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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...

Nonspecific binding of UFH to plasma proteins most likely contributes to the variable anti-IIa response to UFH in patients with thromboembolic disease. Although DS also binds to plasma proteins, the clinical significance of this finding is unclear. In contrast, because LMWH does not bind to plasma proteins, the anti-IIa activity of LMWH should be just as predictable as its anti-Xa activity.

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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