Adenosine Diphosphatase Activity Research Articles

Safety, Tolerability, and Pharmacodynamics of AZD3366 (Optimized Human CD39L3 Apyrase) Alone and in Combination With Ticagrelor and Acetylsalicylic Acid: A Phase 1, Randomized, Placebo-Controlled Study.

ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y12 receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease. We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the exvivo thromboelastometric parameters. AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in exvivo thromboelastometric parameters. URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.

A putative adenosine kinase family protein possesses adenosine diphosphatase activity

Adenosine kinase is a potential target for development of new types of drugs. The COG1839 family has been defined as "adenosine-specific kinase" family based on structural analysis and the adenosine-binding ability of a family member, PAE2307. However, there has been no experimental evidence with regard to the enzymatic function of this protein family. Here we measured the enzymatic activity of TTHA1091, a COG1839 family protein from Thermus thermophilus HB8. The phosphorylation of adenosine by TTHA1091 was undetectable when ATP or ADP were used as phosphate donor. However, the degradation of ADP to AMP was detected, indicating that this protein possessed adenosine diphosphatase (ADPase) activity. The (ADPase) activity was inhibited by divalent cations and was specific to ADP and CDP. Thus, this study provides the first experimental evidence for the enzymatic function of the "adenosine-specific kinase" family and suggests a need to reexamine its functional annotation.

Atherosclerosis and atherosensitivity in two southwest Algerian desert rodents, Psammomys obesus and Gerbillus gerbillus, and in Rattus norvegicus.

Cardiovascular disease, including atherosclerosis, is the leading cause of death in patients with diabetes worldwide; thus, it is a major medical concern. The endothelium contributes to the control of many vascular functions, and clinical observations show that it is a primary target for diabetic syndrome. To get better insight into the mechanisms underlying atherosclerosis, we studied the interspecific differences in the arterial metabolisms of two, Psammomys obesus and Gerbillus gerbillus, as well as Rattus norvegicus (Wistar rat), well known for its atheroresistance. Twenty-two enzymatic activities and six macromolecular substances were histochemically compared in the two desert species and in Wistar aortas (abdominal and thoracic) and arteries (femoral and caudal) embedded in a common block. In the healthy adult rodents, enzyme activities were very intense. They demonstrated that aortic myocytes are capable of various synthesis and catabolism processes. However, considering the frequency of atherosclerosis and its phenotypes, significant differences appeared between the species studied. Our comparative study shows that aortic atherosensitive animals have several common metabolic characteristics, which are found in Psammomys rich in metachromatic glycosaminoglycans (involved in the inhibition of lipolysis and in calcification of the organic matrix), reduced activity in enzymes related to the Krebs cycle (weakening energetic power), and low lipolytic enzyme, adenosine triphosphatase, and adenosine diphosphatase activities. However, the most fundamental pathophysiological difference is the low lipolytic power of the aorta of Psammomys when compared to Wistar rats. This characteristic determines its atherosensitivity and makes this animal model more applicable to the experimental development of atherosclerosis.

Retinopathy in Monkeys with Spontaneous Type 2 Diabetes

Type 2 diabetes occurs spontaneously in rhesus monkeys and shows an extraordinary similarity to human diabetes in clinical features and relative time course. The purpose of this study was to investigate clinically and histopathologically the ocular changes in these monkeys. Ophthalmoscopic examinations were performed on aged normal and diabetic monkeys. Retinas from 16 diabetic monkeys and 6 nondiabetic monkeys were incubated postmortem for adenosine diphosphatase (ADPase) activity (labels viable retinal blood vessels) and flat-embedded in JB-4. Tissue sections were cut through areas of interest. Cotton-wool spots, intraretinal hemorrhages, and hard exudates in the macula were observed by ophthalmoscopy in some diabetic monkeys. Dot/blot hemorrhages, cotton-wool spots, and small nonperfused areas were the earliest histologically documented changes in the retinas. Large nonperfused areas extending from optic disc to midfovea were observed in four diabetic monkeys. Formation of small intraretinal microvascular abnormalities (IRMAs) and microaneurysms were associated with the areas of nonperfusion. There were apparent fluid-filled spaces in the outer plexiform layer in three of these maculas, suggesting macular edema. There was a significant correlation between the occurrence of retinopathy and hypertension (P = 0.037 for systolic pressure; P = 0.019 for diastolic pressure). In elastase-digested retinas, the ratio of pericytes to endothelial cells was 0.66:1 in diabetic and 0.64:1 in nondiabetic (P = 0.75) retinas. This is the first detailed analysis of retinopathy in a colony of spontaneous type 2 diabetic monkeys. Monkeys with type 2 diabetes have many of the angiopathic changes associated with human diabetic retinopathy. Hypertension correlates with the severity of the diabetic retinopathy.

