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Fibrinogen-clotting activity of proteinase H, the hemorrhagic zinc-metalloproteinase from Crotalus adamanteus (eastern diamondback rattlesnake) venom
322
Abstracts
with a mol. wt of 34,000 by SDS-PAGE and its isoelectric point was 5.8. The enzyme acted on fibrinogen to form fibrin and specific to chromogenic substrate, T1637. This enzyme contained abundant asparagine or aspartic acid, and there was little tyrosine or methionine. The protease showed higher activity toward TAME than thrombin but had no caseinolytic activity. Enzyme activity was strongly inhibited by PMSF, moderately by benzamidine and soybean trypsin inhibitor, indicating that it is a serine protease inhibitor. The 1.5 kb cDNA for the enzyme was cloned from a lZAP cDNA library derived from the venom glands. The amino acid sequence of the enzyme exhibited significant homology to those of mammalian serine proteases (trypsin, pancreatic kallikrein and thrombin) and other thrombin-like enzymes such as ancrod, batroxobin and flavoxobin. Supported
by a Genetic
Engineering
Grant
from the Ministry
of Education.
Fibrolase, an effective thrombolytic agent in arterial and venous thrombosis animal models. F. S. Markland (Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, Los Angeles, CA 90033, U.S.A.). Fibrolase is a fibrinolytic metalloproteinase. of mol. wt 23,000 isolated from southern copperhead snake venom. The enzyme acts directly on fibrin clots and does not require plasminogen or any other blood-borne intermediates for activity. The enzyme is not inhibited by blood serine proteinase inhibitors. In collaboration with J. J. Bookstein, University of California, San Diego, we showed that the enzyme lyses acute clots in the renal arteries of rabbits and subacute clots in rabbit iliac veins. There was no histological or gross evidence of hemorrhaging and no side-effects were observed. Recently, in collaboration with B. R. Lucchesi, University of Michigan, we investigated the efficacy of recombinant fibrolase to lyse a thrombus formed in the carotid artery of the dog. Five anesthetized dogs were instrumented for the measurement of systemic pressure and right/left carotid artery blood flow velocity. Electrolytic injury was initiated in both the right and left carotid artery. Thirty minutes after both arteries were occluded, fibrolase (4 mg/kg in 3 ml) was infused proximal to the thrombus in the left carotid artery only. Physiological saline (identical rate as fibrolase infusion) was infused proximal to the thrombus in the right carotid artery, simultaneously. In the artery infused with fibrolase, five out of five dogs exhibited reflow within 6 + 1 min of infusion (P < 0.05) vs vehicle-treated artery (Fisher’s Exact test). In the corresponding arteries that received vehicle, the occlusion was maintained in each dog throughout the experimental protocol. Five minutes after the end of fibrolase infusion, a GPIIb/IIIa antibody, 7E3 (0.8 mg/kg i.v.), was administered to prevent reocclusion of the patent artery. In four out of five dogs the vessel treated with fibrolase followed by 7E3 administration remained patent for the entire 2 hr observation period. Our findings indicate that the administration of fibrolase at the site of an occlusive arterial or venous thrombus can achieve rapid thrombolysis and restoration of blood flow. In the carotid arterial model, rethrombosis occurs in the absence of adjunctive therapy and can be prevented by the administration of a platelet glycoprotein IIb/IIIa receptor antibody, 7E3. Supported CA.
in part by NIH Grant
#HL31389.
Recombinant
fibrolase
was provided
by Chiron
Corp.,
Emeryville,
Fibrinogen-clotting activity of proteinase H, the hemorrhagic zinc-metalloproteinase from Crotalus adamanteus (eastern diamondback rattlesnake) venom. S. G. Anderson and C. L. Ownby (Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A.). Proteinase H, the sole hemorrhagic factor in the venom of the eastern diamondback rattlesnake, was first isolated from crude venom by Kurecki and Kress in 1985. In the current study, proteinase H was isolated in a simplification of the method published by Kurecki and Kress and was found to be a fibrinogen-clotting enzyme. To our knowledge this is the first time such activity has been reported for a snake venom hemorrhagin. The fibrinogen-clotting activity of proteinase H was quantified by monitoring the increase in turbidity of bovine fibrinogen in aqueous solution at 445 nm. The effects of EDTA, PMSF and deglycosylation with PNGaseF on the fibrinogen-clotting activity of proteinase H were investigated. Fibrinopeptide release was determined by RP-HPLC and the structure of fibrin clots formed by proteinase H treatment were investigated by transmission electron microscopy. The results of this study indicate that proteinase H is a fibrinogen-clotting enzyme which causes the polymerization of bovine fibrinogen into fibrin strands that are similar in appearance to those formed by treatment with thrombin. Kurecki,
T. and Kress,
L. F. (1985) Toxicon 23, 657.
Abstracts
with a mol. wt of 34,000 by SDS-PAGE and its isoelectric point was 5.8. The enzyme acted on fibrinogen to form fibrin and specific to chromogenic substrate, T1637. This enzyme contained abundant asparagine or aspartic acid, and there was little tyrosine or methionine. The protease showed higher activity toward TAME than thrombin but had no caseinolytic activity. Enzyme activity was strongly inhibited by PMSF, moderately by benzamidine and soybean trypsin inhibitor, indicating that it is a serine protease inhibitor. The 1.5 kb cDNA for the enzyme was cloned from a lZAP cDNA library derived from the venom glands. The amino acid sequence of the enzyme exhibited significant homology to those of mammalian serine proteases (trypsin, pancreatic kallikrein and thrombin) and other thrombin-like enzymes such as ancrod, batroxobin and flavoxobin. Supported
by a Genetic
Engineering
Grant
from the Ministry
of Education.
Fibrolase, an effective thrombolytic agent in arterial and venous thrombosis animal models. F. S. Markland (Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, Los Angeles, CA 90033, U.S.A.). Fibrolase is a fibrinolytic metalloproteinase. of mol. wt 23,000 isolated from southern copperhead snake venom. The enzyme acts directly on fibrin clots and does not require plasminogen or any other blood-borne intermediates for activity. The enzyme is not inhibited by blood serine proteinase inhibitors. In collaboration with J. J. Bookstein, University of California, San Diego, we showed that the enzyme lyses acute clots in the renal arteries of rabbits and subacute clots in rabbit iliac veins. There was no histological or gross evidence of hemorrhaging and no side-effects were observed. Recently, in collaboration with B. R. Lucchesi, University of Michigan, we investigated the efficacy of recombinant fibrolase to lyse a thrombus formed in the carotid artery of the dog. Five anesthetized dogs were instrumented for the measurement of systemic pressure and right/left carotid artery blood flow velocity. Electrolytic injury was initiated in both the right and left carotid artery. Thirty minutes after both arteries were occluded, fibrolase (4 mg/kg in 3 ml) was infused proximal to the thrombus in the left carotid artery only. Physiological saline (identical rate as fibrolase infusion) was infused proximal to the thrombus in the right carotid artery, simultaneously. In the artery infused with fibrolase, five out of five dogs exhibited reflow within 6 + 1 min of infusion (P < 0.05) vs vehicle-treated artery (Fisher’s Exact test). In the corresponding arteries that received vehicle, the occlusion was maintained in each dog throughout the experimental protocol. Five minutes after the end of fibrolase infusion, a GPIIb/IIIa antibody, 7E3 (0.8 mg/kg i.v.), was administered to prevent reocclusion of the patent artery. In four out of five dogs the vessel treated with fibrolase followed by 7E3 administration remained patent for the entire 2 hr observation period. Our findings indicate that the administration of fibrolase at the site of an occlusive arterial or venous thrombus can achieve rapid thrombolysis and restoration of blood flow. In the carotid arterial model, rethrombosis occurs in the absence of adjunctive therapy and can be prevented by the administration of a platelet glycoprotein IIb/IIIa receptor antibody, 7E3. Supported CA.
in part by NIH Grant
#HL31389.
Recombinant
fibrolase
was provided
by Chiron
Corp.,
Emeryville,
Fibrinogen-clotting activity of proteinase H, the hemorrhagic zinc-metalloproteinase from Crotalus adamanteus (eastern diamondback rattlesnake) venom. S. G. Anderson and C. L. Ownby (Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A.). Proteinase H, the sole hemorrhagic factor in the venom of the eastern diamondback rattlesnake, was first isolated from crude venom by Kurecki and Kress in 1985. In the current study, proteinase H was isolated in a simplification of the method published by Kurecki and Kress and was found to be a fibrinogen-clotting enzyme. To our knowledge this is the first time such activity has been reported for a snake venom hemorrhagin. The fibrinogen-clotting activity of proteinase H was quantified by monitoring the increase in turbidity of bovine fibrinogen in aqueous solution at 445 nm. The effects of EDTA, PMSF and deglycosylation with PNGaseF on the fibrinogen-clotting activity of proteinase H were investigated. Fibrinopeptide release was determined by RP-HPLC and the structure of fibrin clots formed by proteinase H treatment were investigated by transmission electron microscopy. The results of this study indicate that proteinase H is a fibrinogen-clotting enzyme which causes the polymerization of bovine fibrinogen into fibrin strands that are similar in appearance to those formed by treatment with thrombin. Kurecki,
T. and Kress,
L. F. (1985) Toxicon 23, 657.