Plasma fibrinogen γ'' chain content in the thrombotic microangiopathy syndrome

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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). Results: 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). CONCLUSIONS: 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.     

Thrombosis risk modification in transgenic mice containing the human fibrinogen thrombin-binding γ' chain sequence

Summary.  Background and objectives:  Thrombin binding activity in murine fibrin (Antithrombin I) is restricted to its E domains inasmuch as murine γ' chains (mu-γ') do not bind thrombin. This feature prompted us to produce a 'gain-of-function' transgenic mouse in which the wild-type (WT) C-terminal mu-γ' chain fibrinogen sequence had been replaced with the C-terminal thrombin-binding human γ' sequence. Results:  This procedure resulted in a murine fibrinogen species containing chimeric hu-γ' chains (hu-γ' fibrinogen). As anticipated, thrombin bound to WT fibrin at a single class of sites, whereas thrombin binding to heterodimeric hu-γ'-containing fibrin was increased, reflecting its content of hu-γ' chains. In an electrolytically-induced femoral vein thrombosis injury model, we found no differences in the volume of thrombus generation between WT and heterozygous hu-γ' mice. However, heterozygous factor (F) V Leiden (FVL^+/−) mice developed greater thrombus volumes than did WT controls ( P  < 0.01). In doubly heterozygous FVL^+/−, hu-γ' mice, thrombus formation was reduced to WT levels ( P  < 0.05). Conclusions:  Murine hu-γ' fibrinogen down-regulates venous thrombosis in the presence of another known thrombosis risk factor, FV Leiden. This finding indicates that hu-γ' chain-containing fibrinogen is a thrombosis risk modifier.     

Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen γ''/γA mRNA ratio

BACKGROUND: Fibrinogen gamma haplotype 2 (FGG-H2) is associated with reduced fibrinogen gamma' levels and fibrinogen gamma'/total fibrinogen ratios and with an increased deep-venous thrombosis (DVT) risk. Two FGG-H2 tagging single nucleotide polymorphisms (SNPs), 9615C>T and 10034C>T, are located in the region of alternative FGG pre-mRNA processing. 10034C>T is located in a GT-rich downstream sequence element (DSE) that comprises a putative cleavage stimulation factor (CstF) binding site. OBJECTIVES: To investigate the functionality of SNPs 9615C>T and 10034C>T, and the importance of the DSE containing 10034C>T. METHODS: Different minigene constructs containing FGG exon 9, intron 9, exon 10 and the 3' region were transiently transfected into HepG2 cells and quantitative real-time polymerase chain reaction was used to measure relative polyadenylation (pA) signal usage (pA1/pA2 ratio). RESULTS: Compared with the reference construct CC (9615C-10034C; FGG-H1; pA1/pA2 ratio set at 100%), the pA1/pA2 ratio of construct TT (9615T-10034T; FGG-H2) was 1.4-fold decreased (71.5%, P = 0.015). The pA1/pA2 ratio of construct CT (9615C-10034T) was almost 1.2-fold decreased (85.3%, P = 0.001), whereas the pA1/pA2 ratio of construct TC (9615T-10034C) did not differ significantly from the reference construct (101.6%, P = 0.890). Functionality of the putative CstF binding site was confirmed using constructs in which this site was deleted or its sequence altered by point mutations. CONCLUSIONS: SNP 10034C>T is located in a GT-rich DSE involved in regulating the usage of the pA2 signal of FGG, which may represent a CstF binding site. We propose that the 10034C>T change is the functional variation in FGG-H2 that is responsible for the reduction in the fibrinogen gamma'/total fibrinogen ratio and the increased DVT risk.     

