We previously demonstrated that when platelets are in motion and in proximity to endothelial cells, they become unresponsive to agonists (
Cultured endothelial cells derived from human umbilical veins or bovine aorta produce a potent inhibitor of platelet aggregation. The inhibitor is synthesized from sodium arachidonate or prostaglandin endoperoxides by a microsomal enzyme system. Tranylcypromrine, a specific antagonist of prostacyclin synthetase, suppresses production of the inhibitor by endothelial cells. The inhibitor, which is ether extractable, has been identified using a two-step thin-layer radiochromatographic procedure and a synthetic prostaglandin I2 standard. With this procedure, we have shown that human and bovine endothelial cells convert sodium [3H]arachidonate to radiolabeled prostaglandin I2 and 6-keto-prostaglandin Fla, as well as prostaglandin E2. Thus, endothelial cells may be non-thrombogenic in vivo because they synthesize and release prostaglandin I2, a potent inhibitor of platelet aggregation.Fragments of blood vessel walls acting upon prostaglandin (PG) endoperoxides or arachidonic acid produce an unstable factor that prevents platelet aggregation and release (1, 2). This factor, recently identified as (5Z)-9-deoxy-6,9-a-epoxy A5-PGFIa, (prostacyclin L-glutamine (2 mM). Cells were grown in T-25 or T-75 flasks (Corning) at 370 under 5% C02/95% air until confluence. Both primary cultures and serial passages were utilized. Initial cultures of bovine endothelial cells were kindly provided by Alan Quarfoot and Francois Booyse (13) and were cultured in RPMI 1640 medium containing 10-20% heat-inactivated fetal calf serum and penicillin, streptomycin, and L-glutamine as above. Cells were harvested from culture flasks by treatment with 0.1% collagenase-0.01% EDTA for 10 min at 370, washed three times in buffer A [137 mM NaCl/4 mM KCl/11 mM glucose/10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), pH 7.5] and finally resuspended in the same buffer at 6 X 106 cells per ml.Culture and Preparation of Other Cells. Human fibroblasts were grown in tissue culture, harvested with trypsin (0.25%) for 10 min, washed similarly, and resuspended in buffer A at the same protein concentration as the endothelial cells. Peripheral blood mixed leukocytes (70-80% neutrophils) were separated from heparin-treated whole blood by dextran sedimentation, washed, and resuspended in-buffer A at a similar protein concentration.Platelet Aggregation Experiments. Platelet-rich plasma (PRP) was prepared from venous blood drawn into 'ho volume of 3.2% trisodium citrate by methods previously described (14) and was kept tightly capped at 220 under 5% C02/95% air until use. Compounds being tested (0.1 ml) were added to 0.4 ml of PRP in the cuvette of a Payton aggregation module (Payton Associates, Buffalo, NY). Aggregating agents used included arachidonic acid (as the sodium salt in 0.1 M Na2COA), adenosine diphosphate (ADP) and collagen (Sigma Chemical Co., St. Louis, MO), and bovine thrombin (Parke Davis Co., Detroit, MI). Synthetic PGI2 (15) final volume of 0.5 ml in an aggregation module cuvette at 370 Abbreviations: PG, prostaglandin; ...
Excessive platelet accumulation and recruitment, leading to vessel occlusion at sites of vascular injury, present major therapeutic challenges in cardiovascular medicine. Endothelial cell CD39, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ATP and ADP released from activated platelets, thereby abolishing recruitment. Therefore, a soluble form of CD39, retaining nucleotidase activities, would constitute a novel antithrombotic agent. We designed a recombinant, soluble form of human CD39, and isolated it from conditioned media from transiently transfected COS-1 cells and from stably transfected Chinese hamster ovary (CHO) cells. Conditioned medium from CHO cells grown under serum-free conditions was subjected to anti-CD39 immunoaffinity column chromatography, yielding a single approximately 66-kD protein with ATPase and ADPase activities. Purified soluble CD39 blocked ADP-induced platelet aggregation in vitro, and inhibited collagen-induced platelet reactivity. Kinetic analyses indicated that, while soluble CD39 had a Km for ADP of 5.9 microM and for ATP of 2.1 microM, the specificity constant kcat/Km was the same for both substrates. Intravenously administered soluble CD39 remained active in mice for an extended period of time, with an elimination phase half-life of almost 2 d. The data indicate that soluble CD39 is a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses.
