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.

Association Between Hemostatic Profile and Migraine: A Mendelian Randomization Analysis.

OBJECTIVE: To assess support for a causal relationship between hemostatic measures and migraine susceptibility using genetic instrumental analysis. METHODS: Two-sample Mendelian randomization instrumental analyses leveraging available genome-wide association study (GWAS) summary statistics were applied to hemostatic measures as potentially causal for migraine and its subtypes, migraine with aura (MA) and migraine without aura (MO). Twelve blood-based measures of hemostasis were examined, including plasma level or activity of 8 hemostatic factors and 2 fibrinopeptides together with 2 hemostasis clinical tests. RESULTS: There were significant instrumental effects between increased coagulation factor VIII activity (FVIII; odds ratio [95% confidence interval] 1.05 [1.03, 1.08]/SD, p = 6.08 × 10-05), von Willebrand factor level (vWF; 1.05 [1.03, 1.08]/SD, p = 2.25 × 10-06), and phosphorylated fibrinopeptide A level (1.13 [1.07, 1.19]/SD, p = 5.44 × 10-06) with migraine susceptibility. When extended to migraine subtypes, FVIII, vWF, and phosphorylated fibrinopeptide A showed slightly stronger effects with MA than overall migraine. Fibrinogen level was inversely linked with MA (0.76 [0.64, 0.91]/SD, p = 2.32 × 10-03) but not overall migraine. None of the hemostatic factors was linked with MO. In sensitivity analysis, effects for fibrinogen and phosphorylated fibrinopeptide A were robust, whereas independent effects of FVIII and vWF could not be distinguished, and FVIII associations were potentially affected by pleiotropy at the ABO locus. Causal effects from migraine to the hemostatic measures were not supported in reverse Mendelian randomization. However, MA was not included due to lack of instruments. CONCLUSIONS: The findings support potential causality of increased FVIII, vWF, and phosphorylated fibrinopeptide A and decreased fibrinogen in migraine susceptibility, especially for MA, potentially revealing etiologic relationships between hemostasis and migraine.

Serological Assessment of the Quality of Wound Healing Processes in Crohn's Disease.

BACKGROUND AND AIMS: Crohn's disease (CD) is a chronic inflammatory condition characterized by continuous mucosal damage and ongoing wound healing of the intestines. The fibrinolytic system is involved in early parts of the wound healing process. Fibrin is a key mediator of primary blood clot formation and is formed by cross-linking of fibrinogen. To gain insights into the dynamics of wound healing in CD patients we investigated the conversion of fibrinogen into fibrin by the pro-peptide FPA, the amount of factor XIII cross-linked fibrin and total fibrin clot. METHODS: Serum samples of 35 CD patients, 15 non-inflammatory bowel disease (non-IBD) patients and 39 age-matched healthy controls were analyzed for three novel neo-epitope markers: D-fragment and D-dimer, reflecting the degradation of total fibrin clot and factor XIII cross-linked fibrin, as well as FPA, reflecting synthesis of fibrin. RESULTS: Crohn's disease patients had a significantly lower D-dimer level (p=0.0001) compared to healthy controls. Crohn's disease and non-IBD patients had a significantly higher level of FPA (p<0.0001) and D-fragment/D-dimer ratio (p<0.0001 and p=0.02). FPA, D-dimer and D-fragment/D-dimer ratio could distinguish CD patients from healthy controls with area under the curve of 0.92 (95% CI 0.83-0.97), 0.78 (95% CI 0.67-0.87) and 0.85 (95% CI 0.75-0.93), respectively. CONCLUSION: Wound healing parameters were clearly changed in CD patients. FPA levels were higher in CD patients as compared to healthy controls, indicating more ongoing wound healing. D-dimer levels were lower in CD patients than in healthy controls, indicating impaired wound healing due to poor quality of factor XIII cross-linked fibrin and clot resolution.

