Mechanisms of Platelet Response to Monosodium Urate Crystals Mark Ginsberg, MD, Peter Henson, PhD, Jan Henson, and Franklin Kozin, MD
The mechanisms of urate-crystal-induced release of platelet constituents has been studied morphologically and biochemically. Urate crystals provoked an earlv energydependent release of the dense-body constitutents serotonin, ADP, and ATP from washed platelets. Concurrently, platelet ultrastructure showed evidence of shape change, contractile wave, and aggregation. These are tvpical morphologic concomitants of platelet secretion. By 30 minutes' incubation, urate-induced platelet Iysis occurred, as shown by loss of the cytoplasmic enzyme lactic dehydrogenase (LDH) and ultrastructurally by disruption of platelet membrane integrity. Cytochalasin B inhibited the urate-crystal-induced shape change, aggregation, and disruption of cell membranes. Platelet degranulation was not inhibited and the initial component of serotonin release was not affected. Cvtochalasin B also abrogated crvstal-induced LDH loss. Thus, the initial crystal-induced serotonin release does not depend on platelet lINsis. It is concluded that urate-crvstal-induced release of serotonin, ATP, and ADP represents an example of platelet secretion. (Am J Pathol 94:549-568, 1979)
MONOSODIUMI URATE CRYSTALS play an important role in the production of gouty inflammation.' Platelets, which are a potent source of phlogistic substances, have been shown to respond to urate crystals. Specifically, these crystals, like other particulates,2" provoke release of platelet constituents in suspensions of washed platelets and in plateletrich plasma. 12 In the case of washed platelets, two phases of release were identified: a) an early secretory phase in which dense body constituents are released and b) a later lytic phase in wshich all cellular constituents are lost from the cells. It was suggested in that study that the first phase of release was a secretory phenomenon because selective release of serotonin without loss of cvtoplasmic enzymes was observed and this phase of release was inhibited by agents which deplete the metabolic pool of ATP in the cells. Energy-dependent secretion which precedes lysis has been described in From the Scripps Clinic and Research Foundation, La Jolla. Califomia: the National Jewish Hospital. Denver, Colorado, and the Medical College of Wisconsin, Milwaukee. Wisconsin. Supported by Grants HL 16411, Al 7007. and AM 18074. This is publication 1564 of the Research Institute of Scripps Clinic. Dr. Ginsberg is the recipient of Clinical Investigator Award ANM00393. Dr. Kozin is the recipient of Clinical Investigator Asward AM0048.3. Accepted for publication Nosember 3, 1978. Address reprint requests to Mlark H. Ginsberg, MD, Department of Immunopathology. Research Instit,ite of Scripps Clinic. 10666 North Torrey Pines Road, La Jolla, CA 920:37. 0002-9440/79/0308-0549$01 .00 549 © American Association of Pathologists
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other cell types13 and has been termed "pseudo cytotoxic" secretion by us. It may be asked whether this is true "secretion" or is an early leakage of certain granule components preceding cell lysis. One purpose of this work was to examine the ultrastructure of platelets undergoing the uratecrystal-induced release reaction to determine whether this response is morphologically similar to the cell's response to other secretion-inducing agents. A second purpose was to determine whether urate-crystal-induced serotonin release required (and was a consequence of) cell lysis, ie, could serotonin secretion be demonstrated in the absence of platelet lysis? In this report, morphologic and biochemical approaches have been used to answer these questions. Materials and Methods Chemicals
The following were obtained from Sigma Chemical Co. (St. Louis): NADH (disodium salt grade II), phenolphthalein glucuronic acid (sodium salt), sodium pyruvate, and once recrystallized bovine serum albumin. Uric acid was from ICN Nutritional Biochemicals Division (International Chemical and Nuclear Corp., Cleveland) and was recrystallized prior to use. Tritiated serotonin was obtained from Amersham-Searle (Arlington Heights, Ill) and was prepared and stored as previously described.12 Cytochalasin B was purchased from Imperial Chemicals Ltd. (Macclesfield, UK) and was dissolved in 1% dimethyl sulfoxide prior to dilution in buffer. Urate Crystals
