Leukocyte XII Regulates Venous Thrombosis Risk

Introduction: Previous studies show that Factor XII (XII) participates in the inflammatory response. XII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. XII deficient patients have reduced leukocyte migration into skin windows. In vitro, purified XIIa corrects neutrophil aggregation and degranulation defects in XII-deficient plasma. Recent studies show that leukocytes initiate and propagate venous thrombosis in vivo. We examined the contribution of XII in the inflammatory response and venous thrombosis.

Methods & Results: Sterile punch biopsy wounds were created on wild type (WT) and F12^-/- mice. On Days 2 and 5, there was a ~3-fold decrease in CD11b-stained cells in F12^-/- wounds vs. WT. On the thioglycolate (TG)-induced sterile peritonitis assay, lavage fluid from F12^-/- mice contained significantly less peritoneal exudative cells (PEC)] on days 1 and 7, (p<0.008). To determine the contribution of XII in WBC function, we used XII siRNA (Alnylam Pharmaceuticals) to create plasma XII deficiency in WT mice. After tail vein injection, plasma XII was reduced to < 5% within 24 h (T_1/2 plasma XII: 6.7 h). In the TG assay, even though plasma XII is decreased to less than 5%, PEC migration is the same as in WT mice. These data suggest that the reduced leukocyte migration observed in F12^-/- mice is related to altered leukocyte function. On adoptive bone marrow (BM) transplantation (BMT) experiments, WT BM transplanted into KO hosts corrects the leukocyte migration defect on the TG assay. These data suggest that there is a pool of XII associated with BM cells that is functionally distinct than plasma-derived, hepatic XII. F12 cDNA is found in leukocytes and shares sequence homology to hepatic XII. Immunofluorescence confirms XII antigen on murine BM-derived and human peripheral blood WBCs. No XII antigen is observed in BM-derived leukocytes from F12^-/- mice. When WBC are activated with fMLF, XII antigen translocates to the external membrane. F12^-/- PMNs have reduced chemotaxis to fMLF and adherence to several integrin-binding glycoproteins. pAktS⁴⁷³ mediates neutrophil cell migration, integrin activation, and cytoskeletal assembly. Normal and F12^-/- PMNs exhibit pAktS⁴⁷³ in response to fMLF and XII. Histologically, F12^-/- wounds show a smaller wound gap and a greater percentage of wound re-epithelialization than WT controls. Inferior vena cava (IVC) thrombosis induced by 90% restriction to flow at 24h contains a smaller thrombus in F12^-/- than WT mice (p<0.04). Histologically, IVC thrombi from WT mice contain abundant neutrophils that are adherent to the wall and trapped within a dense fibrin network (Fig 1). siRNA treatment results in less-occlusive thrombi (n.s) with an adequate neutrophil content but a finer fibrin network (Fig 1). F12^-/- thrombi are non-occlusive and contain significantly less adherent neutrophils (Fig 1). XII itself is integrally a part of neutrophil extracellular traps (NETs) in the forming thrombus and F12^-/- mice have reduced NETs at sites of occlusion. WT BM transplanted into F12^-/- hosts corrects the thrombus weight and degree of inflammation in F12^-/- mice to normal. Likewise, F12^-/- BM into WT hosts, reduces thrombus weight and degree of inflammation.

Conclusions: Leukocyte XII has a dual role in neutrophil function. We hypothesize that signaling by leukocyte XII contributes to neutrophil trafficking in sites of inflammation and venous stasis. At these sites, neutrophils become indispensable for activation of both the extrinsic and intrinsic pathways of coagulation during the early formation of intraluminal fibrin and for subsequent thrombus propagation by NETs and the activation of circulating XII. Defining the signaling pathway of XII in leukocytes will further our understanding as to the mechanism(s) by which these cells cooperate to initiate and propagate venous thrombosis in vivo.