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Increasing the fibrin specificity of t-PA enhances its activity towards platelet rich clots
112
POSTER PRESENTATIONS
P-9: Regulation of Fibrinolysis
312 EFFECT OF PLATELET ON CLOT LYSIS BY UROKINASE-TYPE PLASMINOGEN ACTIVATOER Yuasa H. Okada K, Matsuo 0. Dept. of Physiology, Kinki University School of Medicine, Osakasayama, Japan. Resistance of a platelet-rich thrombus to lysis by plasminogen activators limits the effectiveness of thrombolytic therapy for thromboembolic diseases. Arterial thrombi contain a large number of platelet in which active plasminogen activator inhibitor-l (PAI-1) is contained. Since platelet expresses plasminogen binding site on its surface, potential role of Blood platelet in lysis of thrombi induced was investigated. was collected by veinpuncture from healthy volanteer donors into acid citrate anticoagulant. The platelet-rich plasma (PRP) and the platelet-poor plasma (PPP) were prepared by centrifugation of titrated whole blood. The washed-out platelet was obtained by gel-filtration of PRP. Clot produced by plasminogen containing fibrinogen and platelet, or by PRP or PPP, reacted by thrombin/CaC12 and urokinase-type
INCREASING THE FIBRIN SPECIFICITY OF t-PA ENHANCES ITS ACTIVITY TOWARDS PLATELET RICH CLOTS Canio J. Refino, Julie M. Badillo. Nicholas F. Paoni. Bruce A. Keyt. Hung V. Neuven. and William F. Bennett. Genentech Inc., South San Francisco, CA. USA We recently demonstrated that variants of t-PA with increased fibrin specificity and resistance to PAI-I inhibition had greater potency towards platelet enriched plasma (PEP) clots relative to whole blood clots in vivo (Thromh Haemosrus 1993; 70(2) & PNAS 1994 in press). To further evaluate one t-PA variant (T103N, N117Q, KHRR 296-299 AAAA or TNK) and examine the role of platelets in clot lysis, we compared TNK to t-PA in a series of experiments. Human platelet poor plasma (PPP) with 125I fibrinogen, *platelets, or @AI-I was aliqouted into microtiter plate wells following Ca++ and thrombin addition. After 1 hour at 37°C PEP clots had retracted to 25% of the PPP clots diameter. The clots then had an overlay of PPP containing t-PA or tPA variants added to the wells. Clot Lysis was quantitated by the release of tz51 FDPS to the fluid phase at various times (1150 minutes). Lysis of PPP clots induced by t-PA
314 THE AMOUNT OF ACUTELY
RELEASABLE TISSUETYPE PLASMINOGEN ACTIVATOR (tPA) CLOSELY CORRELATES WITH THE RATE OF CONSTITUTIVE tPA SECRETION. Schrauwen Y, de Vries REM, Kooistra T and Emeis JJ Gaubius Laboratory, TNO-PG, Leiden, The Netherlands.
tPA can be secreted into the circulation from the endothelium by both a constitutive and a regulated pathway (acute release). The latter process occurs only following endothelial cell stimulation, and gives rise to very rapid (local) increases in tPA concentration; it is thought important in speeding up thrombolysis. We have developed a very sensitive ELISA for tPA antigen to study acute release of tPA from human endothelial cells in culture. Upon addition of thrombin active tPA (range: lo-200 pg/2.10s endothelial cells) is rapidly released (maximal release within one minute) into the medium and disappears concomitantly from an intracellular source. On average,
plasminogen activator (u-PA). The clot lysis assay was performed on 96-well micro plate. In order to express u-PA activity in this assay, the time to reach half of the maximum OD (T1/2) of was measured. In the lysis of fibrin clot and PRP clot, Tl/2 was significant prolonged in the presence of various concentrations of platelet in a dose-dependent manner. Although the plasminogen antigen was 75 pg/ 4.5 X lo6 platelets, the activated plasminogen on platelet by U-PA was 0.77rg/ 2.88 X lo6 platelets. Further, the anti-plasminogen IgG had no effect on the fibrin clot lysis, indicating that plasminogen bound on platelets exhibit& little effect on clot lysis. In the lysis of fibrin clot and PRP clot, Tl/2 was significant shortened in the presence of anti-PAIIgG in a dose-dependent manner. Activation of exogenous plasminogen by u-PA was significantly enhanced on platelet. These data suggest that the effect of platelet on clot lysis was not due to plasminogen but PAI- in platelet.
