrinogen was slightly impaired. These results suggest that the reduced level of gammaDelta319,320 fibrinogen in the plasma of the Otsu patient arises from modestly impaired secretion of this variant fibrinogen.

Fibrin(ogen)-αMβ2Interactions Regulate Leukocyte Function and Innate Immunity In Vivo

In addition to its well-characterized role in hemostasis, fibrin(ogen) has been proposed to be a central regulator of the inflammatory response. Multiple in vitro studies have demonstrated that this hemostatic factor can alter leukocyte function, including cell adhesion, migration, cytokine and chemokine expression, degranulation, and other specialized processes. One important link between fibrin(ogen) and leukocyte biology appears to be the integrin receptor alpha(M)beta(2)/Mac-1, which binds to immobilized fibrin(ogen) and regulates leukocyte activities. Although it is well established that fibrin(ogen) is a ligand for alpha(M)beta(2), the precise molecular determinants that govern this interaction are only now becoming clear. A novel line of mice expressing a mutant form of fibrinogen (Fib gamma(390-396A)) has revealed that gamma chain residues 390-396 are important for the high-affinity engagement of fibrinogen by alpha(M)beta(2) and leukocyte function in vivo. Fibrinogen gamma(390-396A) failed to support alpha(M)beta(2)-mediated adhesion of primary neutrophils, monocytes, and macrophages, and mice expressing this fibrinogen variant were found to exhibit a major defect in the host inflammatory response following acute challenges. Most notably, Fib gamma(390-396A) mice display a profound impediment in Staphylococcus aureus elimination by leukocytes following intraperitoneal inoculation. These findings have positively established the physiological importance of fibrin(ogen) as a ligand for alpha(M)beta(2) and illustrate that the fibrin(ogen) gamma chain residues 390-396 constitute a critical feature of the alpha(M)beta(2) binding motif. Finally, the Fib gamma(390-396A) mice represent a valuable system for better defining the contribution of fibrin(ogen) to the inflammatory response in the absence of any confounding alteration in clotting function.

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Pathophysiologic roles of the fibrinogen gamma chain.

Fibrinogen binds through its gamma chains to cell surface receptors, growth factors, and coagulation factors to perform its key roles in fibrin clot formation, platelet aggregation, and wound healing. However, these binding interactions can also contribute to pathophysiologic processes, including inflammation and thrombosis. This review summarizes the latest findings on the role of the fibrinogen gamma chain in these processes, and illustrates the potential for therapeutic intervention. Novel gamma chain epitopes that bind platelet integrin alpha IIbbeta3 and leukocyte integrin alphaMbeta2 have been characterized, leading to the revision of former dogma regarding the processes of platelet aggregation, clot retraction, inflammation, and thrombosis. A series of studies has shown that the gamma chain serves as a depot for fibroblast growth factor-2 (FGF-2), which is likely to play an important role in wound healing. Inhibition of gamma chain function with the monoclonal antibody 7E9 has been shown to interfere with multiple fibrinogen activities, including factor XIIIa crosslinking, platelet adhesion, and platelet-mediated clot retraction. The role of the enigmatic variant fibrinogen gamma chain has also become clearer. Studies have shown that gamma chain binding to thrombin and factor XIII results in clots that are mechanically stiffer and resistant to fibrinolysis, which may explain the association between gammaA/gamma' fibrinogen levels and cardiovascular disease. The identification of new interactions with gamma chains has revealed novel targets for the treatment of inflammation and thrombosis. In addition, several exciting studies have shown new functions for the variant gamma chain that may contribute to cardiovascular disease.

