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Nanoparticles have been assessed in preclinical models of atherosclerosis for detection of plaque complexity and treatment. However, their successful clinical translation has been hampered by less than satisfactory plaque detection, and lack of a general strategy for assessing the translational potential of nanoparticles. Herein, nanoparticles based on comb-copolymer assemblies were synthesized through a modular construction approach with precise control over the conjugation of multiple functional building blocks for in vivo evaluation. This high level of design control also allows physicochemical properties to be varied in a controllable fashion. Through conjugation of c-atrial natriuretic factor (CANF) peptide and radiolabeling with (64)Cu, the (64)Cu-CANF-Comb nanoparticle was assessed for plaque imaging by targeting natriuretic peptide clearance receptor (NPRC) in a double-injury atherosclerosis model in rabbits. The extended blood circulation and improved binding capacity of (64)Cu-CANF-Comb nanoparticles afforded sensitive and specific detection of NPRC upregulated in atherosclerotic lesions by positron emission tomography (PET) at intervals during the progression of disease. Ex vivo tissue validation using autoradiography and immunostaining on human carotid endarterectomy specimens demonstrated specific binding of (64)Cu-CANF-Comb to human NPRC receptors. Taken together, this study not only shows the potential of NPRC-targeted (64)Cu-CANF-Comb nanoparticles for increased sensitivity to an epitope that increases during atherosclerosis plaque development, but also provides a useful strategy for the general design and assessment of translational potential of nanoparticles in cardiovascular imaging.

Antithrombotic therapy is essential to prevent thrombotic reocclusion in patients with acute myocardial infarction, stroke, or venous thromboembolism. However, current antithrombotic drugs cause bleeding that limits dose and clinical effectiveness. Previously, we reported that APT102 (AZD3366), a human apyrase optimized to scavenge extracellular ADP and ATP, exhibited potent antiplatelet efficacy without bleeding in experimental myocardial infarction and stroke. Here we describe APT402, an optimized fusion of APT102 and annexin V that uniquely inhibits both platelet and coagulation components of thrombus growth. APT402 preferentially bound to injured vessels and thrombus and prevented thrombotic occlusion in arterial and venous thrombosis models, without increasing bleeding. Thus, APT402 breaks the confounding link between antithrombotic efficacy and bleeding, pioneering a safe and effective approach to prevent and treat a broad range of thrombotic diseases, and may be particularly useful in clinical conditions associated with high bleeding risks.

Calcium-independent phospholipase A2γ (iPLA2γ) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA2γ in cardiac myocytes, we generated cardiac myocyte-specific iPLA2γ knock-out (CMiPLA2γKO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA2γKO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA2γKO mice demonstrated attenuated Ca(2+)-induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA2γKO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA2γKO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-at-risk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca(2+) by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA2γ, these results are consistent with salvage of myocardium after I/R by iPLA2γ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion.

Sphingosine-1-phosphate receptor (S1PR) activation plays a key role in vascular inflammatory response. Here, we report in vivo validation of [ 11 C]TZ3321, a potent S1PR1 radioligand, for imaging vascular inflammation in a rat model of carotid injury. The right common carotid artery of male adult Sprague-Dawley rats was injured by balloon overinflation that denuded the endothelium and distended the vessel wall. Animals received a 60-minute micro-positron emission tomography (micro PET) scan with [ 11 C]TZ3321 at 72 hours after injury. Ex vivo autoradiography was also conducted. The expression and cellular location of S1PR1 were examined by immunohistological analysis. Three-dimensional (3D) reconstruction of the first 100-second microPET/computed tomography (CT) image indicated the location of bilateral common carotid arteries. [ 11 C]TZ3321 displayed significantly higher accumulation (standardized uptake values: 0.93 ± 0.07 vs 0.78 ± 0.09, n = 6, P = .001) in the injured carotid artery than in the contralateral side. Increased tracer uptake in the injured artery was confirmed by autoradiography (photostimulated luminescence measures: 85.5 ± 0.93 vs 71.48 ± 6.22, n = 2). Concordantly, high S1PR1expression was observed in infiltrated inflammatory cells in the injured artery. Our studies demonstrate [ 11 C]TZ3321 microPET is able to detect the acute upregulation of S1PR1 expression in inflamed carotid artery. Therefore, [ 11 C]TZ3321 has potential to be a PET radiotracer for detecting early inflammatory response and monitoring therapeutic efficacy of vascular inflammation.

Purpose Safe, sensitive, and non-invasive imaging methods to assess the presence, extent, and turnover of myocardial fibrosis are needed for early stratification of risk in patients who might develop heart failure after myocardial infarction. We describe a non-contrast cardiac magnetic resonance (CMR) approach for sensitive detection of myocardial fibrosis using a canine model of myocardial infarction and reperfusion. Methods Seven dogs had coronary thrombotic occlusion of the left anterior descending coronary arteries followed by fibrinolytic reperfusion. CMR studies were performed at 7 days after reperfusion. A CMR spin-locking T 1 ρ mapping sequence was used to acquire T 1 ρ dispersion data with spin-lock frequencies of 0 and 511 Hz. A fibrosis index map was derived on a pixel-by-pixel basis. CMR native T 1 mapping, first-pass myocardial perfusion imaging, and post-contrast late gadolinium enhancement imaging were also performed for assessing myocardial ischemia and fibrosis. Hearts were dissected after CMR for histopathological staining and two myocardial tissue segments from the septal regions of adjacent left ventricular slices were qualitatively assessed to grade the extent of myocardial fibrosis. Results Histopathology of 14 myocardial tissue segments from septal regions was graded as grade 1 (fibrosis area, 50% of field, n = 1). A dramatic difference in fibrosis index (183%, P 1 ρ (9%, P = 0.14), native T 1 (5.5%, P = 0.12), and perfusion (− 21%, P = 0.05). Conclusion A non-contrast CMR index based on T 1 ρ dispersion contrast was shown in preliminary studies to detect and correlate with the extent of myocardial fibrosis identified histopathologically. A non-contrast approach may have important implications for managing cardiac patients with heart failure, particularly in the presence of impaired renal function.

Objective: Natriuretic peptide receptor-C (NPR-C/NPR-3) is a cell surface protein involved in vascular remodelling that is up-regulated in atherosclerosis. NPR-C expression has not been well characterized in human carotid artery occlusive lesions. We hypothesized that NPR-C expression correlates with intimal features of vulnerable atherosclerotic carotid artery plaque. Methods: To test this hypothesis, we evaluated NPR-C expression by immunohistochemistry (IHC) in carotid endarterectomy (CEA) specimens isolated from 18 patients. The grade, location, and co-localization of NPR-C in CEA specimens were evaluated using two tissue analysis techniques. Results: Relative to minimally diseased CEA specimens, we observed avid NPR-C tissue staining in the intima of maximally diseased CEA specimens (65%; p=0.06). Specifically, maximally diseased CEA specimens demonstrated increased NPR-C expression in the superficial intima (61%, p=0.17), and deep intima (138% increase; p=0.05). In the superficial intima, NPR-C expression significantly co-localized with vascular smooth muscle cells (VSMCs) and macrophages. The intensity of NPR-C expression was also higher in the superficial intima plaque shoulder and cap regions, and significantly correlated with atheroma and fibroatheroma vulnerable plaque regions (β=1.04, 95% CI=0.46, 1.64). Conclusion: These findings demonstrate significant NPR-C expression in the intima of advanced carotid artery plaques. Furthermore, NPR-C expression was higher in vulnerable carotid plaque intimal regions, and correlate with features of advanced disease. Our findings suggest that NPR-C may serve as a potential biomarker for carotid plaque vulnerability and progression, in patients with advanced carotid artery occlusive disease.

