Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin

Abstract

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γA/γA isoform of fibrin(ogen) and the γA/γ′ variant with an extended γ-chain. Thrombin binds to the γ′-chain and forms a higher affinity interaction with γA/γ′-fibrin(ogen) than γA/γA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 μm) even though it does not interact with the γ′-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γA/γA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2–5 μm, the α(17–51) and Bβ(1–42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.

Reduced Plasminogen Binding and Delayed Activation Render γ′-Fibrin More Resistant to Lysis than γA-Fibrin

“…Peptide elution was monitored by measuring absorbance at 205 nm, and FPA and FPB were quantified by calculating the areas of each peak visualized on the elution chromatogram and comparing them with the concentrations of FPA and FPB released from clots incubated with thrombin under the same conditions for 60 min. The identities of FPA and FPB were confirmed by mass spectrometry as described previously (22).…”

“…Quantification of Thrombin-mediated FPA and FPB Release from ␥Ј-Fg and ␥ A -Fg-FPA and FPB were quantified using high performance liquid chromatography as described previously (6,22), with slight modifications. Briefly, a series of clots was prepared by incubating 10 nM thrombin with solutions consisting of 5 M ␥Ј-Fg or ␥ A -Fg in HBS containing 5 mM CaCl 2 and 0.01% Tween 80 to a final volume of 750 l for up to 60 min at 37°C.…”

Exogenous Procoagulant Factors as Therapeutic and Biotechnological Tools

“…Batroxobin is a component present on venom from B. atrox moojeni that acts as thrombin-like enzyme [81,82,85]. This serine proteinase only releases fibrinopeptide A by specific cleavage of Arg 16 -Gly 17 bond in the Aα-chain of fibrinogen, and it is not inhibited by antithrombin or heparin cofactor II [82,86].…”

An international collaborative study to calibrate the WHO 2nd International Standard for Ancrod (15/106) and the WHO Reference Reagent for Batroxobin (15/140): communication from the SSC of the ISTH

“…Ancrod and batroxobin are both thrombin‐like snake venom serine proteases produced by Calloselasma rhodostoma and Bothrops atrox moojeni , respectively. Details of the mechanism of fibrinogen binding and action have been published for batroxobin and ancrod . Because ancrod and batroxobin produce fibrin that is readily degraded they promote fibrinogen depletion and have been investigated as potential treatments to reduce normal clot formation under various circumstances.…”