The purification and properties of human plasminogen

Alkjærsig, Norma

doi:10.1042/bj0930171

Cited by 88 publications

(33 citation statements)

References 18 publications

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“…The interval between the completion of the preparation of plasminogen-1251 and its injection into the dogs was only a few minutes. The first blood sample of about [3][4] were intravenously injected, and the plasma plasminogen-125I activity (x), the radioactivity unbound to proteins (z), and the radioactivity remaining in the body (1-u) were determined daily for the period of 7 days. On the 3rd day from the start of the experiment, 1909 plough U of human urokinase (Calbiochem) were intravenously injected.…”

Section: Methodsmentioning

confidence: 99%

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Plasminogen-125I responses in dogs to a single injection of urokinase and typhoid vaccine and to vascular injury

Takeda¹

1972

J. Clin. Invest.

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Section: Methodsmentioning

confidence: 99%

“…The supernate was discarded and the precipitate was dissolved in 20 ml of 0.01 M phosphate buffer (ph 8.0). The crude plasminogen preparation was then dialyzed at 3°C against 8 liters of 0.01 M phosphate buffer (ph 8.0) for 4 hr. The dialysate was spun at 3°C for 5 min at 3000 g and the insoluble material was discarded.…”

Section: Methodsmentioning

confidence: 99%

Plasminogen-125I responses in dogs to a single injection of urokinase and typhoid vaccine and to vascular injury

Takeda¹

1972

J. Clin. Invest.

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“…Subsequent studies have shown that 6-aminohexanoic acid and lysine cause extensive conformational alterations in Glu-plasminogen [3,4] (and references in [4]); Deutsch and Mertz [5] utilized the strong affinity of plasminogen for lysine-Sepharose in the development of a convenient method for purification of plasminogen.…”

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confidence: 99%

Studies on the Lysine-Binding Sites of Human Plasminogen. The Effect of Ligand Structure on the Binding of Lysine Analogs to Plasminogen

Winn

Hochschwender

et al. 1980

Eur J Biochem

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A method is described for measuring relative binding constants of lysine and analogs of lysine to plasminogen and plasminogen 'kringle' fragments. Plasminogen or kringle fragments adsorbed to lysine-Sepharose are eluted with increasing concentrations of lysine or other ligands, the concentration of ligand required to elute 50% of the protein being taken as a measure of the binding constant. The method is simple and is not dependent on monitoring conformational changes. We confirm earlier reports that the best ligands for the lysine binding sites of plasminogen are w-amino acids containing five or six carbons. We show further that both Glu-plasminogen (the native form with N-terminal glutamic acid) and Lys-plasminogen (a degraded form with N-terminal lysine), as well as the heavy chain fragments, kringle 4 and kringle 1 + 2 + 3, have very similar properties with regard to binding specificity for w-amino acids. For all species optimal binding is observed when the distance between the amino and carboxyl carbon is about 0.68 nm. The binding of ligands is decreased by the presence of polar atoms on the a and p positions of the carbon chain of amino acids.Arginine binds relatively weakly at the lysine site and there does not appear to be a separate arginine binding site in plasminogen.It has been known for over 20 years that amino acids such as 6-aminohexanoic acid have antifibrinolytic properties [l], one of which probably results from inhibition of the binding of plasminogen to fibrin [2]. Subsequent studies have shown that 6-aminohexanoic acid and lysine cause extensive conformational alterations in Glu-plasminogen [3,4] (and references in [4]); Deutsch and Mertz [5] utilized the strong affinity of plasminogen for lysine-Sepharose in the development of a convenient method for purification of plasminogen.