Recrystallized uric acid was neutralized 14 to form needle-shaped negatively birefringent crystals. All crystals were heated 200 C for 2 hours to remove possible pyrogens and were suspended in 0.001 M phosphate-buffered 0.15 M NaCI, pH 7.4, prior to use. Platelet Preparations
All preparations were made in plastic apparatus. Buffers
The following buffers were employed in platelet preparation: a) Tris-buffered saline (TBS): NaCl, 7 g/liter; Tris, 3.6 g/liter; glucose, 1 g/liter; pH 7.4; b) Tyrode's solution: NaCI, 8 g/liter; KCI, 0.195 g/liter; NaHCO3, 1.02 g/liter; MgC12.6H20, 0.213 g/liter; CaC12 (anhydrous), 0.145 g/liter; glucose, 1 g/liter; pH 7.4; c) Tyrode's without calcium: the same as described in item b but without CaC12 and with pH adjusted to 6.5. Albumin Density Gradient Washed Platelets
Blood from normal donors who had taken no medication in the previous week was drawn through a 19-gauge needle into 1/6 part acid citrate dextrose."1 Platelet-rich plasma (PRP) was isolated by centrifugation at 200g for 15 minutes at room temperature.12 Washed platelets were prepared from this PRP according to Walsh 16 but with use of Tyrode's solution without Ca++, pH 6.5, as buffer in all washes. After the third wash, the platelets were suspended at 2 X 109/ml in Tyrode's solution without Ca++, pH 6.5. Immediately before use they were diluted 1:10 with protein-free Tyrode's solution.
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Tris-Buffered Saline Washed Platelets
In studies in which serotonin release was measured, platelets were prepared with Trisbuffered saline. Platelets prepared in this fashion do not aggregate well in response to ADP 171S" and their use decreases the likelihood of aggregation-induced secretion " contributing to the observed serotonin release. PRP from blood anticoagulated with 7.7 mM EDTA was incubated at 37 C for 30 minutes with 10 ;Ci 'H-serotonin.'2 The PRP was then sedimented at lOOOg at 8 C for 15 minutes, resuspended in TBS, pH 7.4, recentrifuged, and resuspended twice more before being suspended in TBS, pH 7.4. Morphologic Studies
Platelets were prewarmed at 37 C for 10 minutes before addition of urate crystals to a final concentration of 2 mg/ml. Incubations were performed with horizontal shaking on a Dubnoff metabolic incubator and were terminated by addition of 6X volume of prewarmed (37 C) 2% glutaraldehyde in isotonic buffered saline, approximately 500 mOsm/ liter." The mixture was incubated at 37 C for 30 minutes and at 4 C for 1 hour. Washed cell pellets were then postfixed at 4 C in 1% OsO in isotonic buffered saline for 30 minutes, dehydrated in graded alcohol, embedded in Epon, and sectioned with an LKB ultramicrotome (LKB Instruments Inc., Rockville, Md). Grids stained with uranyl acetate and lead citrate were examined in a Hitachi Hu 11E electron microscope. Measurement of Constituent Release
Labeled platelet suspensions (0.8 ml) in polyethylene tubes were preincubated at 37 C for 10 minutes; then 0.2 ml of buffer or crystals was added and the mixture was incubated with continuous shaking at 37 C in a Dubnoff metabolic incubator. At appropriate times, tubes were placed into iced methanol for 2 to 5 minutes and subsequently centrifuged at 12,500g at 0 C for 20 minutes. Supernates were removed for assays and 1 ml of buffer was added to the pellets, which were then sonicated at settings "7", 4 s, four times in a Sonifier (Bronwill Scientific, Rochester, NY: model 1850) cell disrupter. The sonicate was centrifuged at 12,500g for 20 minutes and the platelet extract was taken for assays. Percent release of any component was defined using the following equation: Activity in crystal-treated supernate - Activity in buffer-treated supemate x 100 Activity in buffer-treated pellet Less than 10% of total platelet radioactivity was noted in control supernates. Assays
Radioactivity was assayed by mixing 100 gl of sample with 900 Al of water and adding 10 ml of Bray's solution before counting in a Packard Tri-Carb liquid scintillation counter (Packard Instrument Co., Inc., Downers Grove, Ill). Lactic dehydrogenase (LDH) was assayed by a standard technique, and 0-glucuronidase was assayed by hydrolysis of phenolphthalein glucuronic acid as previously described.One hundred microliters of ethanol-EDTA extracts of supernatants and platelet pellets were prepared for assay of ATP and ADP as recommended by Holmsen et al.2' Light emission after addition of 500 Mg of firefly lantern extract was measured in a Packard TnCarb liquid scintillation counter as suggested by Stanley and Williams.
