Kinetic characterization of inhibition of human thrombin with DNA aptamers by turbidimetric assay.

A sensitive turbidimetric method for detecting fibrin association was used to study the kinetics of fibrinogen hydrolysis with thrombin. The data were complemented by high-performance liquid chromatography (HPLC) measurements of the peptide products, fibrinopeptides released during hydrolysis. Atomic force microscopy (AFM) data showed that the fibril diameter is the main geometric parameter influencing the turbidity. The turbidimetric assay was validated using thrombin with the standard activity. To study thrombin inhibitors, a kinetic model that allows estimating the inhibition constants and the type of inhibition was proposed. The kinetic model was used to study the inhibitory activity of the two DNA aptamers 15-TBA (thrombin-binding aptamer) and 31-TBA, which bind to thrombin exosites. For the first time, 31-TBA was shown to possess the competitive inhibition type, whereas the shortened aptamer 15-TBA has the noncompetitive inhibition type.

[Functional analysis for dysfibrinogenemias, Toyama and Adachi, which have a mutation of Aalpha16Arg-->His (CGT-->CAT) with aberrant fibrinopeptide A release].

We found and identified four heterozygous dysfibrinogenemias with AalphaR16H(CGT-->CAT) mutation in two families by coagulation tests and direct sequence analysis for PCR-amplified DNA fragments. Two dysfibrinogens were designated as fibrinogen Toyama and Adachi, according to the place of residence of proposituses, respectively. Patients' fibrinogen purified from plasma using immunoaffinity-chromatography was subjected to thrombin- or batroxobin-catalyzed fibrin polymerization, fibrinopeptide A (FPA) release, and clottability test. AalphaR16H-fibrinogen showed impaired thrombin or batroxobin-catalyzed fibrin polymerization in comparison with normal control fibrinogen. It is interesting that the period of protofibril formation of Toyama propositus was longest in those of four affected people. The clottability of AalphaR16H-fibrinogen was 66-70% with thrombin and higher than with batroxobin, 35-50%. In the same condition with fibrin polymerization, thrombin and batroxobin did not cleave the Aalpha16H-17G peptide-bonding, resulting in no release of variant FPA. From these results, we speculated that elongation of the two-stranded protofibril formation would be terminated by participation of the heterodimer fibrinogen molecules composed with a normal and an aberrant Aalpha-chain, and it would result in a decrease in fibrin polymerization. We speculated that the difference in the extent of impairment of fibrin polymerization among the patients might be caused by the different amount of heterodimers. Moreover, we also speculated that batroxobin-induced clottability was lower than thrombin-induced clottability, because batroxobin cannot induce the so-called "B-knob-b-hole" interaction, which enhances fibrin formation.

A novel variant fibrinogen, AαE11del, demonstrating the importance of AαE11 residue in thrombin binding.

INTRODUCTION: We identified a novel heterozygous AαE11del variant in a patient with congenital dysfibrinogenemia. This mutation is located in fibrinopeptide A (FpA). We analyzed the effect of AαE11del on the catalyzation of thrombin and batroxobin and simulated the stability of the complex structure between the FpA fragment (AαG6-V20) peptide and thrombin. MATERIALS AND METHODS: We performed fibrin polymerization and examined the kinetics of FpA release catalyzed by thrombin and batroxobin using purified plasma fibrinogen. To clarify the association between the AαE11 residue and thrombin, we calculated binding free energy using molecular dynamics simulation trajectories. RESULTS: Increasing the thrombin concentration improved release of FpA from the patient's fibrinogen to approximately 90%, compared to the previous 50% of that of normal fibrinogen. Fibrin polymerization of variant fibrinogen also improved. In addition, greater impairment of variant FpA release from the patient's fibrinogen was observed with thrombin than with batroxobin. Moreover, the calculated binding free energy showed that the FpA fragment-thrombin complex became unstable due to the missing AαE11 residue. CONCLUSIONS: Our findings indicate that the AαE11 residue is involved in FpA release in thrombin catalyzation more than in batroxobin catalyzation, and that the AαE11 residue stabilizes FpA fragment-thrombin complex formation.

