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83 Thrombolytic properties of poorly immunogenic variants of recombinant staphylokinase
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ORAL PRESENTATIONS: Thrombolytic therapy
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82 Thrombolytic activity of u-PAJt-PA chimera
(K2tu-PA) evaluated with arterial type (Chandler) thrombi *COLUCCI M, *CA VALLO P, *MONTEDORO G, **MELE A, and *SEMERARO N *Dept. of Biomedical Sciences, University of Bari, and **Menarini Ricerehe, Pomezia, Italy K2tu-PA is a hybrid plasminogen activator obtained by fusion of the 2 "d kringle of t-PA to the catalytic domain of prou-PA at the plasmin cleavage site. At variance with other similar chimeras, it is resistant to proteolysis by thrombin and cannot be converted to mini-urokinase by plasmin. Moreover, in the singlechain form, it is insensitive to PAl- 1. The availability of in vitro model thrombi approaching arterial thrombi formed in vivo prompted us to evaluate the thrombolytic activity of K2tu-PA as compared to melanoma t-PA and recombinant prou-PA. Aliquots of 1 mi blood were mixed with ~25I-fibrinogen (100,000 cpm), thromboplastin (thromborel TM, 1/1000) and CaCI2 (0.025 M), and aspirated in plastic tubes (40 cm length, 0.4 cm internal diameter). The latter were sealed to form a closed loop and rotated for 60 rain at 20 rpm at room temperature. The resulting thrombi, composed ofa platelet-rich "head" and a fibrin-rich "tail", were washed and submersed in autologous plasma. Lysis experiments were carried out at 37 °C after addition of the thrombolytic agent. At high con-
centrations (0.5 and 1 lag/ml) both K2tu-PA and t-PA produced rapid and extensive thrombolysis. However, at lower concentrations (0.03-0.12 lag/ml) the hybrid PA caused markedly more extensive lysis than t-PA. For example, with the lowest concentration, clot lysis after 24 h amounted to 45% in the presence of K2tu-PA but only to 7% in the presence of t-PA. Interestingly, the thrombolytic activity of low concentrations of K2tu-PA was clearly reduced if static clots were used instead of Chandler thrombi. When rPAI-1 (2 lag/ml) was added to the plasma surrounding the thrombus, t-PA (0.5 lag/ml) induced almost negligible lysis (<5% at 4h, 20% after 24 h) whereas K2tu-PA produced considerable lysis (30% and 87% at 4 and 24 h, respectively). As compared to prou-PA, the hybrid PA induced faster and more extensive lysis under all experimental conditions. These data makes K2tu-PA a promising alternative to current thrombolytic agents.
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83 Thrombolytic properties of poorly
immunogenic variants of recombinant staphylokinase *COLLEN D, *HEYMANS S, *LAROCHE Y, **LACRO1XH, and ***STOCKX L *Centerfor Transgene Technology and Gene Therapy, Flanders lnteruniversity Institute for Biotechnology, Leuven. Belgium, **Department of Vascular Surgery, and ***Department of Radiology, University Hospitals Gasthuisberg, Leuven Belgium Selected variants of recombinant staphylokinase (SakSTAR), including SakSTAR (K74Q, E80A, D82A, K 130T, K 135R), SakSTAR(E65D, K74R, E80A, D82A, KI30T, K135R) and SakSTAR (K35A, E65D, K74Q, E80A, D82A, KI30T, KI35R) with intact specific activity and reduced reactivity with antibodies induced in patients treated with wild-type SakSTAR were studied in order to delineate their thrombolytic potency and immunogenicity. In a ~2sI-fibrin-labeled pulmonary embolism model in the hamster, the doses giving 50% clot lysis in 90 minutes were 25 lag/kg for SakSTAR, 25 lag/kg for SakSTAR (K74Q, E80A, D82A, K130T, KI35R), 32 lag/kg for SakSTAR (E65D, K74R, E80A, D82A, K130T, K135R) and 18 lag/kg for SakSTAR (K35A, E65D, K74Q, E80A, D82A, K130T, K135R). Following 100 lag/kg bo-
