One of the major physiological functions of the proteolytic enzyme plasmin is the degradation and solubilization of fibrin, the major constituent of blood clots. Plasmin has a broad trypsin-like specificity and the production of plasmin from its precursor plasminogen is precisely regulated (reviewed in Refs. 1-5). One way in which plasmin activity is localized to the fibrin clot involves a fibrin-specific mechanism for the conversion of plasminogen to plasmin by tissue-type plasminogen activator (t-PA).1 For example, recent studies have shown that by virtue of its ability to bind both t-PA and plasminogen, fibrin acts as a template that promotes the formation of a t-PA⅐fibrin⅐plasminogen ternary complex. The catalytic efficiency of t-PA-dependent conversion of plasminogen to plasmin is determined by the stability of the ternary complex (6). Thus, fibrin is both a substrate of plasmin and a template for plasmin production. Fibrin also plays a role in the plasmin-dependent stimulation of plasmin formation. For example, the partial proteolysis of fibrin results in the transient generation of new carboxyl-terminal lysine residues that act as high affinity binding sites for the lysine-binding sites of plasminogen (7, Recently, the endothelial cell-surface Ca 2ϩ -binding protein, annexin II, has also been shown to stimulate the t-PA-dependent formation of plasmin from plasminogen (13,14). Annexin II was originally identified as an intracellular Ca 2ϩ -and phospholipid-binding protein and subsequent studies suggested that this protein was potentially involved in the regulation of membrane trafficking events such as exocytosis or endocytosis (reviewed in Ref. 15). Annexin II can exist in cells as both a monomer or as a heterotetramer. The heterotetramer, called annexin II tetramer (AIIt) consists of two annexin II molecules and two molecules of an 11-kDa regulatory subunit referred to as the p11 light chain. The binding of the p11 light chain regulates many of the in vitro activities of annexin II and the biochemical properties of AIIt are distinct from the annexin II monomer (16,17). In many cells such as Madin-Darby canine kidney cells, bovine intestinal epithelial cells, and calf pulmonary arterial endothelial cells, 90 -95% of the total cellular annexin II is present in the heterotetrameric form (18,19). Annexin II and AIIt have been shown to exist on the extracellular surface of many cells although the relative extracellular distribution of the two forms of the protein has not been quantified (13, 20 -23).In the present report, we have compared the kinetics of annexin II and AIIt-dependent activation of t-PA-mediated plasminogen activation. These experiments establish the presence of AIIt on the HUVEC surface and that AIIt is a potent in vitro activator of t-PA-mediated plasminogen activation. EXPERIMENTAL PROCEDURESMaterials-Fibrinogen was obtained from Sigma and further purified by gel permeation chromatography on Superose 12 to remove
In this paper, we have characterized the regulation of plasmin activity by annexin II tetramer (AIIt). Plasmin activity was measured by a fibrin lysis assay in which a fibrin polymer was produced from purified components and the extent of polymer lysis was determined by following changes in turbidity. Extrinsic lysis of the fibrin polymer, initiated by addition of tissue plasminogen activator (t-PA), was totally blocked if AIIt was present during fibrin polymer formation. Furthermore, fibrin polymer formed in the presence of AIIt was resistant to extrinsic lysis initiated by addition of plasmin. AIIt bound to fibrin polymer under conditions in which polymer lysis was inhibited. Plasmin-dependent extrinsic lysis of the fibrin polymer was also blocked if AIIt was present in the incubation medium, and under these conditions the amidolytic activity of plasmin, measured with an artificial substrate, was inhibited about 5-fold. In contrast, in the absence of fibrin, and at an AIIt/plasmin molar ratio of 526, the amidolytic activity of plasmin was inhibited by only 22.3% +/- 7.4% (mean +/- SD, n = 5) by AIIt. Plasmin-dependent fibrinolysis was only slightly inhibited if fibrin polymer was formed in the presence of annexins I, II, V, or VI. These results identify AIIt as an in vitro regulator of plasmin activity.
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