Results Following addition of urate crystals to washed platelet suspensions there is rapid, active release of serotonin. After 10 minutes of incubation,
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lysosomal /3-glucuronidase and cytoplasmic lactic dehydrogenase loss begins.12 This suggests that platelet lysis begins at this time. To further analyze these phases of platelet-urate-crystal interactions, ultrastructural studies were performed. Morphology of Platelet-Urate-Crystal Interaction
The unstimulated platelets (Figure 1) were predominantly discoid, had undilated to slightly dilated canaliculi, and contained both dense bodies and a-granules. One minute after addition of urate crystals, the cells had changed shape, becoming spherical with multiple pseudopodia (Figures 2 and 3). Strikingly, pseudopodia (Figure 3) appeared to surround crystal artifacts which appeared as electron-lucent areas or as needle-shaped electron-dense structures with lucent cores. In some planes of section, the crystals appeared to lie in membrane-lined compartments within the platelet cytoplasm, but this may merely reflect a tangential section through a partially "surrounded" crystal. Centralization of platelet granules was observed, and in some cells this had progressed to fusion of some granules near the center of the cell (Figure 2). White 23 has termed this the "contractile wave" and demonstrated it in association with the platelet "release reaction." At 5 minutes of incubation, platelet aggregates were observed (Figure 4). Crystal artifacts appeared to lie within these aggregates. In some cases the artifacts were obviously in the open canalicular system. In other cases, perhaps due to the plane of section, the crystals appeared to be within membrane-lined compartments in the platelet cytoplasm. At 30 minutes of incubation, when substantial loss of cytoplasmic enzymes has occurred, there was striking evidence of loss of platelet internal structure and of platelet plasma membrane integrity, suggesting cell death (Figure 5). Within these cells, crystal artifacts often appeared to lie free in the platelet cytoplasm. Additional evidence of cell lysis was observed in platelet aggregates in which membrane fragments and free mitochondria were observed along with intact cells and trapped urate crystals (Figure 6). These morphologic data indicated that the urate crystal may stimulate platelets in the usual sense, ie, produce shape change, aggregation, and "contracile wave" in addition to provoking platelet lysis. To further explore the role of urate-induced platelet lysis in serotonin release, the effects of cytochalasin B,24'25 an agent which inhibits urate crystal-induced neutrophil lysis, on the urate-platelet interaction were studied.
