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In vitro and in vivo penetration of experimentally produced clots by monoclonal antibodies
68
POSTER
PRESENTATIONS
187 IN
VITRO
AND
IN
VW0
P-6: Experimental
Animal
PENETRATION
OF
EXPERIMENTALLY PRODUCED CLOTS BY MONOCLONAL ANTIBODIES ’ McEvoy F J, Edaell T A, Webbon P M and ‘Gaffnev PJ Royal Veterinary College, Hawkeshead Lane, Hatfield, AL9 2TA, UK, *National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QG, UK Antifibrin monoclonal antibodies show potential as clot targeting agents for diagnosis and possibly therapy in thrombotic disease. To be effective the antibody must bind to the fibrin component of the clot. The ability of the antifibrin mab 1HlO to penetrate clots in viva was investigated. Two heterologous thrombus models were used, in sheep and rabbits. In each case human fibrinogen was mixed with the animal’s own blood and clotted in an isolated vein segment using bovine thrombin. The clots in sheep were observed radiographically and found to be occlusive for a mean of 4.2 * 1 .l days and thereafter appeared
188 CHANGES IN FIBRINOLYSIS,
SEROTONIN AND PLATELET AGGREGATION IN RATS INDUCED BY FOOT SHOCK AND RESTRAINT STRESS. Takada A. Malvszko J. Urano T. Ihara H and Department of Physiology, Takada Y Hamamatsu University School of Medicine, Hamamatsu, JAPAN 431-31 Foot shock given to rats induced inhibition of Euglobulin lysis time (ELT) fibrinolysis. prolonged soon after the application of .shock due to decrease in t-PA activity and increase in PAI activity. ELT returned to normal after Foot shock induced a decline in 24 hours. platelet blood whole induced collagen caused stress Restraint aggregation. expressed by fibrinolysis enhanced shortened ELT due to increase in t-PA and
189
EFFECI-S OF PAI- INHIBITION ON CLOT-LYSIS IN EkDOTOXIN-TREATED RATS Abrahamsson T, Nerme V, Akerblom B, Leznehed A, Westin Eriksson A, Cardiovascular Pharmacology, Astra HBssle AB, S451 83 Miilndal, Sweden. The aim of this study was to investigate the effect of PAI- inhibition on clot-lysis in endotoxin-treated anesthetised rats. The PAIinhibitor studied was PRAP-7, a Fab-fragment of a polyclonal antibody against human PAI-1. The potency of PRAP-I to inhibit rat PAI- in vitro was measured from the effect of the antibody on plasma PAI- activity, using the chromogenic substrate assay SPECTROLYSFU/fibrin. It was shown that rat PAI- activity could be determined in a reliable way using this assay, although developed for human plasma and PAI-1. When added to rat plasma, PRAP-1 decreased rat PAI-I activity with a pIC50-vaiue (-log konc, M) of 6.6 + 0.15 (mean + SD, n = 5). The corresponding value for inhibition of I’AI-1 activity in human plasma was 7.8 f 0.42 (n = 2). Clot formation and lysis were studied in viva by giving the coagulant enzyme batroxobin (20 U/kg) intravenously (iv) to anesthetised rats, pretreated with endotoxin (10 kg/k iv, 2.5 h before bahoxobin), or to untreated animals. In addition, l&-fibrinogen was given iv just prior to batroxobin and the 125 I-concentration was then measured in
Studies
partially occlusive. When targeted in their occlusive phase 13’1 labelled mab accumulated in the clot reaching a maximum ratio of 1.8 + 0.42 when compared to counts in homologous sheep clots in the contralateral limb. In an attempt to confirm that total occlusivity would not prevent antibody access to the clot, heterologous clots were created in occluded rabbit jugular veins. The mab 1 HlO was administered via a peripheral vein and the clots were recovered 1, 6 and 24 hours later. These were examined by immunofluorescence for the presence of mab and in a further series of similar experiments lz51 labelled mab 1HlO was used and detected using autoradiography. Both sets of experiment indicated that penetration of occlusive clots occurred at one hour and that considerable accumulation was present at 6 and 24 hours. The results suggest that circulating antibody can readily gain access to experimentally produced clots in occluded veins.
ELT prolonged decrease in PA1 activity. due to decreased fibrinolytic activity 5 hrs aggregation later. Whole blood platelet by restraint seemed not to be influenced Serotonin (SHT) level, a marker of a stress. severity of stress, increased after footshock application with a concomitant rise in SHIAA, After restraint stress, its major metabolite. both SHT and SHIAA did not differ from Those results show that two kinds controls. stress influence fibrinolysis and of the serotonergic system in different ways. the These may contribute to changes development of atherosclerosis and thrombosis observed after different kinds of stress.