A Thrombin-Activatable Factor X Variant Corrects Hemostasis in a Mouse Model for Hemophilia A.

Engineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors.

Epidemiological and genetic associations of activated factor XII concentration with factor VII activity, fibrinopeptide A concentration, and risk of coronary heart disease in men.

BACKGROUND: The relations of plasma activated factor XII (FXIIa) concentration and a common polymorphism (C46T) of the factor XII gene with hemostatic status and risk of coronary heart disease (CHD) were examined by prospective surveillance. METHODS AND RESULTS: Genotyping for the C46T variant was performed in 2624 men 50 to 61 years of age who were free of CHD at baseline. The genotype distribution was as follows: CC, 56.7%; CT; 36.9%; and TT, 6.6%. Plasma FXIIa was measured by ELISA on 1745 samples collected 1 year after baseline; median levels were (ng/mL) CC, 2.0; CT, 1.4; and TT, 0.8 (P:<0.0001). Respective values for plasma fibrinopeptide A (FPA, nmol/L) were 1.52, 1.35, and 1.15 (P:<0.0001); for factor VII coagulant activity (FVIIc, % standard), 114.5, 116.2, and 109.3 (P:=0.02). Group differences in FVIIc were unchanged by adjustment for body mass index and serum triglycerides. Whereas CHD incidence did not differ significantly by genotype, rates (per 1000 person-years) by thirds of FXIIa distribution were for <1.5 ng/mL, 7. 2; for 1.5 to 2.0 ng/mL, 7.2; and for >2.0 ng/mL, 13.6. Respective hazard ratios with the low third as reference group were 1.01 and 1. 96 (P:=0.007), which were essentially unchanged after allowance for genotype, blood lipids, blood pressure, body mass index, FVIIc, and FPA. CONCLUSIONS: The C46T polymorphism is a determinant of FXIIa, FPA, and possibly FVIIc, suggesting that FXII influences the activity state of the coagulation pathway and FPA cleavage from fibrinogen in vivo. Plasma FXIIa is increased in middle-aged men at high risk of CHD.

Thrombin-bound structures of designed analogs of human fibrinopeptide A determined by quantitative transferred NOE spectroscopy: a new structural basis for thrombin specificity.

The mutation of Gly12 to Val12 in the A alpha chains of human fibrinogen Rouen is associated with a delayed proteolytic release of fibrinopeptide A (FpA or A alpha 1 to 16 of fibrinogen) by thrombin, leading to a bleeding disorder. Analogs of FpA and FpA Rouen have been designed that include a Pro15 to replace Val15 in natural FpA and to mimic the frequent occurrences of a proline residue at equivalent positions of other protein substrates of thrombin. The Pro15 analogs of FpA and FpA Rouen bind specifically to the active site of thrombin as shown by thrombin-induced differential line broadening and transferred nuclear Overhauser effects (transferred NOEs). Pro15 is well tolerated by the thrombin-bound structures of both FpA and FpA Rouen in solution, resulting in enhanced conformational stabilities of the thrombin-FpA complexes. The Val12 mutation in FpA Rouen causes backbone conformational changes in residues Val12 and Gly13 accompanied by an expansion of the hydrophobic cluster of FpA to accommodate the bulky side-chain of Val12. The single turn of helical structure between residues Asp7 and Glu11 is stabilized by hydrogen bonds from the side-chain carboxylate of Asp7 to the exposed backbone NH groups of Ala10 and/or Leu9 (N-capping), and by hydrogen bonds between the exposed backbone carbonyl groups of residues Phe8 and Leu9, and the backbone NH groups of Gly12/Val12 and Gly13 (C-capping). The bound structure of FpA Rouen may be further stabilized by a non-polar (i,i + 4) interaction between the aromatic side-chain of Phe8 and the aliphatic side-chain of Val12. Despite these optimized intrapeptide interactions, the thrombin-peptide interactions are highly dynamic as indicated by the fast rate of dissociation (koff > 100 s-1) of the peptide ligands from the thrombin complexes. Sequence comparison between mammalian fibrinopeptides A and B suggests that the specificity of thrombin is dictated by a four-residue consensus motif, Phe(P4)-Xxx(P3)-Pro(P2)-Arg(P1) or FXPR, when Xxx at P3 can be a charged or a neutral polar residue capable of specific interactions with residues near the active site of thrombin.

