Mice lacking factor XII (fXII) or factor XI (fXI) are resistant to experimentallyinduced thrombosis, suggesting fXIIa activation of fXI contributes to thrombus formation in vivo. It is not clear whether this reaction has relevance for thrombosis in primates. In 2 carotid artery injury models (FeCl 3 and Rose Bengal/laser), fXII-deficient mice are more resistant to thrombosis than fXI-or factor IX (fIX)-deficient mice, raising the possibility that fXII and fXI function in distinct pathways. Antibody 14E11 binds fXI from a variety of mammals and interferes with fXI activation by fXIIa in vitro. In mice, 14E11 prevented arterial occlusion induced by FeCl 3 to a similar degree to total fXI deficiency. 14E11 also had a modest beneficial effect in a tissue factor-induced pulmonary embolism model, indicating fXI and fXII contribute to thrombus formation even when factor VIIa/tissue factor initiates thrombosis. In baboons, 14E11 reduced plateletrich thrombus growth in collagen-coated grafts inserted into an arteriovenous shunt. These data support the hypothesis that fXIIa-mediated fXI activation contributes to thrombus formation in rodents and primates. Since fXII deficiency does not impair hemostasis, targeted inhibition of fXI activation by fXIIa may be a useful antithrombotic strategy associated with a low risk of bleeding complications. (Blood. 2010;116(19):3981-3989) IntroductionInitiation of fibrin formation by contact activation requires proteolytic conversion of plasma factor XII (fXII) to the protease factor XIIa (fXIIa) on a surface. 1-3 FXIIa activates the next zymogen in the coagulation cascade, factor XI (fXI), to factor XIa (fXIa), which in turn converts factor IX (fIX) to factor IXa␤ (fIXa␤). This series of reactions, referred to as the intrinsic pathway of coagulation, drives thrombin generation and fibrin formation in the activated partial thromboplastin time (aPTT) assay used by clinical laboratories. A role for fIX in hemostasis is not in question, as its deficiency causes the severe bleeding disorder hemophilia B. However, the importance of the intrinsic pathway, as a whole, to clot formation and stability at a site of injury is probably limited, as fXII deficiency is not associated with abnormal bleeding, 1,2 and fXI-deficient patients have a variable hemorrhagic disorder with milder symptoms than hemophiliacs. 2,4 Current models of thrombin generation address these phenotypic differences by incorporating additional mechanisms for protease activation. Thus, fIX is activated by the factor VIIa/tissue factor complex in addition to fXIa, 3,5 while fXI can be activated by thrombin. 3,6 Mice lacking fXII, like their human counterparts, do not have a demonstrable bleeding abnormality, 7 supporting the premise that fXIIa activation of fXI is not required for hemostasis. 8 Given this, it was surprising to observe that mice lacking fXII 9 or fXI 10 were resistant to arterial thrombotic occlusion. While this suggested contact activation might play an important role in pathologic coagulation, if not hemostasis...

• Factor XII can contribute to thrombus formation in human and nonhuman primate blood.• An antibody that blocks factor XII activation (15H8) produces an antithrombotic effect in a primate thrombosis model.The plasma zymogens factor XII (fXII) and factor XI (fXI) contribute to thrombosis in a variety of mouse models. These proteins serve a limited role in hemostasis, suggesting that antithrombotic therapies targeting them may be associated with low bleeding risks.Although there is substantial epidemiologic evidence supporting a role for fXI in human thrombosis, the situation is not as clear for fXII. We generated monoclonal antibodies (9A2 and 15H8) against the human fXII heavy chain that interfere with fXII conversion to the protease factor XIIa (fXIIa). The anti-fXII antibodies were tested in models in which anti-fXI antibodies are known to have antithrombotic effects. Both anti-fXII antibodies reduced fibrin formation in human blood perfused through collagen-coated tubes. fXII-deficient mice are resistant to ferric chloride-induced arterial thrombosis, and this resistance can be reversed by infusion of human fXII. 9A2 partially blocks, and 15H8 completely blocks, the prothrombotic effect of fXII in this model. 15H8 prolonged the activated partial thromboplastin time of baboon and human plasmas. 15H8 reduced fibrin formation in collagen-coated vascular grafts inserted into arteriovenous shunts in baboons, and reduced fibrin and platelet accumulation downstream of the graft. These findings support a role for fXII in

Abstract-We previously reported that adenosine A 2B receptor activation stimulates angiogenesis. Because hypoxia is a potent stimulus for the release of both adenosine and angiogenic factors, we tested the hypothesis that hypoxia alters the expression of adenosine receptors toward an "angiogenic" phenotype. Key Words: adenosine Ⅲ hypoxia Ⅲ receptors, purinergic Ⅲ endothelium Ⅲ vasculature Ⅲ muscle, smooth Ⅲ endothelium-derived factors T he purine nucleoside adenosine is an intermediate product of adenine nucleotide metabolism. Adenosine serves as a "retaliatory metabolite" in situations where oxygen supply is decreased or energy consumption is increased. Under these conditions, adenosine is released into the extracellular space and signals to restore the balance between energy supply and demand. Four extracellular G proteincoupled receptors, namely, A 1 , A 2A , A 2B , and A 3 , mediate adenosine actions. A 2B receptors have a lower affinity compared with other subtypes and require micromolar concentrations of adenosine for their stimulation. 1 Such high levels of extracellular adenosine can be reached during hypoxia, ischemia, inflammation, and injury. 2 The low affinity of A 2B receptors suggests that they are primarily engaged under these pathophysiologic conditions. A 2B receptors regulate various pathological processes, including mast cell activation, 3 vasodilation, 4 inhibition of cardiac fibroblast 5 and vascular smooth muscle growth, 6 stimulation of endothelial cell (EC) growth, 7 and angiogenesis. 8 -10 Stimulation of angiogenesis appears to be an important function of A 2B receptors. We have previously shown that A 2B receptors upregulate the production of angiogenic factors in human mast cells, retinal ECs, and human microvascular endothelial cells (HMEC-1s) under normoxic conditions. 8 -10 Tissue hypoxia is a powerful stimulus for the expression of genes associated with angiogenesis and, as mentioned previously, it is during hypoxia that adenosine levels increase to concentrations that engage A 2B receptors. Therefore, we tested the hypothesis that hypoxia would also modulate expression of adenosine receptor subtypes toward an "angiogenic" A 2B phenotype.

