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145. Fibrinolysis and atherogenesis: the role of macrophage cathepsins
3rd International Fibrinogen Symposium
ing properties and, furthermore, be able to pass through conformational changes induced both mechanically and electrostatically. Indeed, with application of a 1.25 mmol/l [Ca2+]o solution, measuring of the 39.1 nm long, cell membrane integral proteoheparan sulfate sensor yielded a shortening of the macromolecule by 8.9 nm, whereas Na ÷ ions effected a molecular elongation by 13.7 nm. The Ca2÷induced contraction of the sensor leads to a decrease in Na ÷ binding, and the Na÷-induced extension to a promotion in Na ÷ binding. Considering the Bjerrum length for Na ÷, 12 Na ÷ ions per macromolecule are released into the blood through the Caz÷ contraction of the helical spring, while the extension of the spring caused by sodium enables the additional binding of 19 Na ÷ ions from the blood. This means that Na ÷ ions amplify the sensitivity of the flow sensor for Na ÷ in an autocatalytic process. On the other hand, Ca2÷ ions impair the sensitivity of the sensor to bind Na ÷. Since less Na ÷ ions are bound after the Ca2÷ shortening of the heparan sulfate chains, Caz÷ ions are obviously capable of adjusting the susceptibility of the sensor via a conformational change and, in this way, of controlling the signal transduction cascade that regulates the blood flow. Under pathophysiological conditions, e.g. in arteriosclerosis, the flow-dependent dilatation may be blunted or even reversed to a flow-dependent constriction. One reason for this is an impaired flow sensor function which, through calcification of certain oversulfated domains, loses its sensitivity to flow and blood sodium.
45
Subendothelial collagen type I-III is a very strong agonist of platelet-dependent thrombus formation in arteries. The antithrombotic action of rabbit polyclonal antibodies to rat collagen type I-III and their chemically synthetized conjugate with monoclonals to human recombinant twochain/one-chain urokinase type plasminogen activator (rtcuPA/rscu-PA), cross reacting with rat tcu-PA/scu-PA was studied both in vitro and in vivo. Anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombi-like structures induced by rat collagen immobilized with the polystirol surface in a condition mimics the high shear rate in the large elastic-type arteries. Monoclonals to human tcu/scu-PA did not influence the collagen-induced platelet activation in
vitro.
The short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the plateletdependent thrombus formation in the arterio-venous shunt in rats by 56+4% (P<0.001) as well as by the bispecific conjugate (44_+4%, P<0.001). The treatment of collagen-adsorbed conjugate by rtcu-PA did not increase the antithrombotic effect of bifunctional antibodies. The present data suggest that the local administration of the anticollagen antibodies at the site of vascular wall injury may be the efficient tool for the prevention of plateletdependent thrombus formation in arteries after thrombolysis or percutaneous transluminal coronary angioplasty.
143. Fibrin a n d t h r o m b i n in atherogenesis E. B. Smith
Department of Clinical Biochemistry, University of Aberdeen, Foresterhill, Aberdeen AB9 2ZD, UK It is increasingly clear that fibrin plays a major role in atherogenesis. Fibrin can be demonstrated in most atherosclerotic lesions, and in experimental studies it stimulates smooth muscle cell migration. It is also the source of fibrin degradation products (FDP), which are mitogenic. However, it is not clear to what extent the fibrin in lesions is derived from incorporation of mural thrombus, or is formed within the lesion from flbrinogen, which is invariably present. Formation of fibrin requires thrombin; we have now demonstrated free c~-thrombin in extracts of lesions, showing that the potential for fibrinformation is present. Furthermore, tx-thrombin is a potent mitogen, and modulator of cellular behaviour. FDP are also present in all plaque extracts but the amounts of immunologically detectable plasminogen/plasmin is extremely low. Incubation at pH7 of minced intima failed to generate additional FDP, and zymography failed to demonstrate a plasmin band. By contrast, incubation of minced intima at pH4 generated increased low molecular mass FDP; this was inhibited by pepstatin, suggesting that the flbrinolytic agent was cathepsin D. Experiments are now in progress to compare the mitogenicity of FDP generated by plasmin and cathepsin D.
