István Léránt

Flow Rate–Modulated Dissolution of Fibrin With Clot-Embedded and Circulating Proteases

The efficiency of plasmin, miniplasmin, and neutrophil leukocyte elastase in fibrin digestion is well characterized in static systems. Since in vivo the components of the fibrinolytic system are permanently exposed to flow, we have developed two in vitro models and studied the effect of shear forces on fibrin dissolution with these proteases. Cylindrical nonocclusive fibrin clots are perfused at various flow rates through their preformed axial channel, and dissolution of fibrin is followed by measuring the absorbance of degradation products released into the circulating fluid phase. In one experimental setting, fibrin surface is degraded with enzymes applied in the recirculating fluid phase; in another setting, clots containing gel-embedded proteases are perfused with enzyme-free buffer. As shear rate at fibrin surface is changed from 25 to 500 s(-1), the rate of product release by recirculated enzymes increases 2.8-, 2.9-, and 4-fold for plasmin, miniplasmin, and porcine pancreatic elastase, respectively. Buffer-perfused fibrin containing gel-embedded plasmin or miniplasmin is disintegrated by shear forces at a relatively early stage of dissolution, and this disassembly is related to the formation of fragment Y (150 kDa) and fragment D (100 kDa) fibrin degradation products. Fibrin clots degraded by incorporated polymorphonuclear leukocyte elastase, which yields different degradation products, do not disassemble abruptly, even at the highest shear rate (500 s(-1)). Our results suggest that fibrin surface degradation is accelerated with increasing shear rate and that plasmin or miniplasmin embedded in the clot promotes the release of particular clot remnants into the circulating phase, whereas polymorphonuclear leukocyte elastase does not.

Modulation of plasminogen activation and plasmin activity by methylglyoxal modification of the zymogen

The effect of methylglyoxal on the plasminogen-plasmin system is studied. Treatment of plasminogen with methylglyoxal at a 20-fold molar excess results in covalent modification of the molecule as evidenced by the decreased number of NH(2) side chains, arginine side chain residues and the new band in the non-tryptophan dependent fluorescent spectrum. This structural modification is associated with profound functional alterations: the rate of activation by streptokinase, tissue-type plasminogen activator, urokinase-type plasminogen activator and trypsin decreases and the amidolytic activity of the generated plasmin is impaired. Plasmin treatment with methylglyoxal on the other hand does not alter its steady-state kinetic parameters on a peptidyl-anilide synthetic substrate, indicating that modification susceptible side chains are sensitive to methylglyoxal only in the zymogen. Our data suggest that in vivo fibrinolysis could be impaired under pathological conditions, e.g. increased methylglyoxal formation in diabetes mellitus.

Impaired inactivation by antithrombin and hirudin and preserved fibrinogen-clotting activity of thrombin in complex with antithrombin antibody from a patient with antiphospholipid syndrome

Immunoglobulin G (IgG) isolated from the blood plasma of a patient with secondary antiphospholipid syndrome (APS) expresses fibrinogen-clotting and amidolytic activity (the thrombin activity in 20 micromole IgG is equivalent to approximately 5 nmole pure thrombin), and activates factor XIII. Hirudin (1 microM) decreases the intrinsic thrombin activity of the APS IgG by only 25%, whereas it inhibits completely pure thrombin with equivalent activity. Under conditions, when antithrombin inactivates 60% of the thrombin activity in the presence of normal IgG, the APS IgG protects almost completely the added thrombin against inactivation by antithrombin. Heparin, however, partially relieves this protective effect and at the same time it facilitates the inhibition of the intrinsic thrombin activity by antithrombin. The APS IgG reduces the thrombin activity in protein C activation assay by 50% compared to the activity in the presence of normal IgG. All described properties are related to the Fab fragment of the antibody. The IgG preserving the fibrin-generating activity of thrombin with concomitant protection against inhibitors unravels a new aspect of the thrombotic mechanism in APS. This condition is probably rare: only one out of 23 examined patients with primary or secondary APS expresses IgG with the described properties.

Conditions of formation of the heparin-fibronectin-collagen complex and the effect of plasmin

The formation and composition of the insoluble heparin-fibronectin-collagen complex and its degradation by proteolysis was investigated. At fixed concentrations of the other molecular components of the complex, the maximal rate of complex formation, measured turbidimetrically, was reached at a concentration of 4 microM heparin and 0.9 microM collagen, while the rate of complex formation was linearly related to concentrations of fibronectin as high as 3 microM. Heparin was incorporated into the complex in a saturable manner, and was released in active anticoagulant form by plasmin but not by urokinase. The complex formation was inhibited by 5 mM calcium or 250 mM NaCl as well as by polybrene or spermin. It is suggested that fibronectin binds both heparin and collagen cooperatively to form an insoluble ternary complex of the extracellular matrix.

Association of thrombin, plasmin, thrombin-antithrombin III complex and plasmin-antithrombin III complex with isolated hepatocytes

The interaction of thrombin, plasmin or their antithrombin III complexes with isolated mouse hepatocytes was studied. Plasmin bound to hepatocytes in a concentration-dependent manner with an apparent Kd of 6.4.10(-8) M, attaining equilibrium within 10 min, and the interaction was inhibited by 6-amino-n-hexanoic acid. Plasmin treated with diisopropylfluorophosphate (DFP) bound to the cells in similar way as the untreated form of the enzyme. Thrombin bound also to hepatocytes, in a concentration-dependent manner, with a Kd of 5.4.10(-8) M reaching a steady state after 180 min. Thrombin inactivated with DFP, however, was inhibited in its binding to these cells. These data suggest that, whereas the kringle domains of plasmin are responsible for the enzyme-cell interaction, the active center of thrombin may be involved in the binding of this enzyme to hepatocytes. Plasmin-antithrombin III and thrombin-antithrombin III complexes were also associated with hepatocytes in a time-dependent manner, reaching a plateau after 180 min, and the two complexes competed in the interaction. While the interaction of active proteinases plasmin or thrombin with hepatocytes did not result in their internalization, the antithrombin III complexes were taken up by the cells, and thrombin-antithrombin III complex was degraded. These results indicate that hepatocytes may participate in the elimination of proteinase-antithrombin III complexes from the plasma, while the association of plasmin and thrombin with hepatocytes could imply distinct biological importance.

interaction of antithrombin III and thrombinantithrombin III complex with cultured aortic endothelial cells

The binding of antithrombin III, thrombin, thrombin-antithrombin III complex to endothelial cells was investigated. While the rate of the binding of thrombin to these cells was very rapid, that of antithrombin III was relatively slow and the thrombin-antithrombin III complex was intermediate. Binding kinetics indicated that antithrombin III, like thrombin, showed high affinity to endothelial cells; with a Kd of 3 X 10(-8) M and with 5 X 10(4) binding sites per cell. The dissociation of the inhibitor molecule was also rapid, i.e., approximately 70% bound antithrombin III was released in 2 minutes. Heparin, in a 100-fold molar excess to antithrombin III, or the modification of lysine residues of the inhibitor involved in the interaction with heparin, did not influence the association of antithrombin III with endothelial cells. In addition, antithrombin III did not compete with thrombin blocked in its active center for binding to endothelial cells. It is suggested that the binding sites of endothelial cells are different for thrombin and antithrombin III, and antithrombin III does not bind to these cells through its heparin binding domain.