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.

Keywords: Batroxobin; Blood Coagulation Factors; Fibrin; Fibrinogen; Fibrinopeptides; Snake Venom; Thrombin.

FIGURE 1.

Integrity of thrombin and batroxobin as assessed by SDS-PAGE analysis. Thrombin (lane 2) and batroxobin (lane 4) were subjected to SDS-PAGE analysis on a 4–15% polyacrylamide gradient gel under nonreducing conditions. The molecular weights of the mobility markers are shown on the left (lane 1).

FIGURE 2.

SPR analysis of the interaction of FPR-batroxobin and FPR-thrombin with immobilized γ_A/γ_A- or γ_A/γ′-fibrinogen. γ_A/γ_A- (●) and γ_A/γ′-fibrinogen (▴) were adsorbed on individual flow cells to ∼6000–8000 RU. Increasing concentrations (0–15 μm) of FPR-thrombin (A) or FPR-batroxobin (B) were successively injected into flow cells for 2 min, followed by a 4-min wash to monitor dissociation. The amount of protease bound at equilibrium (Req) after background correction is plotted against the input FPR-protease concentration. The insets show representative sensorgrams for the interaction of FPR-thrombin (A) and FPR-batroxobin (B) with γ_A/γ_A-fibrinogen in concentrations up to 15 and 6.4 μm, respectively. Data points represent the mean ± S.D. of 2- 3 experiments, and the lines represent nonlinear regression analyses.

FIGURE 3.

Binding of ¹²⁵I-YPR-batroxobin and ¹²⁵I-YPR-thrombin to γ_A/γ_A- or γ_A/γ′-fibrin clots. 20 nm ¹²⁵I-YPR-thrombin (A) or 40 nm ¹²⁵I-YPR-batroxobin (B) was added to microcentrifuge tubes containing 0–8 μm γ_A/γ_A- (●) or γ_A/γ′-fibrinogen (▴), and 10 nm α-thrombin was used to initiate clotting. After incubation for 60 min, clots were pelleted by centrifugation, and free ¹²⁵I-YPR-protease in the supernatant was used to calculate the bound fraction. Data are plotted as the percentage of ¹²⁵I-YPR-protease bound to fibrin clots versus the fibrinogen concentration. Data points represent the mean ± S.D. of two experiments, each performed in duplicate, and the lines represent nonlinear regression analyses.

FIGURE 4.

Effect of γ′-peptide-directed IgG on ¹²⁵I-YPR-batroxobin and ¹²⁵I-YPR-thrombin binding to γ_A/γ_A- or γ_A/γ′-fibrin clots. The binding of 40 nm ¹²⁵I-YPR-thrombin (●/▴) or ¹²⁵I-YPR-batroxobin (○/Δ) to clots formed from 1 μm γ_A/γ_A- (circles) or γ_A/γ′-fibrinogen (triangles) was assessed in the absence or presence of γ′-peptide-directed IgG up to 8 μm. Clots were generated with thrombin and pelleted, and clot-bound ¹²⁵I-YPR-protease was determined. The percentage of ¹²⁵I-YPR-protease bound in the absence of antibody is plotted versus antibody concentration. The main figure illustrates the percentage of ¹²⁵I-YPR-protease bound to γ_A/γ′-fibrin clots, and the inset shows the percentage of ¹²⁵I-YPR-protease bound to γ_A/γ_A-fibrin clots. Symbols represent the mean ± S.D. of two experiments, each performed in duplicate, and the lines represent nonlinear regression analyses of the data.

FIGURE 5.

Diffusion of ¹²⁵I-YPR-batroxobin and ¹²⁵I-YPR-thrombin from γ_A/γ_A- or γ_A/γ′-fibrin clots. Clots were formed around plastic loops by incubating 9 μm γ_A/γ_A- (●/○) or γ_A/γ′-fibrinogen (■/□) with 100 nm thrombin and 30 nm fXIII in the presence of 20 nm ¹²⁵I-YPR-thrombin (A) or 40 nm ¹²⁵I-YPR-batroxobin (B) and then immersed in solutions containing TBS-Ca (●/■) or 2 m NaCl and 5 mm EDTA (○/□). At the indicated time points, 0.5-ml aliquots of the bathing solutions were removed and counted for radioactivity to quantify bound ¹²⁵I-YPR-protease. Data are plotted as the percentage of clot-bound ¹²⁵I-YPR-protease versus time, and lines represent nonlinear regression analyses. Data points represent the mean ± S.D. of three separate determinations.