Untargeted Metabolomics Differentiates l-Carnitine Treated Septic Shock 1-Year Survivors and Nonsurvivors.

l-Carnitine is a candidate therapeutic for the treatment of septic shock, a condition that carries a ≥40% mortality. Responsiveness to l-carnitine may hinge on unique metabolic profiles that are not evident from the clinical phenotype. To define these profiles, we performed an untargeted metabolomic analysis of serum from 21 male sepsis patients enrolled in a placebo-controlled l-carnitine clinical trial. Although treatment with l-carnitine is known to induce changes in the sepsis metabolome, we found a distinct set of metabolites that differentiated 1-year survivors from nonsurvivors. Following feature alignment, we employed a new and innovative data reduction strategy followed by false discovery correction, and identified 63 metabolites that differentiated carnitine-treated 1-year survivors versus nonsurvivors. Following identification by MS/MS and database search, several metabolite markers of vascular inflammation were determined to be prominently elevated in the carnitine-treated nonsurvivor cohort, including fibrinopeptide A, allysine, and histamine. While preliminary, these results corroborate that metabolic profiles may be useful to differentiate l-carnitine treatment responsiveness. Furthermore, these data show that the metabolic signature of l-carnitine-treated nonsurvivors is associated with a severity of illness (e.g., vascular inflammation) that is not routinely clinically detected.

Fibrinopeptide A induces C-reactive protein expression through the ROS-ERK1/2/p38-NF-κB signal pathway in the human umbilical vascular endothelial cells.

Atherosclerosis is a chronic inflammatory disease of the arterial wall. Inflammation causes endothelial injury and dysfunction, which is an initial step of atherosclerosis. Fibrinopeptide A (FPA) is a biomarker of the activation of the coagulation system, and a high concentration of FPA in the blood occurs in patients with ischemic cardiocerebrovascular diseases. The present research observed that FPA stimulated the generation of C-reactive protein (CRP), IL-1ß, and IL-6 in human umbilical vascular endothelial cells (HUVECs); and anti-IL-1 ß and anti-IL-6 neutralizing antibodies did not alter FPA-induced CRP expression in HUVECs. The subchronic administration of FPA into rats increased the plasma FPA and CRP levels. Further studies showed that FPA stimulated superoxide anion generation, activated ERK1/2 and p38, promoted nuclear factor κB (NF-κB) nuclear translocation, and raised the NF-κB level in the nuclei of HUVECs. Antioxidant N-acetylcysteine (NAC), complex II inhibitor thenoyltrifluoroacetone (TTFA), and NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibited FPA-stimulated generation of superoxide anion, and NAC reduced FPA-induced expressions of the phosphorylated ERK1/2 and p38. NAC, TTFA, DPI, inhibitors of ERK1/2, p38, and NF-κB all downregulated FPA-induced CRP expression. These results indicate that FPA induces CRP expression in HUVECs via the ROS-ERK1/2/p38-NF-κB signal pathway. Moreover, this is the first report that FPA produces a proinflammatory effect on the vascular endothelial cells.

Impact of fibrinogen carbamylation on fibrin clot formation and stability.

Carbamylation is a non-enzymatic post-translational modification induced upon exposure of free amino groups to urea-derived cyanate leading to irreversible changes of protein charge, structure and function. Levels of carbamylated proteins increase significantly in chronic kidney disease and carbamylated albumin is considered as an important biomarker indicating mortality risk. High plasma concentrations and long half-life make fibrinogen a prime target for carbamylation. As aggregation and cross-linking of fibrin monomers rely on lysine residues, it is likely that carbamylation impacts fibrinogen processing. In this study we investigated carbamylation levels of fibrinogen from kidney disease patients as well as the impact of carbamylation on fibrinogen cleavage by thrombin, fibrin polymerisation and cross-linking in vitro. In conjunction, all these factors determine clot structure and stability and thus control biochemical and mechanical properties. LC-MS/MS analyses revealed significantly higher homocitrulline levels in patient fibrinogen than in fibrinogen isolated from control plasma. In our in vitro studies we found that although carbamylation does not affect thrombin cleavage per se, it alters fibrin polymerisation kinetics and impairs cross-linking and clot degradation. In addition, carbamylated fibrin clots had reduced fiber size and porosity associated with decreased mechanical stability. Using mass spectroscopy, we discovered that N-terminally carbamylated fibrinopeptide A was generated in this process and acted as a strong neutrophil chemoattractant potentially mediating recruitment of inflammatory cells to sites of fibrin(ogen) turnover. Taken together, carbamylation of fibrinogen seems to play a role in aberrant fibrin clot formation and might be involved in haemostatic disorders associated with chronic inflammatory diseases.

[The clotting tests and molecular markers in evaluating of coagulation alterations against the background of anti-thrombotic prevention by Dabigatran after large orthopedic operations].

