Using a combination of biochemical and proteomic analysis, we find that the coagulation protease factor Xa binds to annexin 2 on the endothelial cell surface. Annexin 2 interaction is required for signal transduction (), whereas the procoagulant function of factor Xa is independently regulated (). Only the Xaβ form of factor Xa binds to annexin 2, and this binding occurs independently of C-terminal lysine residues (). It appears that autoproteolysis is important for binding, because the factor Xa–specific inhibitor Nap5 or inactivation with a chloromethyl-ketone inhibitor reduced annexin 2 binding ( and ). The interaction of factor Xa with annexin 2 is also specific, because annexin 1 does not bind factor Xa and factor X does not bind annexin 2 (). Annexin 2 has previously been found to function as a receptor for the fibrinolytic zymogen plasminogen (Plg).22
Recent studies also indicate that the anti-angiogenic factor angiostatin may bind annexin 2.36
Neither Plg nor angiostatin competes for factor Xa binding to annexin 2 (), and EACA does not inhibit annexin 2/factor Xa coprecipitation (), indicating that the factor Xa/annexin 2 interaction is distinct from that of other annexin 2 ligands. Interestingly, previous work has shown that autoproteolysis or plasmin-mediated cleavage of Xaα to Xaβ exposes a Plg binding site and inhibits coagulation. 7
This result, in combination with our studies here, indicates a possible regulatory mechanism in which Xaβ is the primary regulator of signal transduction when TF is not present.
In the presence of TF, factor Xa mediates signal transduction through the activation of PAR-1 and PAR-2.12,19
In the absence of TF, annexin 2 mediates factor Xa signaling () predominantly through PAR-1. Interestingly, in the presence of TF, the overall level of factor Xa signaling does not change. However, the annexin 2–dependent components of factor Xa signaling are completely abolished. The coagulation-independent, signaling-specific regulation of factor Xa by annexin 2 that we observe in vitro is also replicated in primary mouse cells ( and ). Whether this TF effect is attributable to the regulation of factor Xa autoproteolysis or by influencing factor Xa–Gla domain interactions with annexin 2, perhaps in some combination of PAR-1 and PAR-2, remains to be fully elucidated.
Recent in vitro and in vivo studies have demonstrated the importance of annexin 2 in the regulation of fibrinolysis and neoangiogenesis. Higher annexin 2 expression was observed in cells from acute promyelocytic leukemia patients when compared with other leukemic cells. This resulted in increased tissue plasminogen activator–dependent plasmin generation and may account for the increased hemorrhagic complications associated with acute promyelocytic leukemia. 37
Recombinant annexin 2 restored plasmin generation in HUVECs impaired by plasminogen activator inhibitor-1 or homocysteine, and injection of annexin 2 enhanced the patency of thrombosed arteries in a rat carotid artery thrombosis model.38
Increased annexin 2 on the surface of aortic endothelial cells is associated with increased plasmin activity in diabetic rat aortas.39
In vivo studies have demonstrated that annexin 2–knockout mice displayed enhanced fibrin deposition in the microvasculature and improper clearance of thrombi as well as inefficient tissue plasminogen activator–dependent plasmin generation.25
In 3 different angiogenesis models, fibroblast growth factor–stimulated cornea, oxygen-primed neonatal retina, and aortic ring explants, annexin 2–null mice demonstrated significantly reduced neovascularization and capillary sprouting, respectively.25
The decrease in aortic ring sprouting was attributable to impaired matrix metalloproteinase (MMP)-9 and MMP-13 activation.25
Factor Xa has also been shown to play a role in the activation of MMPs, including conversion of pro–MMP-2 to MMP-2 in vascular smooth muscle cells40
and promotion of MMP-1 release in gingival fibroblasts.41
Although only a small amount of factor Xa is necessary to initiate coagulation, 42,43
studies have shown that higher levels of factor Xa can be generated under certain pathologic circumstances,44,45
in addition to the physiologic conversion of factor X to factor Xa that occurs under TF-independent circumstances via the intrinsic pathway. These observations indicate a potential physiological relevance for the results we observe in vitro regarding factor Xa binding to annexin 2 in the absence of TF. Previous work indicates that annexin 2 plays a predominant role in cell invasion, fibrin homeostasis, and fibrin degradation, but not in blood coagulation.25,37
The coagulation-independent regulation of factor Xa signaling that we demonstrate for annexin 2 is consistent with these observations. IL-6, IL-8, and MCP-1 expression have been shown to be increased by factor Xa in HUVECs,46
and PAR-1 has been demonstrated to upregulate cyclooxygenase-2 in HUVECs.47
To determine the role of annexin 2 in regulating these downstream signaling functions, we will further characterize factor Xa signaling in TF expressing and nonexpressing cells isolated from annexin 2 knockout mice.
Factor Xa–mediated signal transduction via PAR-1 and PAR-2 has been shown to be cell type–dependent using endothelial cells and fibroblasts from PAR-1 and PAR-2 knockout mice.12
PAR-2 was demonstrated to be the primary mediator of factor Xa–induced phosphoinositide hydrolysis and ERK1/2 phosphorylation in the murine endothelial cells, whereas in murine fibroblasts the majority of signaling was regulated by PAR-1.12
PAR-2 activation has also been shown to play a role in P-selectin–dependent leukocyte rolling resulting in microvascular inflammation.48
Whether the interaction of factor Xa and annexin 2 is relevant to some of the aforementioned hemostatic, angiogenic, and inflammatory functions remains to be elucidated. Future mechanistic studies are required to determine the differences in factor Xa signaling via PAR-1 and PAR-2 and how annexin 2 binding affects these processes. In conclusion, our work proposes a novel role for annexin 2 as an endothelial cell surface receptor for factor Xa that regulates factor Xa–mediated signal transduction via PAR-1 in a coagulation-independent manner.