Effect of Purified Vipera Palestinae Hemorrhagin on Blood Coagulation and Platelet Function^[*]

Summary

Purified Vipera palestinae hemorrhagin (VPH) impairs thrombin formation, fibrinogen clottability, FSF activity, platelet clot retracting activity, ADP- and connective tissue-induced platelet aggregation and connective tissue - induced platelet ADP release. The effects of VPH became manifest or increased in intensity on incubation with the respective substrates prior to measurement of their activities. Inactivation of the VPH protease by DFP resulted in complete abolishment of the first four and partial inhibition of the last two of the above VPH activities.

Administration of VPH to guinea pigs caused widespread hemorrhages, associated with moderate hypofibrinogenemia but normal platelet count, clotting time and clot retraction. DFP-treated VPH caused hemorrhage without hypofibrinogenemia.

^* This investigation was supported by the U.S.Army through its European Research Office, Contract No. D AJA 37-69-C-0252.

• References

• 1 Flexner S, Noguchi H. Univ. Pennsylvania. M. Bull. 15: 354 1902-1903 cit. ref. 2.
• 2 Taube H. N, Essex H. E. Pathologie changes in the tissues of the dog following injections of rattle snake venom. Arch. Pathol. 24: 43 1937;
• 3 Rechnic J, Trachtenberg P, Casper J, Moroz Ch, de Vries A. Afibrinogenemia and thrombocytopenia in guinea pigs following injection of Echis colorata venom. Blood 20: 735 1962;
• 4 Djaldetti M, Joshua H, Bessler H, Rosen M, Gutglas H, de Vries A. Coagulation disturbance in the dog following Echis colorata venom inoculation. Hémostase 4: 323 1964;
• 5 Rosenfeld G. Fibrinolysis by snake venoms. Sangre (Barcelona) 9: 352 1964;
• 6 Reid H. A. Defibrination by Agkistrodon rhodostoma venom. In: Russell F. E. Animal Toxins. 323. Pergamon Press; Oxford and New York: 1967
• 7 Houssay B. A. Classification des actions des venins de serpents sur l’organisme animal. CR. Soc. Biol. 105: 308 1930;
• 8 Kellaway C. H. Animal poisons. Ann. Rev. Biochem. 8: 541 1939;
• 9 Zeller E. A. Enzymes of snake venoms and their biological significance. Advanc. Enzymol. 5: 459 1948;
• 10 Porges N. Snake venoms, their biochemistry and mode of action. Science 117: 47 1953;
• 11 Slotta K. Chemistry and biochemistry of snake venoms. Fortsch Chem. Org. Nat. 12: 406 1955;
• 12 Kaiser E, Michl H. Die Biochemie der tierischen Gifte. 1st Ed.. 134. Franz Deuticke; Wien: 1958
• 13 Maeno H, Mitsuhashi S, Sawai Y, Okonogi T. Studies on Habu snake venom: Enzymatic studies on the proteinase of Habu snake venom. Jap. J. Microbiol. 3: 131 1959;
• 14 Ohsaka A, Ikezawa H, Kondo H, Kondo S, Uchida N. Haemorrhagic activities of Habu venom and their relation to lethal toxicity, proteolytic activities and other pathological activities. Brit. J. exp. Pathol. 41: 478 1960;
• 15 Twanaga S, Omori T, Oshima G, Suzuki T. Studies on snake venoms. XVI. Demonstration of a proteinase with hemorrhagic activity in the venom of Agkistrodon halys blomhoffii. J. Biochem. (Tokyo) 57: 392 1965;
• 16 Tu A. T, Toom P. M, Ganthavorn S. Hemorrhagic and proteolytic activities of Thailand snake venoms. Biochem. Pharmacol. 16: 2125 1967;
• 17 Tu A. T, Honima M, Hong B. S. Hemorrhagic, myonecrotic, thrombotic and proteolytic activities of viper venoms. Toxicon 6: 175 1969;
• 18 Leffkowitz M. Snakes and snake bite in Israel. Dapim Refuiim 13: 309 1954;
• 19 Efrati P. Snake bite (vipera): Clinical picture and pathophysiology. Dapim Refuiim 13: 330 1954;
• 20 Efrati P, Reif L. Clinical and pathological observations on sixty-five cases of viper bite in Israel. Amer. J. trop. Med. Hyg. 2: 1085 1953;
• 21 Grotto L, Moroz Ch, de Vries A, Goldblum N. Isolation of Vipera palestinae hemorrhagin and distinction between its hemorrhagic and proteolytic activities. Biochem. biophys. Acta (Amst.) 133: 356 1967;
• 22 Jerushalmy Z, Zucker M. B. Some effects of fibrinogen degradation products (FDP) on blood platelets. Thrombos. Diathes. haemorrh. (Stuttg.) 15: 413 1966;
• 23 Loewy A. G, Dunathan K, Kriel R, Wolfinger H. L. Fibrinase. I. Purification of substrate and enzyme. J. biol. Chem. 236: 2625 1961;
• 24 Brecher G, Cronkite E. P. Morphology and enumeration of human blood platelets. J. appi. Physiol. 3: 365 1950;
• 25 Wintrobe M. M. Clinical Hematology. 6th edition. 329. Lea & Febiger; Philadelphia: 1967
• 26 Lee R. I, White P. D. A clinical study of the coagulation time of blood. Amer. J. med. Sci. 145: 495 1913;
• 27 Biggs R, Macfarlane R. G. Human blood coagulation and its disorders. Blackwell Scientific publications; Oxford: 1962
• 28 Biggs R, Douglas A. S. Thromboplastin generation test. J. clin. Path. 6: 23 1953;
• 29 Ratnoff O. B, Menzie G. A new method for the determination of fibrinogen in small samples of plasma. J. Lab. clin. Med. 37: 316 1951;
• 30 Latallo Z. S, Budzynski A. Z, Lipinski B, Kowalski E. Inhibition of thrombin and fibrin polymerization: two activities derived from plasmin digested fibrinogen. Nature (Lond.) 203: 1184 1964;
• 31 Lowry O. H, Rosebrough N. J, Farr A. L, Randall R. J. Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265 1951;
• 32 Latallo Z. S, Fletcher A. P, Alkjaersig N, Sherry S. Influence of pH, ionic strength, neutral ions and thrombin on fibrin polymerization. Amer. J. Physiol. 202: 675 1962;
• 33 Scheidegger J. J. Une micro-méthode de l’immunoélectrophorèse. Int. Arch. Allergy 7: 103 1955;
• 34 Ouchterlony O. Antigen-antibody reaction in gels: Acta path, microbiol. scand. 26: 507 1949;
• 35 Davis B. J. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404 1964;
• 36 Donnelly T. H, Laskowski M, Notley N, Scheraga H. A. Equilibria in the fibrinogen-fibrin conversion. II. Reversibility of the polymerization steps. Arch. Biochem. 56: 369 1955;
• 37 Lorand L, Rule N. G, Ong H. H, Furlanetto R, Jacobsen A, Downey J, Oner N, Bruner-Lorand J. Amine specificity in transpeptidation. Inhibition of fibrin cross-linking. Biochemistry 7: 1214 1968;
• 38 Cohen I, Djaldetti M, Karp M, de Vries A. Thrombocytosis with impaired platelet adhesiveness and platelet factor 3 availability. New Istanbul Contr. Clin. Sc. 9: 20 1966;
• 39 Hovig T. Release of platelet-aggregating substance (adenosine diphosphate) from rabbit blood platelets induced by saline ‘extract’ of tendons. Thrombos. Diathes. haemorrh. (Stuttg.) 9: 264 1963;
• 40 Haslam R. J. Role of adenosine diphosphate in the aggregation of human blood-platelets by thrombin and by fatty acids. Nature (Lond.) 202: 765 1964;
• 41 Adam H. In: Methods of Enzymatic Analysis. 573 Edit. Bergmeyer H. U. Academic Press; New York: 1963