Background: Maintenance of cardiolipin molecular speciation by remodeling directly regulates mitochondrial bioenergetic efficiency. Results: Transgenic expression of cardiolipin synthase accelerates cardiolipin remodeling, improves mitochondrial function, modulates mitochondrial signaling, and attenuates mitochondrial dysfunction during diabetes. Conclusion: Cardiolipin synthase integrates multiple aspects of mitochondrial bioenergetic and signaling functions. Significance: Cardiolipin synthase expression attenuates mitochondrial dysfunction in diabetic myocardium.

Sensitive, specific, and noninvasive detection of angiogenesis would be helpful in discovering new strategies for the treatment of cardiovascular diseases. Recently, we reported the (64)Cu-labeled C-type atrial natriuretic factor (CANF) fragment for detecting the upregulation of natriuretic peptide clearance receptor (NPR-C) with PET on atherosclerosis-like lesions in an animal model. However, it is unknown whether NPR-C is present and overexpressed during angiogenesis. The goal of this study was to develop a novel CANF-integrated nanoprobe to prove the presence of NPR-C and offer sensitive detection with PET during development of angiogenesis in mouse hind limb.We prepared a multifunctional, core-shell nanoparticle consisting of DOTA chelators attached to a poly(methyl methacrylate) core and CANF-targeting moieties attached to poly(ethylene glycol) chain ends in the shell of the nanoparticle. Labeling of this nanoparticle with (64)Cu yielded a high-specific-activity nanoprobe for PET imaging NPR-C receptor in a mouse model of hind limb ischemia-induced angiogenesis. Histology and immunohistochemistry were performed to assess angiogenesis development and NPR-C localization.(15)O-H(2)O imaging showed blood flow restoration in the previously ischemic hind limb, consistent with the development of angiogenesis. The targeted DOTA-CANF-comb nanoprobe showed optimized pharmacokinetics and biodistribution. PET imaging demonstrated significantly higher tracer accumulation for the targeted DOTA-CANF-comb nanoprobe than for either the CANF peptide tracer or the nontargeted control nanoprobe (P0.05, both). Immunohistochemistry confirmed NPR-C upregulation in the angiogenic lesion with colocalization in both endothelial and smooth muscle cells. PET and immunohistochemistry competitive receptor blocking verified the specificity of the targeted nanoprobe to NPR-C receptor.As evidence of its translational potential, this customized DOTA-CANF-comb nanoprobe demonstrated superiority over the CANF peptide alone for imaging NPR-C receptor in angiogenesis.

Aim: To develop a fibrin-specific urokinase nanomedicine thrombolytic agent. Materials & Methods:In vitro fibrin-clot dissolution studies were utilized to develop and characterize simultaneous coupling and loading of anti-fibrin monoclonal antibody and urokinase onto perfluorocarbon nanoparticle (NP) surface. In vivo pharmacokinetics and fibrin-specific targeting of the nanolytic agent was studied in dogs. Results: Simultaneous coupling of up to 40 anti-fibrin antibodies and 400 urokinase enzymes per perfluorocarbon NP produced an effective targeted nanolytic agent with no significant surface protein–protein interference. Fibrin clot dissolution was not improved by increasing homing capacity from 10 to 40 antibodies/NP, but increasing enzymatic payload from 100 to 400/NP resulted in maximized lytic effect. Fluorescent microscopy showed that rhodamine-labeled urokinase nanoparticles densely decorated the intraluminal thrombus in canine clots in vivo analogous to the fibrin pattern, while an irrelevant-targeted agent had negligible binding. Conclusion: This agent offers a vascularly constrained, simple to administer, low-dose nanomedicine approach that may present an attractive alternative for treating acute stroke victims.

Cardiovascular disease is the leading cause of death worldwide. PET has the potential to provide information on the biology and metabolism of atherosclerotic plaques. Natriuretic peptides (NPs) have potent antiproliferative and antimigratory effects on vascular smooth-muscle cells (VSMCs) and, in atherosclerosis, participate in vascular remodeling, in which the expression of NP clearance receptors (NPR-Cs) is upregulated both in endothelium and in VSMCs.We investigated the potential of a C-type atrial natriuretic factor (C-ANF) to image developing plaque-like lesions in vivo. C-ANF was functionalized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and labeled with (64)Cu for noninvasive PET in a hypercholesterolemic rabbit with atherosclerotic-like lesions induced by air desiccation of a femoral artery, followed by balloon overstretch of the developing neointima. Histopathology and immunohistochemistry were performed to assess plaque development and NPR-C localization.(64)Cu-DOTA-C-ANF uptake in the atherosclerotic region was visible on small-animal PET images, with the highest target-to-background ratio (3.59 +/- 0.94) observed after the air desiccation-induced injury. Immunohistochemistry and immunofluorescence staining showed NPR-C near the luminal surface of the plaque and in VSMCs. PET and immunohistochemistry competitive blocking studies confirmed receptor-mediated tracer uptake in the plaque. With blocking, PET tracer localization of atherosclerotic to control arteries was decreased from 1.42 +/- 0.02 to 1.06 +/- 0.06 (P0.001).We demonstrated that (64)Cu-DOTA-C-ANF is a promising candidate tracer for in vivo PET of NPR-Cs on atherosclerotic plaques.

The goal of the study is to develop a noninvasive magnetic resonance imaging (MRI)-based biomechanical imaging technique to address biomechanical pathways of atherosclerotic progression and regression in vivo using a 3D fluid-structure interaction (FSI) model. Initial in vivo study was carried out in an early plaque model in pigs that underwent balloon-overstretch injury to the left carotid arteries. Consecutive MRI scans were performed while the pigs were maintained on high cholesterol (progression) or normal chow (regression), with an injection of a plaque-targeted contrast agent, Gadofluorine M. At the end of study, the specimens of carotid arterial segments were dissected and underwent dedicated mechanical testing to determine their material properties. 3D FSI computational model was applied to calculate structure stress and strain distribution. The plaque structure resembles early plaque with thickened intima. Lower maximal flow shear stress correlates with the growth of plaque volume during progression, but not during regression. In contrast, maximal principle structure stress/stain (stress-P1 and strain-P1) were shown to correlate strongly with the change in the plaque dimension during regression, but moderately during progression. This MRI-based biomechanical imaging method may allow for noninvasive dynamic assessment of local hemodynamic forces on the development of atherosclerotic plaques in vivo.

Understanding the oxygen consumption of the left ventricular myocardium provides important insight into the relationship between myocardial oxygen supply and demand. In other territories, cardiac magnetic resonance has been utilized to measure myocardial oxygen consumption with a blood level oxygen dependent (BOLD) technique. The BOLD technology requires repetitive sampling of stationary tissues and is frequently implemented in areas such as the brain. A limitation to utilizing BOLD cardiac magnetic resonance techniques in the heart has been cardiac motion. In this study, we document a methodology for acquiring BOLD images in the heart and demonstrate the utility of the technique for identifying associations between myocardial oxygen consumption and blood flow.