Diversity of primary structures of the carboxy-terminal regions of mammalian fibrinogen A alpha-chains. Characterization of the partial nucleotide and deduced amino acid sequences in five mammalian species; rhesus monkey, pig, dog, mouse and Syrian hamster.

The partial amino acid sequences of fibrinogen A alpha-chains from five mammalian species have been inferred by means of the polymerase chain reaction (PCR). From the genomic DNA of the rhesus monkey, pig, dog, mouse and Syrian hamster, the DNA fragments coding for alpha-C domains in the A alpha-chains were amplified and sequenced. In all species examined, four cysteine residues were always conserved at the homologous positions. The carboxy- and amino-terminal portions of the alpha-C domains showed a considerable homology among the species. However, the sizes of the middle portions, which corresponded to the internal repeat structures, showed an apparent variability because of several insertions and/or deletions. In the rhesus monkey, pig, mouse and Syrian hamster, 13 amino acid tandem repeats fundamentally similar to those in humans and the rat were identified. In the dog, however, tandem repeats were found to consist of 18 amino acids, suggesting an independent multiplication of the canine repeats. The sites of the alpha-chain cross-linking acceptor and alpha 2-plasmin inhibitor cross-linking donor were not always evolutionally conserved. The arginyl-glycyl-aspartic acid (RGD) sequence was not found in the amplified region of either the rhesus monkey or the pig. In the canine alpha-C domain, two RGD sequences were identified at the homologous positions to both rat and human RGDS. In the Syrian hamster, a single RGD sequence was found at the same position to that of the rat. Triplication of the RGD sequences was seen in the murine fibrinogen alpha-C domain around the homologous site to the rat RGDS sequence. These findings are of some interest from the point of view of structure-function and evolutionary relationships in the mammalian fibrinogen A alpha-chains.

Fibrinopeptides A and B of baboons (Papio anubis, Papio hamadryas, and Theropithecus gelada): their amino acid sequences and evolutionary rates and a molecular phylogeny for the baboons.

Amino acid sequences of fibrinopeptides A and B from savanna baboons, Papio anubis and Papio hamadryas, and highland baboon, Theropithecus gelada, were established. The sequences of the fibrinopeptides A from the three baboons were identical: (sequence: see text) The fibrinopeptides B were composed of 9 residues and demonstrated the sequence: (sequence see text) where X3 = Arg in P. anubis, His in P. hamadryas, and Gly in Th. gelada. Position-3 of the B peptides was the only replacement site observed among the 25 amino acid residues in both fibrinopeptides from the baboons. Based on these sequences, a molecular phylogeny for the three species of baboons was deduced. The evolutionary rates of the peptides B of the baboons and macaques were also estimated. It was observed that the fibrinopeptides changed at an uneven rate during the evolution of old-world monkeys, i.e., baboons and macaques.

Fibrinopeptides A and B of Japanese monkey (Macaca fuscata) and patas monkey (Erythrocebus patas): their amino acid sequences, restricted mutations, and a molecular phylogeny for macaques, guenons, and baboons.

Amino acid sequences of fibrinopeptides A and B from the macaque, Macaca fuscata (Japanese monkey) and the guenon, Erythrocebus patas (patas monkey) were established. Fibrinopeptides A of the monkeys had a sequence identical with those of baboons: Ala-Asp-Thr-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg. Fibrinopeptides B were 9-residue, "short," peptides with the sequences Asn-Glu-Glu-Ser-Leu-Phe-Ser-Gly-Arg for M. fuscata and Asn-Glu-Glu-Val-Leu-Phe-Gly-Gly-Arg for E. patas. The sequence of the B peptide of M. fuscata differed from that of a close-related species, M. mulatta (rhesus monkey), at a single site, Leu (M.f.)----Pro (M.m.). A single replacement between the B peptides of E. patas and Cercocebus aethiops (green monkey), Val (E.p.)----Gly (C.a.), was detected. A phylogenic relationship of macaques, guenons, and baboons, named Cercopithecinae (Old World monkey), was deduced from the sequence data. A selective rather than random amino acid replacement was observed in the B peptides of these Old World monkeys, suggesting a restricted mutation of their fibrinopeptides during primate evolution.

