Factor XII Stimulates ERK1/2 and Akt through uPAR, Integrins, and the EGFR to Initiate Angiogenesis.

Abstract 3040

Poster Board II-1016

The physiologic function of factor XII (XII) is not known. New interest in XII has occurred because XII KO mice are protected from thrombosis. Exposed arterial collagen, aggregated protein, RNA, and platelet polysomes are recently recognized entities in developing thrombus that promote XII autoactivation to XIIa increasing thrombus formation, independent of a role in hemostasis. However, these observations do not indicate a constitutive, physiologic function for XII. We sought a physiologic function for zymogen XII. XII has been recognized to stimulate MAP kinase. Cleaved high molecular weight kininogen (HKa) is known to be antiangiogenic. Further, XII and HK mutually block each others binding to endothelial cells. Both XII and HKa bind urokinase plasminogen activator receptor (uPAR) at an overlapping site. We investigated if XII stimulates cells by interacting with uPAR and if this activity influences angiogenesis. XII (3-200 nM) with 0.05 mM Zn ion induces ERK1/2 (MAPK44 and 42) and Akt (Ser473) phosphorylation in endothelial cells. XII-induced phosphorylation of ERK1/2 or Akt is a zymogen activity, not an enzymatic event. ERK1/2 or Akt phosphorylation is blocked upstream by PD98059 or Wortmannin or LY294002, respectively. The uPAR signaling region for XII is on domain 2 adjacent to uPAR's integrin binding site. HKa or peptides from HKa's domain 5 inhibit XII-induced ERK1/2 and Akt phosphorylation. A beta-1-integrin peptide that binds uPAR, antibody 6S6 to beta-1-integrin, or the EGFR inhibitor AG1478 blocks XII-induced phosphorylation of ERK1/2 and Akt. XII induces endothelial cell proliferation and 5-bromo-2'deoxy-uridine incorporation. XII stimulates aortic sprouting in normal but not uPAR deficient mouse aorta and this mechanism is blocked by PD98059, LY294002, AG1478, or HKa. XII also induces angiogenesis in matrigel plugs. Finally, XII knockout mice have reduced constitutive and wound-induced blood vessel number on initial biopsy and 9 days after wound healing, respectively. In sum, XII initiates outside-in signaling mediated by uPAR, beta-1-integrin, and the EGFR leading to HUVEC proliferation, growth, and angiogenesis. XII is a constitutive proangiogenic protein.