During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or ␣-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX. (Blood. 2009;114:452-458) IntroductionThe plasmas of placental and marsupial mammals contain factor XI (fXI), 1 the zymogen of a protease (fXIa) that contributes to fibrin formation and stability through activation of factor IX (fIX). [2][3][4] Congenital fXI deficiency is associated with a variable traumainduced bleeding disorder in humans and other species. [5][6][7][8] The mechanism by which fXI is converted to fXIa during blood coagulation has been a topic of recent debate. 9,10 When blood is exposed to a surface in vitro, the process of contact activation converts factor XII (fXII) to the protease fXIIa, which then activates fXI. 3,4 Substances, such as RNA, 11 polyphosphates, 12 and collagen, 13 induce pathologic coagulation in mice in a fXIIdependent manner 13,14 and may represent physiologic surfaces for fXII activation. However, the contribution of fXIIa-mediated fXI activation to normal hemostasis is unclear, as fXII deficiency, unlike fXI deficiency, is not associated with abnormal bleeding in any species in which it has been identified. 4 This key observation supports hypotheses proposing that fXI is either activated during hemostasis by a protease other than fXIIa or that auxiliary mechanisms for fXI activation compensate in the absence of fXII. 3,[15][16][17] Candidates for fXI-activating proteases include ␣-thrombin, 15,16 meizothrombin, 18 and fXIa (autoactivation). 15,16 Thrombin has received much attention in this regard. Several laboratories have presented evidence suggesting that a protease generated early in coagulation, such as thrombin, converts fXI to fXIa. [19][20][21][22][23] This hypothesis has been challenged by a recent study that did not find evidence for fXI activation in thrombin or tissue facto...

Adenosine provokes bronchoconstriction in asthmatics through acute activation of mast cells, but its potential role in chronic inflammation has not been adequately characterized. We hypothesized that adenosine up-regulates Th2 cytokines in mast cells, thus promoting IgE synthesis by B lymphocytes. We tested this hypothesis in human mast cells (HMC-1) expressing A2A, A2B, and A3 adenosine receptors. The adenosine analog 5′-N-ethylcarboxamidoadenosine (NECA) (10 μM) increased mRNA expression of IL-1β, IL-3, IL-4, IL-8, and IL-13, but not IL-2 and IFN-γ. Up-regulation of IL-4 and IL-13 was verified using RT-PCR and ELISA; 10 μM NECA increased IL-13 concentrations in HMC-1 conditioned medium 28-fold, from 7.6 ± 0.3 to 215 ± 4 pg/ml, and increased IL-4 concentrations 6-fold, from 19.2 ± 0.1 to 117 ± 2 pg/ml. This effect was mediated by A2B receptors because neither the selective A2A agonist 2-p-(2-carboxyethyl)phenethylamino-NECA nor the selective A3 agonist N6-(3-iodobenzyl)-N-methyl-5′-carbamoyladenosine reproduced it, and the selective A2B antagonist 3-isobutyl-8-pyrrolidinoxanthine prevented it. Constitutive expression of CD40 ligand on HMC-1 surface was not altered by NECA. Human B lymphocytes cocultured for 12 days with NECA-stimulated HMC-1 produced 870 ± 33 pg IgE per 106 B cells, whereas lymphocytes cocultured with nonstimulated HMC-1, or cultured alone in the absence or in the presence of NECA, produced no IgE. Thus, we demonstrated induction of IgE synthesis by the interaction between adenosine-stimulated mast cells and B lymphocytes, and suggest that this mechanism is involved in the amplification of the allergic inflammatory responses associated with asthma.

Factor XI (FXI) is the zymogen of a plasma protease, factor XIa (FXIa), that contributes to thrombin generation during blood coagulation by proteolytic activation of several coagulation factors, most notably factor IX (FIX). FXI is a homolog of prekallikrein (PK), a component of the plasma kallikrein-kinin system. While sharing structural and functional features with PK, FXI has undergone adaptive changes that allow it to contribute to blood coagulation. Here we review current understanding of the biology and enzymology of FXI, with an emphasis on structural features of the protein as they relate to protease function.

When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.

Objective During coagulation, factor IX (FIX) is activated by two distinct mechanisms mediated by the active proteases of either factors VII (FVIIa) or XI (FXIa). Both coagulation factors may contribute to thrombosis; factor XI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. Approach and Results We have reported that reducing FXI levels with FXI antisense oligonucleotides (ASOs) produces antithrombotic activity in mice, and that administration of FXI ASOs to primates decreases circulating FXI levels and activity in a dose- and time-dependent manner. Here we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present report, ASO-mediated reduction of FXI plasma levels by ≥50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. Conclusion These results indicate that reducing FXI levels using ASOs is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.