Objective: Gadofluorine M has been reported to enhance early atherosclerotic plaque signals in magnetic resonance imaging (MRI). The aim of this study was to examine the use of Gadofluorine M to monitor the progression of advanced plaques in a rabbit model. Methods: Focal advanced atherosclerosis was induced in the right femoral arteries of 6 New Zealand white rabbits using a combination of cholesterol-enriched diet, and sequential air-desiccation, and balloon-overstretch injury. MRI with conventional 3 contrasts (T1, T2, and proton density [PD]) was performed to monitor the progression of the atherosclerotic plaques with 2 MRI scans separated by 4 to 8 weeks. Gadofluorine M was given intravenously to the rabbits 24 hours before the first MRI scans, and before (n = 3) or during (n = 3) the second MRI scan. The left femoral arteries were used as a control. Histopathologic images localized individual plaque components. Results: The advanced plaque displayed multilayered neointima that included foam cells, smooth muscle cells, and extracellular matrix. The separate image contrasts offered similar T1-weighted enhancement patterns, but the combination of all 3 contrasts helped to delineate plaque and lumen boundaries. Gadofluorine M strongly enhanced neointima areas with an image contrast (contrast-to-noise ratio [CNR]) of approximately 15, versus 2 in the control femoral arterial wall. With improved images, significant changes in neointima and total plaque volumes over the 4 to 8 weeks between scans could be identified. Gadofluorine M remained within the plaques with significant image enhancements (contrast-to-noise ratio = 5.8) for 2 months after a single injection. Conclusion: This preliminary study in rabbits indicated that Gadofluorine M provides specific enhancements of components associated with advanced atherosclerotic plaques and may help to monitor the progression of the plaque in a rabbit model of atherogenesis.

In this study, porcine carotid arteries were subjected to balloon overstretch injury followed by local delivery of paramagnetic nanoparticles targeted to alphavbeta3-integrin expressed by smooth muscle cells or collagen III within the extracellular matrix. Carotid T1-weighted angiography and vascular imaging was performed at 1.5T. While MR angiograms were indistinguishable between control and targeted vessel segments, alphavbeta3-integrin-and collagen Ill-targeted nanoparticles spatially delineated patterns and volumes of stretch injury. In conclusion, MR molecular imaging with alphavbeta3-integrin or collagen Ill-targeted nanoparticles enables the non-invasive, three-dimensional characterization of arterial pathology unanticipated from routine angiography.

Before molecular imaging with MRI can be applied clinically, certain problems, such as the potential sparseness of molecular epitopes on targeted cell surfaces, and the relative weakness of conventional targeted MR contrast agents, must be overcome. Accordingly, the conditions for diagnostic conspicuity that apply to any paramagnetic MRI contrast agent with known intrinsic relaxivity were examined in this study. A highly potent paramagnetic liquid perfluorocarbon nanoparticle contrast agent ( approximately 250 nm diameter, >90,000 Gd3+/particle) was imaged at 1.5 T and used to successfully predict a range of sparse concentrations in experimental phantoms with the use of standard MR signal models. Additionally, we cultured and targeted the smooth muscle cell (SMC) monolayers that express "tissue factor," a glycoprotein of crucial significance to hemostasis and response to vascular injury, by conjugating an anti-tissue factor antibody fragment to the nanoparticles to effect specific binding. Quantification of the signal from cell monolayers imaged at 1.5 T demonstrated, as predicted via modeling, that only picomolar concentrations of paramagnetic perfluorocarbon nanoparticles were required for the detection and quantification of tissue factor at clinical field strengths. Thus, for targeted paramagnetic agents carrying high payloads of gadolinium, it is possible to quantify molecular epitopes present in picomolar concentrations in single cells with routine MRI.

Objective— Neointima formation occurs in arteries in response to mechanical or chemical injury and is responsible for substantial morbidity. In this work, the role of the intracellular linker protein Grb2 in the pathogenesis of neointima formation was examined. Grb2 is a critical signaling protein that facilitates the activation of the small GTPase ras by receptor tyrosine kinases. Methods and Results— Cultured rat aortic smooth muscle cells were treated with an antisense morpholino to Grb2 and these cells showed a reduced proliferative response to platelet-derived growth factor stimulation. Grb2 −/− mice do not survive embryonic development. Grb2 +/− mice appear normal at birth and are fertile but have defective signaling in several tissues. Cultured smooth muscle cells derived from Grb2 +/− mice grew at a much slower rate than cells derived from Grb2 +/+ mice. Grb2 +/− and Grb2 +/+ mice were subjected to carotid injury. After 21 days, Grb2 +/+ mice developed robust neointima formation that, in some cases, resulted in an occlusive lesion. In contrast, Grb2 +/− mice were resistant to the development of neointima Conclusions— Grb2 is an essential component of the signaling cascade resulting in neointima formation after arterial injury.

Although mice deficient in various genes are providing greater insight into the mechanisms of restenosis after angioplasty, there have been limitations with murine models not simulating human vascular disease. To develop a more clinically applicable model of primary atherosclerosis and restenosis following angioplasty of the primary lesion, we fed apolipoprotein E-deficient mice a Western diet and occluded the left common carotid artery for 2 days. Three weeks after flow was restored, the temporarily occluded carotids demonstrated atherosclerotic lesions containing foam cells, cholesterol clefts, necrotic cores, and fibrous capsules. The atherosclerotic carotids in other animals underwent angioplasty with a beaded probe, resulting in plaque and medial layer disruption. Three weeks after angioplasty, although there was significant neointimal lesion formation, the luminal narrowing did not change significantly secondary to overall vessel enlargement (positive remodeling). Neointimal lesions were composed of smooth-muscle cells and extracellular matrix observed adjacent to the original atherosclerotic plaques. Similarly, even at 3 months after the angioplasty the lumen was maintained despite greater neointimal lesion formation caused by progressive positive remodeling. This new murine model of primary atherosclerosis and postangioplasty intimal hyperplasia and remodeling mimics the human disease pattern of postangioplasty intimal hyperplasia. Used in transgenic animals, this model will likely facilitate understanding of the mechanisms of restenosis in humans.

Murine myocardium contains diminutive amounts of calcium-independent phospholipase A2 (iPLA2) activity (

ZK-807834 (also known as CI-1031) is a small molecule that potently and selectively inhibits Factor Xa. Studies in animals have shown that ZK-807834 attenuates thrombosis and thrombus progression after fibrinolysis and exhibits an increased antithrombotic-to-bleeding risk ratio compared with conventional agents. The present study describes the human in vitro anticoagulant and pharmacodynamic profile ZK-807834. Consistent with its selective inhibition of Factor Xa, ZK-807834 in the range of 0.3-0.5 microM prolonged prothrombin time (PT) and activated partial thromboplastin time (aPPT) twofold without affecting thrombin time (TT). Intersubject variability of in vitro anticoagulant activity was nominal and gender-independent. ZK-807834 inhibited Factor Xa in clot-bound prothrombinase with an average IC50 of 10+/-7 (S.D.) nM. ZK-807834 exhibited no direct effect on ADP- or collagen-induced platelet aggregation. Based on the potency and specificity, ZK-807834 may represent an important advance in the development of selective and safe antithrombotics.