_
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_
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MONOSODIUM URATE CRYSTALS
by Urate Crystals
Effects of Cyhaasin B on Reease of Platet Costue
Biochemical Studies
When platelets were preincubated with cytochalasin B prior to addition of urate crystals, serotonin release at 10 minutes of incubation was unaltered (Text-figure 1). This serotonin release occurs during the initial "'secretory phase" of urate stimulation of platelets. Serotonin release at 30 minutes of incubation, however, was partially inhibited. The amount of inhibition appeared to correspond to the increment in release from 10 to 30 minutes, ie, during the so-called lytic phase. Our explanation of this is that the cytochalasin B inhibited the lytic component of serotonin release. This possibilitv was substantiated by the concurrent abolition of urateinduced LDH loss by cytochalasin B. Thus, cytochalasin B inhibited crvstal-induced platelet lysis but did not inhibit the "secretory phase" of urate-crystal-induced serotonin release. Morphologic Studies
The influence of cytochalasin B on the morphologic aspects of uratecrystal-induced serotonin release was also assessed. Cytochalasin-Btreated unstimulated platelets were somewhat "'fatter" disks than un-
TE.XT-FIGLRE 1-Effect of cvtochalasin B on platelet response to urate crvstals. Cytochalasin B was preincubated w%ith platelets at the indicated final concentration. After 10 minutes, 0.8 mg/ml urate crystals was added, and the percent release was determined after 10 (lower panel) and 30 (upper panel) minutes. Solid circles, serotonin, open circles, ,-glucuronidase; triangles, lactic dehvdrogenase. Error bars indicate ± 1 standard error of triplicate determinations. Where no bars appear, the error is within the point.
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treated cells and showed a dilated open canalicular system (Figure 7). One minute after addition of crystals to cytochalasin-B-treated platelets, crystal artifacts were observed in relation to the platelet plasma membranes. The cells, however, remained discoid and did not show centralization of granules in spite of intact microtubules (Figures 8 and 9). In 15 of 75 untreated platelets examined, crystals were observed in membrane-lined structures apparently within the platelet cytoplasm. In contrast, this was observed in only 3 of 75 cytochalasin-B-treated cells. At 30 minutes of incubation, discrete, intact, discoid platelets in contact with urate crystals were observed. Many of these cells were completely degranulated (Figure 10). Thus, cytochalasin B inhibited urate-crystal-induced shape change, aggregation, and lysis but did not inhibit urate-induced platelet degranulation. Release of ATP and ADP
To determine whether urate crystals induced an active secretion of dense body constituents or merely caused selective exchange of serotonin, the release of two other dense body constituents was assayed. As shown in Text-figure 2, ADP and ATP release occurred concurrently with serotonin release. This indicates that urate crystals provoke release of a number of dense granule constituents, including two for which no reuptake mechanism is known. Discussion
The data provided in this report indicate that urate crystals release platelet constituents by at least two mechanisms, ie, secretion and lysis.
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Urate crystals (0.8 mg/ml) were added to suspensions of washed platelets. Percent release of serotonin (triangles), ADP (open circles), and ATP (solid circles) were determined as described in Materials and Methods. Mean ± SEM of trip-
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MONOSODIUM URATE CRYSTALS
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The evidence that urate crystals induce platelet secretion is as follows: 1. Urate crystals induced a platelet morphologic response typical of the reaction to the other secretion-inducing agents. 2. Urate-induced platelet lysis could be abrogated by cytochalasin B as measured morphologically and biochemically, without abrogation of serotonin release. 3. The initial (secretory) phase of platelet serotonin release occurred without evident loss in platelet membrane integrity as assessed morphologically or biochemically. 4. Other dense body constituents were released by urates concurrently with serotinin. This rules out the possibility that urates act merely to reverse serotonin reuptake. 5. Agents which deplete cellular energy inhibited the early secretory phase of urate-induced serotonin release.12 Urate crystals avidly bind IgG,ft and IgG-coated urate crystals are also potent platelet stimuli. This raises the possibility that IgG in the platelet suspensions may mediate urate-induced secretion. Recent work," has established that this is not the case and has suggested that the urate crystal surface provides a direct trigger for platelet secretion. Thus, urate crystals are polyanions which apparently directly trigger platelet secretion. The mechanism by which they do so is under study. By both morphologic and biochemical criteria, prolonged incubation of urate crystals with platelets leads to cell lysis. We previously noted 12 that metabolic inhibitors decreased urate-induced platelet lysis. The present studies demonstrate that cytochalasin B, an agent which disrupts microfilaments, also abrogates crystal-induced platelet lysis. This suggests a role for cellular contractile elements in this phenomenon. Cytochalasin B is known to inhibit platelet aggregation," shape change," and apparent particle uptake.' These three effects were observed on platelet response to urate crystals. Thus, it is not possible to ascertain which, if any, of these platelet responses is critical for lysis. What is clear is that some active response of the platelet is required for urate-induced lysis. Since cytochalasin B did not inhibit urate-induced platelet secretion, secretion is not the active response which leads to urate-induced platelet lysis. Thus, urate crystals release platelet constituents by at least two mechanisms: 1) By active secretion of dense body constituents. Since this is followed by, but not dependent on, lysis, it appears to be a true case of secretion. 2) Urate crystals also induce platelet lysis. This requires an active platelet response, possibly involving cellular contractile mechanisms. The relative roles of each mechanism of release of platelet constituents by urate crystals in the gouty patient remains to be assessed.