Fibrinogen New Orleans: hereditary dysfibrinogenemia with an A alpha chain abnormality.

An abnormal fibrinogen (Fibrinogen New Orleans, or FNO) has been found in a 30 year-old woman, her mother and daughter, but not her father. The propositus suffered mild bleeding, but not thrombo-embolism or abnormal wound healing. Plasma and purified fibrinogen from the propositus caused a prolongation in the clotting time of normal plasma. FNO had increased anodal migration when studied by immunoelectrophoresis. We have been able to follow the release of fibrinopeptides A and B using sensitive SDS-PAGE. In FNO, release of the A peptide was markedly delayed, whereas the cleavage of B peptide was much less delayed, compared to that of A peptide.

Production of a recombinant antithrombotic and fibrinolytic protein, PLATSAK, in Escherichia coli.

The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin. Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets. We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids. The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3' fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography. The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin. The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.

Hypodysfibrinogenaemia due to production of mutant fibrinogen alpha-chains lacking fibrinopeptide A and polymerisation knob 'A'.

Inherited disorders of fibrinogen are rare and affect either the quantity (hypofibrinogenaemia and afibrinogenaemia) or the quality of the circulating fibrinogen (dysfibrinogenaemia) or both (hypodysfibrinogenaemia). Extensive allelic heterogeneity has been found for all these disorders: in congenital afibrinogenaemia for example more than 40 mutations, the majority in FGA , have been identified in homozygosity or in compound heterozygosity. Numerous mutations have also been identified in patients with hypofibrinogenaemia, many of these patients are in fact heterozygous carriers of afibrinogenaemia mutations. Despite the number of genetic analyses performed, the study of additional patients still allows the identification of novel mutations. Here we describe the characterization of a novel FGA intron 2 donor splice-site mutation (Fibrinogen Montpellier II) identified in three siblings with hypodysfibrinogenaemia. Functional analysis of RNA produced by the mutant minigene in COS-7 cells revealed that the mutation led to the in-frame skipping of exon 2. Western blot analysis of COS-7 cells expressing an exon 2 deleted FGA cDNA revealed that an alpha-chain lacking exon 2, which codes in particular for fibrinopeptide A and polymerisation knob 'A', has the potential to be assembled into a hexamer and secreted. Analysis of precipitated fibrinogen from patient plasma showed that the defect leads to the presence in the circulation of alpha-chains lacking knob 'A' which is essential for the early stages of fibrin polymerisation. Fibrin made from purified patient fibrinogen clotted with thrombin displayed thinner fibers with frequent ends and large pores.

Incorporation of fibrin molecules containing fibrinopeptide A alters clot ultrastructure and decreases permeability.

Previous studies have shown that a heterozygous mutation in the fibrinogen Aalpha chain gene, which results in an Aalpha R16C substitution, causes fibrinolytic resistance in the fibrin clot. This mutation prevents thrombin cleavage of fibrinopeptide A from mutant Aalpha R16C chains, but not from wild-type Aalpha chains. However, the mechanism underlying the fibrinolytic resistance is unclear. Therefore, this study investigated the biophysical properties of the mutant fibrin that contribute to fibrinolytic resistance. Fibrin clots made from the mutant fibrinogen incorporated molecules containing fibrinopeptide A into the polymerised clot, which resulted in a 'spiky' clot ultrastructure with barbed fibrin strands. The clots were less stiff than normal fibrin and were cross-linked slower by activated FXIII, but had an increased average fiber diameter, were more dense, had smaller pores and were less permeable. Protein sequencing showed that unclottable fibrinogen remaining in the supernatant consisted entirely of homodimeric Aalpha R16C fibrinogen, whereas both cleaved wild-type alpha chains and uncleaved Aalpha R16C chains were in the fibrin clot. Therefore, fibrinolytic resistance of the mutant clots is probably a result of altered clot ultrastructure caused by the incorporation of fibrin molecules containing fibrinopeptide A, resulting in larger diameter fibers and decreased permeability to fibrinolytic enzymes.

