With existing optical imaging techniques three-dimensional (3-D) mapping of microvascular perfusion within tissue beds is severely limited by the efficient scattering and absorption of light by tissue. To overcome these limitations we have developed a method of optical angiography (OAG) that can generate 3-D angiograms within millimeter tissue depths by analyzing the endogenous optical scattering signal from an illuminated sample. The technique effectively separates the moving and static scattering elements within tissue to achieve high resolution images of blood flow, mapped into the 3-D optically sectioned tissue beds, at speeds that allow for perfusion assessment in vivo. Its development has its origin in Fourier domain optical coherence tomography. We used OAG to visualize the cerebral microcirculation, of adult living mice through the intact cranium, measurements which would be difficult, if not impossible, with other optical imaging techniques.

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...

The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)-dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti-human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and ␤-thromboglobulin (␤TG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in D-dimer release from thrombi, and prevented the occlusion of 2-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (32 mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagencoated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface. (Blood. 2009;113:936-944) IntroductionBlood coagulation during hemostasis is initiated by the tissue factor (TF)/factor VIIa complex (the extrinsic pathway) that activates factors IX and X, and ultimately produces thrombin at sites of vascular injury. 1 In thrombosis, intravascular blood coagulation may also be initiated by the extrinsic pathway. 2,3 However, impairment of the TF/factor VIIa pathway does not provide full protection from thrombosis, since symptomatic factor VII deficient subjects can develop concurrent thrombosis and severe bleeding. 4 The functions of the contact proteins (factor XI, factor XII, prekallikrein, and high-molecular-weight kininogen) in hemostasis are less clear. The physiologic role of factor XI (FXI) has been difficult to determine because of the variable bleeding disorder associated with FXI deficiency, 5 and because monospecific FXI inhibitors have not been widely available for experimental investigation. FXI activation is thought to proceed through thrombin-and/or factor XIIdependent mechanisms, and activated FXI (FXIa) contributes to sustained thrombin generation after initiation of blood clotting by activating factor IX. These activities ultimately promote coagulation, platelet activation, and preservation of fibrin clot integrity. 6,7 Thrombin also increases the density of fibrin networks 8 and indirectly inhibits fibrinolysis through activation of carboxypeptidase B (thrombin-activatable fibrinolysis inhibitor, TAFI). 9 Thus, FXI may support thrombus propagation and clot stability by increasing thrombin generation. 10,11 Compelling circumstantial evidence suggests a contributory role for FXI in the p...

Thrombosis is initiated by tissue factor (TF), a coagulation cofactor/receptor expressed in the vessel wall, on myeloid cells, and on microparticles (MPs) with variable procoagulant activity. However, the molecular pathways that generate prothrombotic TF in vivo are poorly defined. The oxidoreductase protein disulfide isomerase (PDI) is thought to be involved in the activation of TF. Here, we found that in mouse myeloid cells, ATPtriggered signaling through purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7 receptor; encoded by P2rx7) induced activation (decryption) of TF procoagulant activity and promoted release of TF + MPs from macrophages and SMCs. The generation of prothrombotic MPs required P2X7 receptor-dependent production of ROS leading to increased availability of solvent-accessible extracellular thiols. An antibody to PDI with antithrombotic activity in vivo attenuated the release of procoagulant MPs. In addition, P2rx7 -/-mice were protected from TF-dependent FeCl 3 -induced carotid artery thrombosis. BM chimeras revealed that P2X7 receptor prothrombotic function was present in both hematopoietic and vessel wall compartments. In contrast, an alternative anti-PDI antibody showed activities consistent with cellular activation typically induced by P2X7 receptor signaling. This anti-PDI antibody restored TF-dependent thrombosis in P2rx7 -/-mice. These data suggest that PDI regulates a critical P2X7 receptor-dependent signaling pathway that generates prothrombotic TF, defining a link between inflammation and thrombosis with potential implications for antithrombotic therapy.