SummaryInjury to the arterial wall initiates a cascade of events including platelet deposition and an increase in procoagulant activity of the vessel wall that is associated with intimal thickening and vascular wall remodeling. This study was designed to characterize the effects of aurintricarboxylic acid (ATA), an inhibitor of von Willebrand factor function, on vascular procoagulant activity and the development of intimal thickening after balloon-induced injury to the rabbit aorta.Treatment with ATA, aspirin, or the combination of agents at doses that attenuated platelet aggregation decreased platelet deposition and procoagulant activity bound to the vessel wall after injury. Treatment with ATA reduced the intimal thickening observed 2 weeks after injury. Surprisingly, aspirin treatment had no effect on intimal thickening. These data indicate that inhibition of platelet deposition, while it is able to attenuate local thrombin elaboration, is not alone sufficient to attenuate subsequent intimal thickening that occurs in response to arterial injury.

In patients with acute myocardial infarction undergoing reperfusion therapy to restore blood flow through blocked arteries, simultaneous inhibition of platelet P2Y12 receptors with the current standard of care neither completely prevents recurrent thrombosis nor provides satisfactory protection against reperfusion injury. Additionally, these antiplatelet drugs increase the risk of bleeding. To devise a different strategy, we engineered and optimized the apyrase activity of human nucleoside triphosphate diphosphohydrolase-3 (CD39L3) to enhance scavenging of extracellular adenosine diphosphate, a predominant ligand of P2Y12 receptors. The resulting recombinant protein, APT102, exhibited greater than four times higher adenosine diphosphatase activity and a 50 times longer plasma half-life than did native apyrase. Treatment with APT102 before coronary fibrinolysis with intravenous recombinant human tissue-type plasminogen activator in conscious dogs completely prevented thrombotic reocclusion and significantly decreased infarction size by 81% without increasing bleeding time. In contrast, clopidogrel did not prevent coronary reocclusion and increased bleeding time. In a murine model of myocardial reperfusion injury caused by transient coronary artery occlusion, APT102 also decreased infarct size by 51%, whereas clopidogrel was not effective. These preclinical data suggest that APT102 should be tested for its ability to safely and effectively maximize the benefits of myocardial reperfusion therapy in patients with arterial thrombosis.

Injury to an artery induces formation of a platelet-rich thrombus, while stasis or trauma to a vein induces a fibrin-rich thrombus. We have implemented preparations for evolving both platelet-rich and fibrin-rich thrombi simultaneously in rabbits for use to define the efficacy of novel antithrombotic agents. For platelet-rich thrombosis, a carotid artery and contralateral jugular vein were dissected and an arteriovenous shunt inserted distally to prevent cerebral infarction during thrombus formation. The shunted artery was then instrumented with a proximal Doppler probe for measuring flow velocity and a distal transluminal needle electrode. Electrical injury to the artery was induced by application of 250 microA of anodal current to the indwelling needle electrode. Thrombotic occlusion was consistently observed within 60 min, permitting measurements of the effects on the incidence and time of occlusion of antithrombotic agents administered over 2 h. For fibrin-rich thrombosis, an external jugular vein was dissected, including the distal bifurcation. One of the branches was catheterized and a copper wire with cotton threads attached was advanced through the catheter into the superior vena cava, allowing exposure of the threads to flowing blood. A 25- to 30-mg thrombus was formed within 2 h, permitting reliable measurements of effects on thrombus weight of antithrombotic agents administered during this interval. Implementing both arterial and venous thrombosis simultaneously did not change measurements compared with either method alone. This approach may facilitate recognition of differences in efficacy of selected agents against thrombi of diverse composition.

BACKGROUND Production of oxygen free radicals, and activation of neutrophils and plasma complement contribute to myocardial reperfusion injury, but the role of coagulation has not been assessed. OBJECTIVE To characterize tissue-factor-mediated generation of thrombin and its association with tissue injury during reperfusion from normothermic ischemia of isolated, Langendorf-perfused rabbit hearts. METHODS Activation of coagulation was assessed by addition of 12% rabbit plasma and human fibrinogen to Krebs-Henseleit-buffer perfusate with measurement of levels of human fibrinopeptide A (hFPA) in the heart effluent as an index of thrombin-mediated formation of fibrin. RESULTS Concentrations of hFPA in the effluent were minimal during non-ischemic perfusion (5 +/- 5 ng/ml, n=6) and during 50 min of ischemia (13 +/- 3 ng/ml, n=6), but increased markedly during the first 20 min of reperfusion (to 41 +/- 29 ng/ml, P=0.03 versus before reperfusion). Addition to the perfusate of 10 microg/ml recombinant human tissue-factor-pathway inhibitor, the physiologic inhibitor of tissue-factor-mediated coagulation, abolished increases in the level of hFPA after reperfusion. However, indexes of myocardial injury manifested during reperfusion, including decrease in recovery of left ventricular pressure developed, increase in left ventricular end-diastolic pressure, and increase in activity of creatine kinase in the heart effluent, were not improved by anticoagulation with recombinant human tissue-factor-pathway inhibitor. CONCLUSION Our results do not support the hypothesis that coagulation plays a major role in ischemia/reperfusion injury of Langendorf-perfused rabbit hearts.

Inhibition of factor Xa (FXa) may interrupt thrombus progression. This study compared the antithrombotic activity of a novel FXa inhibitor, ZK-807834 [MW, 527 D; Ki (human FXa), 0.11 nM], with recombinant tick anticoagulant peptide [rTAP; MW, 6,685 D; Ki, (human FXa) = 0.28 nM], and DX-9065a [MW 445 D, Ki (human FXa), 40 nM] in rabbits with arterial thrombosis induced by electrical vascular injury. ZK-807834 also was compared with low molecular weight heparin (LMWH; MW, 5,500 D) during venous thrombosis induced by placing a copper wire and threads in the vena cava. Inhibitors were administered as an i.v. bolus and 2-h infusion. Total dosages of ZK-807834,or =0.7 micromol/kg (n = 18); rTAP,or =1 micromol/kg (n = 18); or DX-9065a,or =11 micromol/kg (n = 18) decreased the incidence of arterial thrombotic occlusion compared with control animals (p0.05). However, five of six animals given the lowest effective dosage of rTAP and four of six animals given DX-9065a bled from a surgical incision5 min, but only two of six animals given ZK-807834 bled5 min. Venous clot weights were reduced compared with controls for dosages of ZK-807834or =0.007 micromol/kg (n = 36) or LMWHor =0.2 micromol/kg (n = 18). Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were unchanged from baseline at the minimally effective dose of ZK-807834, whereas aPTT was increased twofold at the effective dose of LMWH. Thus ZK-807834 may be useful to attenuate thrombosis at lower dosages and with less perturbation of systemic hemostasis compared with available agents.

RATIONALE AND OBJECTIVES Molecular imaging with targeted contrast agents enables tissues to be distinguished by detecting specific cell-surface receptors. In the present study, a ligand-targeted acoustic nanoparticle system is used to identify angioplasty-induced expression of tissue factor by smooth muscle cells within carotid arteries. METHODS Pig carotid arteries were overstretched with balloon catheters, treated with tissue factor-targeted or a control nanoparticle system, and imaged with intravascular ultrasound before and after treatment. RESULTS Tissue factor-targeted emulsions bound and increased the echogenicity and gray-scale levels of overstretched smooth muscle cells within the tunica media, versus no change in contralateral control arteries. Expression of stretch-induced tissue factor in carotid artery media was confirmed by immunohistochemistry. CONCLUSIONS The potential for abnormal thrombogenicity of balloon-injured arteries, as reflected by smooth muscle expression of tissue factor, was imaged using a novel, targeted, nanoparticulate ultrasonic contrast agent.