Pathologic features of vascular endothelial growth factor-induced retinopathy in the nonhuman primate

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent ischemia-upregulated angiogenic protein that has been implicated in diabetic retinopathy. Intravitreal VEGF injections have not previously been shown to produce preretinal neovascularization. The purpose of this study was to further characterize the angiopathic changes that occur after intravitreal injections in a nonhuman primate and determine if preretinal neovascularization develops. DESIGN: Experimental animal study. METHODS: Vascular endothelial growth factor 165 was injected into the eyes of normal cynomolgus monkeys at regular intervals. As a control, normal eyes were injected with phosphate buffered saline. Color photography and fluorescein angiography were performed at regular intervals. The retinas were incubated for adenosine diphosphatase (ADPase) activity to visualize retinal vessels. The retinas were flat-embedded and areas of potential preretinal neovascularization were identified en bloc and serially sectioned. RESULTS: Areas of capillary nonperfusion and vessel dilation and tortuousity were seen by angiography. In serial sections, the nonperfused areas were found to be associated with endothelial cell hyperplasia in vessel lumens. Preretinal neovascularization originating only from superficial veins and venules was observed throughout peripheral retina, but was not seen in the posterior pole. Lacunae-like veins were subdivided by the process of intussusception and endothelial cell bridging. Arterioles demonstrated endothelial cell hyperplasia and microaneurysms. CONCLUSION: Intraocular injections of VEGF were sufficient to produce preretinal neovascularization in the nonhuman primate. Most vasculopathic structures were associated with endothelial cell hyperplasia. These results demonstrate that VEGF alone can produce many features of both nonproliferative and proliferative diabetic retinopathy including the previously undescribed development of preretinal neovascularization. This well-characterized VEGF-induced primate model of retinal neovascularization may be useful as a means of testing new treatments for retinal neovascularization.

Targeting platelet aggregation: CD39 gene transfer augments nucleoside triphosphate diphosphohydrolase activity in injured rabbit arteries

Background. CD39, the major endothelial nucleoside triphosphate diphosphohydrolase (NTPDase), plays an important role in local thromboregulation. We hypothesized that balloon injury (BI) leads to an acute reduction in arterial NTPDase activity that could be restored by a targeted gene delivery strategy. Methods. Recombinant adenoviral vectors containing human CD39 (Ad-CD39) or β-galactosidase (Ad-LacZ) were used. Endothelial (ECs) and smooth muscle cells (SMCs) were infected in vitro and NTPDase activity measured. New Zealand white rabbits (N = 28) underwent bilateral iliofemoral artery balloon injury, followed by incubation with Ad-CD39, Ad-LacZ, or vehicle. Explanted vessels were analyzed for NTPDase activity and localization of CD39 expression by immunohistochemistry. Deposition of fluorescent-labeled platelets was studied 3 days after injury and vector treatment. Results. In vitro, Ad-CD39 infection resulted in a greater than 40-fold increase in adenosine diphosphatase activity in ECs and a 3-fold increase in SMCs. In vivo, CD39 transgene expression localized to the luminal aspect of Ad-CD39-treated vessels. BI resulted in an acute reduction in vessel wall NTPDase activity (P <.05). Ad-CD39 augmented NTPDase activity when compared with vehicle or Ad-LacZ (P <.05). Platelet deposition on the injured arterial surface was modest and not different between Ad-CD39- and Ad-LacZ-treated vessels. Conclusions. BI decreases native NTPDase activity, which can be augmented by adenovirus-mediated gene transfer of CD39. Further studies are required to determine whether targeted delivery of CD39 could convey thromboprotective properties to an injured vessel. (Surgery 2001;130:296-303.)

