Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution.

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 noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 microM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [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 10(2) M(-1) x 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 cross-linking 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.

[1]  James Inglese,et al.  Chemokine receptor-ligand interactions measured using time-resolved fluorescence. , 1998, Biochemistry.

[2]  E. Regoeczi.  Occlusion of Plasma Proteins by Human Fibrin: Studies using Trace‐Labelled Proteins , 1968, British journal of haematology.

[3]  M. London,et al.  Non-covalent associations of proteins in plasma: self-, mixed fibrin(ogen), mixed protein-non-protein associations. , 1997, Clinical biochemistry.

[4]  J. Hermans,et al.  Assembly of fibrin. A light scattering study. , 1979, The Journal of biological chemistry.

[5]  M. Straume,et al.  Monte Carlo method for determining complete confidence probability distributions of estimated model parameters. , 1992, Methods in enzymology.

[6]  R. McBane,et al.  Fibrinogen, Fibrin and Crosslinking in Aging Arterial Thrombi , 2000, Thrombosis and Haemostasis.

[7]  I. Brooks,et al.  Determination of rate and equilibrium binding constants for macromolecular interactions by surface plasmon resonance. , 1994, Methods in enzymology.

[8]  G. Siefring,et al.  Fibrinoligase-catalyzed cross-linking of myosin from platelet and skeletal muscle. , 1979, Archives of biochemistry and biophysics.

[9]  R. Karlsson,et al.  Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. , 1991, Journal of immunological methods.

[10]  A. Veis,et al.  Factor XIIIa-catalyzed cross-linking of platelet and muscle actin. Regulation by nucleotides. , 1980, Biochimica et biophysica acta.

[11]  J. Koretz.  Hybridization and reconstitution of thick-filament structure. , 1982, Methods in enzymology.

[12]  D. Cove,et al.  Platelet contractile proteins: separation and characterization of the actin and myosin-like components. , 1975, Journal of mechanochemistry & cell motility.

[13]  K Kolev,et al.  Regulation of fibrinolytic activity of neutrophil leukocyte elastase, plasmin, and miniplasmin by plasma protease inhibitors. , 1994, The Journal of biological chemistry.

[14]  S L Diamond,et al.  Inner clot diffusion and permeation during fibrinolysis. , 1993, Biophysical journal.

[15]  K. Kolev,et al.  Myosin as cofactor and substrate in fibrinolysis , 1997, FEBS letters.

[16]  M. Carr,et al.  Fibrin has larger pores when formed in the presence of erythrocytes. , 1987, The American journal of physiology.

[17]  K. Kolev,et al.  Quantitative Comparison of Fibrin Degradation with Plasmin, Miniplasmin, Neurophil Leukocyte Elastase and Cathepsin G , 1996, Thrombosis and Haemostasis.

[18]  B. Persson,et al.  Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins , 1991 .

[19]  L. Lorand,et al.  Structural origins of fibrin clot rheology. , 1999, Biophysical journal.

[20]  E. Angles-cano,et al.  A spectrophotometric solid-phase fibrin-tissue plasminogen activator activity assay (SOFIA-tPA) for high-fibrin-affinity tissue plasminogen activators. , 1986, Analytical biochemistry.

[21]  N. Shibata,et al.  Reorganization of contractile elements in the platelet during clot retraction. , 1984, Journal of ultrastructure research.

[22]  Allen P. Minton,et al.  The effect of volume occupancy upon the thermodynamic activity of proteins: some biochemical consequences , 2004, Molecular and Cellular Biochemistry.

[23]  D. Deutsch,et al.  Plasminogen: Purification from Human Plasma by Affinity Chromatography , 1970, Science.

[24]  S. Skarlatos,et al.  Phospholipid loss in dying platelets , 1993, Virchows Archiv. B, Cell pathology including molecular pathology.

[25]  L. Lorand,et al.  Human plasma factor XIII: subunit interactions and activation of zymogen. , 1993, Methods in enzymology.

[26]  S. Lowey,et al.  Preparation of myosin and its subfragments from rabbit skeletal muscle. , 1982, Methods in enzymology.

[27]  J. Fox.  3 – Platelet Contractile Proteins , 1986 .

[28]  Shin Lin,et al.  Identification of fibrinogen derivatives in the Triton-insoluble residue of human blood platelets. , 1983, Cell motility.

[29]  J C Haselgrove,et al.  Three-dimensional reconstruction of fibrin clot networks from stereoscopic intermediate voltage electron microscope images and analysis of branching. , 1995, Biophysical journal.

[30]  M. Morrison.  Lactoperoxidase-catalyzed iodination as a tool for investigation of proteins. , 1980, Methods in enzymology.

[31]  A. Laudano,et al.  Studies on synthetic peptides that bind to fibrinogen and prevent fibrin polymerization. Structural requirements, number of binding sites, and species differences. , 1980, Biochemistry.

[32]  Michael L. Johnson,et al.  [16] Nonlinear least-squares analysis , 1985 .

[33]  F. Castellino,et al.  Modulation of fibrin assembly and polymerization by the beta-amyloid of Alzheimer's disease. , 1996, Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.

[34]  R. Colman.  Hemostasis and thrombosis: Basic principles and clinical practice , 1987 .

[35]  Magnus Strandh,et al.  Detection and characterization of weak affinity antibody antigen recognition with biomolecular interaction analysis , 1997, Journal of molecular recognition : JMR.

[36]  C. Francis,et al.  Structural studies of fibrinolysis by electron microscopy. , 1998, Blood.

[37]  J. Torbet.  Fibrin assembly in human plasma and fibrinogen/albumin mixtures. , 1986, Biochemistry.

[38]  B. Persson,et al.  Studies of interactions with weak affinities and low‐molecular‐weight compounds using surface plasmon resonance technology , 1998, Journal of molecular recognition : JMR.

[39]  A Liljeborg,et al.  Native fibrin gel networks observed by 3D microscopy, permeation and turbidity. , 1989, Biochimica et biophysica acta.

[40]  Paul,et al.  Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin. , 1985, The Journal of biological chemistry.

[41]  K. Kolev,et al.  Flow rate-modulated dissolution of fibrin with clot-embedded and circulating proteases. , 1998, Circulation research.

[42]  K. Tenekedjiev,et al.  Functional Evaluation of the Structural Features of Proteases and Their Substrate in Fibrin Surface Degradation* , 1997, The Journal of Biological Chemistry.

[43]  Michael A. Malcolm,et al.  Computer methods for mathematical computations , 1977 .