Changes in fibrinogen and fibrin induced by a peptide analog of fibrinogen γ365–380

BACKGROUND: The effects of synthetic peptides with sequences derived from the gamma-chain of fibrinogen on the functional properties of fibrinogen and fibrin were investigated. METHODS: Methods included thrombelastography, clot turbidity measurement, clot elasticity measurement, platelet aggregation, and scanning transmission electron microscopy (STEM). RESULTS: Peptide gamma369-380 (NH(2)-WATWKTRWYSMK-COOH) showed the greatest impact on fibrin structure, compared with the 76 other overlapping dodecapeptides. Addition of this peptide, or peptide gamma365-380 (NH(2)-NGIIWATKTREWYSMK-COOH) to a mixture of fibrinogen and thrombin resulted a shorter clotting time, higher clot turbidity, lower clot elastic modulus, a higher degree of D-trimer and D-tetramer formation, and impaired plasmin proteolysis of the clot. In STEM, fibrin formed in the presence of peptide gamma369-380 consisted of a more extensive array of linear fibrils typically consisting of 20 or more molecules. Fibrils were better organized than those from non-peptide containing mixtures. CONCLUSIONS: Replacement of the tryptophan residue gamma376 massively reduced the effect of the peptide on fibrin structure. Binding of the peptide to fibrinogen induces conformational changes, which result in accelerated clotting and increased lateral association of fibrin protofibrils. The results imply a relevant functional role of sites interacting with peptide gamma369-380 region in the fibrinogen molecule.     

Elevated plasma fibrinogen γ'' concentration is associated with myocardial infarction: effects of variation in fibrinogen genes and environmental factors

BACKGROUND: Fibrinogen gamma', a fibrinogen gamma-chain variant generated via alternative mRNA processing, has been associated with susceptibility to thrombotic disease. OBJECTIVE: The present case-control study searched for potential determinants of the plasma fibrinogen gamma' concentration and examined the relationship between this variant and risk of myocardial infarction (MI). PATIENTS AND METHODS: The Stockholm Coronary Artery Risk Factor study, comprising 387 postinfarction patients and 387 healthy individuals, was employed. The fibrinogen gamma (FGG) 9340T > C [rs1049636], fibrinogen alpha (FGA) 2224G > A [rs2070011] and fibrinogen beta (FGB) 1038G > A [rs1800791] polymorphisms were determined. The plasma fibrinogen gamma' concentration was measured by enzyme-linked immunosorbent assay. The multifactor dimensionality reduction method was used for interaction analyses on risk of MI. RESULTS: The FGG 9340T > C and FGA 2224G > A polymorphisms, total plasma concentrations of fibrinogen, insulin and high-density lipoprotein, and gender appeared to be independent determinants of plasma fibrinogen gamma' concentration in patients, and the corresponding determinants in controls included FGG 9340T > C and FGA 2224G > A polymorphisms and plasma fibrinogen concentration. An elevated plasma fibrinogen gamma' concentration proved to be an independent predictor of MI [adjusted odds ratio (OR) (95% CI): 1.24 (1.01, 1.52)]. The plasma fibrinogen gamma' concentration was involved in a high-order interaction with total plasma fibrinogen and the FGG 9340T > C and FGA 2224G > A polymorphisms, associated with a further increased risk of MI [OR (95% CI): 3.22 (2.35, 4.39)]. CONCLUSIONS: Plasma fibrinogen gamma' concentration influences the risk of MI, and this relationship seems to be strengthened by the presence of an elevated total plasma fibrinogen concentration and the FGG 9340T and FGA 2224G alleles.     

Development of fibrinogen γ-chain peptide-coated, adenosine diphosphate-encapsulated liposomes as a synthetic platelet substitute

Summary.  Background : The dodecapeptide HHLGGAKQAGDV (H12), corresponding to the fibrinogen γ-chain carboxy-terminal sequence (γ400-411), is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated H12-coated nanoparticles (polymerized albumin or liposome) as platelet function-supporting synthetic products. Objectives : To strengthen the hemostatic ability of H12-coated particles as a platelet substitute, we exploited installation of a drug delivery function by encapsulating adenosine diphosphate (ADP) into liposomes [H12-(ADP)-liposomes]. Methods and results : Via selective interaction with activated platelets through GPIIb/IIIa, H12-(ADP)-liposomes were capable of augmenting agonist-induced platelet aggregation by releasing ADP in an aggregation-dependent manner. When intravenously injected into rats, liposomes were readily targeted to sites of vascular injury as analyzed on computed tomography. In fact, comparable to fresh platelets, liposomes exhibited considerable hemostatic ability for correcting prolonged bleeding time in a busulphan-induced thrombocytopenic rabbit model. In addition, the liposomes showed no activating or aggregating effects on circulating platelets in normal rabbits. Conclusion : H12-(ADP)-liposome may thus offer a promising platelet substitute, being made with only synthetic materials and exerting hemostatic functions in vivo via reinforcement of primary thrombus formation by residual platelets in thrombocytopenia at sites of vascular injury, but not in circulation.     