Abstract —Intravenous infusion of recombinant tissue factor pathway inhibitor (rTFPI) for 24 hours decreases neointimal thickening and luminal stenosis 1 month after balloon-induced injury to the carotid arteries in minipigs. This study was designed to determine whether the effect of rTFPI is accounted for by early decreases in procoagulant activity and thrombosis on the injured vessel wall. Vascular injury was induced by balloon hyperinflations in both carotid arteries of anesthetized pigs given no anticoagulant as a control (n=16), an intravenous infusion for 24 hours of rTFPI (0.5 mg/kg bolus and 25 μg · kg −1 · min −1 , n=14), or an intravenous infusion of unfractionated heparin (100 U · kg −1 · h −1 , n=19). Accumulation of radiolabeled autologous platelets was markedly decreased over 24 hours on injured arteries from animals given rTFPI (0.6×10 6 /cm 2 ) compared with controls (2.5×10 6 /cm 2 , P =0.0004). Deposition of radiolabeled fibrin was also decreased in rTFPI-treated animals (269±266 μg/cm 2 ) compared with controls (2389±1673 μg/cm 2 , P =0.04). Similar effects were observed with heparin. However, factor Xa activity, assayed after 24 hours by incubation of the injured arterial segments with the chromogenic substrate S-2222, was decreased more markedly on arteries from rTFPI-treated animals (0.14±0.13 OD) than those from heparin-treated animals (0.29±0.18 OD) compared with controls (0.47±0.24 OD, P =0.0007). In addition, arteries from rTFPI-treated animals showed a 4-fold lower induction of tissue factor protein compared with controls ( P =0.0002). Attenuation of procoagulant activity and tissue factor–mediated thrombin generation in response to injury may account for the promising results with rTFPI in the porcine angioplasty model.

BACKGROUND Prolonged intravenous infusions of recombinant tissue factor pathway inhibitor (rTFPI) have been shown to attenuate markedly neointimal formation and stenosis after balloon-induced injury to the carotid arteries in minipigs. DESIGN Because local delivery of rTFPI to the injury site would be clinically advantageous, we designed this study to compare the local delivery and retention of rTFPI in balloon-injured arteries using three catheter-based systems. METHODS Similar amounts (range 3-4.5 mg) of a mixture of 125I-labeled and unlabeled rTFPI were delivered by either passive diffusion at moderate pressure (5 x 10(5) Pa with the LocalMed InfusaSleeve, or 4 x 10(5) Pa with the SciMed Dispatch device), or facilitated diffusion combining lower pressure (2 x 10(5) Pa) and electrical current (3.5 mA/cm2; e-MED, iontophoresis) to balloon-injured carotid arteries in anesthetized rabbits. RESULTS Comparable amounts of rTFPI were retained on the injured vessels immediately after delivery (t = 0) with the LocalMed (628 +/- 68 micrograms/g per cm2, n = 4), SciMed (522 +/- 167 micrograms/g per cm2, n = 4), and e-MED (497 +/- 142 micrograms/g per cm2, n = 4) catheters (NS). However, rTFPI was decreased by 37% after 24 h compared with t = 0 (P < 0.02) in the e-MED group, but was increased 1.5-fold (P = 0.02) and 1.3-fold in the SciMed and LocalMed groups, respectively, presumably because of redistribution of rTFPI from remote endothelial or perivascular sites. Retention of rTFPI was six to nine times higher for injured compared with non-injured arteries, and persisted for at least 48 h after delivery with the LocalMed catheter. CONCLUSIONS Sustained, marked retention of rTFPI delivered locally at the site of balloon-induced arterial injury appears to result from catheter-based systems that use passive diffusion at moderate pressure.

Abstract —This study was designed to characterize the relative roles of bound Xa/Va and thrombin activity in vascular wall procoagulant activity after balloon-induced injury and the extent to which intravenous aspirin and heparin attenuate procoagulant activity associated with the vascular wall. Abdominal aortic injury was induced in rabbits by overinflation and multiple passages of a 4F embolectomy catheter. Rabbits were killed 15 minutes or 4, 8, 24, 48, 72, 96, or 120 hours after injury. Aortic segments were incubated ex vivo to define bound procoagulant activity. Thrombin activity bound to the aorta was detected by 4 hours after injury and was most marked over the first 24 hours, as estimated by increases in concentration of fibrinopeptide A during incubation of segments with recalcified barium-adsorbed plasma or activity against the thrombin-synthetic substrate S-2238. Based on comparison with purified human thrombin incubated under the same conditions, a maximum of 0.04 to 0.1 nmol/L per square centimeter of thrombin activity was associated with the vascular wall during the first 24 hours and remained detectable for 72 hours. In contrast, bound Xa/Va complex activity to injured segments was detected within 15 minutes and induced activation of prothrombin added to recalcified barium-adsorbed plasma incubated with injured segments for 96 hours. Aspirin (15 mg/kg) administered 30 minutes before injury attenuated 111 In-platelet deposition at 4 hours by 67%, with an associated decrease in bound Xa/Va and thrombin activity at 15 minutes and 4 hours. However, intravenous heparin did not attenuate bound Xa/Va activity at 15 minutes or thrombin activity at 15 minutes and 4 hours. Platelet-dependent bound Xa/Va activity occurs rapidly after arterial injury and may promote thrombin elaboration for up to 96 hours. Bound thrombin activity and de novo thrombin elaboration on the vascular wall may play an important role in the progression of thrombosis and vascular wall remodeling.

This study was designed to investigate the effects of contrast agents on MR images of balloon-injured carotid arteries containing atherosclerotic-like lesions. We have evaluated an intravascular contrast agent, MS-325 (METASYN INC., Cambridge, MA) and an extravascular contrast agent, Optimark, (Mallinckrodt Medical Inc., St. Louis, MO) on MR angiograms obtained 4 weeks after balloon hyperinflation-induced injury of the left common carotid artery in 12 hypercholesterolemic minipigs. High in-plane resolution (.8 x .4 mm2), thin slice (1 mm) time-of-flight gradient echo sequences were used to acquire the MR angiographic images. Vascular lumen definition was compared before and after a single bolus intravenous injection of a contrast agent. Digital subtraction angiograms were obtained from all pigs after MR imaging. High grade stenosis developed in 1 of the 12 pigs and five pigs had complete occlusion of the injured vessel. The remaining pigs exhibited essentially no visible stenoses as assessed either by MR angiography or digital subtraction angiography. The vessel walls of the stenosed and occluded vessels were visible after the injection of either intravascular or extravascular contrast agent. Histologic analyses showed well developed neovascularization in the neointima or occlusive thrombosis. We conclude that the observed contrast-enhanced vessel wall is caused by an increased vascular supply associated with thrombosis and neointimal thickening that leads to an accumulation of contrast agent in the abnormal vessel walls after the injection of the T1-shortening paramagnetic contrast agent.

