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|Title:||Interactions of vampire bat plasminogen activator, tissue-type plasminogen activator, and TNK-variants with fibrin, fibrinogen, and the fibrin fragment (DD)E|
|Authors:||Stewart, Ronald J.|
|Advisor:||Weitz, Jeffrey I.|
|Keywords:||Medical Sciences;Medical Sciences|
|Abstract:||<p>Fibrinolysis is the process by which blood clots are solubilized. For the purposes of intravascular fibrinolysis, tissue-type plasminogen activator (t-PA) catalyzes the rate-limiting step by converting the zymogen, plasminogen (Pg), to the active enzyme, plasmin. Plasmin then degrades the fibrin meshwork, generating soluble fibrin degradation products. Because the catalytic efficiency of Pg activation by t-PA is higher in the presence of fibrin than that in the presence of fibrinogen(Fg), t-PA is designated a fibrin-specific Pg activator. Despite this designation, t-PA causes systemic plasminemia and fibrinogenolysis when given to patients. Recently, it was demonstrated that the fibrin-specificity of t-PA is compromised because (DD)E, a major soluble degradation product of crosslinked fibrin, stimulates Pg activation to the same extent as fibrin. This project was initiated to gain a better understanding of how (DD)E compromises the fibrin-specificity of t-PA by characterizing the interactions of t-PA and Pg with fibrin, (DD)E and Fg. t-PA binds to fibrin through two classes of sites, one high affinity, finger-dependent site, and one low affinity, kringle-dependent site. In contrast, t-PA binds (DD)E and Fg solely through its second kringle domain, because these interactions are blocked by lysine or its analogues. Both t-PA and Pg bind (DD)E with affinities similar to those for fibrin, thereby explaining why (DD)E stimulates t-PA-mediated Pg activation to the same extent as fibrin. t-PA primarily binds to carboxy-terminal lysines on (DD)E because the affinity of t-PA for (DD)E is reduced 160-fold when (DD)E is exposed to carboxypeptidase B (CPB) or the active form of thrombin activatable firbinolysis inhibitor (TAFIa), an endogenous CPB-like enzyme. In contrast, the affinity of Pg for (DD)E is reduced only 2- to 4-fold when (DD)E is exposed to CPB or TAFIa, suggesting that Pg binds primarily internal lysine residues on (DD)E. t-PA and Pg have weak affinity for Fg, thereby explaining why Fg is a poor stimulator of Pg activation by t-PA. These studies demonstrate that the fibrin-specificity of t-PA is compromised by its kringle-dependent interactions with (DD)E and, to a lesser extent, Fg. The limited fibrin-specificity of t-PA has prompted the development of Pg activators with greater selectivity for fibrin. Two such agents are the activator isolated from the saliva of the vampire bat (b-PA), and a bioengineered t-PA variant, TNK-t-PA. b-PA is structurally distinct from t-PA in that b-PA lacks a lysine-binding kringle. TNK-t-PA was designed to have a longer half-life than t-PA, resistance to inhibition by plasminogen activator inhibitor-1, and increased fibrin-specificity over t-PA. When the fibrin-specificities of t-PA, b-PA, and TNK-t-PA were compared, the hierarchy of fibrin-specificity (b-PA > TNK-t-PA > t-PA) correlated with the ratio of their activity in the presence of fibrin relative to (DD)E. Whereas all activators have similar activities in the presence of fibrin, they are distinguished by their activity in the presence of (DD)E. b-PA, the most fibrin-specific, exhibits minimal stimulation by (DD)E, whereas t-PA, the least fibrin-specific, exhibits the greatest stimulation by (DD)E. Stimulation by (DD)E, in turn, reflects the affinity of the activator for (DD)E. b-PA does not bind (DD)E, presumably because it lacks a lysine-binding kringle. t-PA binds (DD)E with high affinity, whereas TNK-t-PA has ∼9-fold lower affinity for (DD)E than t-PA. These studies highlight the importance of (DD)E in compromising the fibrin-specificity of t-PA. Furthermore, they suggest that the fibrin-specificity of t-PA or t-PA variants could be improved by abolishing their lysine-binding properties.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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