Purification and Characterization of Lupus Anticoagulant Like Protein from Agkistrodon Halys Brevicaudus Venom

Summary

By means of CM-Sephadex C-25, DEAE-Sephadex A-50, Sephadex G-200, and Sephadex G-75 chromatographies, a lupus anticoagulant like protein (LALP) from Agkistrodon halys brevicaudus was purified. On SDS-PAGE, the purified LALP had a molecular weight of 25,500 daltons under non-reducing condition and 15,000 daltons under reducing condition. The isoelectric point was pH 5.6. Its N terminal amino acid sequencing revealed a mixture of 2 sequences: DCP(P/S)(D/G)WSSYEGH(C/R)Q(Q/K). It was devoid of phospho-lipaseA, fibrino(geno)lytic, 5′-nucleotidase, L-amino acid oxidase, phosphomonoesterase, phosphodiesterase and thrombin-like activities, which were found in crude venom. In the presence of LALP, PT, aPTT, and dRVVT of human plasma were markedly prolonged and its effects were concentration-dependent but time-independent. The inhibitory effect of LALP on the plasma clotting time was enhanced by decreasing phospholipid concentration in TTI test. The individual clotting factor activity was not affected by LALP when higher dilutions of LALP-plasma mixture were used for assay. Russell’s viper venom time was shortened when high phospholipid confirmatory reagent was used. Therefore, the protein has lupus anticoagulant property.

• References

• 1 Seegers WH, Ouyang C. Snake and blood coagulation. In: Snake Venoms Lee CY. ed Handbook of Experimental Pharmacology. Berlin: Springer-Verlag; 1979. 52 684-750
• 2 Teng CM, Seegers WH. Agkistrodon acutus snake venom inhibits pro-thrombinase complex formation. Thromb Res 1981; 23: 255-263
• 3 Ouyang C, Teng C. Purification and properties of the anticoagulant principle of Agkistrodon acutus venom. Biochim Biophys Acta 1972; 278: 155-162
• 4 Ouyang C, Teng CM. In vivo effects of the purified thrombin-like and anticoagulant principles of Agkistrodon acutus (Hundred pace snake) venom. Toxicon 1978; 16: 583-593
• 5 Cheng HC, Ouyang C. Isolation of coagulant and anticoagulant principles from the venom of Agkistrodon acutus. Toxicon 1967; 4: 235-243
• 6 Whitaker JR. Determination of molecular weights of proteins by gel filtration on Sephadex. Anal Chem 1963; 35: 1950-1953
• 7 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 8 Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976; 72: 248-254
• 9 Tan NH, Tan CS. Acidimetric assay for phospholipase A using egg yolk suspension substrate. Analyt Biochem 1988; 170: 282-288
• 10 Ouyang C, Teng CM. Fibrinogenolytic enzymes of Trimeresurus mucro-squammatus venom. Biochim Biophys Acta 1976; 420: 298-308
• 11 Dixon TF, Purdom M. Serum 5’-Nucleotidase. J Clin Pathol 1954; 7: 341-343
• 12 Bjork W. Purification of phosphodiesterase from Bothrops atrox venom, with special consideration of the elimination of monophophatases. J Biol Chem 1963; 238: 2487-2490
• 13 Wellner D, Lichtenberg LA. Assay of amino acid oxidase. In: Methods in Enzymology Tabor H, Tabor CW. (eds) New York: Academic Press; 1971. 17 593-596
• 14 Bom GVR, Cross MJ. The aggregation of blood platelets. J Physiol (Lond) 1963; 168: 178-195
• 15 Lian EC-Y, Deykin D. Diagnosis of von Willebrand’s disease - A comparative study of diagnostic tests on nine families with von Willebrand’s disease and the differential diagnosis from hemophilia and thrombocy-topathy. Am J Med 1976; 60: 344-356
• 16 Schleider MA, Nachman RL, Jaffe EA, Coleman M. A clinical study of the lupus anticoagulant. Blood 1976; 48: 499-509
• 17 Machin SJ, Mackie IJ. Haemostasis. In: Laboratory Haematology, An Account of Laboratory Techniques Chanarin I. (ed) Churchill Livingstone; New York: 1989: 272-280
• 18 Exner E. et al Guideline for testing and revised cirteria for lupus anticoagulant. Thromb Haemost 1991; 65: 320-322
• 19 Thiagarajan P, Pengo V, Shapiro SS. The use of the dilute Russell’s viper venom time for the diagnosis of lupus anticoagulants. Blood 1986; 68: 869-874
• 20 Thiagarajan P, Shapiro SS, Demarco L. Monoclonal immunoglobulin M lambda coagulation inhibitor with phospholipid specificity: Mechanism of a lupus anticoagulant. J Clin Invest 1980; 66: 397-405
• 21 Pengo V, Thiagarajan P, Shapiro SS, Heine MJ. Immunological specificity and mechanism of action of IgG lupus anticoagulants. Blood 1987; 70: 69-76
• 22 Vermylen J, Amout J. Is the antiphopholipid syndrome caused by antibodies directed against physiologically relevant phopholipid-protein complexes?. J Lab Clin Med 1992; 120: 10-12
• 23 Oosting JD, Derksen RHWM, Entjes HTI, Bouma BN, De Groot PG. Lupus anticoagulant activity is frequently dependent on the presence of β₂ glycoprotein I. Thromb Haemost 1992; 67: 499-502