Submitted October 24, 2002
Accepted January 14, 2003
Myosin: a non-covalent stabilizer of fibrin in the process of clot dissolution
Krasimir Kolev, Kiril Tenekedjiev, Katalin Ajtai, Ilona Kovalszky, Judit Gombas, Balazs Varadi, and Raymund Machovich*
Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
1st Department of Pathology, Semmelweis University, Budapest, Hungary
Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN, USA
* Corresponding author; email: mr{at}puskin.sote.hu.
Myosin modulates the fibrinolytic process as a cofactor of the tissue-plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible non-covalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 µM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (GPRP) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 x 102M-1.s-1 and a dissociation rate constant of 3.07 x 10-4 s-1 are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer/myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer/myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect: myosin is not covalently attached to the clot by the time of complete crosslinking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked and the myosin-fibrin clot is relatively resistant to plasmin.