Ischemia-related processes associated with the generation of inflammatory molecules such as reactive oxygen species (ROS) are difficult to detect at the acute stage before the physiologic and anatomic evidence of tissue damage is present. Evaluation of the inflammatory and healing response early after an ischemic event in vivo will aid in treatment selection and patient outcomes. We introduce a novel near-infrared hydrocyanine molecular probe for the detection of ROS as a marker of tissue response to ischemia and a precursor to angiogenesis and remodeling. The synthesized molecular probe, initially a non-fluorescent hydrocyanine conjugated to polyethylene glycol, converts to a highly fluorescent cyanine reporter upon oxidation.The probe was applied in a preclinical mouse model for myocardial infarction, where ligation and removal of a portion of the femoral artery in the hindlimb resulted in temporary ischemia followed by angiogenesis and healing.The observed increase in fluorescence intensity was approximately sixfold over 24 h in the ischemic tissue relative to the uninjured control limb and was attributed to the higher concentration of ROS in the ischemic tissue.These results demonstrate the potential for non-invasive sensing for interrogating the inflammatory and healing response in ischemic tissue.

Background: We have previously shown that preincubation of whole blood clots with recombinant tissue factor pathway inhibitor (rTFPI) attenuates clot-associated procoagulant activity assessed ex vivo. This study was undertaken to determine whether a single local application of rTFPI induces similar attenuation of the procoagulant activity on preclotted Dacron grafts implanted in an artery in vivo. Methods: Dacron grafts (4 mm × 4 cm long) were preclotted in porcine blood and incubated with either rTFPI (5 mg/ml) or arginine-phosphate buffer for 15 minutes. Grafts were implanted end-to-end in the femoral arteries of 10 pigs, with one rTFPI-treated and one buffer-treated graft implanted in each animal. Animals did not undergo anticoagulation either before or after graft implantation. Radiolabeled porcine fibrinogen was injected intravenously, and the grafts underwent perfusion for 1 hour. A subgroup of animals (n = 7) also had infusion of radiolabeled autologous platelets at the time of administration of radiolabeled fibrinogen. Results: Fibrin(ogen) deposition was decreased in rTFPI-treated grafts by 36% ± 7% (mean + SEM) compared with buffer-treated grafts (p = 0.001). Platelet deposition was also reduced in the rTFPI-treated grafts by 31% ± 15%, although the reduction did not reach statistical significance (p = 0.10). The extent of rTFPI-mediated attenuation of fibrin(ogen) versus platelet deposition varied independently among animals. Conclusions: Clot-directed anticoagulant effects of rTFPI appear to be useful for substantially decreasing the thrombogenicity of Dacron grafts immediately after their implantation. Chronic studies to determine whether the decreases in thrombogenicity result in improved long-term graft patency appear warranted.(J Vasc Surg 1996;24:865-70.)

Criteria for the retrospective diagnosis of acute myocardial infarction rely heavily on increases in lactate dehydrogenase (LD) isoenzymes. However, increases of LD isoenzyme activities are not specific for myocardial injury. Recently, increased concentrations of cardiac troponin I (cTnI) have been shown to be highly specific for myocardial damage and to have sensitivity comparable with that of creatine kinase MB isoenzyme for detecting cardiac injury. Furthermore, increases of cTnI persist in plasma for at least several days. The present study was designed to determine the relative sensitivities of cTnI and LD isoenzymes over time for the diagnosis of infarction. The results indicate that cTnI values are at least as sensitive as LD isoenzymes: 90% of patients with myocardial infarction had above-normal concentrations of cTnI on the 4th day after admission to the coronary care unit. Criteria based on cTnI should improve the accuracy of retrospective diagnoses of acute myocardial infarction.

Purpose: Graft procoagulant activity is determined by thrombin (IIa) and activated factor X (Xa) that binds to thrombus. Thrombus-associated factor Xa and thrombin are resistant to antithrombin III-dependent therapy (heparin). To avoid complications and costs associated with systemic administration, we evaluated whether locally applied antithrombotic agents inhibit prosthetic graft procoagulant activity under no-flow and low-flow conditions.Methods: Four-millimeter-diameter collagen-coated grafts were preclotted in recalcified human plasma, washed, immersed in antithrombotic agents (either 100 nm hirudin, 20 μm D-Phe-L-Pro-L-Arg chloromethylketone, 5 μm tick anticoagulant peptide or 5 or 10 μg/ml tissue factor pathway inhibitor) or saline solution, and extensively rewashed. Grafts were exposed to recalcified plasma either in multiwell plates or underwent perfusion at 1 ml/min flow rate. Fibrinopeptide A, which reflects fibrin elaboration, was measured as a marker of thrombin activity.Results: Inhibitors reduced fibrinopeptide generation at 8 minutes by 55% (tissue factor pathway inhibitor), 57% (hirudin), or 63% (tick anticoagulant peptide and D-Phe-L-Pro-L-Argchloromethylketone) compared with the control agents (p < 0.05). Under low-flow conditions tissue factor pathway inhibitor and hirudin reduced fibrinopeptide generation at 13 minutes by 61% and 49%, respectively, when compared with control agents ( p < 0.05).Conclusions: Graft-associated inhibitors targeted at factors IIa, Xa, or tissue factor/VIIa/Xa complex effectively reduce procoagulant activity on prosthetic grafts. The success of local application of antithrombotic agents in attenuating early fibrin formation suggests that this strategy could favorably influence acute graft patency, and we speculate these agents may improve long-term graft patency as well. (J Vasc Surg 1996;24:624-31.)

Thrombin activity is increased in the setting of acute myocardial infarction (AMI) and has been shown to increase further after the administration of thrombolytic therapy for acute infarction. This increase in thrombin activity may play an important role in the 15% to 25% rate of failure to achieve initial reperfusion and in the 5% to 15% rate of early reocclusion after initially successful thrombolysis. To investigate potential mechanisms of thrombin formation in vivo, to understand better the balance of coagulation and fibrinolysis during treatment with recombinant tissue-type plasminogen activator (rt-PA), and to investigate the role of hemostatic markers as predictors of clinical events, we measured 3 markers of procoagulant activity: fibrinopeptide A (FPA), thrombin-antithrombin III complexes (TAT), and prothrombin fragment 1.2 (F1.2), and a marker of fibrinogenolytic activity (Bβ1–42) in patients enrolled in the Thrombolysis in Myocardial Infarction (TIMI)-5 study. This trial was a randomized, dose-ranging, pilot trial of hirudin versus heparin as adjunctive antithrombotic therapy with rt-PA administered to patients with AMI. Correlation of markers at 1 hour with clinical outcomes revealed that increased FPA and TAT levels were associated with increased mortality and TIMI grades 0, 1, or 2 flow at 90 minutes; increased F1.2 levels were associated with TIMI grade 0 or 1 flow at 90 minutes; and increased levels of all 3 procoagulant markers were associated with hemorrhagic events. Late (12 to 24 hours) increases in F1.2, TAT, and Bβ1–42 may be predictive of recurrent ischemia. These results suggest that selected markers of procoagulant and fibrinogenolytic activity may be useful in predicting clinical outcomes in patients treated with thrombolytic therapy for AMI.

