Tissue-Type Plasminogen Activator

Collen, D; Lijnen, H.R.

doi:10.1159/000215363

Cited by 26 publications

(5 citation statements)

References 26 publications

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“…Thrombin releases fibrinopeptide A (A␣ [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] ) and fibrinopeptide B (B␤ [1][2][3][4][5][6][7][8][9][10][11][12][13][14] ) from Fg, thereby generating fibrin monomers. These aggregate to form twostranded protofibrils that are arranged in a half-staggered fashion, whereby the E domain of a fibrin monomer of one strand is noncovalently associated with D domains of two adjacent fibrin monomers on the opposite strand.…”

Section: Fig 3 Model Of (Dd)e Formation and Degradationmentioning

confidence: 99%

Thrombin-activable Fibrinolysis Inhibitor Attenuates (DD)E-mediated Stimulation of Plasminogen Activation by Reducing the Affinity of (DD)E for Tissue Plasminogen Activator

Stewart¹,

Fredenburgh

Rischke

et al. 2000

Journal of Biological Chemistry

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A complex of D-dimer noncovalently associated with fragment E ((DD)E), a degradation product of crosslinked fibrin that binds tissue plasminogen activator (t-PA) and plasminogen (Pg) with affinities similar to those of fibrin, compromises the fibrin specificity of t-PA by stimulating systemic Pg activation. In this study, we examined the effect of thrombin-activable fibrinolysis inhibitor (TAFI), a latent carboxypeptidase B (CPB)-like enzyme, on the stimulatory activity of (DD)E. Incubation of (DD)E with activated TAFI (TAFIa) or CPB (a) produces a 96% reduction in the capacity of (DD)E to stimulate t-PA-mediated activation of Glu-or Lys-Pg by reducing k cat and increasing K m for the reaction; (b) induces the release of 8 mol of lysine/mol of (DD)E, although most of the stimulatory activity is lost after release of only 4 mol of lysine/mol (DD)E; and (c) reduces the affinity of (DD)E for Glu-Pg, Lys-Pg, and t-PA by 2-, 4-, and 160-fold, respectively. Because TAFIaor CPB-exposed (DD)E produces little stimulation of Glu-Pg activation by t-PA, (DD)E is not degraded into fragment E and D-dimer, the latter of which has been reported to impair fibrin polymerization. These data suggest a novel role for TAFIa. By attenuating systemic Pg activation by (DD)E, TAFIa renders t-PA more fibrin-specific. Intravascular fibrinolysis is initiated when plasminogen (Pg)1 is converted to plasmin by tissue-type plasminogen activator (t-PA) (1, 2). Plasmin then degrades fibrin, yielding soluble fibrin degradation products. Through a positive feedback mechanism, fibrin enhances its own degradation by stimulating t-PA-mediated Pg activation. To potentiate this reaction, fibrin acts as a template onto which both t-PA and Pg bind (3). The activator and its substrate bind to independent sites on intact fibrin because the t-PA interaction is primarily mediated by its fibronectin finger-like domain, whereas Pg binding is kringle-dependent (3-5). As a functional consequence of t-PA and Pg interaction with fibrin, the catalytic efficiency of t-PAmediated Pg activation is 2-3 orders of magnitude greater in the presence of fibrin than in its absence (1,3,6). In contrast to the potent stimulatory effect of fibrin, fibrinogen (Fg) produces only a 25-fold enhancement in the catalytic efficiency of Pg activation by t-PA (1, 7). Because t-PA preferentially activates Pg in the presence of fibrin rather than Fg, it is designated a fibrin-specific Pg activator. When cross-linked fibrin is solubilized by plasmin, a major degradation product is (DD)E, a complex of D-dimer (DD) noncovalently associated with fragment E (8, 9). Recently, we demonstrated that (DD)E compromises the fibrin specificity of t-PA, because this soluble fragment is as potent as fibrin at stimulating Pg activation by t-PA (10, 11). Like fibrin, (DD)E binds t-PA and Pg with high affinity (5,10,12). In contrast to its predominantly finger-dependent interaction with fibrin,

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Section: Fig 3 Model Of (Dd)e Formation and Degradationmentioning

confidence: 99%

Thrombin-activable Fibrinolysis Inhibitor Attenuates (DD)E-mediated Stimulation of Plasminogen Activation by Reducing the Affinity of (DD)E for Tissue Plasminogen Activator