Recombinant fibrinogen, γ275Arg→Cys, exhibits formation of disulfide bond with cysteine and severely impaired D:D interactions

Analysis of dysfibrinogens has provided useful information aiding our understanding of molecular defects in fibrin polymerization. We have already reported impaired fibrin polymerization in a variant fibrinogen (gammaArg275Cys), the Cys being located in the D:D interface. Since this substitution occurred in a heterozygous individual, interpretation of the functional analysis was complicated. We tried to resolve this complication by synthesizing a recombinant variant fibrinogen. A variant gamma-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen Aalpha- and Bbeta-chains. The recombinant variant fibrinogen (gamma275C) was purified using an immunoaffinity column, and we compared its structure and functions with those of normal recombinant fibrinogen (gamma275R) and plasma variant fibrinogen. Mass analyses showed the existence of disulfide-linked Cys in both patient and recombinant variant fibrinogens. Functional analyses indicated that both fibrin polymerization and gamma-gamma dimer formation were markedly impaired in the variant fibrinogen. The impairments were much more pronounced in gamma275C than in plasma variant fibrinogen. In addition, scanning electron microscopic observation of fibrin clots made from gamma275C revealed less dense fibrin fiber bundles and larger fiber diameter than in those made from gamma275R, and also the existence of many aberrant fibrin fibers with tapered ends. These results indicate that gammaArg275 has an important residue affecting the structure and function of the gamma-chain C-terminal domain. However, the variant D:D interface can interact with that of the normal fibrinogen existing in a heterozygous patient with dysfibrinogenemia.

Calcium-binding site beta 2, adjacent to the "b" polymerization site, modulates lateral aggregation of protofibrils during fibrin polymerization.

Structural analysis of recombinant fibrinogen fragment D revealed that the calcium-binding site (beta2-site) composed of residues BbetaAsp261, BbetaAsp398, BbetaGly263, and gammaGlu132 is modulated by the "B:b" interaction. To determine the beta2-site's role in polymerization, we engineered variant fibrinogen gammaE132A in which calcium binding to the beta2-site was disrupted by replacing glutamic acid at gamma132 with alanine. We compared polymerization of gammaE132A to normal fibrinogen as a function of calcium concentration. Polymerization of gammaE132A at concentrations of calcium <or=1 mM exhibited an uncharacteristic 2-3-fold increase in lateral aggregation and fiber thickness compared to normal fibrinogen, while polymerization of variant and normal were indistinguishable at 10 mM calcium. These results suggest that the beta2-site controls the extent of lateral aggregation. That is, when the calcium anchor (beta2-site) is eliminated before "B:b" interactions occur then lateral aggregation is enhanced. We solved structures of fragment D of gammaE132A fibrinogen (rfD-gammaE132A) with and without Gly-His-Arg-Pro-amide (GHRPam) and found no change to the global structure. X-ray diffraction data showed GHRPam binding in the "a" and "b" polymerization sites and that calcium could still bind to the beta2-site of gammaE132A fibrinogen at 70 mM calcium. We found that the gamma2 calcium-binding site (in loop gamma294-301) did not have calcium bound in the structure of fragment D of gammaE132A fibrinogen with GHRPam bound (rfD-gammaE132A+GH). Analysis of structures rfD-gammaE132A+GH and rfD-BbetaD398A+GH indicated that differences in calcium occupation of the gamma2-site resulted from minor conformational changes provoked by crystal packing and GHRPam binding to the "a" site did not directly modulate calcium binding to this site.

Influence of gamma' fibrinogen splice variant on fibrin physical properties and fibrinolysis rate.

A splice variant of fibrinogen, gamma', has an altered C-terminal sequence in its gamma chain. This gammaA/gamma' fibrin is more resistant to lysis than gammaA/gammaA fibrin. Whether the physical properties of gamma' and gammaA fibrin may account for the difference in their fibrinolysis rate remains to be established. Mechanical and morphological properties of cross-linked purified fibrin, including permeability (Ks, in cm2) and clot stiffness (G', in dyne/cm2), were measured after clotting gammaA and gamma' fibrinogens (1 mg/mL). gamma'/gamma' fibrin displayed a non-significant decrease in the density of fibrin fibers and slightly thicker fibers than gammaA/gammaA fibrin (12+/-2 fiber/10(-3) nm3 versus 16+/-2 fiber/10(-3) nm3 and 274+/-38 nm versus 257+/-41 nm for gamma'/gamma' and gammaA/gammaA fibrin, respectively; P=NS). This resulted in a 20% increase of the permeability constant (6.9+/-1.7 10(-9) cm2 versus 5.5+/-1.9 10(-9) cm2, respectively; P=NS). Unexpectedly, gamma' fibrin was found to be 3-times stiffer than gammaA fibrin (72.6+/-2.6 dyne/cm2 versus 25.1+/-2.3 dyne/cm2; P<0.001). Finally, there was a 10-fold decrease of the fibrin fiber lysis rate. Fibrinolysis resistance that arises from the presence of gammaA/gamma' fibrinogen in the clot is related primarily to an increase of fibrin cross-linking with only slight modifications of the clot architecture.