plasma (repeated sampling), as well as in the lung and kidney (30 min after batroxobin). Moreover, an immunohistochemical technique was used to demonstrate the occurence of fibrin-clots in the lungs, and the density of the clots was determined by morphometri. Endotoxin treatment caused an increase in plasma PAI- activity from 15 + 5 to 214 f 83 U/ml (after 3 h), (n = IO). In these rats, as well as in control animals, batroxobin produced a marked fibrin deposition in the lungs. The fibrin-clots were rapidly lysed in control animals, while clot-lysis was markedly attenuated in the endotoxin-treated rats. This inhibition of fibrinolysis was dosedependently antagonized by PRAP-1 (iv bolus + infusion: 600 pg/kg + 120 pg/kg/min; 1200 pg/kg + 240 pg/kg/min). The high dose of the antibody dccrcascd the occurence of fibrin-clots in the lungs from 74 i 26 to 23 + 20% of the visual fields counted, p
POSTER
PRESENTATIONS
187 IN
VITRO
AND
IN
VW0
P-6: Experimental
Animal
PENETRATION
OF
EXPERIMENTALLY PRODUCED CLOTS BY MONOCLONAL ANTIBODIES ’ McEvoy F J, Edaell T A, Webbon P M and ‘Gaffnev PJ Royal Veterinary College, Hawkeshead Lane, Hatfield, AL9 2TA, UK, *National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QG, UK Antifibrin monoclonal antibodies show potential as clot targeting agents for diagnosis and possibly therapy in thrombotic disease. To be effective the antibody must bind to the fibrin component of the clot. The ability of the antifibrin mab 1HlO to penetrate clots in viva was investigated. Two heterologous thrombus models were used, in sheep and rabbits. In each case human fibrinogen was mixed with the animal’s own blood and clotted in an isolated vein segment using bovine thrombin. The clots in sheep were observed radiographically and found to be occlusive for a mean of 4.2 * 1 .l days and thereafter appeared
188 CHANGES IN FIBRINOLYSIS,
SEROTONIN AND PLATELET AGGREGATION IN RATS INDUCED BY FOOT SHOCK AND RESTRAINT STRESS. Takada A. Malvszko J. Urano T. Ihara H and Department of Physiology, Takada Y Hamamatsu University School of Medicine, Hamamatsu, JAPAN 431-31 Foot shock given to rats induced inhibition of Euglobulin lysis time (ELT) fibrinolysis. prolonged soon after the application of .shock due to decrease in t-PA activity and increase in PAI activity. ELT returned to normal after Foot shock induced a decline in 24 hours. platelet blood whole induced collagen caused stress Restraint aggregation. expressed by fibrinolysis enhanced shortened ELT due to increase in t-PA and
189
EFFECI-S OF PAI- INHIBITION ON CLOT-LYSIS IN EkDOTOXIN-TREATED RATS Abrahamsson T, Nerme V, Akerblom B, Leznehed A, Westin Eriksson A, Cardiovascular Pharmacology, Astra HBssle AB, S451 83 Miilndal, Sweden. The aim of this study was to investigate the effect of PAI- inhibition on clot-lysis in endotoxin-treated anesthetised rats. The PAIinhibitor studied was PRAP-7, a Fab-fragment of a polyclonal antibody against human PAI-1. The potency of PRAP-I to inhibit rat PAI- in vitro was measured from the effect of the antibody on plasma PAI- activity, using the chromogenic substrate assay SPECTROLYSFU/fibrin. It was shown that rat PAI- activity could be determined in a reliable way using this assay, although developed for human plasma and PAI-1. When added to rat plasma, PRAP-1 decreased rat PAI-I activity with a pIC50-vaiue (-log konc, M) of 6.6 + 0.15 (mean + SD, n = 5). The corresponding value for inhibition of I’AI-1 activity in human plasma was 7.8 f 0.42 (n = 2). Clot formation and lysis were studied in viva by giving the coagulant enzyme batroxobin (20 U/kg) intravenously (iv) to anesthetised rats, pretreated with endotoxin (10 kg/k iv, 2.5 h before bahoxobin), or to untreated animals. In addition, l&-fibrinogen was given iv just prior to batroxobin and the 125 I-concentration was then measured in
Studies
partially occlusive. When targeted in their occlusive phase 13’1 labelled mab accumulated in the clot reaching a maximum ratio of 1.8 + 0.42 when compared to counts in homologous sheep clots in the contralateral limb. In an attempt to confirm that total occlusivity would not prevent antibody access to the clot, heterologous clots were created in occluded rabbit jugular veins. The mab 1 HlO was administered via a peripheral vein and the clots were recovered 1, 6 and 24 hours later. These were examined by immunofluorescence for the presence of mab and in a further series of similar experiments lz51 labelled mab 1HlO was used and detected using autoradiography. Both sets of experiment indicated that penetration of occlusive clots occurred at one hour and that considerable accumulation was present at 6 and 24 hours. The results suggest that circulating antibody can readily gain access to experimentally produced clots in occluded veins.
ELT prolonged decrease in PA1 activity. due to decreased fibrinolytic activity 5 hrs aggregation later. Whole blood platelet by restraint seemed not to be influenced Serotonin (SHT) level, a marker of a stress. severity of stress, increased after footshock application with a concomitant rise in SHIAA, After restraint stress, its major metabolite. both SHT and SHIAA did not differ from Those results show that two kinds controls. stress influence fibrinolysis and of the serotonergic system in different ways. the These may contribute to changes development of atherosclerosis and thrombosis observed after different kinds of stress.
plasma (repeated sampling), as well as in the lung and kidney (30 min after batroxobin). Moreover, an immunohistochemical technique was used to demonstrate the occurence of fibrin-clots in the lungs, and the density of the clots was determined by morphometri. Endotoxin treatment caused an increase in plasma PAI- activity from 15 + 5 to 214 f 83 U/ml (after 3 h), (n = IO). In these rats, as well as in control animals, batroxobin produced a marked fibrin deposition in the lungs. The fibrin-clots were rapidly lysed in control animals, while clot-lysis was markedly attenuated in the endotoxin-treated rats. This inhibition of fibrinolysis was dosedependently antagonized by PRAP-1 (iv bolus + infusion: 600 pg/kg + 120 pg/kg/min; 1200 pg/kg + 240 pg/kg/min). The high dose of the antibody dccrcascd the occurence of fibrin-clots in the lungs from 74 i 26 to 23 + 20% of the visual fields counted, p