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References 1. Wyngaarden JB: The etiology and pathogenesis of gout. Arthritis and Allied Conditions, Eighth edition. Edited by JL Hollander, DJ McCarty. Philadelphia, Lea & Febiger, 1972, p 1079 2. Glynn MF, Movat HZ, Murphy EA, Mustard JF: Study of platelet adhesiveness and aggregation, with latex particles. J Lab Clin Med 65:179-201, 1965 3. Hardisty RM, Hutton RA: The kaolin clotting time of platelet rich plasma. Br J Haematol 11:258-268, 1965 4. Movat HZ, Weiser WJ, Glynn MF, Mustard JF: Platelet phagocytosis and aggregation. J Cell Biol 27:531-543, 1965 5. Spaet TH, Cintron J: Studies on platelet factor-3 availability. Br J Haematol 11:269-275, 1965 6. Mustard JF, Glynn MF, Nishizawa EE, Packham MA: Platelet-surface interactions: Relationship to thrombosis and hemostasis. Fed Proc 26:106-114, 1967 7. Mueller-Eckhardt C, Lulscher EF: Immune reactions of human blood platelets. II. The effect of latex partieles coated with gamma globulin in relation to complement activation. Thromb Diath Haemorrh 20:168-179, 1968 8. Morris CDW: Observations of the effect of glass beads on platelet aggregation and its relationship to platelet stickiness. Thromb Diath Haemorrh 20:345-353, 1968 9. Jobin F, Lapointe F, Gagnon F: Platelet reactions and immune processes. VI. The effect of immunoglobulins and other plasma proteins on platelet surface interactions. Thromb Diath Haemorrh 25:86-97, 1971 10. Pfueller SL, Lulscher EF: Studies of the mechanisms of the human platelet release reaction induced by immunologic stimuli. II. The effect of Zymosan. J Immunol 112:1211-1218, 1974 11. Zucker MB, Grant RA: Aggregation and release reaction induced in human blood platelets by zymosan. J Immunol 112:1219-1230, 1974 12. Ginsberg MH, Kozin F, O'Malley M, McCarty DJ: Release of platelet constituents by monosodium urate crystals. J Clin Invest 60:999-1007, 1977 13. Henson PM, Ginsberg MH, Morrison DC: Mechanisms of mediator release by inflammatory cells. Membrane Fusion. Edited by G. Poste, GL Nicholson. New York, Elsevier North-Holland, 1978, pp 407-508 14. McCarty DJ, Faires JS: A comparison of the duration of local anti-inflammatory effect of several adrenocorticosteroid esters: A bioassay technique. Curr Ther Res 5:284-290, 1963 15. Aster RH, Jandl JH: Platelet sequestration in man. J Clin Invest 43:843-855, 1964 16. Walsh PN: Albumin density gradient separation and washing of platelets and the study of platelet coagulant activities. Br J Haematol 22:205-217, 1972 17. Lages B, Scrutton MC, Holmsen H: Studies on gel-filtered human platelets: Isolation and characterization in a medium containing no added Ca2+, MG2+, or K+ J Lab Clin Med 85:811-825, 1975 18. Ginsberg MH: Unpublished observations 19. Charo IF, Feinman RD, Detwiler TC: Interrelationships of platelet aggregation and secretion. J Clin Invest 60:866-873, 1977 20. Morel FM, Baker RF, Wayland H: Quantitation of human red blood cell fixation by glutaraldehyde. J Cell Biol 48:91-100, 1971 21. Holmsen H, Storm E, Day HJ: Determination of ATP and ADP in blood platelets: A modification of the firefly luciferase assay for plasma. Anal Biochem 46:489-501, 1972 22. Stanley PE, Williams SG: Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal Biochem 29:381-392, 1969