Endothelial CD39 metabolizes ADP released from activated platelets. Recombinant soluble human CD39 (solCD39) potently inhibited ex vivo platelet aggregation in response to ADP and reduced cerebral infarct volumes in mice following transient middle cerebral artery occlusion, even when given 3 hours after stroke. Postischemic platelet and fibrin deposition were decreased and perfusion increased without increasing intracerebral hemorrhage. In contrast, aspirin did not increase postischemic blood flow or reduce infarction volume, but did increase intracerebral hemorrhage. Mice lacking the enzymatically active extracellular portion of the CD39 molecule were generated by replacement of exons 4-6 (apyrase-conserved regions 2-4) with a PGKneo cassette. Although CD39 mRNA 3′ of the neomycin cassette insertion site was detected, brains from these mice lacked both apyrase activity and CD39 immunoreactivity. Although their baseline phenotype, hematological profiles, and bleeding times were normal, cd39 -/-mice exhibited increased cerebral infarct volumes and reduced postischemic perfusion. solCD39 reconstituted these mice, restoring postischemic cerebral perfusion and rescuing them from cerebral injury. These data demonstrate that CD39 exerts a protective thromboregulatory function in stroke.J. Clin. Invest. 109:1031-1040 (2002. DOI:10.1172/JCI200210649. maintain blood fluidity. These include local generation of nitric oxide, release of eicosanoids, and ectoapyrase activity. CD39, a transmembrane protein originally identified on lymphoid cells, whose extracellular portion exhibits apyrase activity (9), is a highly conserved, constitutively expressed enzyme that strongly inhibits platelet aggregation (10,11). Following transfection of CD39 into COS cells, the cells acquire the ability to inhibit ADP-induced platelet aggregation, establishing CD39 as a prime thromboregulator (10,12). Recently, a recombinant, soluble form of human CD39 (including a secretion leader but lacking transmembrane domains) was isolated from stably transfected CHO cells (11). This soluble CD39 (solCD39) preparation blocked aggregation induced by ADP and several other agonists in vitro, and circulated in mice with a half-life of approximately 2 days (11).The present studies test the hypothesis that augmentation of endogenous CD39 would inhibit ADP-mediated autoamplification of platelet recruitment in distal microvessels and thereby reduce thrombosis following stroke. Since solCD39 does not interfere with primary GPIb-mediated platelet adhesion at the site of vessel damage, solCD39 administration should not, in theory, prevent a layer of platelets from forming at the site of injury or interfere with hemostatic mechanisms that prevent intracerebral hemorrhage. Our studies examine the thromboregulatory role of endogenous CD39 in stroke and the ability of solCD39 to inhibit microvascular thrombosis and confer cerebroprotection in stroke without inducing intracerebral hemorrhage. MethodsMurine platelet aggregation. C57BL/6 mice (6-8 weeks old) were obtained...
Blood platelets represent the first line of host defense when normal vessels are injured. Platelet adhesion to subendothelium, aggregation, and further platelet recruitment culminate in hemostatic plug formation, which is accompanied by the consolidating effect of fibrin deposition on and between platelets. The process is multicellular in that erythrocytes promote and neutrophils inhibit platelet plug formation. Endothelial cells in proximity possess three protective mechanisms (thrombo-regulators) for limiting the size of the hemostatic plug-ADPase, eicosanoids, endothelium-dependent relaxing factor/NO. We propose that in advanced atherosclerotic blood vessels such as coronary arteries, an ulcer or fissure in the fibrous cap of the atheroma serves as an agonist that transforms the platelet into a major prothrombotic offender. Induction of excessive platelet activation overcomes the normal thromboregulatory mechanisms. Erythrocytes further activate platelets, even in the presence of aspirin, and neutrophil blockage of platelet reactivity is insufficient to prevent impending vascular occlusion. Appreciating that multiple cell types and metabolic pathways are involved in modulation of platelet reactivity in vascular occlusion is a relatively recent concept. Strategies designed to restore processes such as thromboregulation may serve to improve therapeusis in thrombosis, which at present is far from optimal.
Thrombopoietic cells may differentially promote or inhibit tissue vascularization by releasing both pro-and antiangiogenic factors. However, the molecular determinants controlling the angiogenic phenotype of thrombopoietic cells remain unknown. Here, we show that expression and release of thrombospondins (TSPs) by megakaryocytes and platelets function as a major antiangiogenic switch. TSPs inhibited thrombopoiesis, diminished bone marrow microvascular reconstruction following myelosuppression, and limited the extent of revascularization in a model of hind limb ischemia. We demonstrate that thrombopoietic recovery following myelosuppression was significantly enhanced in mice deficient in both TSP1 and TSP2 (TSP-DKO mice) in comparison with WT mice. Megakaryocyte and platelet levels in TSP-DKO mice were rapidly restored, thereby accelerating revascularization of myelosuppressed bone marrow and ischemic hind limbs. In addition, thrombopoietic cells derived from TSP-DKO mice were more effective in supporting neoangiogenesis in Matrigel plugs. The proangiogenic activity of TSP-DKO thrombopoietic cells was mediated through activation of MMP-9 and enhanced release of stromal cell-derived factor 1. Thus, TSP-deficient thrombopoietic cells function as proangiogenic agents, accelerating hemangiogenesis within the marrow and revascularization of ischemic hind limbs. As such, interference with the release of cellular stores of TSPs may be clinically effective in augmenting neoangiogenesis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.