Differential catalytic properties and vascular topography of murine nucleoside triphosphate diphosphohydrolase 1 (NTPDase1) and NTPDase2 have implications for thromboregulation

Abstract Nucleoside triphosphate diphosphohydrolases (NTPDases) are a recently described family of ectonucleotidases that differentially hydrolyze the γ and β phosphate residues of extracellular nucleotides. Expression of this enzymatic activity has the potential to influence nucleotide P2 receptor signaling within the vasculature. We and others have documented that NTPDase1 (CD39, 78 kd) hydrolyzes both triphosphonucleosides and diphosphonucleosides and thereby terminates platelet aggregation responses to adenosine diphosphate (ADP). In contrast, we now show that NTPDase2 (CD39L1, 75 kd), a preferential nucleoside triphosphatase, activates platelet aggregation by converting adenosine triphosphate (ATP) to ADP, the specific agonist of P2Y1 and P2Y12 receptors. We developed specific antibodies to murine NTPDase1 and NTPDase2 and observed that both enzymes are present in the cardiac vasculature; NTPDase1 is expressed by endothelium, endocardium, and to a lesser extent by vascular smooth muscle, while NTPDase2 is associated with the adventitia of muscularized vessels, microvascular pericytes, and other cell populations in the subendocardial space. Moreover, NTPDase2 represents a novel marker for microvascular pericytes. Differential expression of NTPDases in the vasculature suggests spatial regulation of nucleotide-mediated signaling. In this context, NTPDase1 should abrogate platelet aggregation and recruitment in intact vessels by the conversion of ADP to adenosine monophosphate, while NTPDase2 expression would promote platelet microthrombus formation at sites of extravasation following vessel injury. Our data suggest that specific NTPDases, in tandem with ecto-5′-nucleotidase, not only terminate P2 receptor activation and trigger adenosine receptors but may also allow preferential activation of specific subsets of P2 receptors sensitive to ADP (eg, P2Y1, P2Y3, P2Y12) and uridine diphosphate (P2Y6).

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Role of Ca2+ in Mediating Plant Responses to Extracellular ATP and ADP

International Journal of Molecular Sciences ◽

10.3390/ijms19113590 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3590 ◽

Cited By ~ 8

Author(s):

Greg Clark ◽

Stanley Roux

Keyword(s):

Cytosolic Calcium ◽

Defense Responses ◽

Receptor Activation ◽

Extracellular Atp ◽

Extracellular Nucleotides ◽

Nucleoside Triphosphate ◽

Plant Responses ◽

Downstream Signaling ◽

Nucleoside Triphosphate Diphosphohydrolase

Among the most recently discovered chemical regulators of plant growth and development are extracellular nucleotides, especially extracellular ATP (eATP) and extracellular ADP (eADP). Plant cells release ATP into their extracellular matrix under a variety of different circumstances, and this eATP can then function as an agonist that binds to a specific receptor and induces signaling changes, the earliest of which is an increase in the concentration of cytosolic calcium ([Ca2+]cyt). This initial change is then amplified into downstream-signaling changes that include increased levels of reactive oxygen species and nitric oxide, which ultimately lead to major changes in the growth rate, defense responses, and leaf stomatal apertures of plants. This review presents and discusses the evidence that links receptor activation to increased [Ca2+]cyt and, ultimately, to growth and diverse adaptive changes in plant development. It also discusses the evidence that increased [Ca2+]cyt also enhances the activity of apyrase (nucleoside triphosphate diphosphohydrolase) enzymes that function in multiple subcellular locales to hydrolyze ATP and ADP, and thus limit or terminate the effects of these potent regulators.