144. Antibodies to collagen could inhibit thrombus formation in arteries I. P. Stepanova 1, G. V. Bashkov 2, S. P: D o m o g a t s k y 1
I Cardiology Research Centre and 2National Haematology Scientific Center, Russian Academy of Medical Sciences, Moscow, Russia © Pearson Professional Ltd 1996
145. Fibrinolysis and atherogenesis: the role of macrophage cathepsins C. M. Stirk 1'2, E. B. Smith 1, S. J. McNally 1, D. S. E. McCallion 2 , W. T. Melvin 2 , P. J. Gaffney 3 , W. D. Thompson 1
1Departments of Pathology, and 2Molecular and Cell Biology, University of Aberdeen, Aberdeen, AB9 2ZD, 3National Institute for Biological Standards and Control, Potters Bar EN6 3EQG, UK A variety of antibodies raised against human fibrin epitopes have been tested for immunohistochemical application to paraffin sections of various examples of acute and chronic inflammation in human tissues, and the results compared with a series of 20 coronary artery atherosclerotic plaques. Immunostaining was optimal for all antibodies using microwave heat treatment rather than trypsin for antigen exposure. DAKO rabbit polyclonal antihuman fibrinogen was taken as a current commercial standard, giving considerable background connective tissue staining. Very clear fibtin mesh staining with no background was given by the mouse monoclonal 5F3 raised against the fibrin E knot (P. Gaffney). Of the antisera raised against 6 synthetic peptides, the 2 most interesting showed a surprising specificity for fibtin only inside macrophages. The explanation offered is that these epitopes are only exposed to antibody detection as a result of fibrin digestion by the recently described non plasmin, intracellular cathepsin pathway exclusive to the macrophage. The majority of plaques showed some fibrin within foamy macrophages, but this was negative or weak on immunostaining with anti fibrins [~ 15-27 and y 54-62. This histological evidence supports the in vitro evidence that fibrin removal by macrophages in plaques is abnormal, and may relate to the admixture with oxidised lipid. Fibrinolysis :(1998) ,10, Suppl. 1, 1-58
ing properties and, furthermore, be able to pass through conformational changes induced both mechanically and electrostatically. Indeed, with application of a 1.25 mmol/l [Ca2+]o solution, measuring of the 39.1 nm long, cell membrane integral proteoheparan sulfate sensor yielded a shortening of the macromolecule by 8.9 nm, whereas Na ÷ ions effected a molecular elongation by 13.7 nm. The Ca2÷induced contraction of the sensor leads to a decrease in Na ÷ binding, and the Na÷-induced extension to a promotion in Na ÷ binding. Considering the Bjerrum length for Na ÷, 12 Na ÷ ions per macromolecule are released into the blood through the Caz÷ contraction of the helical spring, while the extension of the spring caused by sodium enables the additional binding of 19 Na ÷ ions from the blood. This means that Na ÷ ions amplify the sensitivity of the flow sensor for Na ÷ in an autocatalytic process. On the other hand, Ca2÷ ions impair the sensitivity of the sensor to bind Na ÷. Since less Na ÷ ions are bound after the Ca2÷ shortening of the heparan sulfate chains, Caz÷ ions are obviously capable of adjusting the susceptibility of the sensor via a conformational change and, in this way, of controlling the signal transduction cascade that regulates the blood flow. Under pathophysiological conditions, e.g. in arteriosclerosis, the flow-dependent dilatation may be blunted or even reversed to a flow-dependent constriction. One reason for this is an impaired flow sensor function which, through calcification of certain oversulfated domains, loses its sensitivity to flow and blood sodium.
45
Subendothelial collagen type I-III is a very strong agonist of platelet-dependent thrombus formation in arteries. The antithrombotic action of rabbit polyclonal antibodies to rat collagen type I-III and their chemically synthetized conjugate with monoclonals to human recombinant twochain/one-chain urokinase type plasminogen activator (rtcuPA/rscu-PA), cross reacting with rat tcu-PA/scu-PA was studied both in vitro and in vivo. Anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombi-like structures induced by rat collagen immobilized with the polystirol surface in a condition mimics the high shear rate in the large elastic-type arteries. Monoclonals to human tcu/scu-PA did not influence the collagen-induced platelet activation in
vitro.
