Feedback Regulation of Endothelial Cell Surface Plasmin Generation by PKC-dependent Phosphorylation of Annexin A2

Abstract

In response to blood vessel injury, hemostasis is initiated by platelet activation, advanced by thrombin generation, and tempered by fibrinolysis. The primary fibrinolytic protease, plasmin, can be activated either on a fibrin-containing thrombus or on cells. Annexin A2 (A2) heterotetramer (A2⅐p11) 2 is a key profibrinolytic complex that assembles plasminogen and tissue plasminogen activator and promotes plasmin generation. We now report that, in endothelial cells, plasmin specifically induces activation of conventional PKC, which phosphorylates serine 11 and serine 25 of A2, triggering dissociation of the (A2⅐p11) 2 tetramer. The resulting free p11 undergoes ubiquitinmediated proteasomal degradation, thus preventing further translocation of A2 to the cell surface. In vivo, pretreatment of A2 ؉/؉ but not A2 ؊/؊ mice with a conventional PKC inhibitor significantly reduced thrombosis in a carotid artery injury model. These results indicate that augmentation of fibrinolytic vascular surveillance by blockade of serine phosphorylation is A2-dependent. We also demonstrate that plasmin-induced phosphorylation of A2 requires both cleavage of A2 and activation of Toll-like receptor 4 on the cell surface. We propose that plasmin can limit its own generation by triggering a finely tuned "feedback" mechanism whereby A2 becomes serine-phosphorylated, dissociates from p11, and fails to translocate to the cell surface.The human hemostatic system represents a coordinated balance between procoagulant and anticoagulant/profibrinolytic activities. Together, these systems prevent blood loss while maintaining blood flow. Plasmin, the major protease of the fibrinolytic system, acts to digest insoluble fibrin into soluble fibrin degradation products. Plasmin generation is modulated by plasminogen activators and plasminogen activator inhibitors and is further regulated by binding of plasminogen and its activators to cell surface receptors (1). Membrane-associated annexin A2 (A2) 3 is part of a heterotetrameric complex in which the N termini of two copies of A2 bind to two copies of the S100 family protein, S100A10 (p11) (2). A2, a member of the annexin superfamily, consists of a highly conserved C-terminal phospholipid-binding core domain and an N-terminal ligandinteracting domain (3). Complex formation with p11 increases the affinity of A2 for calcium and phospholipids, thereby directing it to cellular membranes (4). Components of the (A2⅐p11) 2 tetramer, moreover, specifically bind tissue plasminogen activator (t-PA) and plasminogen and strongly enhance plasmin generation (5-8, 52).Several lines of evidence identify the (A2⅐p11) 2 complex as a significant regulator of fibrin balance in vivo. First, A2 Ϫ/Ϫ microvascular endothelial cells lack t-PA cofactor activity, and the fibrin content of highly perfused A2 Ϫ/Ϫ tissues is ϳ2-fold greater than that observed in wild type controls (9). In addition, arterial injury in A2 Ϫ/Ϫ mice is followed by a 2-fold increase in thrombotic vascular occlusion with an equivalent reduction in blood flo...

A novel pathway for human endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies

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cAMP-induced secretion of endothelial von Willebrand factor is regulated by a phosphorylation/dephosphorylation switch in annexin A2

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Tissue plasminogen activator regulates Purkinje neuron development and survival

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