FIGURE 6.

Effect of FPR-thrombin on batroxobin binding to γ_A/γ_A-fibrinogen or γ_A/γ_A-fibrin clots. A, effect of FPR-thrombin on γ_A/γ_A-fibrinogen binding to immobilized FPR-batroxobin was determined using SPR. γ_A/γ_A-fibrinogen (2.5 μm) was incubated with FPR-thrombin concentrations up to 40 μm in the absence (●) or presence (■) of 80 μm hirudin, and the mixture was then injected into flow cells containing immobilized FPR-batroxobin. Binding in the presence of competitors was normalized relative to that determined in their absence. Data are plotted as the percentage of γ_A/γ_A-fibrinogen bound versus the FPR-thrombin concentration. B, binding of ¹²⁵I-YPR-batroxobin (■) and ¹²⁵I-YPR-thrombin (●) to γ_A/γ_A-fibrin clots was measured in the absence or presence of FPR-thrombin concentrations up to 25 μm. Clots were formed by incubating 2 or 0.06 μm γ_A/γ_A- fibrinogen with 20 nm ¹²⁵I-YPR-thrombin or 40 nm ¹²⁵I-YPR-batroxobin, respectively, and 10 nm thrombin. After 60 min, clots were counted for radioactivity to quantify bound ¹²⁵I-YPR-protease. Data are plotted as the percentage of ¹²⁵I-YPR-protease bound versus the FPR-thrombin concentration. Data points represent the mean ± S.D. of 3–4 experiments, and the lines represent nonlinear regression analyses.

FIGURE 7.

Clot accretion induced by batroxobin or thrombin bound to γ_A/γ_A- or γ_A/γ′-fibrin clots. Clots were formed around plastic loops by incubating 8.3 μm γ_A/γ_A-fibrinogen (●/○) or γ_A/γ′-fibrinogen (■/□) with 76.5 units/ml of batroxobin (●/■) or 45 nm thrombin (○/□) in the presence of 2 mm CaCl₂, 30 nm fXIII, and 500 cpm of ¹²⁵I-fibrinogen for 30 min at 37 °C. After determining radioactivity, clots were incubated in 1 ml of citrated human plasma containing ∼800,000 cpm/ml of ¹²⁵I-fibrinogen. At intervals, clots were washed twice with 1 ml of HBS and again counted for radioactivity so that fibrin accretion could be determined. The inset provides an expanded view of clot accretion induced by thrombin. Data points represent the mean ± S.D. of three experiments, each performed in triplicate, and the lines represent nonlinear regression analyses. (*, p < 0.001).

FIGURE 8.

Models of binding sites on batroxobin and fibrinogen. A, crystal structure of TSV-PA (Protein Data Bank accession number 1BQY) in complex with Glu-Gly-Arg chloromethyl ketone (green) is shown in space-filling format using PyMOL. The image is rotated clockwise ∼90° from standard view with the active site Ser-195 (magenta) facing left. Hydrophobic (yellow) and basic (blue) residues in batroxobin that constitute a putative exosite are mapped onto the TSV-PA structure. Residues are identified using chymotrypsin numbering system. B, crystal structure of the NH₂ termini of fibrinogen α- and β-chains forming the thrombin-binding site (Protein Data Bank accession number 2A45) is shown in space-filling format. Both the γ-chains and the reciprocal α- and β-chains are omitted for clarity. The α- and β-chain surfaces are white and gray, respectively. Thrombin binding residues α-chain Phe-35 and Asp-38 and β-chain Ala-68, Asp-69, and Asp-71 are shown within a solid black oval with hydrophobic residues in yellow and acidic residues in red. Residues comprising the putative batroxobin-binding site include α-chain Asp-30, Asp-32, Trp-33, Pro-34, and Phe-35 (hatched black oval). The site where the unmapped α1–26 segment exits the α-chain at Αla-27 indicates the vicinity of FpA.