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23. White JG: Electron microscopic studies of platelet secretion. Prog Hemost Thromb 2:49-98, 1974 24. Hoffstein S, Weissmann G: Mechanisms of lysosomal enzyme release from leukocytes. IV. Interaction of monosodium urate crystals with dogfish and human leukocytes. Arthritis Rheum 18:153-165, 1975 25. Spilberg I, Gallacher A, Mendell B: Studies on crystal-induced chemotactic factor II role of phagocytosis. J Lab Clin Med 85:631-636, 1975 26. Kozin F, McCarty DJ: Protein binding to monosodium urate monohydrate, calcium pyrophosphate dihydrate, and silicon dioxide crystals. I. Physical characteristics. J Lab Clin Med 89:1314-1325, 1977 27. Ginsberg MH, Kozin F: Mechanisms of cellular interaction with monosodium urate crystals: IgG-dependent and IgG-independent platelet stimulation. Arthritis Rheum (In press) 28. Haslam RJ, Davidson MML McClenagan MD: Cytochalasin B, the blood platelet release reaction and cyclic GMP. Nature 253:455-457, 1975 29. White JG, Krumweide M: Influence of cytochalasin B on the shape change induced in platelets in the cold. Blood 41:823-832, 1973 30. White JG: Uptake of latex particles by blood platelets: Phagocytosis or sequestration? Am J Pathol 69:439-458, 1972
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GINSBERG ET AL
[Illustrations follow]
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Figure 1-Albumin density gradient washed platelets. (x 56,000)
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The mechanisms of urate-crystal-induced release of platelet constituents has been studied morphologically and biochemically. Urate crystals provoked an earlv energydependent release of the dense-body constitutents serotonin, ADP, and ATP from washed platelets. Concurrently, platelet ultrastructure showed evidence of shape change, contractile wave, and aggregation. These are tvpical morphologic concomitants of platelet secretion. By 30 minutes' incubation, urate-induced platelet Iysis occurred, as shown by loss of the cytoplasmic enzyme lactic dehydrogenase (LDH) and ultrastructurally by disruption of platelet membrane integrity. Cytochalasin B inhibited the urate-crystal-induced shape change, aggregation, and disruption of cell membranes. Platelet degranulation was not inhibited and the initial component of serotonin release was not affected. Cvtochalasin B also abrogated crvstal-induced LDH loss. Thus, the initial crystal-induced serotonin release does not depend on platelet lINsis. It is concluded that urate-crvstal-induced release of serotonin, ATP, and ADP represents an example of platelet secretion. (Am J Pathol 94:549-568, 1979)
MONOSODIUMI URATE CRYSTALS play an important role in the production of gouty inflammation.' Platelets, which are a potent source of phlogistic substances, have been shown to respond to urate crystals. Specifically, these crystals, like other particulates,2" provoke release of platelet constituents in suspensions of washed platelets and in plateletrich plasma. 12 In the case of washed platelets, two phases of release were identified: a) an early secretory phase in which dense body constituents are released and b) a later lytic phase in wshich all cellular constituents are lost from the cells. It was suggested in that study that the first phase of release was a secretory phenomenon because selective release of serotonin without loss of cvtoplasmic enzymes was observed and this phase of release was inhibited by agents which deplete the metabolic pool of ATP in the cells. Energy-dependent secretion which precedes lysis has been described in From the Scripps Clinic and Research Foundation, La Jolla. Califomia: the National Jewish Hospital. Denver, Colorado, and the Medical College of Wisconsin, Milwaukee. Wisconsin. Supported by Grants HL 16411, Al 7007. and AM 18074. This is publication 1564 of the Research Institute of Scripps Clinic. Dr. Ginsberg is the recipient of Clinical Investigator Award ANM00393. Dr. Kozin is the recipient of Clinical Investigator Asward AM0048.3. Accepted for publication Nosember 3, 1978. Address reprint requests to Mlark H. Ginsberg, MD, Department of Immunopathology. Research Instit,ite of Scripps Clinic. 10666 North Torrey Pines Road, La Jolla, CA 920:37. 0002-9440/79/0308-0549$01 .00 549 © American Association of Pathologists