Background Activation of coagulation has been implicated in both acute thrombotic occlusion and restenosis after balloon angioplasty. However, concomitant administration of antithrombotic agents has thus far failed to prevent these complications. Importantly, the factors contributing to procoagulant activity of balloon-injured arteries over time have not been defined. This study was designed to determine the duration of procoagulant activity on the luminal surface of balloon-injured arteries and the relative roles of tissue factor and thrombin in this response. Methods and Results Abdominal aortas in rabbits were subjected to repetitive balloon hyperinflations sufficient to disrupt the internal elastic lamina. Aortas were excised at P ≤.000001 versus uninjured vessels). Preincubation of segments at these intervals with an antibody to tissue factor markedly attenuated the increases in FPA, as did perfusion of segments with plasma depleted of vitamin K–dependent coagulation factors, indicating that the observed increases in FPA in whole plasma did not result from preformed thrombin bound to the injured vessel wall. Conclusions Tissue factor–mediated coagulation appears to be primarily responsible for prolonged procoagulant activity of balloon-injured arteries.

Background Thrombi are known to induce activation of the coagulation system, which may be a mechanism for progression of thrombosis and its recurrence after thrombolysis. This study was designed to characterize the relative role of thrombin and activated factor X (factor Xa) as mediators of procoagulant activity of whole blood clots in blood and plasma. Methods and Results Clots formed from human blood were incubated in recalcified (25 mmol/L CaCl 2 ) citrated plasma or nonanticoagulated blood with increasing concentrations of recombinant desulfatohirudin (hirudin) to inhibit thrombin activity, recombinant tick anticoagulant peptide (TAP) or recombinant tissue factor pathway inhibitor (TFPI) to inhibit factor Xa, or heparin. Fibrinopeptide A (FPA) was assayed serially as an index of procoagulant (thrombin) activity. FPA generation was greatly accelerated by addition of clots to recalcified plasma (from 1251±211 ng/mL after 15 minutes without clot to 5916±1412 ng/mL with clot, n=7, P P 90% inhibition of FPA with 0.05 μmol/L hirudin and 1.0 U/mL heparin) and factor Xa (>90% inhibition of FPA with 1.0 μmol/L TAP and 0.15 μmol/L TFPI) in a concentration-dependent manner. Preincubation of clots with tissue-type plasminogen activator sufficient to induce partial clot lysis increased the rate of thrombin-induced FPA generation by increasing the surface area of clot exposed to plasma. However, procoagulant activity induced by partially lysed clots was attenuated by lower concentrations of both thrombin and Xa inhibitors, presumably because access of the inhibitors to bound procoagulant molecules was facilitated. Comparable results were obtained with incubations in nonanticoagulated blood. Conclusions These results indicate that factor Xa is primarily responsible for the procoagulant activity of clots in vitro and suggest a potential molecular mechanism for the observed efficacy of inhibitors of factor Xa in preventing recurrent thrombosis after coronary thrombolysis.

To determine whether differences in the kinetics of isoforms of MM and MB creatine kinase affect their ability to detect coronary patency in patients treated with thrombolytic agents, we compared MM and MB isoform profiles in 33 consecutive patients. Results were discordant in 13 of the 33 at 1 hour. When the rates of increase of both isoforms were considered, discordance was present in only 10 of the 33 patients. In five patients %MM3 rose rapidly during the second hour and infarct-related vessels were patent. Four of the five without a rapid increase had occluded infarct-related vessels. These data suggest that criteria based on rates of change in %MB2 are more sensitive than those based on %MM3. However, criteria based on %MM3 are more likely to identify patients in need of interventions to maintain coronary patency.

CORONARY ARTERY DISEASE (CAD) manifests as an imbalance between supply and demand of myocardial oxygen. Myocardial O2 consumption (MVO2) represents an important physiological index of CAD and can provide direct evidence of ischemic heart disease. MR blood oxygen level-dependent (BOLD) imaging has been employed as a noncontrast agent approach to determine myocardial oxygenation extraction fraction (OEF) during hyperemia (1), using a segmented multicontrast turbo spin-echo (TSE) sequence. MVO2 can then be determined by Fick’s law: MVO2 = OEF × MBF, where MBF is the microvascular myocardial blood flow. In the prior study, an imaging acquisition dark-blood turbo-spin-echo (DB-TSE) sequence was employed to measure myocardial T2 within a single breath-hold. By acquiring two T2 images with different echo spacing (t) at rest to calculate model parameters, hyperemic T2 can be used to calculate hyperemic OEF through the two-compartment model (1). However, previously each T2 was calculated using only three different TEs due to limited acquisition time. Because the saturation of blood signal using DB-TSE relies on steady electrocardiogram (ECG) triggering, arrhythmias that caused irregular ECG triggering often induced flow artifacts and deteriorated image quality of the T2 maps. Intrascan motion between the two T2 acquisitions at rest might have significant impact in the calculation of model parameters. These factors have contributed to the inaccuracy of OEF estimation (2). T2-prepared sequences (T2prep) have long been used to assess myocardial edema (3,4) and reveal the T2-weighted BOLD effect in hearts (5). The brightblood T2prep sequence is relatively less sensitive to the irregular ECG triggering in terms of flow artifacts. Because the T2prep method employs Hahn spin-echo or 3–4 180° pulses to achieve T2-weighting, relatively larger τ can be used in the T2prep. This may improve the sensitivity of the T2 method to oxygenation changes in the myocardial microvasculature, in which 90% of myocardial blood volume (MBV) resides in the capillaries (6). With fast gradient-echo acquisition, cardiac motion artifacts could be reduced and acquisition speed improved. The latter may help to increase the number of TEs with which to calculate myocardial T2. The aim of this study was to evaluate and validate a newly developed T2prep BOLD imaging sequence for the quantification of hyperemic myocardial MVO2 using positron emission tomography (PET) imaging as the gold standard method. The study was performed in a canine model with and without coronary artery stenosis.

The binding of thrombin to fibrin is thought to be an important mechanism by which thrombi exhibit procoagulant activity; however, the extent to which other procoagulants are associated with thrombi has not been previously defined. This study was designed to determine whether clotting factors other than thrombin are bound to whole-blood clots and can thereby contribute to significant procoagulant activity. Clots formed in vitro from human blood exhibited minimal thrombin activity when incubated in plasma depleted of vitamin K-dependent factors by barium-citrate adsorption, as indicated by increases in the concentration of fibrinopeptide A (FPA), a marker of fibrin formation, to 72 nM after 30 min. Incubation of clots in barium-absorbed plasma repleted with 0.9 microM human prothrombin under the same conditions resulted in marked increases in the concentration of FPA (> 1,000 nM) and clotting by 30 min. The increases in FPA were attributable to activation of the added prothrombin by clot-associated Factor Xa, judging from concomitant increases in the concentration of prothrombin fragment 1.2. Similar results were obtained with thrombi induced in the axillary arteries of dogs by vascular injury and incubated with plasma in vitro. Activation of prothrombin was inhibited in a dose-dependent manner by tick anticoagulant peptide, a direct inhibitor of Factor Xa, at concentrations of 0.5-5.0 microM. Clot-associated Factor Xa activity was resistant to inhibition by anti-thrombin III, judging from the lack of inhibition of prothrombin activation during incubation of clots in plasma containing heparin pentasaccharide, an anti-thrombin III-mediated inhibitor of Factor Xa. Thus, the activity of Factor Xa appears to be an important determinant of the procoagulant activity of whole-blood clots and arterial thrombi, and is resistant to inhibition by anti-thrombin III-dependent inhibitors.