lus injection in hamsters, the variants were cleared from plasma at rates of 1.6 to 4. l ml/min, as compared to 2.2 ml/min for SakSTAR. Intra-arterial administration in groups of 6 patients with peripheral arterial occlusion induced significantly less circulating neutralizing antibodies after 3 to 4 weeks. The median values (15 to 85 percentile ranges), expressed as lag compound neutralized per ml plasma were 9.3 (0.1 to 19) for SakSTAR (K74Q, E80A, D82A, K130T, K135R), 0.75 (0.1 to 1.1) for SakSTAR (E65D, K74R, ESOA, D82A, K130T, KI35R) and 12 (0.9 to 43) for SakSTAR (K35A, E65D, K74Q, E80A, D82A, KI30T, K135R), as compared to 12 (4 to 100) lag/ml in 70 patients given SakSTAR. Overt neutralizing antibody induction (>5 lag compound neutralized per ml plasma) was observed in 56 of 70 patients (80%) given wild-type SakSTAR, but occurred only in 3 of the 6 SakSTAR (K74Q, E80A, D82A, K130T, K135R) patients, in l of the 6 S akSTAR (E65D, K74R, E80A, D82A, K130T, K135R) patients, and in 3 of the 6 SakSTAR (K35A, E65D, K74Q, E80A, D82A, K 130T, K 135R) patients (p < 0.01 versus SakSTAR by 2x4 Chi square analysis. On the basis of the present data, variant SakSTAR (E65D,K74R,E80A, D82A,K130T,K 135R) may constitute a preferred agent for thrombolytic therapy.
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84 Local fibrinolytic treatment in 163 patients
with acute ischemia of lower limb **LOPEZ-FERN.4NDES MF, *MARINI M, *CAO Jl, and *SOLER R Departments of *Haematology and **Radiology, Hospital Juan Canalejo, La Coruna, Spain Introduction. Surgical revascularization as the initial therapy in acute lower limb
ischemia (ALLI) is associated with a high cumulative mortality and amputation rate. Catheter directed delivery of low dose thrombolytic agents offers the possibility for revascularization with a minimum of stress for the patients. The aim of this study is to evaluate the results of the intra-arterial fibrinolytic treatment in ALLI and compare the results in native or bypass graft arteries. Material and methods, 163 patients with ALLI (maximal duration of symptoms 7 days; mean 36.84 hours) were included in this study. The arteries affected were 119 native arteries, 40 bypass grafts and 4 homografts. The clinical symptoms were classified as: viable (n=17), threatened (n=122) and irreversible (n=24). In all eases a basal arteriography was performed. The fibrinolytic treatment was infused through a multiperforated Katzen guide wire placed in the thrombus. A bolus of 250.000 IU of Urokinase followed by a perfusion of 100.000 IU/hour until total lysis was achieved. The patients were evaluated at the end of the treatment, and at 30 days, three months and one year after treatment. Results. Total lysis was achieved in 72.39%, partial lysis in 18.40% and no lysis in 9.20%. There were no complications in 154 patients, and were present in 8 cases:
bleeding (n=5) and sepsis (n=3). Of the patients with viable ischemia total lysis was achieved in 15 cases (88.23%), partial lysis in 2 and a clear clinical improvement in all patients. Total lysis was achieved in 81.15% (n=99) of the treatened ischemia, and partial lysis in 20 cases; the clinical improvement was detected in 102 patients. In patients with irreversible ischemia a total lysis was achieved in 16.66% (n---4),partial lysis in 8 cases and no lysis in 12. After the fibrinolytic treatment a surgical reparative procedure was performed in 38 cases, pereutaneous transluminal angioplasty in 28 cases, sympathectomy in 4 cases, amputation in 17 cases (10.43%); in 76 cases no additional treatment was needed. The follow-up at 30 days after fibrinolytic treatment revealed that 124 patients (76.07%) remained asymptomatic, 7 patients have died, three of them with rethrombosis; three patients have suffered amputation and two patients with rethrombosis were operated on. At three months retrombosis were detected in the 22.72% of the bypasses, in the 8.40% of the native arteries and in three of the four cryopreserved grafts. Conclusions. Intra-arterial fihrinolysis even without associated surgical treatment is
a very useful treatment of ALLI. Patients with viable and threatened ischemia are especially suitable of fihrinolytic treatment, with very good results in short and long time folow-up.