or TNK at plasma concentrations of 0.3 to 9.0 ug/ml were dose dependent and identical for comparable concentrations of the two reagents. PPP clots spiked with active PAI-1, required 6 fold higher concentrations of PAI- to inhibit TNK vs t-PA induced lysis by 50%. With PEP clots, lysis produced by both activators in the concentration range 0.3 to 1 ug/ml were similarly depressed (-50%) compared to PPP clots. At higher activator concentrations (>3ug/ml), there was a paradoxical decrease in the lysis of the t-PA treated clots. In contrast, TNK treated clot lysis continued to increase in a dose dependent fashion. Assay of the plasma phases showed that there was considerable plasminogen depletion in both PPP and PEP wells at high concentrations of t-PA but not TNK. These data suggest that PEP clots are more susceptible than PPP clots to the plasminogen steal phenomena and that at high pharmacological concentrations of t-PA (as might be encountered during front loaded or bolus regimens), increased fibrin specificity may play a more important role than PAI- resistance in sustaininrz” lvsis of . platelet rich thrombi.
33% of the cell extracts is released upon addition of thrombin. A positive correlation between the amount of acutely releasable tPA and constitutive tPA secretion is found (r=0.68, n=17). When sodium butyrate is used to increase tPA synthesis, the constitutive tPA secretion increases 20-fold and the acute release of tPA increases 18fold. These in vitro data agree with in vivo data in the rat: with retinoic acid and cholera toxin the plasma tPA levels increase 1.4-fold and 5.4-fold. resp. and the acute release of tPA is increased then 1.9-fold and 3.7-fold, resp. These results suggest that the amount of tPA which is stored and acutely released in vitro and in vivo, closely correlates with the constitutive secretion (synthesis) of tPA. (Supported by grant 90.75 from the Dutch Heart Foundation).
POSTER PRESENTATIONS
P-9: Regulation of Fibrinolysis
312 EFFECT OF PLATELET ON CLOT LYSIS BY UROKINASE-TYPE PLASMINOGEN ACTIVATOER Yuasa H. Okada K, Matsuo 0. Dept. of Physiology, Kinki University School of Medicine, Osakasayama, Japan. Resistance of a platelet-rich thrombus to lysis by plasminogen activators limits the effectiveness of thrombolytic therapy for thromboembolic diseases. Arterial thrombi contain a large number of platelet in which active plasminogen activator inhibitor-l (PAI-1) is contained. Since platelet expresses plasminogen binding site on its surface, potential role of Blood platelet in lysis of thrombi induced was investigated. was collected by veinpuncture from healthy volanteer donors into acid citrate anticoagulant. The platelet-rich plasma (PRP) and the platelet-poor plasma (PPP) were prepared by centrifugation of titrated whole blood. The washed-out platelet was obtained by gel-filtration of PRP. Clot produced by plasminogen containing fibrinogen and platelet, or by PRP or PPP, reacted by thrombin/CaC12 and urokinase-type
INCREASING THE FIBRIN SPECIFICITY OF t-PA ENHANCES ITS ACTIVITY TOWARDS PLATELET RICH CLOTS Canio J. Refino, Julie M. Badillo. Nicholas F. Paoni. Bruce A. Keyt. Hung V. Neuven. and William F. Bennett. Genentech Inc., South San Francisco, CA. USA We recently demonstrated that variants of t-PA with increased fibrin specificity and resistance to PAI-I inhibition had greater potency towards platelet enriched plasma (PEP) clots relative to whole blood clots in vivo (Thromh Haemosrus 1993; 70(2) & PNAS 1994 in press). To further evaluate one t-PA variant (T103N, N117Q, KHRR 296-299 AAAA or TNK) and examine the role of platelets in clot lysis, we compared TNK to t-PA in a series of experiments. Human platelet poor plasma (PPP) with 125I fibrinogen, *platelets, or @AI-I was aliqouted into microtiter plate wells following Ca++ and thrombin addition. After 1 hour at 37°C PEP clots had retracted to 25% of the PPP clots diameter. The clots then had an overlay of PPP containing t-PA or tPA variants added to the wells. Clot Lysis was quantitated by the release of tz51 FDPS to the fluid phase at various times (1150 minutes). Lysis of PPP clots induced by t-PA
314 THE AMOUNT OF ACUTELY
RELEASABLE TISSUETYPE PLASMINOGEN ACTIVATOR (tPA) CLOSELY CORRELATES WITH THE RATE OF CONSTITUTIVE tPA SECRETION. Schrauwen Y, de Vries REM, Kooistra T and Emeis JJ Gaubius Laboratory, TNO-PG, Leiden, The Netherlands.