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NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female

Biomolecules ◽

10.3390/biom11020147 ◽

2021 ◽

Vol 11 (2) ◽

pp. 147

Author(s):

Romuald Brice Babou Kammoe ◽

Gilles Kauffenstein ◽

Julie Pelletier ◽

Bernard Robaye ◽

Jean Sévigny

Keyword(s):

Smooth Muscle ◽

Ex Vivo ◽

Smooth Muscle Contraction ◽

Receptor Activation ◽

Nucleoside Triphosphate ◽

Nucleotide Receptors ◽

Nerve Terminals ◽

Wild Type ◽

Nucleoside Triphosphate Diphosphohydrolase ◽

Bladder Contraction

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5′-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1−/−) mice compared to wild type controls. The strongest responses were obtained with uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6−/− bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1−/−, and P2ry6−/− female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

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Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies

Blood ◽

10.1182/blood.v76.10.2017.2017 ◽

1990 ◽

Vol 76 (10) ◽

pp. 2017-2023 ◽

Cited By ~ 111

Author(s):

MH Ginsberg ◽

AL Frelinger ◽

SC Lam ◽

J Forsyth ◽

R McMillan ◽

...

Keyword(s):

Platelet Aggregation ◽

Ligand Binding ◽

Cell Activation ◽

Flow Cytometric Analysis ◽

Adenosine Diphosphate ◽

Receptor Activation ◽

Affinity Column ◽

Primary Defect ◽

Fibrinogen Receptor ◽

Fibrinogen Binding

Abstract Normal primary platelet aggregation requires agonist-mediated activation of membrane GPIIb-IIIa, binding of fibrinogen to GPIIb-IIIa, and cellular events after ligand binding. PAC1 monoclonal antibody distinguishes between resting and activated states of GPIIb-IIIa, and other antibodies preferentially recognize GPIIb (PMI-1) or IIIa (anti- LIBS1) after the binding of fibrinogen or fibrinogen-mimetic peptides, such as GRGDSP. Using these antibodies and platelet flow cytometry, we studied two distinct persistent platelet aggregation abnormalities. Platelets from a thrombasthenic variant, which contained near-normal amounts of GPIIb-IIIa, failed to aggregate or bind PAC1 in response to agonists. In addition, GRGDSP, which binds to normal GPIIb-IIIa without prior cell activation, failed to increase the binding of PMI-1 or anti- LIBS1 to the thrombasthenic platelets, suggesting a primary defect in ligand binding. Chromatography of detergent-solubilized platelets on a KYGRGDS affinity column confirmed that the patient's GPIIb-IIIa lacked the fibrinogen binding site. In another patient with myelofibrosis and defective aggregation, PAC1 failed to bind to adenosine diphosphate- stimulated platelets, but did bind when protein kinase C was directly activated with phorbol myristate acetate. Furthermore, the binding of PMI-1 and anti-LIBS1 increased in response to GRGDSP, confirming a defect in agonist-mediated fibrinogen receptor activation rather than in fibrinogen binding or events distal to binding. These studies indicate that this immunochemical approach is useful in classification of clinical abnormalities of platelet aggregation as defects in either (a) fibrinogen receptor activation, (b) fibrinogen binding, or (c) postoccupancy events.

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Zinc-induced platelet aggregation is mediated by the fibrinogen receptor and is not accompanied by release or by thromboxane synthesis

Blood ◽

10.1182/blood.v66.1.213.213 ◽

1985 ◽

Vol 66 (1) ◽

pp. 213-219 ◽

Cited By ~ 29

Author(s):

P Heyns A du ◽

A Eldor ◽

R Yarom ◽

G Marx

Keyword(s):