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“…Plasminogen was measured with the chromogenic substrate S-2251 (20) after activation for 10 min at 370C with 1,000 IU of urokinase/ 5 ,l plasma of which the plasmin inhibitors were first neutralized by acidification (21). EPA was measured with a twosite immunoradiometric assay (22).…”

mentioning

confidence: 99%

Thrombolysis with Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits with Experimental Jugular Vein Thrombosis. EFFECT OF MOLECULAR FORM AND DOSE OF ACTIVATOR, AGE OF THE THROMBUS, AND ROUTE OF ADMINISTRATION

Collen¹,

Stassen²,

Verstraete³

1983

J. Clin. Invest.

253

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A B S T R A C T A simple venous thrombosis model in rabbits was used for the quantitative evaluation of the thrombolytic effect of human extrinsic (tissue-type) plasminogen activator as compared with urokinase.A thrombus was formed in an isolated segment of the jugular vein from a mixture of 125I-labeled fibrinogen, whole rabbit blood, and thrombin. In order to immobilize the thrombus during lysis, it was formed around a woolen thread introduced longitudinally in the lumen of the vein. Thrombotic extension of the clot was prevented by subcutaneous injection of heparin. The extent of thrombolysis was measured as the difference between the radioactivity introduced in the clot and that recovered in the vein segment at the end of the experiment. In control animals the extent of thrombolysis was 5.6±1.4% (n = 5) after 6 h, 14.5±1.7% (n = 10) after 30 h, 16.0±1.5% (n = 11) after 78 h, and 48.1±2.7% (n = 10) after 174 h (mean±SEM).Extrinsic (tissue-type) plasminogen activator, highly purified from the culture fluid of a human melanoma cell line, was administered systemically or locally over a time period of 4 h and the percent thrombolysis measured 2 h after the end of the infusion. One-and two-chain extrinsic plasminogen activator had very similar thrombolytic potency. Systemic infusion resulted in a dose-dependent degree of thrombolysis. The activator-induced thrombolysis, after infusion of 100,000 IU (n1 mg protein), was -75% for fresh clots, 35% for 1-d-old clots, 30% for 3-d-old clots, and 50% for 7-d-old clots. The thrombolytic activity of urokinase was more than five times lower than that of extrinsic plasminogen activator: Infusion of 500,000 IU resulted in -40% lysis of fresh clots and 25% of 1-3-d-old clots, while 7-d-old clots appeared to have become resistent to urokinase. Local infusion resulted in a 5-10 times higher thrombolytic effect of both extrinsic plasminogen activator and urokinase.Thrombolysis with extrinsic plasminogen activator was not associated with systemic activation of the fibrinolytic system as evidenced by unaltered plasma levels of fibrinogen, plasminogen, and a2-antiplasmin.Systemic infusion of urokinase resulted in significant thrombolysis only at doses that were associated with disseminated plasminogen activation. Local infusion of urokinase required a 5-10-fold higher dose than extrinsic plasminogen activator to obtain a similar degree of thrombolysis, which also occurred in the absence of systemic activation of the fibrinolytic system. It is concluded that the extent of thrombolysis by extrinsic plasminogen activator is mainly determined by the dose of activator and its delivery in the vicinity of the thrombus and much less by the age of the thrombus or the molecular form of the activator. Extrinsic plasminogen activator appears to be superior to urokinase because of its higher (5-10-fold) specific thrombolytic activity and the absence of systemic activation of the fibrinolytic system, which results in defibrinogenation and a bleeding tendency. 368J. Clin. Invest.

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The purification and properties of human plasminogen

Cited by 88 publications

References 18 publications

Plasminogen-125I responses in dogs to a single injection of urokinase and typhoid vaccine and to vascular injury

Plasminogen-125I responses in dogs to a single injection of urokinase and typhoid vaccine and to vascular injury

Studies on the Lysine-Binding Sites of Human Plasminogen. The Effect of Ligand Structure on the Binding of Lysine Analogs to Plasminogen

Thrombolysis with Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits with Experimental Jugular Vein Thrombosis. EFFECT OF MOLECULAR FORM AND DOSE OF ACTIVATOR, AGE OF THE THROMBUS, AND ROUTE OF ADMINISTRATION

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