Fibrinogen gamma-chain splice variant γ′ alters fibrin formation and structure

AbstractFibrinogen γA/γ′ results from alternative splicing of mRNA. This variant, which constitutes approximately 8% to 15% of plasma fibrinogen, contains FXIII and thrombin binding sites. Our objective was to investigate whether γA/γ′ differs in fibrin formation and structure from the more common variant γA/γA. Both variants were separated and purified by anion-exchange chromatography. Fibrin formation and clot structure of the variants and unfractionated fibrinogen were investigated by turbidity and scanning electron microscopy (SEM). Thrombin cleavage of fibrinopeptides was analyzed by high-performance liquid chromatography (HPLC). Turbidity analysis showed significantly altered polymerization rates and overall fiber thickness in γA/γ′ clots compared with γA/γA and unfractionated fibrinogen. This finding was consistent with a range of thrombin concentrations. HPLC demonstrated reduced rates of fibrinopeptide B (FpB) release from γA/γ′ fibrinogen compared with γA/γA. Delayed FpB release was associated with delayed lateral aggregation of protofibrils and significant differences were found on SEM, with γA/γ′ clots consisting of smaller diameter fibers and increased numbers of branch points compared with both γA/γA and unfractionated fibrinogen. These results demonstrate that the γA/γ′ splice variant of fibrinogen directly alters fibrin formation and structure, which may help to explain the increased thrombotic risk associated with this variant.

Two electrophoretic fibrinogen variants found in patients with normal coagulation and familial thrombosis

Two electrophoretic fibrinogen variants found in patients with normal coagulation and familial thrombosis

Two novel fibrinogen variants found in patients with pulmonary embolism and their families

The occurrence of dysfibrinogen is quite rare in comparison with other hemostatic defects, specially in cases of venous thrombosis. Fibrinogen is known to have multiple functions, which are not evaluated by simple coagulation testing. We have used gel electrophoresis to search for new mutations. Specimens of purified fibrinogen from 217 consecutive patients with familial or recurrent or early thrombosis and from 490 control subjects were evaluated by electrophoresis. Plasma fibrinogen levels and coagulation-dependent tests (electromechanical and optical coagulometric determinations, immunological measurement, thrombin and Reptilase(R) times) were normal. Two novel familial variants were detected. For a 42-year-old patient, an in-frame 117 base pair insertion in the Aalpha-chain gene caused a 5-kDa mobility shift of the Aalpha chain. This corresponds to a 39 amino acid duplication in the connector domain (fibrinogen Champagne au Mont d'Or). This pattern was also found in the patient's mother and child. A second 31-year-old patient presented an extra band under non-reducing conditions, 30 kDa larger than HMW fibrinogen and reacting with antifibrinogen antibodies (fibrinogen Lozanne). A heterozygous 5909A-->G mutation was found on the Bbeta-chain gene leading to heterozygous Bbeta Tyr236--> stop codon. The predicted truncated Bbeta chain could participate in chain assembly. Two family members were also affected, one of whom had suffered early venous thrombosis. Electrophoretic testing of apparently normal fibrinogens can reveal new variants which may be clinically relevant.