The large orthopedic operations are associated with high risk of development of thrombosis of deep veins of lower extremities. Nowadays, new oral anticoagulants are widely applied for anti-thrombotic prevention. The coagulation alterations against the background of effect of Dabigatran, a direct inhibitor of thrombin were examined in 30 patients underwent endoprosthesis replacement of knee joint. The routine clotting indices, fibrinopeptid A, soluble fibrin-monomeric complexes, D-dimer. The samples of blood were selected before operation, after 30 minutes, and at 1st, 3d, 7th and 14th day after endoprosthesis replacement of knee joint. It is demonstrated that routine clotting tests and also detection of D-dimer and soluble fibrin-monomeric complexes provide no adequate evaluation of coagulation activity in patients underwent large orthopedic operation. The concentration of specific marker of fibrin formation of fibrinopeptid A continues to be increased no less than two weeks after endoprosthesis replacement of knee joint that testifies keeping hyper-coagulation and risk of thrombosis. The intake of Dabigatran etexilate in fixed dosage does not exclude development of thrombosis of deep veins of lower extremities that substantiates point of view concerning usefulness of individualization of anti-thrombotic prevention in case of application of new oral anti-coagulants.

A novel mutation in the fibrinogen Aα chain (Gly13Arg, fibrinogen Nanning) causes congenital dysfibrinogenemia associated with defective peptide A release.

Dysfibrinogenemia is characterized by blood coagulation dysfunction induced by an abnormal molecular structure of fibrinogen. Here, we describe a new case. A 32-year-old female was suspected of having dysfibrinogenemia during routine laboratory screening, based on her decreased functional fibrinogen level, normal fibrinogen antigen level, and prolonged thrombin time. We extracted DNA and performed polymerase chain reaction and DNA sequencing to identify genetic mutation. Fibrin polymerization, the kinetics of the fibrinopeptide release, scanning electron microscopy, mass spectrometric analysis, fibrin cross-linking, sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot were conducted. DNA sequencing identified a heterozygous point mutation, Gly13Arg in Aα chain. Fibrin polymerization was markedly impaired (prolonged lag phase and decreased final turbidity). The rate and extent of fibrinopeptide A release from the patient were abnormal and reduced. The mass spectrometry analysis revealed the presence of mutant fibrinogen chains in the patient's circulation. Electron micrographs revealed abnormal fibrin clots. Fibrin cross-linking was normal. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot showed no difference. We report a new case with a mutation in the fibrinopeptide A region, AαGly13Arg. These results indicated that the functional abnormalities were related to delayed and defective fibrinopeptide A cleavage and likely impaired thrombin binding.

Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme.

Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous expression of a serine protease from Crotalus durissus collilineatus, named collinein-1, in Pichia pastoris, as well as the enzymatic comparative characterization of the toxin in native and recombinant forms. The complementary DNA (cDNA) encoding collinein-1 was amplified from cDNA library of C. d. collilineatus venom gland and cloned into the pPICZαA vector. The recombinant plasmid was used to transform cells of KM71H P. pastoris. Heterologous expression was induced by methanol and yielded 56 mg of recombinant collinein-1 (rCollinein-1) per liter of culture. The native collinein-1 was purified from C. d. collilineatus venom, and its identity was confirmed by amino acid sequencing. The native and recombinant enzymes showed similar effects upon bovine fibrinogen by releasing preferentially fibrinopeptide A. Although both enzymes have induced plasma coagulation, native Colinein-1 has shown higher coagulant activity. The serine proteases were able to hydrolyze the chromogenic substrates S-2222, S-2238, and S2302. Both enzymes showed high stability on different pH and temperature, and their esterase activities were inhibited in the presence of Zn2+ and Cu2+. The serine proteases showed similar k cat/K m values in enzyme kinetics assays, suggesting no significant differences in efficiency of these proteins to hydrolyze the substrate. These results demonstrated that rCollinein-1 was expressed with functional integrity on the evaluated parameters. The success in producing a functionally active recombinant SVSP may generate perspectives to their future therapeutic applications.

Relevance of fibrinolytic protein (D-dimer) and fibrinopeptide A as markers of sickle cell anaemia vaso-occlusive crisis.

AIMS AND OBJECTIVES: To determine the plasma concentration of fibrinolytic protein (D-dimer) and Fibrinopeptide A(FPA) in sickle cell anaemia (SCA) patients in steady state and vaso-occlusive crisis(VOC) for the purpose of determining their clinical value in assessing/or predicting the onset of VOC Subjects and Methods: A total of 25 (14 Males: 11Females) HbSS subjects in VOC , 24 (13M:11F) HbSS subjects in steady state between the ages of 10-40 years old and 30 (17M:13F) healthy HbAA volunteers, of the same age and sex with the subjects were recruited for the study. Haematological parameters{Haemoglobin (Hb), Haematocrit(HCT), White blood cell count(WBC) and Platelets(Plt)}, prothrombin time(PT), activated partial thromboplastin time(APTT), plasma concentrations of D-dimer and FPA were determined. RESULTS: Haemoglobin concentration of 6.22±1.75 g/dl and HCT of 18.45±6.43% for SCA subjects in VOC; Hb of 7.42±1.36 g/dl and HCT of 22.83 ±4.68% in steady state were significantly decreased(p <0.01) compared with Hb(13.0±1.04 g/dl and HCT( 41.09±3.50%) for HbAA controls. However, plasma FPA of 680.99 ± 411.37 ng/ml, WBC of 19.44±14.88 x109/L, Plt of 292.72±148.57 x109/L, APTT of 52.24±5.34sec. for SCA subjects in VOC and Plasma FPA of 449.67 ± 310.01 ng/ml, WBC of 11.84±7.67 x109/L, Plt of 292.72±148.57 x109/L, APTT of 47.76±4.80secs in steady state were significantly increased when compared with FPA(163.52 ± 86.26ng/ml), WBC(5.15±1.24 x109/L), Plt(173.44±59.90 x109/ L), APTT( 37.75±1.41secs) for HbAA controls. CONCLUSION: Fibrinolysis is not significantly increased in SCA either in the steady state or during VOC. Fibrinopeptide A assay appears to be of value in the assessment of VOC in sickle cell anaemia.

Batroxobin binds fibrin with higher affinity and promotes clot expansion to a greater extent than thrombin.

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the Aα- and Bß-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γA/γA isoform of fibrin(ogen) and the γA/γ' variant with an extended γ-chain. Thrombin binds to the γ'-chain and forms a higher affinity interaction with γA/γ'-fibrin(ogen) than γA/γA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 µM) even though it does not interact with the γ'-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γA/γA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2-5 µM, the α(17-51) and Bß(1-42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.

Fibrinopeptide A release is necessary for effective B:b interactions in polymerisation of variant fibrinogens with impaired A:a interactions.

Fibrin polymerisation is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b'. We demonstrated markedly delayed thrombin-catalysed fibrin polymerisation, through B:b interactions alone, of recombinant γD364H -fibrinogen with impaired hole 'a'. To determine whether recombinant variant fibrinogens with no release of fibrinopeptide A (FpA) polymerise similarly to γD364H -fibrinogen, we examined two variant fibrinogens with substitutions altering knob 'A', Aα17A- and Aα17C-fibrinogen. We examined thrombin- or batroxobin-catalysed fibrinopeptide release by HPLC, fibrin clot formation by turbidity and fibrin clot structure by scanning electron microscopy (SEM) and compared the results of the variants with those for γ D364H-fibrinogen. Thrombin-catalysed FpA release of Aα17A-fibrinogen was substantially delayed and none observed for Aα17C-fibrinogen; fibrinopeptide B (FpB) release was delayed for all variants. All variant fibrinogens showed substantially impaired thrombin-catalysed polymerisation; for Aα17A-fibrinogen it was delayed less, and for Aα17C more than for γD364H -fibrinogen. No variants polymerised with batroxobin, which exposed only knob 'A'. The inhibition of variant fibrinogens' polymerisation was dose-dependent on the concentration of either GPRP or GHRP, and both peptides that block holes 'b'. SEM showed that the variant clots from Aα17A- and γD364H-fibrinogen had uniform, ordered fibres, thicker than normal, whereas Aα17C -fibrinogen formed less organised clots with shorter, thinner, and tapered ends. These results demonstrate that FpA release per se is necessary for effective B:b interactions during polymerisation of variant fibrinogens with impaired A:a interactions.

Association between the plasma proteome and plasma α-tocopherol concentrations in humans.

Vitamin E is a lipophilic antioxidant that has been inversely associated with certain chronic diseases; however, the biological processes regulated by this vitamin have not been fully elucidated. The objective of the present study was to examine the association between the most biologically active and abundant form of vitamin E in the circulation, α-tocopherol, and the plasma proteome. Subjects were from the Toronto Nutrigenomics and Health Study and included men and women (n=1,022) who completed a general health and lifestyle questionnaire and 196-item food frequency questionnaire, and provided a fasting blood sample. Plasma α-tocopherol concentrations were measured by high-performance liquid chromatography and 54 plasma proteins were assayed by a mass spectrometry-based multiple reaction monitoring method. Analysis of covariance was used to compare mean concentrations of plasma proteins across tertiles of α-tocopherol. Plasma concentrations of apolipoprotein C-III, fibrinogen alpha, beta, and gamma chains, fibronectin and fibrinopeptide A were significantly and positively associated with plasma α-tocopherol, while intermediate levels of α-tocopherol were significantly associated with higher levels of alpha-1B-glycoprotein (all P<.0009). These findings show that circulating levels of α-tocopherol are significantly associated with specific plasma proteins and suggest novel physiological effects of vitamin E.

