Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 279, Issue 17, Pages 18063-18072Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M310462200
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Funding
- NHLBI NIH HHS [HL17964, HL66197, HL60169] Funding Source: Medline
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Plasmin, the pivotal thrombolytic enzyme, is generated on the surface of many cell types, where urokinase receptor (uPAR)-bound urokinase (uPA) activates cell-bound plasminogen (Plg). It has been reported that neutrophils mediate endogenous thrombolysis involving a uPA-dependent mechanism, and we previously demonstrated that both uPAR and integrin alpha(M)beta(2) recognize uPA to control cell migration and adhesion. In the present study, we report that the alpha(M)beta(2) regulates neutrophil-dependent fibrinolysis. Phorbol 12-myristate 13-acetate (PMA)-stimulated but not resting neutrophils dissolved fibrin clots, and this activity was not only uPA- and Plg-dependent but also alpha(M)beta(2)-dependent. Purified alpha(M)beta(2) directly bound uPA (K-d = 40 nM) and Plg (K-d = 1 muM) in a dose-dependent and saturable manner. In Plg activation assays, addition of purified alpha(M)beta(2), but not a control protein, to a single chain uPA (sc-uPA)/Plg mixture, decreased the K-m from 2 to 0.1 muM, thereby augmenting the overall reaction efficiency by 50-fold. The binding of sc-uPA to alpha(M)beta(2) was critical for the alpha(M)beta(2)-mediated enhancement of plasmin (Plm) generation, because this effect was lost when WT-sc-uPA was replaced with a kringle-less mutant (DeltaK-sc-uPA), which does not bind to alpha(M)beta(2). Plm inactivation by alpha(2)-antiplasmin was significantly delayed when Plm was preincubated with purified, soluble alpha(M)beta(2). When Plg was added to PMA-stimulated neutrophils, both uPA and Plg were co-immunoprecipitated with alpha(M)beta(2). Thus, assembly of Plg and uPA on integrin alpha(M)beta(2) regulates Plm activity and, thereby, plays a crucial role in neutrophil-mediated thrombolysis.
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