Recombinant tissue plasminogen activator in patients with pulmonary embolism: correlation of fibrinolytic specificity and efficacy.

Vaughan, Douglas E.; Goldhaber, Samuel Z.; Kim, J; Loscalzo, Joseph

doi:10.1161/01.cir.75.6.1200

Cited by 28 publications

(15 citation statements)

References 9 publications

(3 reference statements)

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“…This occurs because, like fibrin, (DD)E also binds t-PA and Pg with high affinity, thereby enhancing their interaction (14). As a potent stimulator of t-PA-mediated activation of Pg, (DD)E generated during thrombus dissolution has the potential to induce systemic plasminemia (11,15). These data suggest that the fibrin specificity of an activator not only depends on its relative stimulation by fibrin and Fg but also on the degree to which it is stimulated by (DD)E. Furthermore, activators that have reduced stimulation by (DD)E but retain activity in the presence of fibrin should be more fibrin-specific than t-PA.…”

Section: Tissue-type Plasminogen Activator (T-pa)mentioning

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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Section: Tissue-type Plasminogen Activator (T-pa)mentioning

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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“…Similar analyses were used for the in vivo data at 50 and 100 minutes, a time when fibrinolysis had reached a plateau. The (3,30, 300, and 3,000 IU/mI) to determine whether ultrasound affected the rate and extent of t-PA-induced fibrinolysis (Figure 3). Differences in mean clot lysis caused by effects of ultrasound In paired experiments, clots were incubated with fresh frozen plasma to which t-PA had been added at a final concentration of 300 or 3,000 IU/ml, and one clot in each pair was exposed to intermittent ultrasound ( Figure 4).…”

Section: Toxicity Studiesmentioning

confidence: 99%

Effect of ultrasound on tissue-type plasminogen activator-induced thrombolysis.

et al. 1992

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BACKGROUND The efficacy of fibrinolytic therapy is limited by the small surface area of the clot that is available for the binding of the thrombolytic agent, such as tissue-type plasminogen activator (t-PA). We hypothesized that exposure of the clot to ultrasound during thrombolytic treatment could enhance lysis through perturbation of the thrombus, which would expose additional fibrin binding sites for t-PA. METHODS AND RESULTS Whole human blood clots containing radiolabeled fibrinogen were incubated in vitro for 200 minutes with Tris-albumin buffer containing t-PA at concentrations ranging from 3 to 3,000 IU/ml. In paired experiments, one of the clots also was exposed to intermittent ultrasound (1 MHz, 1.75 W/cm2) throughout the experiment. The ultrasound was delivered as a 2-second exposure followed by a 2-second rest interval. The overall difference in mean clot lysis between thrombi receiving ultrasound and those receiving no ultrasound was significant (p less than 0.001) at all concentrations of t-PA. For clots incubated with t-PA at a concentration of 300 IU/ml, ultrasound increased the percent lysis at 200 minutes from 42 +/- 5% (mean +/- SEM) to 64 +/- 10%. In six paired experiments in a rabbit jugular vein thrombosis model, rabbits received 1 mg t-PA alone or t-PA and intermittent ultrasound (1 MHz, 1.75 W/cm2) for 200 minutes. For rabbits receiving ultrasound and t-PA, lysis was 55 +/- 11% at 100 minutes compared with 30 +/- 12% for rabbits receiving only t-PA. Lysis was 6 +/- 10% for rabbits (n = 4) receiving ultrasound alone. No evidence for tissue damage was noted in rabbits exposed to intermittent ultrasound. CONCLUSIONS Exposure of whole blood clots in vitro to intermittent ultrasound combined with t-PA caused a significant enhancement of thrombolysis compared with t-PA alone. Intermittent ultrasound also showed a trend toward enhancement of t-PA-induced clot lysis in an animal thrombosis model. These data suggest that noninvasive intermittent ultrasound may be a useful adjunct to thrombolytic therapy.

