Isolation and characterization of an anticoagulant protein homologous to botrocetin from the venom of Bothrops jararaca

Abstract

We previously isolated a unique anticoagulant protein named IX/X-bp (factor IX/factor X-binding protein) from the venom of the habu snake Trimeresurus flavoviridis. We recently determined its primary structure and found that this protein had a structure homologous to the carbohydrate-recognition domains of C-type lectins. Most interestingly, a high homology was found between this protein and botrocetin, an inducer of platelet agglutination found in the venom of the jararaca snake Bothrops jararaca. To examine the possible identity of these proteins, we searched for IX/X-bp-like protein(s) in the venom of B. jararaca. When the venom was subjected to DEAE anion-exchange chromatography, such an activity was eluted separately from that of botrocetin. This activity was purified to homogeneity and designated jararaca IX/X-bp. Jararaca IX/X-bp was a disulfide-linked heterodimer consisting of 16- and 15-kDa subunits, being structurally similar to botrocetin. The respective NH2-terminal amino acid sequences were also very similar. Jararaca IX/X-bp had no botrocetin-like activity. However, this protein did have an activity to bind to factors IX and X and protein S in a Ca(2+)-dependent fashion, that resulted in interference with coagulation, while botrocetin did not. The binding to coagulation factors appeared not to be mediated by the lectin-like activity of jararaca IX/X-bp, because a derivative of factor X free of carbohydrates retained the ability to bind. It is concluded, therefore, that the two proteins isolated from the same venom have different biological activities despite the high degree of structural similarity between them.

Isolation and Characterization of Carinactivase, a Novel Prothrombin Activator in Echis carinatus Venom with a Unique Catalytic Mechanism

“…In addition to IX/ X-bp from the habu snake venom, proteins structurally related to the regulatory subunit of CA-1 are widely distributed in viper venoms. We have identified homologues of IX/X-bp in venoms of Bothrops jararaca (27) and Deinagkistrodon acutus (28). Moreover, numerous proteins with structures very similar to IX/X-bp but with totally different pharmacological actions have been found in venoms of various Viperidae snakes, and appear to constitute a unique protein family.…”

Purification, crystallization and preliminary X-ray crystallographic analysis of agkaggregin, a C-type lectin-like protein fromAgkistrodon acutusvenom

“…It has been found that the heterodimers isolated from different snake venoms normally possess different target proteins and therefore that these heterodimers can be grouped into four classes as follows: (i) factor X/IX binding proteins (Atoda & Morita, 1989;Atoda et al, 1995;Sekiya et al, 1993;Chen & Tsai, 1996), (ii) -thrombinbinding protein/inhibitors (Zingali et al, 1993;Castro et al, 1998), (iii) vWF-binding proteins (Andrews et al, 1989;Hamako et al, 1996), and (iv) platelet GP binding proteins (Peng et al, 1991;Taniuchi et al, 1995;Andrews et al, 1996;Kawasaki et al, 1996;Fujimura et al, 1995;Peng et al, 1993;Sakurai et al, 1998). For example, of the snake-venom C-type lectin-like proteins that possess the activity of inducing platelet aggregation, convulxin (Polgar et al, 1997;Jandrot-Perrus et al, 1997), 50 kDa alboaggregin (Andrews et al, 1996) and alboaggregin-A (Peng et al, 1992;Kowalska et al, 1998) are con®rmed to be GPIb-binding proteins, while botrocetin (Fujimura et al, 1991;Sugimoto et al, 1991) and bitiscetin (Hamako et al, 1996) have the potential to bind vWF.…”

Purification, crystallization and preliminary crystallographic analysis of AHP IX-bp, a zinc ion and pH-dependent coagulation factor IX binding protein fromAgkistrodon halysPallas venom

“…Their structures are homologous to each other: a Gla domain is located at the N-terminus followed by two growth-factor domains, one activation peptide or connecting domain and one catalytic domain (Yoshitake et al, 1985;Leytus et al, 1986). A family of coagulation factor IX/X binding proteins (abbreviated IX/X-BPs) has been identi®ed in snake venoms and contains many peptides and proteins that affect haemostasis and thrombosis (Markland, 1998;Matsui et al, 2000;Andrews & Berndt, 2000;Atoda & Morita, 1989;Atoda et al, 1995Atoda et al, , 1998Sekiya et al, 1993;Chen & Tsai, 1996;Xu et al, 2000). Furthermore, this family belongs to a superfamily termed the snake-venom C-type lectin-like proteins.…”