• 42 Brons K, Gormsen J. Studies on platelet factor 3 activity. Scand. J. Haemat. 5: 300 1968;
• 43 Poplawski A, Niewiarowski S. Method for determining antiheparin activity of platelets and erythrocytes. Thrombos. Diathes. haemorrh. (Stuttg.) 13: 149 1965;
• 44 Barnhart M. I. Immunochemistry. In: Blood Clotting Enzymology. 258. Edit. Seegers W. H. Academic Press; New York and London: 1967
• 45 Meaume J. Les venins de serpents agents modificateurs de la coagulation sanguine. Toxicon 4: 25 1966;
• 46 De Vries A, Cohen I. Hemorrhagic and blood coagulation disturbing action of snake venoms. In: Recent advances in Blood Coagulation. 277. Edit. Poller L. J. A. Churchill Ltd.; 1969
• 47 Alexander B, Rimon A, Katchalski E. Action of water-insoluble trypsin derivatives on fibrinogen clottability. Thrombos. Diathes. haemorrh. (Stuttg.) 16: 507 1966;
• 48 Sanchez-Avalos J, Miller S. P. Degradation of fibrinogen by proteolytic enzymes. I. Characterization and isolation of products and their relation to fibrinolytic states. Thrombos. Diathes. haemorrh. (Stuttg.) 19: 499 1968;
• 49 Alkjaersig N, Fletcher A. P, Sherry S. Pathogenesis of the coagulation defect developing during pathological plasma proteolytic states. II. The significance, mechanism and consequences of defective fibrin polymerization. J. clin. Invest. 41: 917 1962;
• 50 Bang U. M, Fletcher A. P, Alkjaersig N, Sherry S. Pathogenesis of the coagulation defect developing during pathological plasma proteolytic states. Ill Demonstration of abnormal clot structure by electron microscopy. J. clin. Invest. 41: 935 1962;
• 51 Lipinski B, Wegrzynowicz Z, Budzynski A. Z, Kopéc M, Batallo Z. S, Kowalski E. Soluble unclottable complexes formed in the presence of fibrinogen degradation products during the fibrinogen-fibrin conversion and their potential significance in pathology. Thrombos. Diathes. haemorrh. (Stuttg.) 17: 65 1967;
• 52 Kowalski E. Fibrinogen derivatives and their biologic activities. Seminars in Hematol. 5: 45 1968;
• 53 Djaldetti M, Cohen I, Joshua H, Bessler H, Lorberbaum O, de Vries A. In vivo and in vitro inactivation of fibrin stabilizing factor by Echis colorata venom. Hémostase 5: 121 1965;
• 54 Cross M. J. Effect of fibrinogen on the aggregation of platelets by adenosine diphosphate. Thrombos. Diathes. haemorrh. (Stuttg.) 12: 524 1964;
• 55 McLean J. R, Maxwell R. E, Hertler D. Fibrinogen and adenosine diphosphate-induced aggregation of platelets. Nature (Lond.) 202: 605 1964;
• 56 Chandler A. B, Stormorken H, Solum N. O, Gaarder A. An attempt to assay the anti-Willebrandfactor. Scand. J. clin. Lab. Invest. 17 (84) 129 1965;
• 57 Solum N. O, Stormorken H. Influence of fibrinogen on the aggregation of washed human blood platelets induced by adenosine diphosphate, thrombin, collagen and adrenaline. Scand. J. clin. Lab. Invest. 17 (Suppl. 84) 170 1965;
• 58 Hovig T. The effect of various enzymes on the ultrastructure, aggregation, and clot retraction ability of rabbit blood platelets. Thrombos. Diathes. haemorrh. (Stuttg.) 13: 84 1965;
• 59 Schmid H. J, Jackson D. P, Conley C. L. Mechanism of action of thrombin on platelets. J. clin. Invest. 41: 543 1962;
• 60 Morse E. E, Jackson D. P, Conley C. L. Role of platelet fibrinogen in the reactions of platelets to thrombin. J. clin. Invest. 44: 809 1965;