Factor XII: a novel target for safe prevention of thrombosis and inflammation.

Plasma protein factor XII (FXII) activates the procoagulant and proinflammatory contact system that drives both the kallikrein-kinin system and the intrinsic pathway of coagulation. When zymogen FXII comes into contact with negatively charged surfaces, it auto-activates to the serine proteaseactivated FXII (FXIIa). Recently, various in vivo activators of FXII have been identified including heparin, misfolded protein aggregates, polyphosphate and nucleic acids. Murine models have established a central role of FXII in arterial and venous thrombosis. Despite its central function in thrombosis, deficiency in FXII does not impair haemostasis in animals and humans. In a preclinical cardiopulmonary bypass system in large animals, the FXIIa-blocking antibody 3F7 prevented thrombosis; however, in contrast to traditional anticoagulants, bleeding was not increased. In addition to its function in thrombosis, FXIIa initiates formation of the inflammatory mediator bradykinin. This mediator increases vascular leak, causes vasodilation, and induces chemotaxis with implications for septic, anaphylactic and allergic disease states. Therefore, targeting FXIIa appears to be a promising strategy for thromboprotection without associated bleeding risks but with anti-inflammatory properties.

Neutrophil secretion products regulate anti-bacterial activity in monocytes and macrophages.

Macrophages represent a multi-functional cell type in innate immunity that contributes to bacterial clearance by recognition, phagocytosis and killing. In acute inflammation, infiltrating neutrophils release a wide array of preformed granule proteins which interfere functionally with their environment. Here, we present a novel role for neutrophil-derived granule proteins in the anti-microbial activity of macrophages. Neutrophil secretion obtained by antibody cross-linking of the integrin subunit CD18 (X-link secretion) or by treatment with N-Formyl-Met-Leu-Phe (fMLP secretion) induced a several-fold increase in bacterial phagocytosis by monocytes and macrophages. This response was associated with a rapid activation of the monocytes and macrophages as depicted by an increase in cytosolic free Ca(2+). Interestingly, fMLP secretion had a more pronounced effect on monocytes than the X-link secretion, while the opposite was observed for macrophages. In addition, polymorphonuclear cells (PMN) secretion caused a strong enhancement of intracellular reactive oxygen species (ROS) formation compared to incubation with bacteria. Thus, secretion of neutrophil granule proteins activates macrophages to increase the phagocytosis of bacteria and to enhance intracellular ROS formation, indicating pronounced intracellular bacterial killing. Both mechanisms attribute novel microbicidal properties to PMN granule proteins, suggesting their potential use in anti-microbial therapy.

Contact system revisited: an interface between inflammation, coagulation, and innate immunity.

The contact system is a plasma protease cascade initiated by factor XII (FXII) that activates the proinflammatory kallikrein-kinin system and the procoagulant intrinsic coagulation pathway. Anionic surfaces induce FXII zymogen activation to form proteolytically active FXIIa. Bacterial surfaces also have the ability to activate contact system proteins, indicating an important role for host defense using the cooperation of the inflammatory and coagulation pathways. Recent research has shown that inorganic polyphosphate found in platelets activates FXII in vivo and can induce coagulation in pathological thrombus formation. Experimental studies have shown that interference with FXII provides thromboprotection without a therapy-associated increase in bleeding, renewing interest in the FXIIa-driven intrinsic pathway of coagulation as a therapeutic target. This review summarizes how the contact system acts as the cross-road of inflammation, coagulation, and innate immunity.

New agents for thromboprotection. A role for factor XII and XIIa inhibition.

Blood coagulation is essential for hemostasis, however excessive coagulation can lead to thrombosis. Factor XII starts the intrinsic coagulation pathway and contact-induced factor XII activation provides the mechanistic basis for the diagnostic aPTT clotting assay. Despite its function for fibrin formation in test tubes, patients and animals lacking factor XII have a completely normal hemostasis. The lack of a bleeding tendency observed in factor XII deficiency states is in sharp contrast to deficiencies of other components of the coagulation cascade and factor XII has been considered to have no function for coagulation in vivo. Recently, experimental animal models showed that factor XII is activated by an inorganic polymer, polyphosphate, which is released from procoagulant platelets and that polyphosphate-driven factor XII activation has an essential role in pathologic thrombus formation. Cumulatively, the data suggest to target polyphosphate, factor XII, or its activated form factor XIIa for anticoagulation. As the factor XII pathway specifically contributes to thrombosis but not to hemostasis, interference with this pathway provides a unique opportunity for safe anticoagulation that is not associated with excess bleeding. The review summarizes current knowledge on factor XII functions, activators and inhibitors.

Superselective intra-arterial umbilical cord blood administration to BM in experimental animals.

Umbilical cord blood (UCB) as a source of hematopoietic stem cells for transplantation is limited by the low number of cells and delayed engraftment. UCB cells are infused i.v. for transplantation, although only a proportion of the cells reach the BM. We investigated whether UCB could be administered safely using superselective intra-arterial (i.a.) injection. We injected human UCB (5 × 10(6)) into the aorta in rats, into the iliac artery in mice and into the femoral nutrient artery (FNA) in rabbits. We used angiography, immunohistochemistry, intravital microscopy and qPCR to assess safety end points and the distribution of injected cells. All animals showed normal behavior. No evidence of organ infarction was noted. UCB injected into the FNA of rabbits did not change the flow rates, measured by angiography. By qPCR, we found significantly higher fold-change values in the injected BM compared with i.v. injection (P=0.0087). Using intravital microscopy we visualized the mouse capillary bed during i.a. injection without cellular congestion. In summary, we show that i.a. infusion of UCB is safe and reaches an eightfold increase in engraftment in the BM compared with i.v. infusion. These studies lay the foundation for clinical trials.

Imaging inflammatory plasma leakage in vivo.

Increased vascular permeability and consequent plasma leakage from postcapillary venules is a cardinal sign of inflammation. Although the movement of plasma constituents from the vasculature to the affected tissue aids in clearing the inflammatory stimulus, excessive plasma extravasation can lead to hospitalisation or death in cases such as influenza-induced pneumonia, burns or brain injury. The use of intravital imaging has significantly contributed to the understanding of the mechanisms controlling the vascular permeability alterations that occur during inflammation. Today, intravital imaging can be performed using optical and non-optical techniques. Optical techniques, which are generally used in experimental settings, include traditional intravital fluorescence microscopy and near-infrared fluorescence imaging. Magnetic resonance (MRI) and radioisotopic imaging are used mainly in the clinical setting, but are increasingly used in experimental work, and can detect plasma leakage without optics. Although these methods are all able to visualise inflammatory plasma leakage in vivo, the spatial and temporal resolution differs between the techniques. In addition, they vary with regards to invasiveness and availability. This overview discusses the use of imaging techniques in the visualisation of inflammatory plasma leakage.