A new 17O-labeled blood contrast agent was injected intravenously in control dogs. Electrocardiogram (ECG)-triggered myocardial T1ρ imaging was performed to obtain spin-locking T1ρ-weighted myocardial signals for the detection of resultant metabolite H217O water in the heart. Bolus and slow injection methods of various doses of the 17O-labeled and 16O-labeled agents were carried out in order to evaluate the sensitivity of this method and determine the optimal injection method. Bolus injection provided approximately 1% signal reduction, whereas slow injection with larger amount of agent yielded 11.9 ± 0.6% signal reduction. Myocardial oxygen consumption rate was determined by a technique to quantify cerebral oxygenation consumption rate previously developed in 17O brain studies. With either injection method, myocardial oxygen consumption rate at rest was 5.0 – 5.6 μmol/g/min. Therefore, it appears feasible to detect metabolically generated HO water in vivo in the heart, using the 17O-labeled blood tracer. Myocardial oxygen consumption rate can then be quantified in vivo, which may open new doors for the assessment of myocardial metabolism. Magn Reson Med 63:1442–1447, 2010. © 2010 Wiley-Liss, Inc.

Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that play prominent roles in regulating vasomotor tone. To identify the role of calcium-independent phospholipase A2β (iPLA2β) in vasomotor function, we measured vascular responses to phenylephrine (PE) and ACh in mesenteric arterioles from wild-type (WT; iPLA2β+/+) mice and those lacking the β-isoform (iPLA2β−/−) both ex vivo and in vivo. Vessels isolated from iPLA2β−/−mice demonstrated increased constriction to PE, despite lower basal smooth muscle calcium levels, and decreased vasodilation to ACh compared with iPLA2β+/+mice. PE constriction resulted in initial intracellular calcium release with subsequent steady-state constriction that depended on extracellular calcium influx. Endothelial denudation had no effect on vessel tone or PE-induced constriction although the dilation to ACh was significantly reduced in iPLA2β+/+vessels. In contrast, vessels from iPLA2β−/−constricted by 54% after denudation, indicating smooth muscle hypercontractility. In vivo, blood pressure, resting vessel diameter, and constriction of mesenteric vessels to PE were not different in iPLA2β−/−vessels compared with WT mouse vessels. However, relaxation after ACh administration in situ was attenuated, indicating an endothelial inability to induce dilation in response to ACh. In cultured endothelial cells, inhibition of iPLA2β with ( S)-(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2 H-pyran-2-one (BEL) decreased endothelial nitric oxide synthase phosphorylation and reduced endothelial agonist-induced intracellular calcium release as well as extracellular calcium influx. We conclude that iPLA2β is an important mediator of vascular relaxation and intracellular calcium homeostasis in both smooth muscle and endothelial cells and that ablation of iPLA2β causes agonist-induced smooth muscle hypercontractility and reduced agonist-induced endothelial dilation.

Objectives. This study was designed to characterize the nature and time course of carboxy-terminal lysine cleavages from the tissue isoform of MB creatine kinase (CK) in vivo. Background. Rapid conversion of the tissue isoform of MM CK to two additional circulating isoforms with one or both carboxyterminal lysines cleaved facilitates early detection of new tissue isoform release after acute myocardial infarction and coronary recanalization. Characterization of changes in plasma MB CK isoform profiles, potentially enhancing specificity even further, has been hindered by difficulties in separating the isoform products and elucidation of carboxy-terminal lysine cleavages underlying their formation. Methods. Isoform species with carboxy-terminal lysine present on B-monomers were separated from those from which lysine had been cleaved by anion exchange chromatography. Carboxyterminal lysine on M-monomers was assayed with the use of a monospecific antibody. Results. MB CK in four pooled plasma samples from among 77 normal subjects exhibited carboxy-terminal lysine on 48 ± 21% (mean ± SEM) of B-monomers and 82 ± 12% of M-monomers. Within the 1st 16 h alter the onset of acute myocardial infarction, virtually all M- and B-monomers exhibited carboxy-terminal lysine, indicating release into plasma and the lack of rapid cleavage of lysine from the tissue isoform. After 20 to 30 h, 43 ± 9% (three pools from 19 patients) of B-monomers and 95 ± 10% of M-monomers exhibited lysine at the carboxyl terminus. After 40 to 50 h, 13 ± 13% (four pools from 34 patients) of B-monomers and 46 ± 19% of M-monomers still retained carboxy-terminal lysine. Conclusions. In contrast to MM CK, the tissue isoform of MB CK undergoes slow cleavage of lysine from both monomers in vivo. Sequential cleavage of lysine first from the carboxyl terminus of B-monomers and subsequently from M-monomers is consistent with generation of at least two additional isoforms. Development of assays capable of resolving all of the isoforms of MB CK that can occur in vivo might increase sensitivity for early detection of new tissue isoform release associated with acute myocardial infarction and coronary recanalization compared with currently available assays that resolve only two species.

Background— A comprehensive evaluation of myocardial ischemia requires measures of both oxygen supply and demand. Positron emission tomography (PET) is currently the gold standard for such evaluations, but its use is limited because of its ionizing radiation, limited availability, and high cost. A cardiac MRI method was developed for assessing myocardial oxygenation. The purpose of this study was to evaluate and validate this technique compared with PET during pharmacological stress in a canine model of coronary artery stenosis. Methods and Results— Twenty-one beagles and small mongrel dogs without coronary artery stenosis (controls) or with moderate to severe acute coronary artery stenosis underwent MRI and PET imaging at rest and during dipyridamole vasodilation or dobutamine stress to induce a wide range of changes in cardiac perfusion and oxygenation. MRI first-pass perfusion imaging was performed to quantify myocardial blood flow and volume. The MRI blood oxygen level-dependent technique was used to determine the myocardial oxygen extraction fraction during pharmacological hyperemia. Myocardial oxygen consumption was determined by the Fick law. In the same dogs, 15 O-water and 11 C-acetate were used to measure myocardial blood flow and myocardial oxygen consumption, respectively, by PET. Regional assessments were performed for both MR and PET. MRI data correlated nicely with PET values for myocardial blood flow ( R 2 =0.79, P R 2 =0.74, P R 2 =0.66, P Conclusions— Cardiac MRI methods may provide an alternative to radionuclide imaging in settings of myocardial ischemia. Our newly developed quantitative MRI oxygenation imaging technique may be a valuable noninvasive tool to directly evaluate myocardial energetics and efficiency.

To validate fast perfusion mapping techniques in a setting of coronary artery stenosis, and to further assess the relationship of absolute myocardial blood volume (MBV) and blood flow (MBF) to global myocardial oxygen demand.A group of 27 mongrel dogs were divided into 10 controls and 17 with acute coronary stenosis. On 1.5-T MRI, first-pass perfusion imaging with a bolus injection of a blood-pool contrast agent was performed to determine myocardial perfusion both at rest and during either dipyridamole-induced vasodilation or dobutamine-induced stress. Regional values of MBF and MBV were quantified by using a fast mapping technique. Color microspheres and (99m)Tc-labeled red blood cells were injected to obtain respective gold standards.Microsphere-measured MBF and (99m)Tc-measured MBV reference values correlated well with the MR results. Given the same changes in MBF, changes in MBV are twofold greater with dobutamine than with dipyridamole. Under dobutamine stress, MBV shows better association with total myocardial oxygen demand than MBF. Coronary stenosis progressively reduced this association in the presence of increased stenosis severity.MR first-pass perfusion can rapidly estimate regional MBF and MBV. Absolute quantification of MBV may add additional information on stenosis severity and myocardial viability compared with standard qualitative clinical evaluations of myocardial perfusion.