Platelet Aggregation ◽

Dense Body ◽

Platelet Rich Plasma ◽

Adenosine Monophosphate ◽

Adenosine Diphosphate ◽

Creatine Phosphate ◽

Calcium Flux ◽

Fibrinogen Receptor ◽

Induced Aggregation

Abstract We demonstrate that zinc (0.1 to 0.3 mmol/L) induces aggregation of washed platelet suspensions. Higher concentrations (1 to 3 mmol/L) of zinc were needed to aggregate platelets in platelet-rich plasma obtained from blood anticoagulated with low-molecular-weight heparin, probably due to the binding of zinc to the plasma proteins. Zinc- induced aggregation of normal washed platelets required added fibrinogen and no aggregation occurred with thrombasthenic platelets or with normal platelets pretreated with a monoclonal antibody (10E5) that blocks the platelet fibrinogen receptor. These data indicate that the platelet membrane fibrinogen receptor-glycoproteins IIb and IIIa mediate the effect of zinc. Zinc-induced aggregation was blocked by the agent TMB-8, which interferes with the internal calcium flux, and by prostacyclin, which elevates platelet cyclic adenosine monophosphate levels. Zinc-induced aggregation was not accompanied by thromboxane synthesis or by the secretion of dense-body serotonin and was not affected by preexposure of platelets to acetylsalicylic acid. Experiments with creatine phosphate/creatine phosphokinase showed that the zinc effect on platelets was independent of extracellular adenosine diphosphate (ADP). Zinc had an additive effect when platelet aggregation was stimulated with subthreshhold concentrations of collagen or ADP. Together with the known effects of nutritional zinc on in vivo bleeding, on platelet aggregation, and on lipid metabolism, the results suggest that zinc may have an important bearing on normal hemostasis, thrombosis, and atherosclerosis.

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Ticlopidine in Its Prodrug Form Is a Selective Inhibitor of Human NTPDase1

Mediators of Inflammation ◽

10.1155/2014/547480 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Joanna Lecka ◽

Michel Fausther ◽

Beat Künzli ◽

Jean Sévigny

Keyword(s):

Enzyme Histochemistry ◽

Physiological Responses ◽

Specific Inhibitor ◽

Selective Inhibitor ◽

Nucleoside Triphosphate ◽

P2 Receptor ◽

Extracellular Nucleotide ◽

Nucleoside Triphosphate Diphosphohydrolase ◽

Liver And Pancreas

Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), like other ectonucleotidases, controls extracellular nucleotide levels and consequently their (patho)physiological responses such as in thrombosis, inflammation, and cancer. Selective NTPDase1 inhibitors would therefore be very useful. We previously observed that ticlopidine in its prodrug form, which does not affect P2 receptor activity, inhibited the recombinant form of human NTPDase1 (Ki=14 μM). Here we tested whether ticlopidine can be used as a selective inhibitor of NTPDase1. We confirmed that ticlopidine inhibits NTPDase1 in different forms and in different assays. The ADPase activity of intact HUVEC as well as of COS-7 cells transfected with human NTPDase1 was strongly inhibited by 100 µM ticlopidine, 99 and 86%, respectively. Ticlopidine (100 µM) completely inhibited the ATPase activity of NTPDase1in situas shown by enzyme histochemistry with human liver and pancreas sections. Ticlopidine also inhibited the activity of rat and mouse NTPDase1 and of potato apyrase. At 100 µM ticlopidine did not affect the activity of human NTPDase2, NTPDase3, and NTPDase8, nor of NPP1 and NPP3. Weak inhibition (10–20%) of NTPDase3 and -8 was observed at 1 mM ticlopidine. These results show that ticlopidine is a specific inhibitor of NTPDase1 that can be used in enzymatic and histochemistry assays.

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Differential requirements for platelet aggregation and inhibition of adenylate cyclase by epinephrine. Studies of a familial platelet alpha 2-adrenergic receptor defect

Blood ◽

10.1182/blood.v71.2.494.494 ◽

1988 ◽

Vol 71 (2) ◽

pp. 494-501 ◽

Cited By ~ 42

Author(s):

AK Rao ◽

J Willis ◽

MA Kowalska ◽

YT Wachtfogel ◽

RW Colman

Keyword(s):

Platelet Aggregation ◽

Adenylate Cyclase ◽

Adrenergic Receptors ◽

Adrenergic Receptor ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Adenosine Diphosphate ◽

Normal Subjects ◽

Camp Levels ◽

Epinephrine Concentration

Abstract We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.

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Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling Events

Thrombosis and Haemostasis ◽

10.1055/s-0039-1688788 ◽

2019 ◽

Vol 119 (07) ◽

pp. 1124-1137 ◽

Cited By ~ 3

Author(s):

Joanne C. Clark ◽

Deirdre M. Kavanagh ◽

Stephanie Watson ◽

Jeremy A. Pike ◽

Robert K. Andrews ◽

...

