Bothrops protease A, a unique highly glycosylated serine proteinase, is a potent, specific fibrinogenolytic agent.
BACKGROUND
The hemostatic system is the major target of snake venom serine proteinases (SVSPs) that act on substrates of the coagulation, fibrinolytic and kallikrein-kinin systems. Bothrops protease A (BPA), the most glycosylated SVSP, is a non-coagulant, thermostable enzyme. A cDNA encoding BPA showed that the protein has a calculated molecular mass of 25 409 Da, implying that approximately 62% of its molecular mass as assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis (67 kDa) is due to carbohydrate moieties.


RESULTS
Here we show that BPA is a potent fibrinogenolytic agent in vitro, as it readily degraded human and rat fibrinogen at a very low enzyme concentration. Partially N-deglycosylated BPA (p-N-d-BPA) generated similar fibrinogen products, but with enhanced fibrinogenolytic activity. In vivo, injection of 0.75 nmoles of BPA in rats completely avoided thrombus formation induced by stasis in the vena cava, or by endothelium injury in the jugular vein. Moreover, it decreased the fibrinogen plasma level and prolonged the recalcification time. Cleavage of fibrinogen in human and rat plasma was observed with native BPA and p-N-d-BPA by electrophoresis followed by western blot using an anti-fibrinogen antibody. BPA did not cause unspecific degradation of plasma proteins and did not cleave isolated albumin, vitronectin and fibronectin at the same concentration used with fibrinogen. Serine proteinase inhibitors failed to inhibit BPA, probably due to steric hindrance caused by its huge carbohydrate moieties.


CONCLUSIONS
To the best of our knowledge, this investigation underscores a new, thermostable, specific defibrinogenating agent that may have an application in the prevention of thrombus formation.
Paes Leme
A F
AF
Laboratório Especial de Toxinologia Aplicada, CAT-CEPID, Instituto Butantan, São Paulo, Brazil.
Prezoto
B C
BC
Yamashiro
E T
ET
Bertholim
L
L
Tashima
A K
AK
Klitzke
C F
CF
Camargo
A C M
AC
Serrano
S M T
SM
D000818:Animals; D005340:Fibrinogen / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000494:pharmacology*; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012910:Snake Venoms / Q000737:chemistry / Q000201:enzymology* / Q000494:pharmacology*; D013379:Substrate Specificity; D013927:Thrombosis / Q000517:prevention & control
D005343:Fibrinolytic Agents; D015842:Serine Proteinase Inhibitors; D012910:Snake Venoms; D005340:Fibrinogen; C038929:Bothrops jararaca protease A; D012697:Serine Endopeptidases