Globular adiponectin induces platelet activation through the collagen receptor GPVI-Fc receptor γ chain complex

Summary.  Background:  The adipocyte-derived cytokine, adiponectin (Ad), exerts potent vascular effects, although the direct effects of Ad on blood platelets are unclear. Objective:  The influence of globular Ad (gAd) on blood platelet function was investigated. Research design and methods:  We measured platelet aggregation and tyrosine phosphorylation signaling events in human and mouse platelets. The ability of gAd to activate Glycoprotein VI (GPVI) activity was determined with a NFAT luciferase reporter assay. Results:  gAd, but not full length Ad, induced rapid aggregation and granule secretion of human and mouse platelets through a pathway that is ablated under conditions of Src kinase inhibition, indicating a tyrosine kinase-dependent mechanism. Consistent with this, gAd stimulates rapid tyrosine phosphorylation of several proteins in human and mouse platelets. The pattern of increase in tyrosine phosphorylation was similar to that induced by collagen, with the tyrosine kinase Syk and PLCγ2 being identified among the list of tyrosine phosphorylated proteins. As collagen activates platelet through the GPVI-Fc receptor γ-chain (FcRγ) complex, we used FcRγ null platelets (which also lack GPVI) to explore the mechanism by which gAd stimulates platelets. Stimulation of tyrosine phosphorylation and platelet aggregation by gAd was abolished in FcRγ null platelets and markedly reduced in the absence of PLCγ2. Further, GPVI was confirmed as a collagen receptor for gAd by increased luciferase activity in Jurkat T-cells transfected with GPVI. Conclusions:  We identify gAd as a novel ligand for GPVI that stimulates tyrosine kinase-dependent platelet aggregation. Our data raise the possibility that gAd may promote unwanted platelet activation at sites of vascular injury.     

Hypofibrinogenemia caused by a nonsense mutation in the fibrinogen Bβ chain gene

Summary.  Congenital hypofibrinogenemia, fibrinogen Tottori II, caused by a nonsense mutation in the fibrinogen Bβ chain gene, was found in a 68-year-old Japanese female. The plasma fibrinogen level was 99.2 mg dL⁻¹ as determined by the thrombin time method. No overt molecular abnormalities were observed in purified patient fibrinogen by SDS–PAGE analysis. After sequencing all exons and exon–intron boundaries of three fibrinogen genes, we found a heterozygous single point mutation of T→G at position 3356 of the patient fibrinogen Bβ chain gene. This nucleotide mutation results in a nonsense mutation (TAT sequence for Bβ 41Tyr to TAG sequence for a translation termination signal). The mutation was confirmed by polymerase chain reaction-restriction fragment length polymorphism analysis, since this nucleotide mutation results in a new NheI recognition sequence at this position. These data indicated that the nonsense mutation of the fibrinogen Bβ chain gene caused a truncated fibrinogen Bβ chain, which may not be assembled in the fibrinogen molecule.     

Novel Aα chain truncation (fibrinogen Perth) resulting in low expression and impaired fibrinogen polymerization