Radiation-induced glomerular thrombus formation and nephropathy are not prevented by the ADP receptor antagonist clopidogrel

Purpose: To assess the effects of kidney irradiation on glomerular adenosine diphosphatase (ADPase) activity and intraglomerular microthrombus formation, and their correlation to the development of renal functional impairment. Methods and Materials: C3H/HenAf-nu + mice were given single-dose or fractionated kidney irradiations. Glomerular ADPase activity was measured using a cerium-based histochemical method. Microthrombus formation within the glomeruli was assessed by a semiquantitative immunohistochemical analysis of fibrinogen/fibrin deposits. Renal function was assessed by the [ 51Cr]EDTA retention assay. Results: The ADPase activity was significantly reduced, to approximately 50% of pretreatment value, 4–40 weeks after 10–16 Gy single-dose irradiation and at 44 weeks after 20 × 2 Gy. No dose-effect relationship was found. An approximately fourfold increase in glomerular fibrinogen/fibrin staining was observed at 1 year after irradiation. This increase was not influenced by treating the mice with daily, oral clopidogrel, a platelet ADP receptor antagonist, which reduced platelet aggregation by more than 75%. Radiation-induced impairment of glomerular filtration was also not affected by the clopidogrel treatment. Conclusion: These data indicate that irradiation significantly reduced glomerular ADPase activity, which correlated with an increased glomerular fibrinogen/fibrin deposition. We were not able to reduce these prothrombotic changes, nor to protect against radiation nephropathy, by pharmacological intervention with an ADP-receptor antagonist.

Aspirin treatment of the low-dose-endotoxin-treated pregnant rat: pathophysiologic and immunohistologic aspects

In the present study, we evaluated the effect of low-dose aspirin (acetylsalicylic acid (ASA); 1.0 mg/kg daily) on blood pressure, albumin excretion, glomerular fibrinogen deposits, and glomerular (basement) membrane-bound adenosine diphosphatase (ecto-ADPase) activity, as well as on glomerular inflammation in pregnant rats infused with low-dose endotoxin (1.0 mg/kg). Rats (day 14 of pregnancy) were infused with endotoxin (ET rats) or saline (control rats) and received ASA in their drinking water. These rats were compared with non-ASA-treated rats. Blood pressure and albumin excretion were measured from day 15 to day 21, and glomerular fibrinogen and ecto-ADPase activity were measured at day 21. Glomerular inflammation was evaluated at various times after the start of the infusion. The results show that treatment with ASA had a significant beneficial effect on hypertension and inflammation induced by endotoxin in pregnant rats, whereas it reduced albumin excretion and glomerular fibrinogen deposits in some of the rats.

Toxicological Effects of Beryllium on Platelets and Vascular Endothelium

Although ample research has described the toxic effects of the metal beryllium on the respiratory apparatus, less is known about its effects on the vascular apparatus, including pulmonary blood vessels. We investigated thein vitroeffects of beryllium on endothelial vascular adenosine diphosphatase activity and prostacyclin production in bovine aortic endothelium, and on nitric oxide release in isolated rabbit arteries. Rabbit and human platelet responsiveness was also evaluated. Beryllium inhibited vascular endothelial adenosine diphosphatase activity, prostacyclin production, and nitric oxide release, thus inducing functional alterations in vascular endothelial cells. It also induced platelet hyperreactivity to arachidonic acid, as shown by a lowering of the threshold of aggregating concentration and by concurrently increasing thromboxane production. In contrast, beryllium left the response to aggregating and nonaggregating concentrations of ADP and collagen unchanged. These findings show that beryllium may impair some vascular endothelial functions and alter the interaction between platelet and endothelial mediators.

Detection of Vascular Endothelial Growth Factor (VEGF) Protein in Vascular and Non-Vascular Cells of the Normal and Oxygen-Injured Rat Retina

Vascular endothelial growth factor (VEGF) is a potent and specific endothelial cell cytokine that can be up-regulated by hypoxia. There is evidence that VEGF is a significant mediator in retinal neovascular diseases and other disorders in which hypoxia is believed to influence the pathogenesis. Here we demonstrate the spatial relationships among areas of retinal non-perfusion, VEGF protein and vascular endothelial cells throughout the retina, and relate these results to cellular distribution of VEGF in cross section. Newborn albino rats were oxygen-injured by cycles of alternating 50% and 10% oxygen for 14 days and then placed in room air. On days 16, 21 and 26, oxygen-injured and control (raised in room air) rats were sacrificed, enucleated and retinas were dissected and fixed for whole mount immunostaining for VEGF or embedding in glycol methacrylate for VEGF immunohistochemistry. Intact eyes taken on days 16 and 20 were processed similarly. Vascular endothelial cells were demonstrated by staining whole-mounted retinas for adenosine diphosphatase (ADPase) activity. Preretinal neovascular growths (i.e., abnormal vessels extending from the retina into the vitreous) were VEGF-positive. There was also a pan-retinal distribution of non-endothelial cells that were VEGF-positive in both room air and oxygen-injured rats, with stronger immunostaining in day 16 oxygen-injured retinas. In cross-section, VEGF staining was confirmed in preretinal growths, normal retinal vessels, cells in the inner nuclear layer (primarily Müller cells) and ganglion cells. Retinas which had been incubated with non-immune IgG or absorbed anti-VEGF antibody showed little or no staining. In conclusion, we have identified cells of the inner retina which express VEGF. The production of VEGF by these cells—in particular, Müller cells—may promote preretinal neovascularization in oxygen-injured eyes. We have found, moreover, that the combination of immunohistochemistry and ADPase staining of whole mount preparations is a unique and powerful tool for evaluating relationships between presumed areas of retinal ischemia, VEGF (and other cytokines) and retinal blood vessels, within an entire retina. This approach can he used to study any proliferative retinal disorders in which VEGF is a potential component of the pathogenesis.

