Integrin alphaMbeta2 orchestrates and accelerates plasminogen activation and fibrinolysis by neutrophils.
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 microm) 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 microm, 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.
Pluskota
Elzbieta
E
Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Soloviev
Dmitry A
DA
Bdeir
Khalil
K
Cines
Douglas B
DB
Plow
Edward F
EF
D002448:Cell Adhesion; D002465:Cell Movement; D004305:Dose-Response Relationship, Drug; D005337:Fibrin / Q000737:chemistry / Q000378:metabolism*; D005341:Fibrinolysin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D016177:Macrophage-1 Antigen / Q000378:metabolism*; D009154:Mutation; D009504:Neutrophils / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D013997:Time Factors; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D016177:Macrophage-1 Antigen; D011994:Recombinant Proteins; D005337:Fibrin; D010958:Plasminogen; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate