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Susan T Lord - One of the best experts on this subject based on the ideXlab platform.
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recombinant fibrinogen studies reveal that thrombin specificity dictates order of Fibrinopeptide release
Journal of Biological Chemistry, 2000Co-Authors: Jennifer L Mullin, Oleg V Gorkun, Cameron G Binnie, Susan T LordAbstract:Abstract During cleavage of fibrinogen by thrombin, Fibrinopeptide A (FpA) release precedes Fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A′β fibrinogen, replacing FpB with a Fibrinopeptide A-like peptide, FpA′ (G14V). Analyses of Fibrinopeptide release from A′β fibrinogen showed that FpA release and FpA′ release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA′ were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA′ release from A′β fibrinogen was unaffected. Thus, with A′β fibrinogen, Fibrinopeptide release from the β chain is similar to Fibrinopeptide release from the α chain. We conclude that the ordered release of Fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A′β fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A′β fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the Fibrinopeptide from the N terminus of the β chain does not affect polymerization of fibrinogen.
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characterization of purified recombinant fibrinogen partial phosphorylation of Fibrinopeptide a
Biochemistry, 1993Co-Authors: Cameron G Binnie, Joann M Hettasch, Elizabeth Strickland, Susan T LordAbstract:: Human fibrinogen has been expressed in Chinese hamster ovary (CHO) cells using a novel two-step procedure which permits efficient synthesis of engineered variant fibrinogens. CHO cells secreting recombinant fibrinogen were grown in roller bottles and maintained in serum-free media for several months. Recombinant protein was purified from media containing 2-4 micrograms/mL fibrinogen using protamine-Sepharose chromatography. Recombinant fibrinogen was identical to plasma fibrinogen when examined on Coomassie-stained SDS gels run under reducing conditions, and on SDS gels when run under nonreducing conditions after partial or complete plasmin degradation, indicating normal chain assembly, disulfide bond formation, and overall protein conformation. Thrombin digestion of purified fibrinogen led to clot formation with release of normal Fibrinopeptides, as identified by HPLC. Fibrinopeptide A released from recombinant fibrinogen was partially phosphorylated (22%), similar to the degree of phosphorylation found for human plasma fibrinogen (20-25%), indicating that partial phosphorylation in inherent in fibrinogen synthesis.
Cameron G Binnie - One of the best experts on this subject based on the ideXlab platform.
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recombinant fibrinogen studies reveal that thrombin specificity dictates order of Fibrinopeptide release
Journal of Biological Chemistry, 2000Co-Authors: Jennifer L Mullin, Oleg V Gorkun, Cameron G Binnie, Susan T LordAbstract:Abstract During cleavage of fibrinogen by thrombin, Fibrinopeptide A (FpA) release precedes Fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A′β fibrinogen, replacing FpB with a Fibrinopeptide A-like peptide, FpA′ (G14V). Analyses of Fibrinopeptide release from A′β fibrinogen showed that FpA release and FpA′ release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA′ were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA′ release from A′β fibrinogen was unaffected. Thus, with A′β fibrinogen, Fibrinopeptide release from the β chain is similar to Fibrinopeptide release from the α chain. We conclude that the ordered release of Fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A′β fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A′β fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the Fibrinopeptide from the N terminus of the β chain does not affect polymerization of fibrinogen.
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characterization of purified recombinant fibrinogen partial phosphorylation of Fibrinopeptide a
Biochemistry, 1993Co-Authors: Cameron G Binnie, Joann M Hettasch, Elizabeth Strickland, Susan T LordAbstract:: Human fibrinogen has been expressed in Chinese hamster ovary (CHO) cells using a novel two-step procedure which permits efficient synthesis of engineered variant fibrinogens. CHO cells secreting recombinant fibrinogen were grown in roller bottles and maintained in serum-free media for several months. Recombinant protein was purified from media containing 2-4 micrograms/mL fibrinogen using protamine-Sepharose chromatography. Recombinant fibrinogen was identical to plasma fibrinogen when examined on Coomassie-stained SDS gels run under reducing conditions, and on SDS gels when run under nonreducing conditions after partial or complete plasmin degradation, indicating normal chain assembly, disulfide bond formation, and overall protein conformation. Thrombin digestion of purified fibrinogen led to clot formation with release of normal Fibrinopeptides, as identified by HPLC. Fibrinopeptide A released from recombinant fibrinogen was partially phosphorylated (22%), similar to the degree of phosphorylation found for human plasma fibrinogen (20-25%), indicating that partial phosphorylation in inherent in fibrinogen synthesis.
Tadayoshi Kosugi - One of the best experts on this subject based on the ideXlab platform.
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habutobin recognizes thr7 in the sequence of Fibrinopeptide a of rabbit fibrinogen
Toxicon, 2000Co-Authors: T Nejime, Kiyohiko Kinjoh, Mariko Nakamura, Kazuhiko Hanashiro, Masanori Sunagawa, Yukinori Eguchi, Tadayoshi KosugiAbstract:Habutobin, a thrombin-like enzyme from Trimeresurus flavoviridis venom, cleaves only the Arg16–Gly17 bond in the rabbit Aα chain and releases Fibrinopeptide A (FPA). To investigate the role of amino acid residues in the rabbit FPA sequence upon habutobin action, we examined the inhibitory effects of FPA and peptides containing partial sequences of FPA on the habutobin action. Fibrinopeptides from rabbit, human, bovine and dog were isolated and rabbit FPA was fragmented using dilute HCl. Rabbit FPA inhibited the action of habutobin although FPA from human, bovine and dog did not. Among the fragments of rabbit FPA, a heptapeptide Aα 3–9, the N-terminal region of rabbit FPA, competitively inhibited the release of FPA by habutobin, whereas the C-terminal hexapeptide of FPA (Aα 11–16) exerted no effect on the habutobin action. Synthetic tripeptides Ser–Thr–Phe corresponding to Aα 6–8 and Ala–Thr–Phe also inhibited the habutobin action, but Ser–Asp–Phe and Ala–Thr–Gly did not. It is concluded that habutobin would recognize the region around Thr7–Phe8 in the sequence of rabbit FPA (Aα 1–16) prior to the cleavage of the Arg16–Gly17 bond.