Long-term retinal vascular abnormalities in an animal model of retinopathy of prematurity.

A conventional criticism of animal models of retinopathy of prematurity (ROP) concerns the common occurrence of rapid spontaneous resolution of retinal vascular sequelae. The purpose of this study was to determine whether animals subjected to a novel variable oxygen exposure protocol would undergo the rapid spontaneous resolution of retinal vascular pathology that is typical of past models. Newborn rats were exposed to an oxygen environment that alternated between 50% and 10% every 24 h for 14 days and then removed to room air, or were raised from birth in room air as controls. To determine early retinal vascular growth rate, both exposed and non-exposed rats were sacrificed between 3 and 28 days of age, after which eyes were enucleated and retinas dissected and stained for adenosine diphosphatase (ADPase) activity to demonstrate the vasculature. Rats were maintained in room air for 2 to 18 weeks after the variable oxygen exposure period for assessment of long-term retinal vascular abnormalities by ADPase histochemistry. The retinal vasculature of oxygen-exposed rats was significantly different from that of room air-raised rats with respect to capillary density, branching frequency, and bifurcation angle. These differences were restricted to the area that was vascularized after removal to room air (the peripheral-most 25% of the retinal area), and they persisted for the duration of the study. We have developed a rat model of ROP using an exposure protocol designed to create systemic oxygen levels that approximate those of premature infants. This model does not demonstrate the complete resolution of vessel abnormalities that historically has limited animal models of ROP.

Adenosine diphosphatase and adenosine triphosphatase activities of maize tonoplast‐enriched vesicles

Abstract Microsomal fraction enriched with tonoplast vesicles was obtained from maize root (Zea mays L. FRB 73) by sucrose step gradient centri‐fugation. The vesicles contained an adenosine diphosphatase (ADPase) activity as determined by the hydrolysis of Mg‐ADP. The ADPase and adenosine triphospha‐tase (ATPase) of the fraction have different sensitivities toward several modifiers. ADPase activity was not sensitive to nitrate, N,N'‐dicyclohexylcarbodiimide (DCCD), diethylstilbestrol (DES), and azide, but partially sensitive to vanadate and molybdate. In contrast, ATPase was very sensitive to nitrate, DCCD, and DES, but not to vanadate, azide, and molybdate. The presence of 2'‐or (3')‐trinitrophenol adenosine 5'‐diphosphate (TNP‐ADP) significantly reduced ADPase activity but not that of the ATPase activity. On the other hand, the addition of TNP‐ATP abolished ATPase activity but not the ADPase activity. Since the influence of these modifiers to the activities are different, the binding and the hydrolysis ...

Reproductive condition, glomerular adenosine diphosphatase activity, and platelet aggregation in the rat: effect of endotoxin.