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“…[16][17][18]34 Because high-molecular-weight cross-linked polymers may occur in such patients, Francis et a122 have suggested lysis of these polymers may be a major source of increased concentrations of XL-FDP after thrombolysis. Increased XL-FDP immunoreactivity after incubation of plasma in vitro with t-PA was thought by these investigators to be due to release of D-dimer fragments from fibrin polymers.…”

Section: Sources Of Xl-fdp In Vivomentioning

confidence: 99%

“…15 Plasma concentrations of XL-FDPs are also consistently increased after administration of fibrinolytic agents to patients with acute myocardial infarction, pulmonary embolism, or deep venous thrombosis. [16][17][18] Whether these elevations in XL-FDP specifically reflect clot lysis is not clear because XL-FDP immunoreactivity also increases with some assays after administration of tissue-type plasminogen activator (t-PA) to normal volunteers. '9 Francis et a120-22 have suggested XL-FDP such as fragment D-dimer may derive from lysis of circulating crosslinked fibrin polymers of high molecular weight that are present in plasma of patients with acute myocardial infarction as well as in plasma of other acutely ill patients.…”

mentioning

confidence: 99%

Validity of enzyme-linked immunosorbent assays of cross-linked fibrin degradation products as a measure of clot lysis.

et al. 1990

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Concentrations of cross-linked fibrin degradation products (XL-FDPs) in plasma, measured by enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies (MAbs) raised against fragment D-dimer of cross-linked fibrin, increase when patients are given fibrinolytic agents. Whether XL-FDPs derive from circulating cross-linked fibrin polymers in plasma, compared with clot-associated fibrin, has been questioned because increases in XL-FDP are measured by some assays after fibrinolysis in vitro in the absence of clot. We characterized the source of XL-FDP immunoreactivity in plasma of patients with acute myocardial infarction and ischemic heart disease and the response to plasminogen activation in vitro induced by pharmacological concentrations of tissue-type plasminogen activator (t-PA) and streptokinase. XL-FDPs were measured with two different ELISA. One, "pan-specific tag ELISA," was based on a capture MAb specific for XL-FDP and a tag MAb that recognizes an epitope exposed in the fragment D region of both fibrin and fibrinogen, whereas the other, "fibrin-specific tag ELISA," was based on a capture and tag MAbs both specific for fibrin. After plasminogen activation was induced in vitro in plasma from patients with myocardial infarction, increased concentrations of XL-FDP were measured by the pan-specific tag ELISA; however, concentrations measured with the fibrin-specific tag ELISA were not increased. To determine the mechanism for this discrepancy, plasma was subjected to immunoadsorption with a MAb specific for fragment D-dimer before and after in vitro activation of the fibrinolytic system and immunoblotting with a fragment D-dimer-specific MAb and with the pan-specific MAb. Increased concentrations of fragment D-dimer, as well as fibrinogen fragment D at high concentrations, were recognized by the specific MAb. Non-cross-linked fragments were also shown by immunoblotting with the pan-specific MAb to coprecipitate with cross-linked fibrin fragments. This suggested the increased concentrations of XL-FDP measured by the panspecific tag ELISA after in vitro activation of the fibrinolytic system were due to detection of non-cross-linked fibrinogen fragments. However, fibrin fragment D-dimer concentrations were found to increase in plasma of 15 patients given t-PA for acute myocardial infarction. We conclude fragment D-dimer in plasma of patients during thrombolysis does not originate from circulating soluble cross-linked fibrin but rather is a marker of solid-phase fibrin dissolution, which may be quantitated with assays based on capture and tag antibodies that do not detect fibrinogen or its degradation products. (Circulation 1990;82:1159-1168

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Recombinant tissue plasminogen activator in patients with pulmonary embolism: correlation of fibrinolytic specificity and efficacy.

Cited by 28 publications

References 9 publications

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

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

Effect of ultrasound on tissue-type plasminogen activator-induced thrombolysis.

Validity of enzyme-linked immunosorbent assays of cross-linked fibrin degradation products as a measure of clot lysis.

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