Fibrinogen Magdeburg I: a novel variant of human fibrinogen with an amino acid exchange in the fibrinopeptide A (Aα 9, Leu→Pro)

Introduction: The exchange of Aα 16, Arg for Cys or His is the most common molecular defect in dysfibrinogenemia directly affecting the thrombin cleavage site involved in fibrinopeptide A (FPA) release. Other amino acid exchanges within the fibrinopeptide A have been only rarely reported. Materials and methods: In clinically asymptomatic dysfibrinogenemic patients with low functional plasma fibrinogen (Fg) levels and prolonged thrombin time but normal or slightly prolonged batroxobin (reptilase) time, mutation analysis was carried out by direct sequencing of the coding regions of the three fibrinogen genes. Isolated fibrinogen was functionally characterized for thrombin- or batroxobin-induced fibrinopeptide release and fibrin formation. Fibrinogen and fibrinopeptides were structurally studied by electrophoretic techniques or high-performance liquid chromatography. Results and conclusions: Molecular analysis revealed heterozygosity for a novel missense mutation T1182C in the FGA gene causing the amino acid exchange Aα 9, Leu→Pro. Fibrin generation induced by thrombin was moderately impaired, whereas batroxobin-induced fibrin formation was almost normal. Release of the abnormal fibrinopeptide A by thrombin was delayed but fibrin monomer aggregation was almost normal. Cleavage of Aα chains by batroxobin was only slightly delayed. Fibrinopeptides A of the patient fibrinogen did not show any gross abnormality in chromatographic behaviour. This new molecular variant designated fibrinogen Magdeburg I supports the view that amino acid residue Leu-9 in the Aα chain as part of a small hydrophobic cluster is involved in the interaction with an apolar binding site of thrombin, thus adding to our understanding of the thrombin–fibrinogen interaction crucial in coagulation.

Fibrinogen γ′ chain binds thrombin exosite II

A high-affinity thrombin-binding site in an alternately processed fibrinogen variant, the gammaA/gamma' isoform, is characterized in this report. The binding site has been shown to be situated between gamma' 414 and 427, and Tyr418 and 422 in this part of the gamma' chain are known to be sulfated. A synthetic peptide corresponding to the gamma' chain carboxyl terminus is shown to bind thrombin with a Kd = 0.63 +/- 0.16 micro mol L-1. Maximum binding of this peptide requires negative charges on Tyr418 and 422. Competitive binding studies with hirudin peptides, heparin and DNA aptamers specific for thrombin exosites I or II indicate thrombin binds to the gamma' peptide via exosite II. Thus, thrombin binding to the gamma' chain leaves exosite I and the active site accessible to substrates. This may explain why fibrin-bound thrombin can retain enzymatic activity, and why fibrin-bound thrombin is heparin-resistant.

Analysis of fibrinogen γ-chain truncations shows the C-terminus, particularly γIle387, is essential for assembly and secretion of this multichain protein

To examine the role of the fibrinogen gamma chain in the assembly and secretion of this multichain protein, we synthesized a series of fibrinogen variants with truncated gamma chains, terminating between residues gamma379 and the C-terminus, gamma411. The variant fibrinogens were synthesized from altered gamma-chain complementary DNAs in cultured Chinese hamster ovary cells. Immunoassays of the culture media demonstrated that only those variants with gamma chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the gamma chain, from 1.4 microg/mL for normal fibrinogen to 0.39 microg/mL for gamma 387 fibrinogen. Immunoassays of cell lysates showed that all variant gamma chains were synthesized, although the levels varied significantly. For variants longer than 386 residues, levels decreased with length but remained near normal. In contrast, expression of the 4 variants with 386 residues or less was about 20-fold reduced. Quantitative reverse transcription-polymerase chain reaction demonstrated that the gamma-chain messenger RNA level was independent from chain length. Western blot analyses showed that lysates expressing variants with 387 residues or more contained species comparable to the known intermediates in fibrinogen assembly, including half-molecules. For shorter variants, these intermediates were not evident. We conclude that residues near the C-terminus of the gamma chain are essential for fibrinogen assembly, and more specifically, that gamma387 is critical. We propose that the loss of residue gamma387 destabilized the structure of gamma chain, preventing assembly of alphagamma and betagamma dimers, essential intermediates in the assembly of normal fibrinogen.

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.