In experiment A, the activity of the glomerular antithrombotic enzyme adenosine diphosphatase (ADPase) and the sensitivity of this enzyme for endotoxin (1.0 microgram/kg BW) in various reproductive conditions of female rats were studied through use of enzyme histochemical methods. In experiment B, the effect of this dose of endotoxin on the thrombotic tendency of the glomeruli in pregnant (Pr) and pseudopregnant (PSP) rats was studied by means of ex vivo alternate perfusion of the kidneys with human platelets and adenosine diphosphate (ADP). In experiment A, cyclic (C), ovariectomized (OVX), Pr, and PSP rats were infused with endotoxin or saline. Three days later (for Pr and PSP rats, Day 8), animals were killed. In intact rats (C, Pr, PSP), the activity of glomerular ADPase was the same; however, the activity decreased after OVX. Endotoxin decreased the activity of glomerular ADPase in Pr rats only. In endotoxin-treated Pr rats, spontaneously formed platelet microaggregates were present in a few glomeruli; in glomeruli of the other groups, microaggregates were not observed. Platelet microaggregates were also present in the venous microvasculature of endotoxin-treated Pr rats and, to a lesser extent, in that of other groups, while saline-treated OVX rats were negative in this respect. In experiment B, Pr and PSP rats were treated as in experiment A; ex vivo kidney perfusion was performed on Day 8. Immediately after perfusion, rats were killed. Only Pr endotoxin-treated rats exhibited significantly increased intraglomerular platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

A New Technique for Visualization of the Human Retinal Vasculature

Adenosine diphosphatase, an endothelial cell ectoenzyme that hydrolyses adenosine 5'-diphosphate to adenosine monophosphate, has been used histochemically to study the human retinal vasculature in flat mounts or as flat-embedded tissue that can be sectioned. In all retinas, reaction product was confined almost exclusively to the vasculature, with arteries having more reaction product than veins or capillaries. Adenosine diphosphatase activity was observed in endothelium, smooth-muscle cells, pericytes, and erythrocytes. In the more elaborate version of the technique, the adenosine diphosphatase-incubated retinas were flat embedded in glycol methacrylate. This allowed study of the retinal vasculature in dual perspective: en bloc with the use of dark-field microscopy and specific areas of interest histologically sectioned for further evaluation. The technique is simple and preserves angioarchitecture because the retinas are intact, and patency of the vasculature can be determined.

Purification and characterization of adenosine diphosphatase from human umbilical vessels.

Adenosine diphosphatase (ADPase) activity was solubilized with a non-ionic detergent, Tween 20, from human umbilical vessels and purified to homogeneity by diethylaminoethyl-Sepharose CL-6B, adenosine 5'-monophosphate-Sepharose 4B, and concanavalin A-Sepharose chromatography. The apparent molecular mass was 75 kDa. The purified enzyme hydrolyzed pyrophosphate bonds of nucleoside di- and triphosphates in the presence of calcium ion. It was insensitive to the adenosine triphosphatase (ATPase) inhibitors, oligomycin and ouabain, and sensitive to sodium azide. Therefore, we concluded that the ADPase activity in human umbilical vessels does not derive from ADPase degrading only ADP but from ATP diphosphohydrolase (EC 3.6.1.5). The broad substrate specificity and the sensitivity to various inhibitors and calcium ion are common to ATP diphosphohydrolase from bovine aorta. However, there might exist some structural difference around the active site, because the antiserum raised in rabbit against the bovine aorta enzyme scarcely inhibited the human umbilical enzyme.

Demonstration of antithrombotic activity of glomerular adenosine diphosphatase [published erratum appears in Blood 1991 Oct 15;78(8):2163

We have demonstrated that reduced glomerular adenosine diphosphatase (ADPase) activity within the rat kidney is associated with an increased thrombotic tendency. To establish a possible causal relationship between these intraglomerular events, experiments were conducted to inhibit adenosine diphosphate (ADP) degradation without influencing other glomerular prothrombotic or antithrombotic mechanisms. Concurrently, we studied intraglomerular platelet aggregation. Two ways of selective inhibition of glomerular ADPase activity were applied: (1) by competitive substrates (ie, uridine diphosphate [UDP]), and (2) by the nondegradable ADP analogue ADP-beta-S. Both strategies were used during ex vivo alternate perfusion of kidneys with platelets and ADP (to test intraglomerular thrombotic tendency). Each group (n = 6) received different substrates or a combination of substrates. A significant increase in platelet aggregation was observed in kidneys after perfusion with platelets and ADP together with the competitive substrate UDP as compared to perfusions with platelets and ADP alone (78.5% +/- 9.8% v 27.9% +/- 11.4% glomeruli staining positive for platelets, P less than .005). In contrast, UDP alone had no effect on platelet aggregation. Other nucleoside polyphosphates (guanosine diphosphate and inosine triphosphate) were also effective as competitive substrates in the ex vivo perfusion model (n = 4). None of these substrates was capable of increasing ADP-induced aggregation when studied in vitro. In addition, ADP- beta-S also increased platelet aggregation in the perfusion model as compared with native ADP (P less than .005). These results show that selective reduction of ADP degradation in intact kidneys strongly promotes the intraglomerular proaggregatory condition. It can be concluded that glomerular ADPase exerts potent antithrombotic activity within the normal rat kidney.

