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Binding of hirudin to meizothrombin
B E Fischer1, U Schlokat, M Himmelspach
1Biomedical Research Center, Immuno AG, Orth an der Donau, Austria.
Abstract:
Prothrombin (coagulation factor II) is the inactive precursor molecule of thrombin (coagulation factor IIa). Proteolytic cleavage of the peptide bond Arg320-Ile321 converts prothrombin into the two-chain thrombin precursor meizothrombin. Meizothrombin hydrolyses peptidyl substrates, but cleavage of fibrinogen is poor. Unfortunately, meizothrombin exhibits a significant autocatalytic activity and thus is not structurally stable in solution. Hirudin, the 65-residue peptide anticoagulant from the salivary gland of the European leech Hirudo medicinalis, is a highly specific and effective thrombin inhibitor. To study the interactions of meizothrombin and hirudin, recombinant prothrombin with active site Asp419 replaced by Asn (D419N-prothrombin) was produced in CHO cells and transformed into D419N-meizothrombin in vitro. D419N-meizothrombin exhibited no proteolytic and autocatalytic activity. D419N-meizothrombin was affinity purified at an immobilized C-terminal hirudin-derived peptide demonstrating the presence and activity of the anion binding exosite. D419N-meizothrombin exhibited binding activity to hirudin immobilized at the solid phase in an ELISA. Incubation of D419N-meizothrombin with hirudin resulted in a significant increase of intrinsic fluorescence. Fluorescence titration of D419N-meizothrombin with hirudin produced a sharp break in the titration curve at the molar equivalence point and a total fluorescence enhancement of 24%. However, the titration curve did not reflect a simple binding mechanism. Incubation of D419N-meizothrombin with fibrinopeptide A and C-terminal hirudin peptide 54-65 did not change fluorescence emission. Trp468 located in the gamma-loop of thrombin was replaced by Phe in the double-mutant D419N/W468F-thrombin. Similar to D419N-thrombin and D419N-meizothrombin, formation of the D419N/W468F-thrombin/hirudin complex resulted a significant increase in intrinsic fluorescence. Apparently, the binding of hirudin induces similar structural changes in both meizothrombin and thrombin. The structural change does not involve the flexible gamma-loop. The results suggest that meizothrombin binds hirudin similar to thrombin.
Insights
This study shows that meizothrombin, a thrombin precursor, binds to hirudin, a potent anticoagulant. This interaction causes structural changes similar to those observed when hirudin binds to active thrombin.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Prothrombin (coagulation factor II) is the inactive precursor to thrombin (coagulation factor IIa).
- Meizothrombin is an intermediate in thrombin generation, exhibiting limited fibrinogenolytic activity and significant autocatalysis.
- Hirudin is a highly specific inhibitor of thrombin derived from the medicinal leech.
Purpose of the Study:
- To investigate the interaction between meizothrombin and hirudin.
- To characterize the binding of hirudin to meizothrombin and compare it to thrombin-hirudin interactions.
- To elucidate the structural changes induced by hirudin binding to meizothrombin.
Main Methods:
- Production of recombinant D419N-prothrombin and its conversion to D419N-meizothrombin, a catalytically inactive mutant.
- Affinity purification of D419N-meizothrombin using immobilized hirudin-derived peptides.
- Enzyme-linked immunosorbent assay (ELISA) to detect D419N-meizothrombin binding to immobilized hirudin.
- Fluorescence spectroscopy to monitor conformational changes upon hirudin binding.
Main Results:
- D419N-meizothrombin, lacking proteolytic and autocatalytic activity, demonstrated binding to immobilized hirudin.
- Fluorescence titration revealed a significant increase in intrinsic fluorescence upon hirudin binding to D419N-meizothrombin, indicating conformational changes.
- Similar fluorescence enhancement was observed with a double mutant D419N/W468F-thrombin, suggesting hirudin binding induces comparable structural alterations in both meizothrombin and thrombin.
- These structural changes do not involve the flexible gamma-loop.
Conclusions:
- Meizothrombin binds to hirudin, mediated by the anion binding exosite.
- Hirudin binding induces conformational changes in meizothrombin that are similar to those induced in thrombin.
- The flexible gamma-loop is not involved in the structural rearrangements upon hirudin binding to meizothrombin.