Keyword(s):

Adenylyl Cyclase ◽

G Protein ◽

Platelet Activation ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Adenosine Diphosphate ◽

Receptor Activation ◽

Inhibitory Effect ◽

Human Platelets ◽

Adenosine Receptor Agonist

Background The G protein-coupled receptor, adenosine A2A, signals through the stimulatory G protein, Gs, in platelets leading to activation of adenylyl cyclase and elevation of cyclic adenosine monophosphate (cAMP) and inhibition of platelet activation. Objective This article investigates the effect of A2A receptor activation on signalling by the collagen receptor glycoprotein (GP) VI in platelets. Methods Washed human platelets were stimulated by collagen or the GPVI-specific agonist collagen-related peptide (CRP) in the presence of the adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed for aggregation, adenosine triphosphate secretion, protein phosphorylation, spreading, Ca2+ mobilisation, GPVI receptor clustering, cAMP, thromboxane B2 (TxB2) and P-selectin exposure. Results NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion to collagen or CRP in the absence or presence of the P2Y12 receptor antagonist, cangrelor and the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA nor forskolin altered clustering of GPVI, elevation of Ca2+ or spreading of platelets on a collagen surface. Further, neither NECA nor forskolin, altered collagen-induced tyrosine phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet activation by the TxA2 mimetic, U46619, but not the combination of adenosine diphosphate and collagen. Conclusion NECA and forskolin have no effect on the proximal signalling events by collagen. They inhibit platelet activation in a response-specific manner in part through inhibition of the feedback action of TxA2.

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Osteoclast ATP receptor activation leads to a transient decrease in intracellular pH

Journal of Cell Science ◽

10.1242/jcs.108.9.3051 ◽

1995 ◽

Vol 108 (9) ◽

pp. 3051-3058

Author(s):

H. Yu ◽

J. Ferrier

Keyword(s):

Intracellular Ph ◽

Proton Pump Inhibitors ◽

Proton Pump ◽

Laser Scanning ◽

Purinergic Receptor ◽

Adenosine Monophosphate ◽

Adenosine Diphosphate ◽

Receptor Activation ◽

Extracellular Medium ◽

Bathing Medium

Application of extracellular adenosine triphosphate (ATP) induces a pulsed decrease in osteoclast intracellular pH (pHi), as measured with seminaphthofluorescein (SNAFL)-calcein on a laser scanning confocal microscope. Adenosine diphosphate also produces a pHi decrease, but adenosine monophosphate, uridine triphosphate, 2-methylthio-ATP, and beta, gamma-methylene-ATP have little effect on pHi. The ATP-induced pHi decrease is largely inhibited by suramin, a P2 purinergic receptor blocker. Clamping intracellular free [Ca2+] ([Ca2+]i) with BAPTA/AM does not affect the ATP-induced pHi change, showing that this pHi decrease is not caused by the increased intracellular [Ca2+]i that is produced by activation of osteoclast purinergic receptors. We show that an increase in [Ca2+]i by itself will produce a pHi increase. The ATP effect is not blocked by inhibition of Na+/H+ exchange by either Na(+)-free bathing medium or amiloride. Two inhibitors of the osteoclast cell membrane proton pump, N-ethylmaleimide and vanadate, produce partial inhibition of the ATP-induced pHi decrease. Two other proton pump inhibitors, bafilomycin and N,N'-dicyclohexylcarbodiimide, have no influence on the ATP effect. None of the proton pump inhibitors but vanadate has a direct effect on pHi. Vanadate produces a transient pHi increase upon application to the bathing medium, possibly as a result of its known effect of stimulating the Na+/H+ exchanger. Inhibition of Cl-/HCO3- exchange by decreasing extracellular Cl- gives a pronounced long-term pHi increase, supporting the hypothesis that this exchange has an important role in osteoclast pHi homeostasis. In Cl(-)-free extracellular medium, there is a greatly reduced effect of extracellular ATP on pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

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Expression of P2Y nucleotide receptors and ectonucleotidases in quiescent and activated rat hepatic stellate cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00294.2003 ◽

2004 ◽

Vol 287 (2) ◽

pp. G417-G424 ◽

Cited By ~ 59

Author(s):

Jonathan A. Dranoff ◽

Mika Ogawa ◽

Emma A. Kruglov ◽

Marianna D. A. Gaça ◽

Jean Sévigny ◽

...

