apyrase and Hemorrhage

Antiplatelet therapy is given to millions of patients and has saved numerous lives. However, it is also associated with complications including fatal bleedings. Clinically used antiplatelet drugs seem to follow the rule of an inherent link of improved anti-thrombotic potency with increased risk of bleeding complications. Therefore, there is an ongoing quest to develop drugs that are able to break this link that has prevented many patients from receiving antiplatelet protection and has resulted in substantial mortality and morbidity. We describe a new antiplatelet approach that is based on an recombinant antibody protein, a drug format that has recently attracted major interest. Two unique components are genetically combined in this molecule: 1) The ecto-nucleoside triphosphate diphosphohydrolase NTPDase CD39, which enzymatically degrades ATP and ADP to AMP, which is then further degraded to adenosine by the endothelially expressed CD73. Thereby, the platelet activating ADP is reduced and replaced by the platelet inhibiting adenosine resulting in a strong antiplatelet effect. 2) A single-chain antibody (scFv) that specifically binds to the activated GPIIb/IIIa receptor and thus allows targeting to activated platelets. The described fusion protein results in strong enrichment of CD39's antiplatelet effect, resulting in potent inhibition of platelet adhesion and aggregation and thrombosis in mice. The activated platelet targeting allows using a low systemic concentration that does not interfere with normal haemostasis and thus does not cause bleeding time prolongation in mice.. We describe a new antiplatelet approach that promises to deliver strong localized antithrombotic effects without associated bleeding problems.

Topics: Animals; Antigens, CD; Apyrase; Drug Design; Hemorrhage; Humans; Mice; Molecular Targeted Therapy; Platelet Aggregation Inhibitors; Rats; Thrombosis; Treatment Outcome

GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI-Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies. GPVI-Fc is a potent inhibitor of atherosclerotic plaque-induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI-Fc to the ectonucleotidase CD39 (fusion protein GPVI-CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion-directed dual antiplatelet therapy that is expected to lack systemic bleeding risks.. GPVI-CD39 effectively stimulated local ADP degradation and, compared with GPVI-Fc alone, led to significantly increased inhibition of ADP-, collagen-, and human plaque-induced platelet aggregation in Multiplate aggregometry and plaque-induced platelet thrombus formation under arterial flow conditions. GPVI-CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride-induced arterial thrombosis, GPVI-CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo.. GPVI-CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI-Fc-mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk-benefit ratio.

Topics: Animals; Antigens, CD; Apyrase; Carotid Artery Diseases; Carotid Artery Injuries; Chlorides; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Ferric Compounds; Fibrinolytic Agents; Glycoproteins; Hemorrhage; Humans; Immunoglobulin Fc Fragments; Male; Mice, Inbred C57BL; Plaque, Atherosclerotic; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Recombinant Fusion Proteins; Thrombosis

Caffeine or ketorolac decrease the risk of retinopathy of prematurity and may act synergistically to improve beneficial effect. Combination of caffeine (Caff) and ketorolac (Keto) to prevent oxygen-induced retinopathy was studied.. Newborn rats exposed to room air (RA) or intermittent hypoxia (IH) consisting of 12% O2 during hyperoxia (50% O2) from birth (P0) had single daily IP injections of Caff from P0-P13 or saline; and/or ocular Keto (Acuvail, 0.45% ophthalmic solution) administered subcutaneously over the eyes from P5-P7. Pups were studied at P14 or placed in RA for recovery from IH (IHR) until P21. Eyes were examined for neovascularization, histopathology, growth factors, and VEGF-signaling genes.. Severe retinal damage noted during IHR in the untreated groups evidenced by hemorrhage, neovascularization, and oxygen-induced retinopathy (OIR) pathologies were prevented with Keto/Caff treatment. Keto and/or Caff treatment in IH also promoted retinal neural development evidenced by eye opening (92%, P < 0.001 vs. 31% in the placebo-treated IH group). No corneal pathologies were noted with Keto.. Caff or Keto given individually reduced retinal neovascularization, but the two drugs given together prevented severe OIR.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Apyrase; Arteries; Body Weight; Caffeine; Central Nervous System Stimulants; Choroid; Citrates; Dinoprost; Drug Synergism; Female; Hemorrhage; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Ketorolac; Neovascularization, Pathologic; Oxygen; Rats; Rats, Sprague-Dawley; Retina; Retinopathy of Prematurity; Signal Transduction; Time Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

Exercise-induced pulmonary hemorrhage (EIPH) is a common disorder of equine athletes. The role of polymorphisms in genes encoding hemostasis-regulatory proteins in horses with abnormal hemorrhage is unknown.. The goal of this study was to evaluate the genes encoding 2 ectonucleotidases, CD39/NTPDase-1 and CD39L1/NTPDase-2, and one ecto-5' nucleotidase, CD73, in horses with abnormal hemorrhage or pathologic changes consistent with EIPH.. Twenty-three horses with histories of abnormal hemorrhage, 8 horses with gastrointestinal signs, and 45 healthy horses were evaluated using polymerase chain reaction-based techniques. Formalin-fixed tissues from 21 horses with pathologic changes consistent with EIPH were also evaluated.. Three single nucleotide polymorphisms (SNPs) were identified in the gene encoding CD39 and one SNP was identified in the gene encoding CD39L1. No SNPs were identified in the gene encoding CD73. CD39 SNPs were identified in 19 of 20 (95%) horses with unexplained hemorrhage and 20 of 21 (95%) horses with pathologic features consistent with EIPH. CD39L1 SNPs were identified in 6 of 20 (30%) horses with unexplained hemorrhage and 8 of 21 (38%) horses with pathologic features consistent with EIPH. CD39 and CD39L1 SNPs were identified in 5 of 8 (62.5%) and one of 8 (12.5%) horses, respectively, presenting with colic or weight loss. CD39 and CD39L1 SNPs were identified in 28 of 45 (62%) and 13 of 45 (28.8%) healthy horses, respectively.. CD39 and CD39L1 are critically important in maintaining normal hemostasis and limiting inflammation. Further studies are needed to evaluate their role in the pathogenesis of equine EIPH.

