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.
Qi Ling, Andrew T. Jacovina, Arunkumar Deora, Maria Febbraio, Ronit Simantov, Roy L. Silverstein, Barbara Hempstead, Willie H. Mark, Katherine A. Hajjar
Submitter: Katherine A. Hajjar | firstname.lastname@example.org
Published January 16, 2004
Our hypothesis that annexin II monomer acts as a catalytic receptor for plasminogen and tissue plasminogen activator (tPA) is based on several observations:  Both plasminogen and tPA interact specifically with annexin II in ligand blots of endothelial cell plasma membrane extracts.  Both proteins bind saturably and specifically with annexin II purified from human tissue.  Purified native annexin II monomer augments the catalytic efficiency of tPA-dependent plasminogen activation in a purified protein system.  Anti-annexin II IgG inhibits a significant proportion of total specific binding of plasminogen and tPA to cultured endothelial cells.  Anti-annexin II IgG inhibits tPA "cofactor" activity contributed by endothelial cells.  Intravascular annexin II monomer, finally, prevents carotid artery thrombosis in rats following oxidative injury. These data suggest that annexin II monomer is necessary for t-PA cofactor activity.
As is the case with any null deletion model system, our results do not rule out a potential contribution from other proteins, such as p11, that may interact with annexin II or modify its activity. Dr. Waisman suggests that p11 is important in cell surface tPA- dependent plasminogen activation, and he notes correctly that reduction of annexin II protein expression is associated with a reduction in p11 in some cultured cell systems. We are currently examining the level of p11 expression in a range of annexin II null tissues, and are conducting further experiments to understand the individual contributions of annexin II and p11 to tPA-dependent plasminogen activation both in vitro and in vivo.
Submitter: David M. Waisman | email@example.com
University of Calgary
Published January 16, 2004
In their recent article, Dr. Hajjar and colleagues clearly and convincingly show that annexin II-null mice demonstrate a remarkable phenotype. The homozygous annexin II-null mice displayed deposition of fibrin in the microvasculature and incomplete clearance of injury-induced arterial thrombi.
Typically, annexin II exists as a heterotetramer (referred to as AIIt) on the surface of most cells. The heterotetramer is composed of two annexin II (p36) and two S100A10 (p11) subunits. The binding of annexin II with p11 is essentially irreversible (low nanomolar Kd). It is a point of controversy as to whether or not annexin II or the p11 binding partner plays a role in plasminogen regulation. The presence of a binding site for tPA and plasminogen at the carboxyl-terminus of p11 plus the demonstration that changes in the extracellular levels of p11 (without a corresponding change in annexin II levels) affect cellular plasmin production has been interpreted by our laboratory as suggesting that p11 is the key plasminogen regulator.
It is interesting to note that that manipulation of annexin II levels in cultured cells has been shown to result in changes in p11 levels. However, it is unclear from Dr. Hajjar’s report whether or not the levels of p11 are affected in the annexin II-null mice. Should the p11 levels be altered in the annexin II-null mice, as is likely, then it would not be possible to discern whether annexin II or p11 plays a role in the phenotype of the annexin II-null mouse. Therefore, the issue of whether annexin II plays a direct role in the phenotype of the annexin II-null mouse will await the development of a p11-null mouse.