[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.

Inferring proteolytic processes from mass spectrometry time series data using degradation graphs.

BACKGROUND: Proteases play an essential part in a variety of biological processes. Besides their importance under healthy conditions they are also known to have a crucial role in complex diseases like cancer. In recent years, it has been shown that not only the fragments produced by proteases but also their dynamics, especially ex vivo, can serve as biomarkers. But so far, only a few approaches were taken to explicitly model the dynamics of proteolysis in the context of mass spectrometry. RESULTS: We introduce a new concept to model proteolytic processes, the degradation graph. The degradation graph is an extension of the cleavage graph, a data structure to reconstruct and visualize the proteolytic process. In contrast to previous approaches we extended the model to incorporate endoproteolytic processes and present a method to construct a degradation graph from mass spectrometry time series data. Based on a degradation graph and the intensities extracted from the mass spectra it is possible to estimate reaction rates of the underlying processes. We further suggest a score to rate different degradation graphs in their ability to explain the observed data. This score is used in an iterative heuristic to improve the structure of the initially constructed degradation graph. CONCLUSION: We show that the proposed method is able to recover all degraded and generated peptides, the underlying reactions, and the reaction rates of proteolytic processes based on mass spectrometry time series data. We use simulated and real data to demonstrate that a given process can be reconstructed even in the presence of extensive noise, isobaric signals and false identifications. While the model is currently only validated on peptide data it is also applicable to proteins, as long as the necessary time series data can be produced.

TA-2, a thrombin-like enzyme from the Chinese white-lipped green pitviper (Trimeresurus albolabris): isolation, biochemical and biological characterization.

Through three chromatographic steps, a new thrombin-like enzyme (TLE), named TA-2, from the venom of the Chinese white-lipped green pitviper (Trimeresurus albolabris) has been isolated and purified to homogeneity. TA-2 was a single-chain glycoprotein with about 6% sugar, pI 3.9 and a molecular weight of 38.8 kD. Its N-terminal sequence (VVGGDECNIN) showed high sequence conformity with many other TLEs. In vitro, it coagulated bovine fibrinogen (108.6 NIH units/mg) and cleaved the Aα and Bß chains of bovine fibrinogen-releasing fibrinopeptide A and B, but did not degrade bovine fibrin; displayed high stability at different temperature, pH, and presence of several divalent cations and inhibitors; also exhibited strong activity towards casein (192.3 units/mg) and high esterase activity upon Nα-p-tosyl-L-arginine methyl ester (11 units/mg); and behaved as a promoter to platelet aggregation induced by ADP or collagen. In vivo, TA-2 caused dose-dependent prolongation of bleeding time in mice, but had no hemorrhagic and edema-inducing activities even at high concentrations.

Fibrinogen residue γAla341 is necessary for calcium binding and 'A-a' interactions.

The fibrinogen γ-module has several important sites relating to fibrinogen function, which include the high affinity calcium binding site, hole 'a' that binds with knob 'A', and the D:D interface. Residue γAla341, which is located in the vicinity of these sites, is altered in three variant fibrinogens: fibrinogen Seoul (γAla341Asp), Tolaga Bay (γAla341Val), and Lyon III (γAla341Thr). In order to investigate the impaired polymerisation of fibrinogens γAla341Asp and γAla341Val to understand the role of γAla341 in fibrin polymerisation and fibrinogen synthesis, we have expressed γAla341Asp and γAla341Val in Chinese hamster ovary (CHO) cells, purified these fibrinogens from the culture media and performed biochemical tests to elucidate their function. Expression in CHO cells was similar for these variants. For both variants the kinetics of thrombin-catalysed FpA release was not different from normal fibrinogen, while FpB release was slower than that of normal. Thrombin-catalysed polymerisation of both variants was dependent on the calcium concentration. At physiologic calcium (1 mM) the variants showed impaired polymerisation with a longer lag period and a slower Vmax than normal fibrinogen. Scanning electron micrographs showed the clots were less organised than normal, having thicker and more twisted fibers, and larger pores. Analysis by SDS-PAGE showed that factor XIIIa-catalysed γ and α chain cross-linking was delayed, and plasmin-catalysed lysis was not reduced by the presence of 5 mM calcium or 5 mM GPRP (Gly-Pro-Arg-Pro). Our data indicate that fibrinogen residue γAla341 is important for the proper conformation of the γ-module, maintaining calcium-binding site and 'A-a' interactions.