Topics: Adenosine Triphosphatases; Animals; Antigens, CD; Apyrase; Gene Expression Regulation, Enzymologic; Genetic Predisposition to Disease; Hemorrhage; Horse Diseases; Horses; Lung Diseases; Physical Exertion; Polymorphism, Single Nucleotide

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.

Topics: Adenosine Diphosphate; Animals; Apyrase; Clopidogrel; Coronary Circulation; Dogs; Fibrinolysis; Hemorrhage; Humans; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion Injury; Piperazines; Platelet Aggregation; Prasugrel Hydrochloride; Risk Factors; Thiophenes; Thrombosis; Ticlopidine; Time Factors; Tissue Plasminogen Activator; Treatment Outcome; Vascular Patency

Topics: Animals; Apyrase; Hemorrhage; Humans; Myocardial Reperfusion Injury; Thrombosis

Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7-/-), and in wild-type mice. P2X7-/- mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre- or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7-/- mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39-/-) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39-/- mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1β release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP-induced hepatotoxicity.

Topics: Acetaminophen; Animals; Antigens, CD; Antipyretics; Apyrase; Cells, Cultured; Chemical and Drug Induced Liver Injury; Hemorrhage; Inflammasomes; Interleukin-1beta; Kupffer Cells; Male; Mice; Mice, Inbred C57BL; NAD; Necrosis; Pyridines; Receptors, Purinergic P2X7; Signal Transduction; Tetrazoles

Widespread hemorrhagic manifestations commonly occur in patients with severe heat stroke. The pathogenesis of hemostatic disorders in these patients is not fully understood, although it is believed to be multifactorial in origin. The present investigation was designed to study the changes in blood platelets caused by heat stress in an experimental model of five merino sheep. The experiments were performed in two groups of five merino sheep each. In one group the sheep were subjected to a combination of heat (elevated environmental temperature) and exertional stress, and allowed to proceed throughout the experiment until a state of near collapse was reached (Task A). In the other group (Task B) the animals were heated in the same manner as those in Task A and also subjected to exertional heat; however, when the temperature reached 43.6 +/- 0.2 degrees C, the critical core temperature (CCT), they were subjected to evaporative cooling in a climatic chamber. Serial changes in the platelet counts and platelet functions were measured throughout the duration of the experiments. At the core temperature (CT) of 42.1 degrees C and above there was a significant impairment of adhesion of platelets to glass beads. During the early phases of elevation of CT, platelets showed hyperaggregation in the presence of different agonists (such as, collagen, ADP, ristocetin); this was followed by hypoaggregation when the CCT was raised above 43.6 +/- 0.2 degrees C. However, these impairments of platelet functions occurring at elevated CT and CCT were found to reverse to normal within 24 hours after the animals were cooled to 39 degrees C. It was also found that the hyperaggregation of platelets to different agonists induced by raised CT could be partially prevented by prior in vitro treatment of platelets with apyrase, a known enzyme destroying of ADP. The results of these experiments indicate that heat stress induced by exposing merino sheep to elevated controlled temperature directly activates the platelets. This may be an important contributing factor in causing altered hemostasis in heat stroke activated directly by heat. This mechanism may be operating in altered hemostasis in heat stroke.

Topics: Animals; Apyrase; Arachidonic Acid; Blood Platelets; Collagen; Heat Stress Disorders; Hemorrhage; Platelet Aggregation; Platelet Count; Platelet Function Tests; Ristocetin; Sheep

The possible involvement of adenosine diphosphate (ADP) in haemostatic platelet aggregation was investigated by determining the duration of primary haemorrhage as standardized bleeding times from punctures of small mesenteric arteries in anaesthetized rats. The bleeding times were highly significantly increased by infusing into the mesenteric arterial blood flowing towards the punctures either the nucleotide-dephosphorylating enzyme apyrase or the ADP-receptor antagonists ATP, adenosine 5'-(beta,gamma-methylene)triphosphonate (AMP-PCP) or 2-methylthioadenosine 5'-(beta,gamma-methylene)triphosphonate (2-MeS-AMP-PCP). The increases in bleeding times could not be accounted for by local vasodilator effects of the agents. It is concluded that the presence of ADP through local release and/or formation at sites of vascular injury contributes significantly to haemostasis, presumably by accelerating platelet aggregation.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Animals; Apyrase; Hemorrhage; Hemostasis; Male; Mesenteric Arteries; Mesenteric Veins; Mice; Phosphoric Monoester Hydrolases; Rats; Rats, Inbred Strains; Receptors, Purinergic; Reference Values