Thrombolysis by human tissue plasminogen activator and urokinase in rabbits with experimental pulmonary embolus

Matsuo, Osamu; Rijken, D.C.; Collen, D.

doi:10.1038/291590a0

Cited by 250 publications

(87 citation statements)

References 4 publications

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“…The first 8 amino acid residues of N-terminal sequence of the fragment P4 were V-V-G-G-C-V-A-H, which were identical with that of plasmin. With reference to the complete primary structure of human plasminogen [28], we deduce that the significant cleavage site was at R561-fl-V562, similar to t-PA [29] and staphylokinase [30], and that this cleavage site produced the catalytic domain (P4) of the activated plasmin. When human plasminogen was incubated with subtilisin Carlsberg, at least ten fragments from plasminogen were separated by SDS-PAGE (photographs not shown), but these fragments had no fibrinolytic activity in the fibrin plate.…”

Section: Degradation Of Plasminogenmentioning

confidence: 93%

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Wang

Лі

et al. 2006

J IND MICROBIOL BIOTECHNOL

150

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Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55°C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS-PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), b-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-PhepNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bb-chains and Aa-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.

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Section: Degradation Of Plasminogenmentioning

confidence: 93%

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Wang

Лі

et al. 2006

J IND MICROBIOL BIOTECHNOL

150

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show abstract

“…Thrombolysis by urokinase is associated with systemic plasminogen activation, the degradation of fibrinogen and the coagulation proteins in circulating blood (5,25). In contrast, the t-PA produced by a human melanoma cell line has been shown to be a more specific and effective thrombolytic agent than urokinase because of its high affinity for fibrin and its thrombus-localized stimulation of plasminogen activator activity without the activation of systemic plasminogen or the degradation of plasma proteins (9,16,20).…”

Section: Introductionmentioning

confidence: 99%

Thrombolytic properties of an inactive proenzyme form of human urokinase secreted from human kidney cells.

Kasai

Arimura

Nishida

et al. 1985

Cell Struct. Funct.

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“…The fibrin-dependency of tissue activators and their fibrinbinding properties guarantee their accumulation and activation on the surface of fibrin clots. These characteristics, together with the recent findings that these molecules are both efficient and specific thrombolytic agents (8) suggest that tissue activator, not urokinase, is the physiologically relevant vascular activator. We have begun to study the PAs produced by cultured endothelial cells since available information suggests that the vascular activator may originate from the endothelium itself (9,10).…”

mentioning

confidence: 99%

Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators.

Levin¹,

Loskutoff²

1982

The Journal of Cell Biology

370

112

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Cell extracts and conditioned media (CM) from cultured bovine aortic endothelial cells (BAEs) were fractionated by PAGE in the presence SDS, and plasminogen activator (PA) activity was localized by fibrin autography. Multiple molecular weight forms of PA were detected in both preparations. Cell-associated PAs had Mr of 48,000, 74,000, and 100,000 while secreted PAs showed Mr of 52,000, 74,000, and 100,000. A broad zone of activity (Mr 80,000-100,000) also was present in both cellular fractions. In addition, PAs of Mr 41,000 and 30,000 appeared upon prolonged incubation or repeated freezing and thawing of the samples, and probably represent degradation products of higher molecular weight forms. This complex lysis pattern was not observed when CM was subjected to isoelectric focusing. Instead, only two classes of activator were resolved, one at pH 8.5, the other at 7.6. Analysis of focused samples by SDS PAGE revealed that the activity at pH 8.5 resulted exclusively from the Mr 52,000 form; all other forms were recovered at pH 7.6. The activity of the Mr 52,000 form was neutralized by anti-urokinase IgG but was not affected by antitissue activator IgG indicating that it is a urokinaselike PA. The activities of the Mr 74,000-100,000 forms were not affected by anti-urokinase. They were blocked by antitissue activator suggesting that all the forms in this group were tissue-type PAs. The multiple forms of PA were differentially sensitive to inactivation by diisopropylfluorophosphate (DFP). Treatment of CM with 10 mM DFP for 2 h at 37 degrees C only partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton PA. The activity of the Mr 100,000 form was not affected by this treatment, or by treatment with 40 mM DFP. Thus, cultured BAEs produce multiple, immunologically distinct forms of PA which differ in size, charge, and sensitivity to DFP. These forms include both urokinaselike and tissue-activator-like PAs. The possibility that one of these forms is a zymogen is discussed.

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Thrombolysis by human tissue plasminogen activator and urokinase in rabbits with experimental pulmonary embolus

Cited by 250 publications

References 4 publications

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Thrombolytic properties of an inactive proenzyme form of human urokinase secreted from human kidney cells.

Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators.

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