Summary.  A young woman with a history of menorrhagia and easy bruising presented with a functional fibrinogen concentration of 1.8 mg mL⁻¹, a gravimetric concentration of 3.3 mg mL⁻¹ and a prolonged thrombin clotting time of 32 s. Both reverse phase analysis and reducing SDS–PAGE revealed a normal profile of Aα, Bβ, and γ chains. However, non-reducing gels revealed a broadened 340-kDa band, while the 305-kDa band was normal, suggesting a C-terminal truncation of the Aα chain. DNA sequencing of all exons and intron boundaries revealed a single heterozygous cytosine deletion at nucleotide 4841 of the Aα gene predicting a frameshift and the incorporation of 23 new residues (LMKLPSSTLPQLEKHSQVSSHLC) before termination after residue 517. In agreement with a predicted mass decrease of 9953 Da, the measured mass of the Aα^Perth chain was 56 242 Da, while that of the normal Aα^A chain was 66 189 Da. Tryptic mapping of isolated Aα chains revealed a new [M + 2H] ion at 607 m z⁻¹, corresponding to the predicted penultimate peptide LPSSTLPQLEK. The variant chain was poorly incorporated into plasma fibrinogen at a ratio of Aα^Perth/Aα^A of 0.15 : 1, suggesting the Aα^Perth chain might be out-competed by normal chains during molecular assembly in the hepatocyte. Despite the low expression, polymerization curves showed a decreased V_max and final turbidity, suggesting the fibrinogen Perth clots are composed of thinner fibers. However, the fibrinolytic rate was very similar to that of the control.     

γ-Glutamyl carboxylase (GGCX) tagSNPs have limited utility for predicting warfarin maintenance dose

BACKGROUND: The pharmacogenetic factors contributing to warfarin dosing are of great interest to clinicians, and may have utility in the management of at-risk patients prescribed warfarin. Gamma-glutamyl carboxylase (GGCX), in its role as a key component of the vitamin K cycle, is a potential candidate gene associated with warfarin treatment. OBJECTIVE: To identify single nucleotide polymorphisms (SNPs) and correlated GGCX tagSNPs and test for association with warfarin maintenance dose. PATIENTS/METHODS: A small discovery population of European-descent individuals (n = 23) were resequenced for GGCX SNPs. Polymorphisms identified with > 5% minor allele frequency (MAF) were genotyped in a larger clinical population of 186 European patients. Univariate, multivariate and haplotype-based linear regression were used to assess the impact of GGCX SNPs on warfarin dose. RESULTS: We identified 37 SNPs in GGCX, of which 21 were present at > 5% MAF. These SNPs were binned, based on linkage disequilibrium, and six informative tagSNPs were identified. A single polymorphism at position 12970 (rs11676382; C/G-11%/89%) was associated with a warfarin maintenance dose across all analysis methods. GGCX-12970 explained 2% of the total variance in warfarin dose, in contrast to 21 and 8%, respectively, for VKORC1 and CYP2C9. CONCLUSIONS: The GGCX-12970 SNP had a small, but significant effect, on warfarin maintenance dose. Other polymorphisms in GGCX previously associated with warfarin dose were not confirmed in this study, suggesting that the effects of GGCX are potentially population/treatment-dependent and will not have broad utility for determining warfarin dosing.     

A monoclonal antibody to the fibrinogen γ-chain alters fibrin clot structure and its properties by producing short, thin fibers arranged in bundles

Summary. Background: We previously reported that hamster monoclonal antibody 7E9, which reacts with the C‐terminus of the γ‐chain of mouse fibrinogen, inhibits factor (F)XIIIa‐mediated cross‐linking, platelet adhesion to fibrinogen, and platelet‐mediated clot retraction; in addition, it facilitates thrombolysis. Objectives: To understand the mechanism(s) by which 7E9 acts, we have now studied the effect of 7E9 IgG, 7E9 F(ab′)₂, and 7E9 Fab on fibrin clot structure using electron microscopy and measurements of clot physical properties. Results: By transmission electron microscopy, 7E9 IgG was found to bind primarily to the ends of the fibrinogen molecule. 7E9 IgG and 7E9 F(ab′)₂, both of which are bivalent, were capable of binding to two fibrinogen molecules simultaneously. Scanning electron microscopy of clots formed in the presence of equimolar concentrations of fibrinogen and 7E9 IgG demonstrated the presence of very short and thin fibers (63% reduction in fiber diameter) arranged in unusual bundles, surrounding large pores. Clots formed in the presence of 7E9 demonstrated a marked increase in permeation (approximately 25‐fold increase in perfusion rate at constant pressure), an approximately 50% reduction in dynamic storage modulus (G'; a reflection of decreased clot stiffness), and an approximately 38% increase in loss tangent (tan δ; a reflection of the clot's ability to undergo irreversible deformation). These clots also showed decreased absorbance at 350 nm, reflecting the clot structure produced by 7E9 IgG. The effects of 7E9 IgG were not observed with control hamster IgG, 7E9 F(ab′)₂, or 7E9 Fab fragments, indicating requirements for both the binding properties and mass of 7E9 IgG. Conclusions: These data indicate that 7E9 antibody affects fibrin clot structure in a way that is consistent with the enhanced fibrinolysis we reported previously. Together with our previous observations, we conclude that 7E9 is directed at a strategically important region of fibrinogen with regard to platelet function, FXIIIa‐mediated cross‐linking, clot retraction, fibrin structure, and fibrinolysis. Thus targeting this region of fibrinogen may have antithrombotic therapeutic potential.     

