Purification, Characterization and cDNA Cloning of a Novel Anticoagulant of the Intrinsic Pathway, (Prolixin-S), from Salivary Glands of the Blood Sucking Bug, Rhodnius prolixus

Summary

The salivary glands of the blood sucking insect, Rhodnius prolixus, have an anticoagulant, prolixin-S, which was reported as a specific inhibitor of intrinsic coagulant pathway. Prolixin-S was purified from the salivary glands extract of Rhodnius prolixus by gel filtration and anion exchange HPLC by assaying prolongation of activated partial thromboplastin time (APTT). The isolated protein specifically inhibited factor IXa-catalyzed activation of factor X in the presence of Ca²⁺ and phospholipids irrespective of the presence or absence of factor Villa. The anticoagulant factor had red color and a specific absorbance peak at 402 nm and thus it was identified as a heme protein. A Rhodnius prolixus salivary gland cDNA library was prepared, screened with an antibody against prolixin-S and its complete cDNA sequence was determined. cDNA and deduced amino acid sequences showed that prolixin-S is a novel anticoagulant of 19,922 Da, which has no sequence homology with any other anticoagulant reported so far.

• References

• 1 Waxman L, Smith DE, Arcuri KE, Vlasuk GP. Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. Science 1990; 248: 593-596
• 2 Hoffmann A, Walsmann P, Riesener G, Paintz M, Markwardt F. Isolation and characterization of a thrombin inhibitor from the tick Ixodes ricinus. Pharmazie 1991; 46: 209-212
• 3 Parker KR, Mant MJ. Effects of Tsetse (Glossina morsitans morsitans Westw) (Diptera: Glossinidae) salivary gland homogenate on coagulation and fibrinolysis. Thromb Haemost 1979; 42: 743-751
• 4 Friedrich R, Kroger B, Biajolan S, Lemaire HG, Hoffken HW, Reushen-bach P, Otte M, Dodt J. A kazal-type inhibitor with thrombin specificity from Rhodnius prolixus. J Biol Chem 1993; 268: 16216-16222
• 5 Hellmann K, Hawkins RI, Prolixin S, prolixin G. Two anticoagulants from Rhodnius prolixus STAL. Nature 1965; 207: 265-267
• 6 Ribeiro JMC, Schneider M, Guimareas JA. Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the bloodsucking bug Rhodnius prolixus. Biochem J 1995; 308: 243-249
• 7 Champagne DE, Nussenzveig RH, Ribeiro JMC. Purification, partial characterization and cloning of nitric oxide-carrying heme proteins (nitro-phorins) from salivary glands of the blood-sucking insect Rhodnius prolixus. J Biol Chem 1995; 270: 8691-8695
• 8 Chinzei Y, Azumi C, Miura K, Matsuoka H, Ando K. Changes in hemolymph proteins and their synthetic activities during development and engorgement, and after juvenile hormone treatment in the kissing bug, Rhodnius prolixus. J Insect Physiol 1994; 40: 491-499
• 9 Scott SF, Shinha D, Seaman FS, Walsh PN, Colman RW. Amidolytic assay of human factor XI in plasma: Comparison with a coagulant assay and a new rapid radioimmunoassay. Blood 1984; 63: 42-50
• 10 Nishimura H, Takeya H, Miyata T, Suehiro K, Okayama T, Niho Y, Iwana-ga S. Factor IX fukuoka: substitution of Asn⁹² by His in the second epidermal growth factor-like domain results in defective interaction with factor VUIa/X. J Biol Chem 1993; 268: 24041-24046
• 11 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-159