Abstract
BackgroundRemoval of C-terminal lysine residues that are continuously exposed in lysing fibrin is an established anti-fibrinolytic mechanism dependent on the plasma carboxypeptidase TAFIa, which also removes arginines that are exposed at the time of fibrinogen clotting by thrombin.ObjectiveTo evaluate the impact of alterations in fibrin structure mediated by constitutive carboxypeptidase activity on the function of fibrin as a template for tissue plasminogen activator-(tPA) induced plasminogen activation and its susceptibility to digestion by plasmin.Methods and resultsWe used the stable carboxypeptidase B (CPB), which shows the same substrate specificity as TAFIa. If 1.5 – 6 μM fibrinogen was clotted in the presence of 8 U/mL CPB, a denser fibrin network was formed with thinner fibers (the median fiber diameter decreased from 138 – 144 nm to 89 – 109 nm as established with scanning electron microscopy). If clotting was initiated in the presence of 5 – 10 μM arginine, a similar decrease in fiber diameter (82 -95 nm) was measured. The fine structure of arginine-treated fibrin enhanced plasminogen activation by tPA, but slowed down lysis monitored using fluorescent tPA and confocal laser microscopy. However, if lysis was initiated with plasmin in CPB-treated fibrin, the rate of dissolution increased to a degree corresponding to doubling of the plasmin concentration.ConclusionThe present data evidence that CPB activity generates fine-mesh fibrin which is more difficult to lyse by tPA, but conversely, CPB and plasmin together can stimulate fibrinolysis, possibly by enhancing plasmin diffusion.
Highlights
Binding of tissue-type plasminogen activator and plasminogen to fibrin is a prerequisite for efficient fibrinolysis, in the course of which the generated plasmin provides a positive feedback loop through exposure of new carboxyl terminal lysines that promote fibrinolysis primarily through plasminogen and plasmin binding [2]
Structural modifications of fibrin related to carboxypeptidase B (CPB) activity or presence of arginine We have previously shown [12], when CPB was used as a stable analogue of TAFIa to evaluate the impact of removal of carboxyl terminal lysines on the kinetics of fibrinolysis, that the presence of CPB during fibrinogen clotting modifies the turbidity of the clots before initiation of lysis, which suggested changes in the structure of fibrin
Our work identified two consequences of CPB activity in fibrin that antagonize the CPB-dependent blockade of the positive feed-back loop in fibrinolysis based on C-terminal lysine exposure: 1) enhancement of plasmin activity when the fluid-borne enzyme attacks the surface of pre-formed clots, probably due to improved diffusion; and 2) fibrin structure-related acceleration of plasminogen activation
Summary
Binding of tissue-type plasminogen activator (tPA) and plasminogen to fibrin is a prerequisite for efficient fibrinolysis (reviewed in [1]), in the course of which the generated plasmin provides a positive feedback loop through exposure of new carboxyl terminal lysines that promote fibrinolysis primarily through plasminogen and plasmin binding [2]. The amplifying effect of C-terminal lysines on plasminogen activation and the protection of the bound plasmin against its major plasma inhibitor α2-plasmin inhibitor is counterbalanced by the action of thrombin activatable fibrinolysis inhibitor (TAFI, or carboxypeptidase U), an exopeptidase that removes basic amino acids A distinct, constitutively active enzyme, carboxypeptidase N (CPN) is present in plasma [5,6] It binds fibrin and can be detected in the structure of plasma clots [7,8]. Removal of C-terminal lysine residues that are continuously exposed in lysing fibrin is an established anti-fibrinolytic mechanism dependent on the plasma carboxypeptidase TAFIa, which removes arginines that are exposed at the time of fibrinogen clotting by thrombin. Conclusion: The present data evidence that CPB activity generates fine-mesh fibrin which is more difficult to lyse by tPA, but CPB and plasmin together can stimulate fibrinolysis, possibly by enhancing plasmin diffusion
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