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

Summary.  Background and objectives : 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 (γArg275Cys), 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. Methods : A variant γ-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen Aα- and Bβ-chains. The recombinant variant fibrinogen (γ275C) was purified using an immunoaffinity column, and we compared its structure and functions with those of normal recombinant fibrinogen (γ275R) and plasma variant fibrinogen. Results : Mass analyses showed the existence of disulfide-linked Cys in both patient and recombinant variant fibrinogens. Functional analyses indicated that both fibrin polymerization and γ–γ dimer formation were markedly impaired in the variant fibrinogen. The impairments were much more pronounced in γ275C than in plasma variant fibrinogen. In addition, scanning electron microscopic observation of fibrin clots made from γ275C revealed less dense fibrin fiber bundles and larger fiber diameter than in those made from γ275R, and also the existence of many aberrant fibrin fibers with tapered ends. Conclusion : These results indicate that γArg275 has an important residue affecting the structure and function of the γ-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.     

The role of γ-carboxylation in the anti-apoptotic function of gas6

Gas6 is a novel member of the vitamin K-dependent family of gamma-carboxylated proteins and is a ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions have been shown to mediate cell survival in vascular endothelium. Although the receptor-binding portion of gas6 lies in the C-terminus, the significance of the N-terminal gamma-carboxylated residues (Gla domain) is not clear. To address this question, this study examines the role of the Gla domain in phospholipid binding as well as in the promotion of cell survival, especially in endothelial cells. The results show that carboxylated gas6 binds to phosphatidylserine-containing phospholipid membranes in an analogous manner to other gamma-carboxylated proteins whereas decarboxylated gas6 does not. The gamma-carboxylation inhibitor warfarin abrogates gas6-mediated protection of NIH3T3 fibroblasts from serum starvation-induced apoptosis. Furthermore, the role of gamma-carboxylation in gas6's survival effect on endothelium is demonstrated directly in that only carboxylated, but not decarboxylated, gas6 protects endothelial cells from serum starvation-induced apoptosis. gamma-carboxylation is also required for both Axl phosphorylation and PI3 kinase activation. Taken together, these findings demonstrate that gamma-carboxylation is necessary not only for gas6 binding to phospholipid membranes, but also for gas6-mediated endothelial cell survival.     

A novel mutation of the ceruloplasmin gene in a patient with heteroallelic ceruloplasmin gene mutation (HypoCPGM)

We found a novel missense mutation in the ceruloplasmin (Cp) gene in a patient with the heteroallelic Cp gene mutation (HypoCPGM). The patient was a 72-year-old woman who came to our hospital with a 1-year history of postural tremor of the hands. The diagnosis was made based on serum Cp and copper readings which were about half the normal levels, as well as MRI tests of her brain which showed characteristics for hereditary ceruloplasmin deficiency (HCD), known to be caused by the homoallelic Cp gene mutation. Polymerase chain reaction (PCR)-direct sequencing analysis of the Cp gene of the patient revealed a novel point mutation, A to T, at nucleotide position 82 in Exon 1. This mutation changes the Ile28 codon (ATT) to a Phe codon (TTT) (missense mutation). PCR-restriction analysis with restriction enzyme Tsp EI for the mutation revealed that both the patient and her son were heterozygotes for the mutation.