Keyword(s):

Mrna Expression ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

P2y Receptors ◽

Extracellular Nucleotides ◽

Nucleoside Triphosphate ◽

P2y Receptor ◽

Rt Pcr ◽

Nucleoside Triphosphate Diphosphohydrolase ◽

Receptor Inhibitor

Extracellular nucleotides regulate a variety of cellular activities, including proliferation of fibrogenic cells outside of the liver. However, the expression of receptors for extracellular nucleotides in hepatic stellate cells (HSC) is unknown. Thus our aims were to investigate the expression of mediators of nucleotide signaling in HSC and to determine whether extracellular nucleotides regulate HSC function. Confocal video microscopy was used to observe nucleotide-induced changes in cytosolic Ca2+ (Cai2+) in live HSC. P2Y receptor subtype expression and ectonucleotidase expression in quiescent and activated HSC were determined using RT-PCR, Northern blot, immunoblot, and confocal immunofluorescence. Functional ectonucleotidase activity was assessed using a colorimetric method. Nucleotide-sensitive procollagen-1 mRNA expression in activated HSC was assessed using real-time RT-PCR. Extracellular ATP increased Cai2+ in HSC; this was inhibited by the P2 receptor inhibitor suramin. Quiescent HSC expressed the P2Y subtypes P2Y2 and P2Y4 and were activated by ATP and UTP, whereas activated HSC expressed the P2Y subtype P2Y6 and were activated by UDP and ATP. Activated but not quiescent HSC expressed the ectonucleotidase nucleoside triphosphate diphosphohydrolase 2, extracellular UDP tripled procollagen-1 mRNA expression in activated HSC, and this was inhibited by the P2Y receptor inhibitor suramin. HSC express functional P2Y receptors and switch the expression of P2Y receptor subtypes on activation. Moreover, HSC differentially regulate nucleoside triphosphate diphosphohydrolase expression after activation. Because activation of P2Y receptors in activated HSC regulates procollagen-1 transcription, P2Y receptors may be an attractive target to prevent or treat liver fibrosis.

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PLATELET PHAGOCYTOSIS AND AGGREGATION

The Journal of Cell Biology ◽

10.1083/jcb.27.3.531 ◽

1965 ◽

Vol 27 (3) ◽

pp. 531-543 ◽

Cited By ~ 103

Author(s):

Henry Z. Movat ◽

William J. Weiser ◽

Michael F. Glynn ◽

James F. Mustard

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Chelating Agent ◽

Adenosine Monophosphate ◽

Adenosine Diphosphate ◽

Inhibit Platelet Aggregation ◽

Platelet Suspension ◽

Latex Particles ◽

Washed Platelet

The addition of latex particles to native (no anticoagulant) or citrated human platelet-rich plasma (PRP), or to a once-washed platelet suspension causes platelet aggregation. This aggregation is associated with phagocytosis of the latex particles by the platelets and appears to be due to release of adenosine diphosphate (ADP) from the platelets. Adenosine and adenosine monophosphate, which are known to inhibit platelet aggregation induced by ADP, also block that induced by latex. These compounds do not prevent the phagocytosis of latex particles by the platelet. The addition of iodoacetate and 2,4-dinitrophenol in appropriate concentrations to the PRP, prior to the addition of the latex, blocks platelet aggregation and phagocytosis. This is also true for the chelating agent ethylenediaminetetraacetate (EDTA). Platelets left in contact with latex for a sufficient period of time show loss of their granules. Leucocytes phagocytose both latex and platelets that had themselves phagocytosed latex. It is concluded that phagocytosis of latex particles by platelets resembles that by white cells, and that in both processes metabolic changes appear to be involved.

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