Stewart¹,

Fredenburgh

Rischke

et al. 2000

Journal of Biological Chemistry

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“…The results were obtained from analysis of 3 thrombin-derived tcu-PA as compared to K , 7.1 pM and k2 1.32 s-' (r = 0.996) for intact scu-PA.…”

Section: Nh2-terminal Amino Acid Sequence Of Thrombin-derived Tcu-pmentioning

confidence: 99%

Activation with plasmin of two-chain urokinase-type plasminogen activator derived from single-chain urokinase-type plasminogen activator by treatment with thrombin

1987

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Thrombin converts single-chain urokinase-type plasminogen activator (scu-PA) to an inactive two-chain derivative (thrombin-derived tcu-PA) by hydrolysis of the Arg-I 56 -Phe-457 peptide bond. In the present study, we show that inactive thrombin-derived tcu-PA (specific activity 1000 IU/mg) can be converted with plasmin to active two-chain urokinase-type plasminogen activator (specific activity 43 000 IU/mg) by hydrolysis of the Lys-158 -Ile-159 peptide bond. This conversion follows Michaelis-Menten kinetics with a Michaelis constant K , of 37 pM and a catalytic rate constant k2 of 0.013 s-'. The catalytic efficiency (kz/K,) for the activation of thrombinderived tcu-PA by plasmin is about 500-fold lower than that for the conversion of intact scu-PA to tcu-PA.tcu-PA, generated by plasmin treatment of thrombin-derived tcu-PA, has similar properties to tcu-PA obtained by digestion of intact scu-PA with plasmin (plasmin-derived tcu-PA); its plasminogen activating potential and fibrinolytic activity in an in vitro plasma clot lysis system appear to be unaltered.These observations confirm that the structure of the NH2-terminal region of the B chain of u-PA is an important determinant for its enzymatic activity, whereas that of the COOH-terminal region of the A chain is not.

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“…serine protease that initiates fibrinolysis by converting plasminogen (Pg) to plasmin (1). Plasmin then solubilizes fibrin, yielding fibrin degradation products.…”

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confidence: 99%

Identification of the Mechanism Responsible for the Increased Fibrin Specificity of TNK-Tissue Plasminogen Activator Relative to Tissue Plasminogen Activator

Stewart¹,

Fredenburgh²,

Leslie³

et al. 2000

Journal of Biological Chemistry

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TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin-specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t-PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K M , whereas K-containing variants have a lower k cat than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA. Tissue-type plasminogen activator (t-PA)1 is a trypsin-like serine protease that initiates fibrinolysis by converting plasminogen (Pg) to plasmin (1). Plasmin then solubilizes fibrin, yielding fibrin degradation products. Through a positive feedback mechanism, fibrin enhances its own degradation by stimulating t-PA-mediated Pg activation (2). To enhance this reaction, fibrin binds t-PA and Pg, thereby increasing the catalytic efficiency of t-PA-mediated Pg activation 1000-fold over that in the absence of fibrin (2-6). In contrast to the potent stimulatory effect of fibrin, fibrinogen (Fg) produces only modest enhancement in the catalytic efficiency of Pg activation by t-PA (2, 7, 8). Because it preferentially activates Pg in the presence of fibrin rather than Fg, t-PA is designated a fibrin-specific plasminogen activator. Despite its apparent fibrin specificity, t-PA causes systemic fibrinogenolysis and ␣ 2 -antiplasmin consumption when given to patients (9, 10). Recently, we demonstrated that the fibrin specificity of t-PA is compromised because (DD)E, the major degradation product of cross-linked fibrin (11), stimulates Pg activation by t-PA to a similar extent as fibrin (11)(12)(13). This occurs because, like fibrin, (DD)E also binds t-PA and Pg with high affinity, thereby enhancing their interaction (14). As a potent stimulato...

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Tissue-Type Plasminogen Activator

Cited by 26 publications

References 26 publications

Thrombin-activable Fibrinolysis Inhibitor Attenuates (DD)E-mediated Stimulation of Plasminogen Activation by Reducing the Affinity of (DD)E for Tissue Plasminogen Activator

Thrombin-activable Fibrinolysis Inhibitor Attenuates (DD)E-mediated Stimulation of Plasminogen Activation by Reducing the Affinity of (DD)E for Tissue Plasminogen Activator

Activation with plasmin of two-chain urokinase-type plasminogen activator derived from single-chain urokinase-type plasminogen activator by treatment with thrombin

Identification of the Mechanism Responsible for the Increased Fibrin Specificity of TNK-Tissue Plasminogen Activator Relative to Tissue Plasminogen Activator

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