Identification and confirmation of increased fibrinopeptide a serum protein levels in gastric cancer sera by magnet bead assisted MALDI-TOF mass spectrometry.

Gastric cancer is the second most common malignancy and prognosis remains dismal. The reasons for the poor prognosis are the lack of sensitive serum markers for early detection and screening of high-risk individuals as well as the limited treatment options in advanced cancer stages. Using MALDI-TOF mass spectrometry after prefractionation of sera with magnet hydrophobic C8 coated beads sera from 14 patients with gastric cancer and 14 healthy controls mass spectra were generated. A peptide fragment was found to be highly elevated in cancer sera and was identified as fibrinopeptide A. To confirm proteome analysis of gastric cancer sera, we then screened a larger series of patients with gastric cancer (n = 99), high-risk individuals (n = 13) and normal controls (n = 111) for fibrinopeptide A serum levels. Interestingly, the mean logarithmic concentrations of serum fibrinopeptide A levels were significantly higher in cancer patients (mean 3.636 +/- 0.3738; p < 0.0001) and high-risk individuals (mean 3.569 +/- 0.4722; p < 0.05) compared to normal controls (mean 3.303 +/- 0.4012). In contrast, we observed no association of fibrinopeptide A levels with tumor stage, tumor location, presence of regional or distant metastasis, and Lauren type of gastric cancer. In conclusion, MALDI-TOF mass spectrometry of prefractionated gastric cancer sera allows the identification of potential biomarkers that may lead to the development of serum based tests for screening of high-risk individuals.

Common structural genes for platelet and plasma fibrinogen.

Fibrinogen from plasma was compared with fibrinogen from platelets using two-dimensional electrophoresis. The source of platelet fibrinogen was isolated alpha-granules, thrombin- and collagen-released platelet material. The B beta- and gamma-chains from the different sources showed similar two-dimensional patterns, while gamma'-chains were not observed in platelet fibrinogen preparations. Furthermore, the A alpha-chain could hardly be identified in platelet preparations. When individual fibrinogen was studied in persons heterozygous for genetic B beta- and gamma-chain variants, the two-dimensional variant pattern could be demonstrated in plasma fibrinogen as well as in platelet fibrinogen. This observation strongly indicates that the structural genes for plasma and platelet fibrinogen B beta- and gamma-chains are identical.

Estimating the reliability of evolutionary trees.

Six protein sequences from the same 11 mammalian taxa were used to estimate the accuracy and reliability of phylogenetic trees using real, rather than simulated, data. A tree comparison metric was used to measure the increase in similarity of minimal trees as larger, randomly selected subsets of nucleotide positions were taken. The ratio of the observed to the expected number of incompatibilities for each nucleotide position (character) is a good predictor of the number of changes required at that position on the minimal (most-parsimonious) tree. This allows a higher weighting of nucleotide positions that have changed more slowly and should result in the minimal length tree converging to the correct tree as more sequences are obtained. An estimate was made of the smallest subset of trees that need to be considered to include the actual historical tree for a given set of data. It was concluded that it is possible to give a reasonable estimate of the reliability of the final tree, at least when several sequences are combined. With the present data, resolving the rodent-primate-lagomorph (rabbit) trichotomy is the least certain aspect of the final tree, followed then by establishing the position of dog. In our opinion, it is unreasonable to publish an evolutionary tree derived from sequence data without giving an idea of the reliability of the tree.

Chromosomal localization of human and rat A alpha, B beta, and gamma fibrinogen genes by in situ hybridization.

In situ hybridization of radiolabeled fibrinogen cDNAs to human and rat metaphase chromosomes has shown that the genes encoding the A alpha, B beta, and gamma fibrinogen subunits are syntenic in both species. Our data localize the human fibrinogen gene cluster to band q31 on chromosome 4, thereby confirming and extending previous map assignments of these genes in man. We have also assigned these genes to the q31----q34 region of rat chromosome 2. This is the first map assignment of these genes in the rat and also the first report to clearly establish linkage of the B beta subunit gene to the A alpha and gamma genes in this species.

Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.