Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12^–/–) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor–mediated (uPAR-mediated) Akt2 phosphorylation at S⁴⁷⁴ (pAktS⁴⁷⁴). Downstream of pAkt2S⁴⁷⁴, FXII stimulation of neutrophils upregulated surface expression of α_Mβ₂ integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12^–/– mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12^–/– hosts was sufficient to correct the neutrophil migration defect in F12^–/– mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

Prolylcarboxypeptidase (PRCP) activates prekallikrein to plasma kallikrein, leading to bradykinin liberation, and degrades angiotensin II. We now identify PRCP as a regulator of blood vessel homeostasis. β-Galactosidase staining in PRCPgt/gt mice reveals expression in kidney and vasculature. Invasive telemetric monitorings show that PRCPgt/gt mice have significantly elevated blood pressure. PRCPgt/gt mice demonstrate shorter carotid artery occlusion times in 2 models, and their plasmas have increased thrombin generation times. Pharmacologic inhibition of PRCP with Z-Pro-Prolinal or plasma kallikrein with soybean trypsin inhibitor, Pro-Phe-Arg-chloromethylketone or PKSI 527 also shortens carotid artery occlusion times. Aortic and renal tissues have uncoupled eNOS and increased reactive oxygen species (ROS) in PRCPgt/gt mice as detected by dihydroethidium or Amplex Red fluorescence or lucigenin luminescence. The importance of ROS is evidenced by the fact that treatment of PRCPgt/gt mice with antioxidants (mitoTEMPO, apocynin, Tempol) abrogates the hypertensive, prothrombotic phenotype. Mechanistically, our studies reveal that PRCPgt/gt aortas express reduced levels of Kruppel-like factors 2 and 4, thrombomodulin, and eNOS mRNA, suggesting endothelial cell dysfunction. Further, PRCP siRNA treatment of endothelial cells shows increased ROS and uncoupled eNOS and decreased protein C activation because of thrombomodulin inactivation. Collectively, our studies identify PRCP as a novel regulator of vascular ROS and homeostasis.

Bradykinin (BK) liberates nitric oxide, prostacyclin, and tissue plasminogen activator from endothelial cells. We hypothesized that BK B2 receptor knockout (KO) mice (BKB2R^-/-) have increased thrombosis risk. Paradoxically, the BKB2R^-/- mice have long bleeding times and delayed carotid artery thrombosis, 78 ± 6.7 minutes, versus 31 ± 2.7 minutes in controls. The mechanism(s) for thrombosis protection was sought. In BKB2R^-/- plasma coagulation, fibrinolysis and anticoagulant proteins are normal except for an increased prekallikrein and decreased factor XI. BKB2R^-/- mice have elevated BK 1-5 (160 ± 75 fmol/mL, vs 44 ± 29 fmol/mL in controls) and angiotensin II (182 ± 41 pg/mL, vs 49 ± 7 pg/mL in controls). Ramipril treatment shortens vessel occlusion time. BKB2R^-/- mice have elevated plasma 6-keto-PGF_1α (666 ± 232 ng/mL, vs 23 ± 5.3 ng/mL in controls) and serum nitrate (61 ± 5.3 μM, vs 24 ± 1.8 μMin controls). Treatment with L-NAME (N^G-mono-methyl-l-arginine ester) or nimesulide shortens the thrombosis time. BKB2R^-/- mice have increased angiotensin receptor 2 (AT₂R) mRNA and protein expression. Treatment with an AT₂R antagonist, PD123 319, normalizes the thrombosis time and nitrate and 6-keto-PGF_1α. The long bleeding times in BKB2R^-/- mice also correct with L-NAME and nimesulide therapy. In BKB2R^-/- mice, angiotensin II binding to an overexpressed AT₂R promotes thromboprotection by elevating nitric oxide and prostacyclin. These investigations indicate a pathway for thrombosis risk reduction via the plasma kallikrein/kinin and renin angiotensin systems. (Blood. 2006;108:192-199)