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...

Ions of structure X[N(O)NO]- display broad-spectrum pharmacological activity that correlates with the rate and extent of their spontaneous, first-order decomposition to nitric oxide when dissolved. We report incorporation of this functional group into polymeric matrices that can be used for altering the time course of nitric oxide release and/or targeting it to tissues with which the polymers are in physical contact. Structural types prepared include those in which the [N(O)NO]- group is attached to heteroatoms in low molecular weight species that are noncovalently distributed throughout the polymeric matrix, in groupings pendant to the polymer backbone, and in the polymer backbone itself. They range in physical form from films that can be coated onto other surfaces to microspheres, gels, powders, and moldable resins. Chemiluminescence measurements confirm that polymers to which the [N(O)NO]- group is attached can serve as localized sources of nitric oxide, with one prototype providing sustained NO release for 5 weeks in pH 7.4 buffer at 37 degrees C. The latter composition, a cross-linked poly-(ethylenimine) that had been exposed to NO, inhibited the in vitro proliferation of rat aorta smooth muscle cells when added as a powder to the culture medium and showed potent antiplatelet activity when coated on a normally thrombogenic vascular graft situated in an arteriovenous shunt in a baboon's circulatory system. The results suggest that polymers containing the [N(O)NO]- functional group may hold considerable promise for a variety of biomedical applications in which local delivery of NO is desired.

Treatment of brain disease with recombinant proteins is difficult due to the blood-brain barrier. As an alternative to direct injections into the brain, we studied whether application of high concentrations of therapeutic enzymes via intrathecal (IT) injections could successfully drive uptake across the ependyma to treat brain disease. We studied IT enzyme replacement therapy with recombinant human iduronidase (rhIDU) in canine mucopolysaccharidosis I (MPS I, Hurler syndrome), a lysosomal storage disorder with brain and meningeal involvement. Monthly or quarterly IT treatment regimens with rhIDU achieved supranormal iduronidase enzyme levels in the brain, spinal cord, and spinal meninges. All regimens normalized total brain glycosaminoglycan (GAG) storage and reduced spinal meningeal GAG storage by 58-70%. The improvement in GAG storage levels persisted three months after the final IT dose. The successful use of enzyme therapy via the CSF represents a potentially useful approach for lysosomal storage disorders.

Thrombin, generated through activation of factor XI (FXI) and/or tissue factor (TF)-factor VIIa, is essential for thrombosis and hemostasis. We investigated the role of FXI-dependent thrombus propagation under arterial flow conditions producing rapid thrombus growth that, after the initiation phase, could limit the availability of TF at the blood/thrombus interface. Thrombosis was initiated by knitted dacron or TF-presenting teflon grafts deployed into arteriovenous shunts in baboons treated with antihu-man FXI antibody (aFXI). Although aFXI did not prevent thrombus initiation, it markedly reduced intraluminal throm-bus growth on both surfaces. The anti-thrombotic effect of aFXI was comparable with that of heparin at doses that significantly prolonged the partial thromboplastin time (APTT), prothrom-bin time (PT), and bleeding time (BT). aFXI also prolonged the APTT, but the PT and BT were unaffected. Thus, anti-thrombotic targeting of FXI might inhibit thrombosis with relatively modest hemo-static impairment versus strategies targeting other coagulation factors. (Blood. 2003;102:953-955)

Reaction of nitric oxide (NO) with L-proline in methanolic sodium methoxide yields a diazeniumdiolate product, C5H7N3O4Na2.CH3OH (PROLI/NO), that can be stabilized in basic solution but that dissociates to proline (1 mol) and NO (2 mol) with a half-life of only 1.8 s at pH 7.4 and 37 degrees C. This kinetic behavior has allowed the generation of highly localized antiplatelet and vasodilatory effects. By infusing solutions containing 4 microM PROLI/NO in 0.1 M sodium hydroxide at the rate of 1 nmol.min-1 immediately upstream from a polyester vascular graft in the unheparinized baboon circulatory system, for example, platelet deposition at the normally thrombogenic graft surface was substantially reduced relative to controls receiving only 0.1 M sodium hydroxide. In a second study, infusion of PROLI/NO into the right atrium of sheep with induced pulmonary hypertension selectively dilalated the lung vasculature, dose-dependently reducing the pulmonary arterial pressure by as much as 9 mmHg with no observable effect on the systemic arterial pressure at an infusion rate of up to 24 nmol.kg-1.min-1. PROLI/NO could also be formulated as an insoluble polymer blend that released NO smoothly for prolonged periods. The results suggest that localized delivery of diazeniumdiolates such as PROLI/NO which generate NO with extreme rapidity on entering the blood stream may hold considerable promise for inhibition of thrombus formation, selective dilation of the vasculature, and other research and clinical applications.

The effects on thrombosis and hemostasis of thrombin-induced activation of endogenous protein C (PC) were evaluated in baboons. Thrombosis was induced by placing into arteriovenous shunts a segment of Dacron vascular graft, which generated arterial platelet-rich thrombus, followed by an expansion region of low-shear blood flow, which in turn accumulated fibrinrich venous-type thrombus. Thrombosis was quantified by "'In-platelet imaging and '25I-fibrinogen accumulation. Intravenous infusion of a-thrombin, 1-2 U/kg-min for 1 h, increased baseline activated PC levels (-5 ng/ml) to 250-500 ng/ml (P < 0.01). The lower thrombin dose, which did not deplete circulating platelets, fibrinogen, or PC, reduced arterial graft platelet deposition by 48% (P < 0.05), and platelet and fibrin incorporation into venous-type thrombus by > 85% (P < 0.01). Thrombin infusion prolonged the activated partial thromboplastin clotting time, elevated fibrinopeptide A (FPA), thrombin-antithrombin III complex (T:AT III), and fibrin D-dimer plasma levels (P < 0.01), but did not affect bleeding times. Thrombin's antithrombotic effects were blocked by infusing a monoclonal antibody (HPC4) which prevented PC activation in vivo, caused shunt occlusion, increased the consumption of platelets and fibrinogen, elevated plasma FPA and T:AT III levels, and reduced factor VIII (but not factor V) procoagulant activity (P < 0.05). We conclude that activated PC is a physiologic inhibitor of thrombosis, and that activation of endogenous PC may represent a novel and effective antithrombotic strategy. (J. Clin. Invest.