tPA can be secreted into the circulation from the endothelium by both a constitutive and a regulated pathway (acute release). The latter process occurs only following endothelial cell stimulation, and gives rise to very rapid (local) increases in tPA concentration; it is thought important in speeding up thrombolysis. We have developed a very sensitive ELISA for tPA antigen to study acute release of tPA from human endothelial cells in culture. Upon addition of thrombin active tPA (range: lo-200 pg/2.10s endothelial cells) is rapidly released (maximal release within one minute) into the medium and disappears concomitantly from an intracellular source. On average,
plasminogen activator (u-PA). The clot lysis assay was performed on 96-well micro plate. In order to express u-PA activity in this assay, the time to reach half of the maximum OD (T1/2) of was measured. In the lysis of fibrin clot and PRP clot, Tl/2 was significant prolonged in the presence of various concentrations of platelet in a dose-dependent manner. Although the plasminogen antigen was 75 pg/ 4.5 X lo6 platelets, the activated plasminogen on platelet by U-PA was 0.77rg/ 2.88 X lo6 platelets. Further, the anti-plasminogen IgG had no effect on the fibrin clot lysis, indicating that plasminogen bound on platelets exhibit& little effect on clot lysis. In the lysis of fibrin clot and PRP clot, Tl/2 was significant shortened in the presence of anti-PAIIgG in a dose-dependent manner. Activation of exogenous plasminogen by u-PA was significantly enhanced on platelet. These data suggest that the effect of platelet on clot lysis was not due to plasminogen but PAI- in platelet.
or TNK at plasma concentrations of 0.3 to 9.0 ug/ml were dose dependent and identical for comparable concentrations of the two reagents. PPP clots spiked with active PAI-1, required 6 fold higher concentrations of PAI- to inhibit TNK vs t-PA induced lysis by 50%. With PEP clots, lysis produced by both activators in the concentration range 0.3 to 1 ug/ml were similarly depressed (-50%) compared to PPP clots. At higher activator concentrations (>3ug/ml), there was a paradoxical decrease in the lysis of the t-PA treated clots. In contrast, TNK treated clot lysis continued to increase in a dose dependent fashion. Assay of the plasma phases showed that there was considerable plasminogen depletion in both PPP and PEP wells at high concentrations of t-PA but not TNK. These data suggest that PEP clots are more susceptible than PPP clots to the plasminogen steal phenomena and that at high pharmacological concentrations of t-PA (as might be encountered during front loaded or bolus regimens), increased fibrin specificity may play a more important role than PAI- resistance in sustaininrz” lvsis of . platelet rich thrombi.
33% of the cell extracts is released upon addition of thrombin. A positive correlation between the amount of acutely releasable tPA and constitutive tPA secretion is found (r=0.68, n=17). When sodium butyrate is used to increase tPA synthesis, the constitutive tPA secretion increases 20-fold and the acute release of tPA increases 18fold. These in vitro data agree with in vivo data in the rat: with retinoic acid and cholera toxin the plasma tPA levels increase 1.4-fold and 5.4-fold. resp. and the acute release of tPA is increased then 1.9-fold and 3.7-fold, resp. These results suggest that the amount of tPA which is stored and acutely released in vitro and in vivo, closely correlates with the constitutive secretion (synthesis) of tPA. (Supported by grant 90.75 from the Dutch Heart Foundation).