Reversible Thrombocytopenia of Functional Platelets after Nose-Horned Viper Envenomation is Induced by a Snaclec

Abstract

Profound and transient thrombocytopenia of functional platelets without bleeding was observed in patients envenomed by Vipera a. ammodytes (Vaa). This condition was rapidly reversed by administration of F(ab)₂ fragments of immunoglobulin G targeting the whole venom, leaving platelets fully functional. To investigate the potential role of snake venom C-type lectin-like proteins (snaclecs) in this process, Vaa-snaclecs were isolated from the crude venom using different liquid chromatographies. The purity of the isolated proteins was confirmed by Edman sequencing and mass spectrometry. The antithrombotic effect was investigated by platelet agglutination and aggregation assays and blood coagulation tests. Using flow cytometry, the platelet activation and binding of Vaa-snaclecs to various platelet receptors was analyzed. Antithrombotic efficacy was tested in vivo using a mouse model of vascular injury. Two Vaa-snaclecs were purified from the venom. One of them, Vaa-snaclec-3/2, inhibited ristocetin-induced platelet agglutination. It is a covalent heterodimer of Vaa-snaclec-3 (α-subunit) and Vaa-snaclec-2 (β-subunit). Our results suggest that Vaa-snaclec-3/2 induces platelet agglutination and consequently thrombocytopenia by binding to the platelet receptor glycoprotein Ib. Essentially, no platelet activation was observed in this process. In vivo, Vaa-snaclec-3/2 was able to protect the mouse from ferric chloride-induced carotid artery thrombosis, revealing its applicative potential in interventional angiology and cardiology.

Fig. 1

Purification of Vaa -snaclec-3/2 from the venom. Size-exclusion chromatography of the crude Vaa venom was performed on a column filled with Sephacryl S-200 superfine. Fraction B2, which inhibited ristocetin-induced platelet agglutination (traced activity), was submitted to cation-exchange chromatography on an SP Sepharose Fast Flow column. The traced activity was concentrated in fraction B, which was further analyzed by two consecutive Q-SFF anion-exchange chromatographies. Fraction A after the second Q-SFF column contained a homogeneous protein sample expressing the traced activity. Structural characterization revealed the purified protein as Vaa -snaclec-3/2. Experimental details can be found in the Materials and Methods section.

Fig. 2

Characterization of Vaa- snaclec-3/2. ( A ) RP-HPLC analysis of the purified Vaa- snaclec-3/2 on a C18 column revealed only one sharp peak. SDS-PAGE analysis of this peak (inset) under nonreducing conditions (NR) revealed a single protein with an apparent molecular mass of 24 kDa. Under reducing conditions (R), the band split into two bands. As structural characterization disclosed, these bands corresponded to α and β subunit of Vaa- snaclec-3/2, Vaa- snaclec-3, and Vaa- snaclec-2, respectively. ( B ) Inhibition of ristocetin-induced platelet agglutination by 50 nM of Vaa -snaclec-3/2.

Fig. 3

Effects of Vaa- snaclec-3/2 on platelets. ( A ) Agglutinates of two or three platelets (arrows) formed in PRP smears after 30 minute-incubation with Vaa -snaclec-3/2. ( B ) Vaa -snaclec-3/2 inhibited ristocetin-induced platelet agglutination dose-dependently. The half-maximal inhibitory concentration of Vaa -snaclec-3/2 was 39.2 nM. At 300 nM, Vaa -snaclec-3/2 did not inhibit collagen-induced platelet agglutination ( C ), ADP-induced platelet agglutination ( D ) nor arachidonic-induced platelet agglutination ( E ). ( F ) As revealed by flow cytometry, Vaa -snaclec-3/2 dose-dependently reduced the binding of fluorescently-labeled monoclonal antibody to human platelet CD42b (GPIb). MFI stands for mean fluorescence intensity. ( G ) The expression of P-selectin (CD62P) on the platelet surface was detected by flow cytometry in less than 1% of platelets after exposure of PRP to Vaa -snaclec-3/2. For experimental details, refer the Materials and Methods section. PRP, platelet-rich plasma.

Fig. 4

Antithrombotic effect of Vaa -snaclec-3/2 in a mouse model of carotid artery thrombosis. Intravenous administration of 50 μg/kg Vaa -snaclec-3/2 in a mouse model ( A ) strongly reduced platelet count (i.e., induced thrombocytopenia) ( p  = 0.01; data represent the mean ± SD) and ( B ) protected the carotid artery from occlusion.