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| 2. |
- Aleshkov, S B, et al.
(författare)
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Biochemical and biophysical studies of reactive center cleaved plasminogen activator inhibitor type 1. The distance between P3 and P1' determined by donor-donor fluorescence energy transfer.
- 1996
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 271:35, s. 21231-8
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Tidskriftsartikel (refereegranskat)abstract
- Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). In accordance with other serpins, PAI-1 is thought to undergo a conformational change upon reactive center cleavage. In this study we have developed methods to produce and purify reactive center cleaved wild-type PAI-1 and characterized this molecular form of PAI-1 by biochemical and biophysical methods. Incubation with Sepharose-bound trypsin caused cleavage only at the P1-P1' bond in the reactive center and resulted in 39- and 4-kDa polypeptides, strongly held together by noncovalent interactions. Circular dichroism measurements suggest that the reactive center cleavage triggers larger conformational changes than the conversion from the active to the latent form. Cleaved PAI-1 did not bind to either PAs or vitronectin but retained the heparin-binding capacity. To study the structure of cleaved PAI-1 by polarized fluorescence spectroscopy and to measure intramolecular distances, we used cysteine substitution mutants to which extrinsic fluorescence probes were attached. These studies revealed increasing orientational freedom of probes in the P3 and P1' positions upon cleavage. Distance measurements based on fluorescence energy transfer between probes in positions P3 and P1' indicate that these residues are separated by at least 68 +/- 10 A in cleaved PAI-1.
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| 3. |
- Fa, M, et al.
(författare)
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Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1 : conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin.
- 1995
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Ingår i: Biochemistry. - 0006-2960 .- 1520-4995. ; 34:42, s. 13833-40
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Tidskriftsartikel (refereegranskat)abstract
- Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)
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| 5. |
- Strandberg, LE, et al.
(författare)
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Anticoagulant effects of low-molecular-weight heparin following thrombolytic therapy in acute myocardial infarction: a dose-finding study
- 1996
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Ingår i: Haemostasis. - : S. Karger AG. - 0301-0147. ; 26:5, s. 247-257
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Tidskriftsartikel (refereegranskat)abstract
- The aim of the present study was to gain clinical experience with different dose levels of dalteparin, a low-molecular-weight heparin, following thrombolytic therapy in acute myocardial infarction. Compared to heparin, dalteparin has a longer half-life and a greater and highly predictable bioavailability, which would suggest dalteparin to be a convenient alternative. Twenty patients with ECG signs of acute transmural myocardial ischemia received streptokinase (1.5 million IU for 60 min) and were allocated to a control group or to open treatment with 50, 75 or 100 IU of dalteparin/kg b.w. s.c. b.i.d., starting 4 h later, for 6 days. Each group consisted of 5 patients. Except for the control group, aspirin was withheld during dalteparin treatment. Anti-factor-Xa (anti-FXa) values increased dose-dependently during the first 24 h and were maintained throughout the study period. On day 6, anti-FXa levels after 100 IU/kg b.w. were 0.79 (0.59-1.00) IU/ml (median, min.-max.) 4 h after administration of dalteparin, and 0.51 (0.34-0.82) IU/ml before the subsequent dose of dalteparin. In conclusion, our results indicate that a dalteparin dose slightly higher than 100 IU/kg b.w. is required in order to obtain the presumed therapeutic range of anti-FXa (0.6-1.0 IU/ml).
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