Molecular Mechanisms of Thrombolytic Therapy

Lijnen, H.R.; Collen, Désiré

doi:10.1159/000215359

Cited by 4 publications

(4 citation statements)

References 41 publications

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“…Heparin has several effects on u-PA, including stimulation of cell secretion (Falcone, 1989), enhancement of proenzyme activation (Lijnen & Collen, 1986;Watahiki et al, 1989), promotion of activity (Andrade-Gordon & Strickland, 1986;PSques et al, 1986;Stephens et al, 1991), modulation of inhibition (Stump et al, 1986b) and possibly a contribution to cell/matrix binding (Stephens et al, 1991). Clearly a definition of the structural determinants of the interaction between u-PA and heparin is an important prerequisite for an understanding of these phenomena.…”

Section: Discussionmentioning

confidence: 99%

“…It has been shown in cell-free systems that heparin stimulates both the activation of pro-u-PA by plasmin (Lijnen & Collen, 1986;Watahiki et al, 1989) and the activation of plasminogen by u-PA (Andrade-Gordon & Strickland, 1986;Piques et al, 1986). These effects appear to be mediated by heparin binding to u-PA, although a heparin-binding site has not been identified in the u-PA structure.…”

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confidence: 99%

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Heparin binding to the urokinase kringle domain

Stephens¹,

Am²,

Ht³

et al. 1992

Biochemistry

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The binding of urokinase to immobilized heparin and dextran sulfate was studied using activity assays of the bound urokinase. The markedly higher binding observed with high M(r) urokinase compared to low M(r) urokinase indicated a role for the amino-terminal fragment (ATF). This was confirmed by the use of inactive truncated urokinase and monoclonal antibodies specific for the ATF in competition assays of urokinase binding. Antibody competition assays suggested a site in the kringle domain, and a synthetic decapeptide Arg-52-Trp-62 from the kringle sequence (kringle numbering convention) was competitive in assays of urokinase binding to dextran sulfate and heparin. Heparin binding to the urokinase kringle was unambiguously demonstrated via 1H NMR spectroscopy at 500 MHz. Effective equilibrium association constants (K(a)*) were determined for the interaction of isolated kringle fragment and low M(r) heparin at pH 7.2. The binding was strong in salt-free 2H2O (K(a)* approximately 57 mM-1) and remained significant in 0.15 M NaCl (K(a)* approximately 12 mM-1), supporting a potential physiological role for the interaction. This is the first demonstration of a function for the kringle domain of urokinase, and it suggests that while the classical kringle structure has specificity for lysine binding, there may also exist a class of kringles with affinity for polyanion binding.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Heparin binding to the urokinase kringle domain

Stephens¹,

Am²,

Ht³

et al. 1992

Biochemistry

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“…2 4aM (Table 1). This value should be compared with the estimate of 65 ,tM that is commonly quoted as the Km of t-PA in the absence of fibrin Lijnen & Collen, 1986) and, in view of the disparity, it is not surprising that the systemic effect of t-PA observed in patients (Topol et al, 1985;Williams et al, 1986) is greater than had been predicted (Sobel et al, 1984). The possibility of an exacerbation of systemic activation by heparin only applies to t-PA, because urokinase causes extensive plasminogen activation, even in the absence of heparin, and streptokinaseplasminogen activity is unaffected.…”

Section: Therapeutic Relevancementioning

confidence: 99%

Kinetic studies on the effect of heparin and fibrin on plasminogen activators

Fears

1988

Biochemical Journal

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1. Possible interactions between fibrin(ogen) and heparin in the control of plasminogen activation were studied in model systems using the thrombolytic agents tissue-type plasminogen activator (t-PA), urokinase and streptokinase.plasminogen activator complex and the substrates Glu- and Lys-plasminogen. 2. Both t-PA and urokinase activities were promoted by heparin and by pentosan polysulphate, but not by chondroitin sulphate or hyaluronic acid. The effect was on Km. 3. In the presence of soluble fibrin (and its mimic, CNBr-digested fibrinogen) the effect of heparin on t-PA was attenuated, although not abolished. In studies using a monoclonal antibody and 6-aminohexanoic acid, it was found that heparin and fibrin did not seem to share a binding site on t-PA. 4. The activity of t-PA B-chain was unaffected by heparin, so the binding site is located on the A-chain of t-PA (and urokinase). 5. Fibrin potentiated the activity of heparin on urokinase. The activity of streptokinase.plasminogen was unaffected by heparin whether or not fibrin was present. 6. If these influences of heparin and fibrin also occur in vivo, then, in the presence of heparin, the relative fibrin enhancement of t-PA will be diminished and the likelihood of systemic activation by t-PA is increased.

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“…We speculate that heparin binds to prekallikrein to form a heparin-prekallikrein complex which undergoes a confor mational change and displays an enzymatic activity corresponding to that of kallikrein. Such a mechanism occurs when streptokinase binds with plasminogen to form a complex which undergoes a transition, exposing an ac tive site in the modified plasminogen moiety [26]. This could explain the reversibility of the enzyme activities by protamine sulfate.…”

Section: Discussionmentioning

confidence: 99%

Effects of Heparin on Proteolytic Activities in Human Plasma

Kongsgaard

Aasen²,

Smith-Erichsen³

et al. 1992

Eur Surg Res

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The influence of unfractionated heparin [average molecular weight (MW) 10,000-15,000 kD] and low-molecular-weight heparin (average MW 400-600 kD) on the plasma kallikrein-kinin and the fibrinolytic system was studied in vitro. Unfractionated heparin added to plasma gave an increase in kalli-krein-like activity with a corresponding decrease of prekalli-krein and functional kallikrein inhibition values. Plasmin-like activity was also increased, but minor changes in plasminogen and no change in antiplasmin values occurred. The changes were less pronounced with low molecular heparin compared with unfractionated heparin. The proteolytic changes were reversed by protamine sulfate but not influenced by the protease inhibitior aprotinin. Gel filtration yielded proteolytic activities able to split the plasma kallikrein substrate S-2305 and, to a minor degree, the plasmin substrate S-2251. The proteolytic activities were not due to complex formation with α₂-macroglobulin. We speculate that heparin binds to prekalli-krein to form a heparin-prekallikrein complex which undergoes conformational changes and displays a kallikrein-like activity with the ability to split small synthetic substrates. Whether it is capable to split natural substrates remains unresolved.

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Molecular Mechanisms of Thrombolytic Therapy

Cited by 4 publications

References 41 publications

Heparin binding to the urokinase kringle domain

Heparin binding to the urokinase kringle domain

Kinetic studies on the effect of heparin and fibrin on plasminogen activators

Effects of Heparin on Proteolytic Activities in Human Plasma

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