The short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the plateletdependent thrombus formation in the arterio-venous shunt in rats by 56+4% (P<0.001) as well as by the bispecific conjugate (44_+4%, P<0.001). The treatment of collagen-adsorbed conjugate by rtcu-PA did not increase the antithrombotic effect of bifunctional antibodies. The present data suggest that the local administration of the anticollagen antibodies at the site of vascular wall injury may be the efficient tool for the prevention of plateletdependent thrombus formation in arteries after thrombolysis or percutaneous transluminal coronary angioplasty.
143. Fibrin a n d t h r o m b i n in atherogenesis E. B. Smith
Department of Clinical Biochemistry, University of Aberdeen, Foresterhill, Aberdeen AB9 2ZD, UK It is increasingly clear that fibrin plays a major role in atherogenesis. Fibrin can be demonstrated in most atherosclerotic lesions, and in experimental studies it stimulates smooth muscle cell migration. It is also the source of fibrin degradation products (FDP), which are mitogenic. However, it is not clear to what extent the fibrin in lesions is derived from incorporation of mural thrombus, or is formed within the lesion from flbrinogen, which is invariably present. Formation of fibrin requires thrombin; we have now demonstrated free c~-thrombin in extracts of lesions, showing that the potential for fibrinformation is present. Furthermore, tx-thrombin is a potent mitogen, and modulator of cellular behaviour. FDP are also present in all plaque extracts but the amounts of immunologically detectable plasminogen/plasmin is extremely low. Incubation at pH7 of minced intima failed to generate additional FDP, and zymography failed to demonstrate a plasmin band. By contrast, incubation of minced intima at pH4 generated increased low molecular mass FDP; this was inhibited by pepstatin, suggesting that the flbrinolytic agent was cathepsin D. Experiments are now in progress to compare the mitogenicity of FDP generated by plasmin and cathepsin D.
144. Antibodies to collagen could inhibit thrombus formation in arteries I. P. Stepanova 1, G. V. Bashkov 2, S. P: D o m o g a t s k y 1
I Cardiology Research Centre and 2National Haematology Scientific Center, Russian Academy of Medical Sciences, Moscow, Russia © Pearson Professional Ltd 1996
145. Fibrinolysis and atherogenesis: the role of macrophage cathepsins C. M. Stirk 1'2, E. B. Smith 1, S. J. McNally 1, D. S. E. McCallion 2 , W. T. Melvin 2 , P. J. Gaffney 3 , W. D. Thompson 1
1Departments of Pathology, and 2Molecular and Cell Biology, University of Aberdeen, Aberdeen, AB9 2ZD, 3National Institute for Biological Standards and Control, Potters Bar EN6 3EQG, UK A variety of antibodies raised against human fibrin epitopes have been tested for immunohistochemical application to paraffin sections of various examples of acute and chronic inflammation in human tissues, and the results compared with a series of 20 coronary artery atherosclerotic plaques. Immunostaining was optimal for all antibodies using microwave heat treatment rather than trypsin for antigen exposure. DAKO rabbit polyclonal antihuman fibrinogen was taken as a current commercial standard, giving considerable background connective tissue staining. Very clear fibtin mesh staining with no background was given by the mouse monoclonal 5F3 raised against the fibrin E knot (P. Gaffney). Of the antisera raised against 6 synthetic peptides, the 2 most interesting showed a surprising specificity for fibtin only inside macrophages. The explanation offered is that these epitopes are only exposed to antibody detection as a result of fibrin digestion by the recently described non plasmin, intracellular cathepsin pathway exclusive to the macrophage. The majority of plaques showed some fibrin within foamy macrophages, but this was negative or weak on immunostaining with anti fibrins [~ 15-27 and y 54-62. This histological evidence supports the in vitro evidence that fibrin removal by macrophages in plaques is abnormal, and may relate to the admixture with oxidised lipid. Fibrinolysis :(1998) ,10, Suppl. 1, 1-58