Echicetin: a snake venom protein that inhibits binding of von Willebrand factor and alboaggregins to platelet glycoprotein Ib

Abstract

Echicetin, a new protein isolated from Echis carinatus venom by reverse phase and ion exchange chromatography specifically inhibited agglutination of fixed platelets induced by several platelet glycoprotein Ib (GPIb) agonists, such as bovine von Willebrand factor (vWF), alboaggregins, and human vWF in the presence of botrocetin. Unlike alboaggregins, echicetin bound to GPIb but did not induce agglutination of washed or fixed platelets. In contrast to disintegrins, it did not block adenosine 5'-diphosphate (ADP)-induced platelet aggregation in the presence of fibrinogen. The apparent molecular weight of echicetin measured on sodium dodecyl sulfate (SDS) gel electrophoresis was 26 Kd under nonreducing conditions. On reduction, echicetin showed 16 and 14-Kd subunits suggesting that the molecule is a dimer. Reduced echicetin retained its binding activity and its inhibitory effect on the agglutination of fixed platelets induced by bovine vWF. 125I-echicetin bound to fixed platelets with high affinity (kd = 30 +/- 1.8 nmol/L) at 45,000 +/- 2,400 binding sites per platelet. The binding was selectively inhibited by a monoclonal antibody to the 45-Kd N-terminal domain of platelet GPIb, but not by monoclonal antibodies to other regions on GPIb. Binding of 125I-bovine vWF to fixed platelets was strongly inhibited by echicetin. In contrast, bovine vWF showed a much weaker inhibitory activity on binding of 125I-echicetin to platelets. The half life of echicetin in blood was approximately 170 minutes with no detectable degradation. Echicetin significantly prolonged the bleeding time of mice, suggesting that it may inhibit vWF binding to GPIb in vivo as well as in vitro.

Pharmacological characterization and antithrombotic effect of agkistin, a platelet glycoprotein Ib antagonist

“…In contrast to crotalin, intravenous administration of agkistin reduced platelet counts (Figure 7). However, this result is consistent with the previous reports that echicetin and jajaraca GPIb‐BP caused transient thrombocytopenia in mice ( Peng et al ., 1993 ; Fujimura et a ., 1995 ). At present, the tendency of these GPIb antagonists in causing thrombocytopenia is a precaution to be taken, however, the exact mechanism remains to be investigated.…”

“…The protein standards used in order of decreasing molecular mass were phosphorylase b (97 kDa), bovine serum albumin (66.2 kDa) ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa) and lysozyme (14 kDa). (B) N‐terminal amino acid sequences of the α‐ and β‐subunits of agkistin compared to those of other reported GPIb‐binding proteins including echicetin ( Peng et al ., 1993 ), agkicetin ( Chen & Tsai, 1995 ) and tokaracetin ( Kawasaki et al ., 1995 ). Single‐letter amino acid code was used.…”

“…On the other hand, snake venoms also contain many components that modulate the GPIb‐vWF axis ( Fujimura et al ., 1996 ). They belong to a member of the C‐type lectin family and fall into three categoies: (1) botrocetin ( Read et al ., 1989 ) and bitiscetin ( Hamako & Marsui, 1996 ) which bind to the A1 domain of vWF, changing its conformation so that it is able to interact with GPIbα; (2) echicetin ( Peng et al ., 1993 ) and many similar proteins ( Fujimura et al ., 1995 ; Kawasaki et al ., 1995 ; Taniuchi and Kawasaki, 1995 ) that bind to GPIbα and block its interaction with vWF and (3) alboaggregin A and B ( Peng et al ., 1991 ) which induce platelet activation by binding to and clustering GPIb. In this study, we characterize a member of C‐type lectin venom protein isolated from Formosan Agkistrodon acutus , called agkistin ( Huang & Yeh, 1994 ) in modulating the interactions of vWF and thrombin toward platelet GPIb by acting as a GPIb antagonist, and show its antithrombotic activity in vivo in a murine experimental model.…”

Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus macrops and T. hageni)

“…Cysteine-rich secretory protein is a single-chain bioactive polypeptide identified in several organisms, including snakes [40], reptiles [41], and mammals [42]. Snake venom cysteine-rich secretory protein can block smooth muscle contraction by blocking l-type calcium channels [43].…”

Tokaracetin, a new platelet antagonist that binds to platelet glycoprotein ib and inhibits von Willebrand factor-dependent shear-induced platelet aggregation

“…However, it also induces the agglutination of fixed human platelets without the need for any cofactor. These authors later reported that echicetin, purified from the venom of Echis carinatus, inhibits the binding of AL-B and vWF to GPIb without inducing platelet agglutination [13]. However, the effects of echicetin on h-SIPA and its N-terminal amino acid sequence have not been reported.…”