Demonstration of Antithrombotic Activity of Glomerular Adenenosine Diphosphatase

Abstract We have demonstrated that reduced glomerular adenosine diphosphatase (ADPase) activity within the rat kidney is associated with an increased thrombotic tendency. To establish a possible causal relationship between these intraglomerular events, experiments were conducted to inhibit adenosine diphosphate (ADP) degradation without influencing other glomerular prothrombotic or antithrombotic mechanisms. Concurrently, we studied intraglomerular platelet aggregation. Two ways of selective inhibition of glomerular ADPase activity were applied: (1) by competitive substrates (ie, uridine diphosphate [UDP]), and (2) by the nondegradable ADP analogue ADP-beta-S. Both strategies were used during ex vivo alternate perfusion of kidneys with platelets and ADP (to test intraglomerular thrombotic tendency). Each group (n = 6) received different substrates or a combination of substrates. A significant increase in platelet aggregation was observed in kidneys after perfusion with platelets and ADP together with the competitive substrate UDP as compared to perfusions with platelets and ADP alone (78.5% +/- 9.8% v 27.9% +/- 11.4% glomeruli staining positive for platelets, P less than .005). In contrast, UDP alone had no effect on platelet aggregation. Other nucleoside polyphosphates (guanosine diphosphate and inosine triphosphate) were also effective as competitive substrates in the ex vivo perfusion model (n = 4). None of these substrates was capable of increasing ADP-induced aggregation when studied in vitro. In addition, ADP- beta-S also increased platelet aggregation in the perfusion model as compared with native ADP (P less than .005). These results show that selective reduction of ADP degradation in intact kidneys strongly promotes the intraglomerular proaggregatory condition. It can be concluded that glomerular ADPase exerts potent antithrombotic activity within the normal rat kidney.

Purification and partial characterization of adenosine diphosphatase activity in bovine aorta microsomes

Anti-aggregatory activities in bovine aorta microsomal fractions were solubilized with Triton X-100 and separated into two fractions by DEAE-Sepharose CL-6B. One fraction strongly inhibited arachidonic acid-induced platelet aggregation, and the other inhibited ADP-induced aggregation. The latter fraction contained ADPase activity. The ADPase activity was further purified by affinity chromatography. The purified enzyme had specific activities of 43.8 and 48.2 μmol of P i/min/mg protein for ADP and ATP, respectively. The enzyme required calcium or magnesium ions and it was insensitive to ATPase inhibitors, namely oligomycin and ouabain, and to adenylate kinase inhibitor, Ap5A. Polyacrylamide gel electrophoretic experiments indicated that only one enzyme was involved. This was confirmed by the parallel behavior of ADPase and ATPase activities throughout all the purification steps. These results suggest that the main anti-aggregatory activity of bovine aorta microsomes for ADP-induced aggregation is due to an ATP diphosphohydrolase (EC 3.6.1.5).

Demonstration of plasma-membrane adenosine diphosphatase activity in rat lung

The adenosine diphosphatase (ADPase) activity of rat lung has been investigated. Subcellular fractionation of lung tissue homogenates by sucrose density gradient centrifugation has shown the ADPase activity to be associated with the plasma membrane. ADPase was solubilised from the membranes and fractionated by ammonium sulphate precipitation to separate a specific, low-K m ADPase from non-specific alkaline phosphatase activity. The solubilised ADPase has a K m of 50 μM at pH 7.5 and appears to be distinct from ATPase.

Properties and subcellular localization of adenosine diphosphatase in rat heart

Some properties and subcellular localization of adenosine diphosphatase (ADPase) activity from rat heart have been investigated. The pH optimum was 7.4, maximal activity was found with 5 m m MgCl 2, and the apparent K m was 20 μ m. ADPase activity was strongly inhibited by NaF and AppNHp, and to a lesser extent by AMP and GppNHp. The enzyme was not inhibited by p-nitrophenylphosphate, β-glycerophosphate, or pyridoxal phosphate. The distribution of ADPase activity in subcellular fractions obtained by differential centrifugation paralelled ouabain-sensitive (Na +-K +)ATPase and 5′-nucleotidase activities, suggesting a plasma membrane-bound localization. The functional significance of ADPase in adenosine production and hemostasis is discussed.