Fibrinogen Sumperk II: dysfibrinogenemia in an individual with two coding mutations.

Fibrinogen­a 340-kDa glycoprotein­plays a crucial role in blood coagulation, platelet aggregation, wound healing, and other physiological processes. A mutation in fibrinogen may lead to congenital dysfibrinogenemia,a rare disease characterized by the functional deficiency of fibrinogen. About 580 cases of abnormal fibrinogens have been reported worldwide; thereof 335 cases in the fibrinogen Aa chain[1]. To our knowledge, only five cases of abnormal fibrinogens with two mutations [2­6] and one case of two different mutations in the same family [7] have been described earlier. A 52-year-old female was examined for bleeding. Routine hemostasis screening resulted in a diagnosis of dysfibrinogenemia. Functional testing revealed prolonged fibrin polymerization, prolonged lysis of the clot, abnormal fibrin morphology,and fibrinopeptides release. Genetic analysis showed two heterozygous nonsense mutations­previously described mutation AaGly13Glu and a novel mutation Aa Ser314Cys. The mutation Aa Gly13-Glu was found in her brother and niece, but there was no evidence in either of the mutation Aa Ser314Cys. While mutation Aa Gly13Glu is responsible for abnormal fibrinopeptide release and prolonged thrombin time, the novel mutation Aa Ser314Cys seems to affect fibrin morphology and fibrinolysis.

Thrombogenicity of a new injectable biocompatible elastomer for aneurysm exclusion, compared to expanded polytetrafluoroethylene in a human ex vivo model.

OBJECTIVES: Customized aortic repair (CAR) is a new concept for endovascular aortic aneurysm repair in which a non-polymerised elastomer is injected to fill the aneurysm sac around a balloon catheter. Amongst other variables, the thrombogenicity of the elastomer should be tested, before further clinical experiments can take place. The aim of this human ex vivo study was to measure the thrombogenicity of the elastomer and to compare it to expanded polytetrafluoroethylene (ePTFE). DESIGN AND MATERIALS: In a validated ex vivo model, non-anticoagulated blood was drawn from the antecubital veins of 10 healthy donors with a 19-gauge needle. It was drawn through elastomer tubes and through ePTFE Gore-Tex vascular grafts, both 60 cm long and with an inner diameter of 3 mm. METHODS: Fibrinopeptide A (FPA) and P-selectin expression was measured in blood samples, collected at the end of the grafts. After the experiments, the deposition of platelets and fibrin onto the grafts was visualised by scanning electron microscopy. RESULTS: For these graft types, a progressive increase in FPA production was observed in time. No significant difference was observed between the elastomer and ePTFE grafts (p > 0.05). No increase in P-selectin expression, and thereby no platelet activation, was observed in the perfusate of either grafts (p > 0.05). By scanning electron microscopy, numerous platelet aggregates were observed on the ePTFE grafts, whereas just a few adhered platelets and no aggregates were observed in the elastomer grafts. CONCLUSIONS: The elastomer in its current formulation has a low thrombogenicity, comparable to ePTFE, making it an ideal substance for endovascular aneurysm sac filling. Further research should clarify the feasibility of CAR in vivo.

Changes in fibrinopeptide A peptides in the sera of rats chronically exposed to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitously distributed endocrine disruptors. To investigate peptide changes in the sera of rats chronically exposed to TCDD and to explore the association of these changes with liver morphology, TCDD was administrated to male rats at doses of 140, 350, and 875 ng/kg/week for 29 weeks. Serum was collected and proteomic analysis was performed using automated Bruker Daltonics ClinProt with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. One peptide at 1740.89 was found to be significantly decreased and further identified with nano LC-MS/MS system. The MS BLAST homology search engine reported the peptide to be a partial sequence of fibrinopeptide A. Liver fatty degeneration and necrosis were assessed by hematoxylin and eosin staining. Liver fatty degeneration and necrosis were both found to be significantly increased after TCDD exposure. Levels of fibrinopeptide A were significantly correlated with liver fatty degeneration and necrosis.