Arunkumar B. Deora

Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo

A central tenet of fibrinolysis is that tissue plasminogen activator-dependent (t-PA- dependent) conversion of plasminogen to active plasmin requires the presence of the cofactor/substrate fibrin. However, previous in vitro studies have suggested that the endothelial cell surface protein annexin II can stimulate t-PA-mediated plasminogen activation in the complete absence of fibrin. Here, homozygous annexin II-null mice displayed deposition of fibrin in the microvasculature and incomplete clearance of injury-induced arterial thrombi. While these animals demonstrated normal lysis of a fibrin-containing plasma clot, t-PA-dependent plasmin generation at the endothelial cell surface was markedly deficient. Directed migration of annexin II-null endothelial cells through fibrin and collagen lattices in vitro was also reduced, and an annexin II peptide mimicking sequences necessary for t-PA binding blocked endothelial cell invasion of Matrigel implants in wild-type mice. In addition, annexin II-deficient mice displayed markedly diminished neovascularization of fibroblast growth factor-stimulated cornea and of oxygen-primed neonatal retina. Capillary sprouting from annexin II-deficient aortic ring explants was markedly reduced in association with severe impairment of activation of metalloproteinase-9 and -13. These data establish annexin II as a regulator of cell surface plasmin generation and reveal that impaired endothelial cell fibrinolytic activity constitutes a barrier to effective neoangiogenesis.

An Annexin 2 Phosphorylation Switch Mediates p11-dependent Translocation of Annexin 2 to the Cell Surface

Annexin 2 is a profibrinolytic co-receptor for plasminogen and tissue plasminogen activator that stimulates activation of the major fibrinolysin, plasmin, at cell surfaces. In human subjects, overexpression of annexin 2 in acute promyelocytic leukemia leads to a bleeding diathesis reflective of excessive cell surface annexin 2-dependent generation of plasmin (Menell, J. S., Cesarman, G. M., Jacovina, A. T., McLaughlin, M. A., Lev, E. A., and Hajjar, K. A. (1999) N. Engl. J. Med. 340, 994–1004). In addition, mice completely deficient in annexin 2 display fibrin accumulation within blood vessels and impaired clearance of injury-induced thrombi (Ling Q., Jacovina, A.T., Deora, A.B., Febbraio, M., Simantov, R., Silverstein, R. L., Hempstead, B. L., Mark, W., and Hajjar, K. A. (2004) J. Clin. Investig. 113, 38–48). Here, we show that endothelial cell annexin 2, a protein that lacks a typical signal peptide, translocates from the cytoplasm to the extracytoplasmic plasma membrane in response to brief temperature stress both in vitro and in vivo in the absence of cell death or cell lysis. This regulated response is independent of new protein or mRNA synthesis and does not require the classical endoplasmic reticulum-Golgi pathway. Temperature stress-induced annexin 2 translocation is dependent on both expression of protein p11 (S100A10) and tyrosine phosphorylation of annexin 2 because annexin 2 release is completely eliminated on depletion of p11, inactivation of tyrosine kinase, or mutation of tyrosine 23. Translocation of annexin 2 to the cell surface dramatically increases tissue plasminogen activator-dependent plasminogen activation potential and may represent a novel stress-induced protein secretion pathway.

Endothelial Cell Annexin A2 Regulates Polyubiquitination and Degradation of Its Binding Partner S100A10/p11

The annexin A2 (A2) heterotetramer, consisting of two copies of A2 and two copies of S100A10/p11, promotes fibrinolytic activity on the surface of vascular endothelial cells by assembling plasminogen and tissue plasminogen activator (tPA) and accelerating the generation of plasmin. In humans, overexpression of A2 by acute promyelocytic leukemia cells is associated with excessive fibrinolysis and hemorrhage, whereas anti-A2 autoantibodies appear to accentuate the risk of thrombosis in patients with anti-phospholipid syndrome. Complete deficiency of A2 in mice leads to a lack of tPA cofactor activity, accumulation of intravascular fibrin, and failure to clear arterial thrombi. Within the endothelial cell, p11 is required for Src kinase-mediated tyrosine phosphorylation of A2, which signals translocation of both proteins to the cell surface. Here we show that p11 is expressed at very low levels in the absence of A2 both in vitro and in vivo. We demonstrate further that unpartnered p11 becomes polyubiquitinated and degraded via a proteasome-dependent mechanism. A2 stabilizes intracellular p11 through direct binding, thus masking an autonomous p11 polyubiquitination signal that triggers proteasomal degradation. This interaction requires both the p11-binding N-terminal domain of A2 and the C-terminal domain of p11. This mechanism prevents accumulation of free p11 in the endothelial cell and suggests that regulation of tPA-dependent cell surface fibrinolytic activity is precisely tuned to the intracellular level of p11.