ORAL PRESENTATIONS: Thrombolytic therapy
/
82 Thrombolytic activity of u-PAJt-PA chimera
(K2tu-PA) evaluated with arterial type (Chandler) thrombi *COLUCCI M, *CA VALLO P, *MONTEDORO G, **MELE A, and *SEMERARO N *Dept. of Biomedical Sciences, University of Bari, and **Menarini Ricerehe, Pomezia, Italy K2tu-PA is a hybrid plasminogen activator obtained by fusion of the 2 "d kringle of t-PA to the catalytic domain of prou-PA at the plasmin cleavage site. At variance with other similar chimeras, it is resistant to proteolysis by thrombin and cannot be converted to mini-urokinase by plasmin. Moreover, in the singlechain form, it is insensitive to PAl- 1. The availability of in vitro model thrombi approaching arterial thrombi formed in vivo prompted us to evaluate the thrombolytic activity of K2tu-PA as compared to melanoma t-PA and recombinant prou-PA. Aliquots of 1 mi blood were mixed with ~25I-fibrinogen (100,000 cpm), thromboplastin (thromborel TM, 1/1000) and CaCI2 (0.025 M), and aspirated in plastic tubes (40 cm length, 0.4 cm internal diameter). The latter were sealed to form a closed loop and rotated for 60 rain at 20 rpm at room temperature. The resulting thrombi, composed ofa platelet-rich "head" and a fibrin-rich "tail", were washed and submersed in autologous plasma. Lysis experiments were carried out at 37 °C after addition of the thrombolytic agent. At high con-
centrations (0.5 and 1 lag/ml) both K2tu-PA and t-PA produced rapid and extensive thrombolysis. However, at lower concentrations (0.03-0.12 lag/ml) the hybrid PA caused markedly more extensive lysis than t-PA. For example, with the lowest concentration, clot lysis after 24 h amounted to 45% in the presence of K2tu-PA but only to 7% in the presence of t-PA. Interestingly, the thrombolytic activity of low concentrations of K2tu-PA was clearly reduced if static clots were used instead of Chandler thrombi. When rPAI-1 (2 lag/ml) was added to the plasma surrounding the thrombus, t-PA (0.5 lag/ml) induced almost negligible lysis (<5% at 4h, 20% after 24 h) whereas K2tu-PA produced considerable lysis (30% and 87% at 4 and 24 h, respectively). As compared to prou-PA, the hybrid PA induced faster and more extensive lysis under all experimental conditions. These data makes K2tu-PA a promising alternative to current thrombolytic agents.
/
83 Thrombolytic properties of poorly
immunogenic variants of recombinant staphylokinase *COLLEN D, *HEYMANS S, *LAROCHE Y, **LACRO1XH, and ***STOCKX L *Centerfor Transgene Technology and Gene Therapy, Flanders lnteruniversity Institute for Biotechnology, Leuven. Belgium, **Department of Vascular Surgery, and ***Department of Radiology, University Hospitals Gasthuisberg, Leuven Belgium Selected variants of recombinant staphylokinase (SakSTAR), including SakSTAR (K74Q, E80A, D82A, K 130T, K 135R), SakSTAR(E65D, K74R, E80A, D82A, KI30T, K135R) and SakSTAR (K35A, E65D, K74Q, E80A, D82A, KI30T, KI35R) with intact specific activity and reduced reactivity with antibodies induced in patients treated with wild-type SakSTAR were studied in order to delineate their thrombolytic potency and immunogenicity. In a ~2sI-fibrin-labeled pulmonary embolism model in the hamster, the doses giving 50% clot lysis in 90 minutes were 25 lag/kg for SakSTAR, 25 lag/kg for SakSTAR (K74Q, E80A, D82A, K130T, KI35R), 32 lag/kg for SakSTAR (E65D, K74R, E80A, D82A, K130T, K135R) and 18 lag/kg for SakSTAR (K35A, E65D, K74Q, E80A, D82A, K130T, K135R). Following 100 lag/kg bo-
lus injection in hamsters, the variants were cleared from plasma at rates of 1.6 to 4. l ml/min, as compared to 2.2 ml/min for SakSTAR. Intra-arterial administration in groups of 6 patients with peripheral arterial occlusion induced significantly less circulating neutralizing antibodies after 3 to 4 weeks. The median values (15 to 85 percentile ranges), expressed as lag compound neutralized per ml plasma were 9.3 (0.1 to 19) for SakSTAR (K74Q, E80A, D82A, K130T, K135R), 0.75 (0.1 to 1.1) for SakSTAR (E65D, K74R, ESOA, D82A, K130T, KI35R) and 12 (0.9 to 43) for SakSTAR (K35A, E65D, K74Q, E80A, D82A, KI30T, K135R), as compared to 12 (4 to 100) lag/ml in 70 patients given SakSTAR. Overt neutralizing antibody induction (>5 lag compound neutralized per ml plasma) was observed in 56 of 70 patients (80%) given wild-type SakSTAR, but occurred only in 3 of the 6 SakSTAR (K74Q, E80A, D82A, K130T, K135R) patients, in l of the 6 S akSTAR (E65D, K74R, E80A, D82A, K130T, K135R) patients, and in 3 of the 6 SakSTAR (K35A, E65D, K74Q, E80A, D82A, K 130T, K 135R) patients (p < 0.01 versus SakSTAR by 2x4 Chi square analysis. On the basis of the present data, variant SakSTAR (E65D,K74R,E80A, D82A,K130T,K 135R) may constitute a preferred agent for thrombolytic therapy.
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84 Local fibrinolytic treatment in 163 patients
with acute ischemia of lower limb **LOPEZ-FERN.4NDES MF, *MARINI M, *CAO Jl, and *SOLER R Departments of *Haematology and **Radiology, Hospital Juan Canalejo, La Coruna, Spain Introduction. Surgical revascularization as the initial therapy in acute lower limb
ischemia (ALLI) is associated with a high cumulative mortality and amputation rate. Catheter directed delivery of low dose thrombolytic agents offers the possibility for revascularization with a minimum of stress for the patients. The aim of this study is to evaluate the results of the intra-arterial fibrinolytic treatment in ALLI and compare the results in native or bypass graft arteries. Material and methods, 163 patients with ALLI (maximal duration of symptoms 7 days; mean 36.84 hours) were included in this study. The arteries affected were 119 native arteries, 40 bypass grafts and 4 homografts. The clinical symptoms were classified as: viable (n=17), threatened (n=122) and irreversible (n=24). In all eases a basal arteriography was performed. The fibrinolytic treatment was infused through a multiperforated Katzen guide wire placed in the thrombus. A bolus of 250.000 IU of Urokinase followed by a perfusion of 100.000 IU/hour until total lysis was achieved. The patients were evaluated at the end of the treatment, and at 30 days, three months and one year after treatment. Results. Total lysis was achieved in 72.39%, partial lysis in 18.40% and no lysis in 9.20%. There were no complications in 154 patients, and were present in 8 cases:
bleeding (n=5) and sepsis (n=3). Of the patients with viable ischemia total lysis was achieved in 15 cases (88.23%), partial lysis in 2 and a clear clinical improvement in all patients. Total lysis was achieved in 81.15% (n=99) of the treatened ischemia, and partial lysis in 20 cases; the clinical improvement was detected in 102 patients. In patients with irreversible ischemia a total lysis was achieved in 16.66% (n---4),partial lysis in 8 cases and no lysis in 12. After the fibrinolytic treatment a surgical reparative procedure was performed in 38 cases, pereutaneous transluminal angioplasty in 28 cases, sympathectomy in 4 cases, amputation in 17 cases (10.43%); in 76 cases no additional treatment was needed. The follow-up at 30 days after fibrinolytic treatment revealed that 124 patients (76.07%) remained asymptomatic, 7 patients have died, three of them with rethrombosis; three patients have suffered amputation and two patients with rethrombosis were operated on. At three months retrombosis were detected in the 22.72% of the bypasses, in the 8.40% of the native arteries and in three of the four cryopreserved grafts. Conclusions. Intra-arterial fihrinolysis even without associated surgical treatment is
a very useful treatment of ALLI. Patients with viable and threatened ischemia are especially suitable of fihrinolytic treatment, with very good results in short and long time folow-up.