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application of a monoclonal antibody to estimate rabbit Fibrinopeptide a released by habutobin
Toxicon, 1994Co-Authors: Kiyohiko Kinjoh, Mariko Nakamura, Tadayoshi KosugiAbstract:Abstract K. Kinjoh , M. Nakamura and T. Kosugi . Application of a monoclonal antibody to estimate rabbit Fibrinopeptide A released by habutobin. Toxicon 32, 1413–1423, 1994.—We reported previously that habutobin, one of the type A thrombin-like enzymes, releases Fibrinopeptide A alone from rabbit fibrinogen. To evaluate the effective action of habutobin in experiments using rabbit for the treatment of thrombosis, we attempted to develop an immunological method for measuring the Fibrinopeptide A level in the circulating blood of rabbit. The purified rabbit Fibrinopeptide A was coupled to keyhole limpet hemocyanin and BALB/c mice were immunized with the resultant Fibrinopeptide A-hemocyanin conjugate. The spleen cells of an immunized mouse were fused with myeloma cells (P3-X63-Ag8-U1). As a result, one hybridoma (a-F-7) was selected, which secreted an antibody against rabbit Fibrinopeptide A. Using this monoclonal antibody, we developed a competitive enzyme-linked immunoassay for estimating rabbit Fibrinopeptide A. It was able to measure rabbit Fibrinopeptide A contained in bentonite defibrinated plasma. This competitive enzyme-linked immunoassay should be useful for determining the Fibrinopeptide A level in the circulating blood of rabbits, using plasma defibrinated by bentonite.
Giuseppe Specchia - One of the best experts on this subject based on the ideXlab platform.
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Fibrinopeptide a excretion in urine a marker of the cumulative thrombin activity in stable versus unstable angina patients
American Journal of Cardiology, 1991Co-Authors: Diego Ardissino, Maria Grazia Gamba, Piera Angelica Merlini, Alberto Rolla, Paolo Barberis, Gloria Demicheli, Sophie Testa, Nicola Bruno, Giuseppe SpecchiaAbstract:Abstract Plasma levels and 24-hour urine excretion of Fibrinopeptide A were measured in a consecutive series of 179 patients with angina pectoris. Sixty-four patients had stable angina and 115 patients had unstable angina. Urine was collected over 24 hours the day before coronary arteriography, and blood samples were taken at the end of urine collection. When the values of Fibrinopeptide A in plasma and in the 24-hour urine specimens were compared, no significant correlation was found in patients with either stable (rs = 0.16, difference not significant) and unstable (rs = 0.07, difference not significant) angina. The concentrations of Fibrinopeptide A in the plasma did not differ significantly when patients with stable angina (range 0.1 to 82.6, median 7.4 ng/mL) were compared with patients with unstable angina (range 0.2 to 61.7, median 14 ng/mL, p = 0.055), whereas Fibrinopeptide A 24-hour urinary excretion was significantly higher in patients with unstable angina (range 0.3 to 38.1, median 11.8 μg/24 hr) than in patients with stable angina (range 0.4 to 38.1, median 3.8 μg/24 hr, p
Agnes Henschen - One of the best experts on this subject based on the ideXlab platform.
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thrombin induced Fibrinopeptide release from a fibrinogen variant fibrinogen sydney i with an aα arg 16 his substitution
FEBS Journal, 2008Co-Authors: Christopher Southan, David A Lane, Wolfram Bode, Agnes HenschenAbstract:Fibrinogen, Purified from a recently identified case of dysfibrinogenaemia, fibrinogen Sydney I, was shown by thrombin digestion, high-performace liquid chromatography (HPLC) and amino acid analysis to be a heterozygous case of an Aα Arg-16 His substitution. Kinetic studies have been carried out on the thrombin-induced release of Fibrinopeptide A (FPA), Fibrinopeptide B (FPB) and the variant peptide [His16]FPA. When thrombin was added to fibrinogen Sydney I at a concentration of 0.2 U/ml release of FPA was rapid and there was a 79-fold reduced rate of release of [His16]FPA, but the rate of release of FPB was not appreciably reduced. In contrast, at lower thrombin concentrations the rate of FPB release was reduced in proportion to the rate of total FPA release, supporting the view that release of Fibrinopeptides is a sequential process. The second-order kinetic constant kcat/Km for hydrolysis of the abnormal Aα chain by thrombin was calculated from Lineweaver-Burk plots to be 16–30-fold less than that for the normal Aα chain. Molecular modelling studies, using a refined model of the trypsin-pancreatic-trypsin-inhibitor complex have been used to suggest how the histidine at the P 1 site can be accommodated within the enzyme hydrophobic active-site pocket.