550
GINSBERG ET AL
American Journal of Pathology
other cell types13 and has been termed "pseudo cytotoxic" secretion by us. It may be asked whether this is true "secretion" or is an early leakage of certain granule components preceding cell lysis. One purpose of this work was to examine the ultrastructure of platelets undergoing the uratecrystal-induced release reaction to determine whether this response is morphologically similar to the cell's response to other secretion-inducing agents. A second purpose was to determine whether urate-crystal-induced serotonin release required (and was a consequence of) cell lysis, ie, could serotonin secretion be demonstrated in the absence of platelet lysis? In this report, morphologic and biochemical approaches have been used to answer these questions. Materials and Methods Chemicals
The following were obtained from Sigma Chemical Co. (St. Louis): NADH (disodium salt grade II), phenolphthalein glucuronic acid (sodium salt), sodium pyruvate, and once recrystallized bovine serum albumin. Uric acid was from ICN Nutritional Biochemicals Division (International Chemical and Nuclear Corp., Cleveland) and was recrystallized prior to use. Tritiated serotonin was obtained from Amersham-Searle (Arlington Heights, Ill) and was prepared and stored as previously described.12 Cytochalasin B was purchased from Imperial Chemicals Ltd. (Macclesfield, UK) and was dissolved in 1% dimethyl sulfoxide prior to dilution in buffer. Urate Crystals
Recrystallized uric acid was neutralized 14 to form needle-shaped negatively birefringent crystals. All crystals were heated 200 C for 2 hours to remove possible pyrogens and were suspended in 0.001 M phosphate-buffered 0.15 M NaCI, pH 7.4, prior to use. Platelet Preparations
All preparations were made in plastic apparatus. Buffers
The following buffers were employed in platelet preparation: a) Tris-buffered saline (TBS): NaCl, 7 g/liter; Tris, 3.6 g/liter; glucose, 1 g/liter; pH 7.4; b) Tyrode's solution: NaCI, 8 g/liter; KCI, 0.195 g/liter; NaHCO3, 1.02 g/liter; MgC12.6H20, 0.213 g/liter; CaC12 (anhydrous), 0.145 g/liter; glucose, 1 g/liter; pH 7.4; c) Tyrode's without calcium: the same as described in item b but without CaC12 and with pH adjusted to 6.5. Albumin Density Gradient Washed Platelets
Blood from normal donors who had taken no medication in the previous week was drawn through a 19-gauge needle into 1/6 part acid citrate dextrose."1 Platelet-rich plasma (PRP) was isolated by centrifugation at 200g for 15 minutes at room temperature.12 Washed platelets were prepared from this PRP according to Walsh 16 but with use of Tyrode's solution without Ca++, pH 6.5, as buffer in all washes. After the third wash, the platelets were suspended at 2 X 109/ml in Tyrode's solution without Ca++, pH 6.5. Immediately before use they were diluted 1:10 with protein-free Tyrode's solution.
Vol. 94, No. 3 March 1979
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Tris-Buffered Saline Washed Platelets
In studies in which serotonin release was measured, platelets were prepared with Trisbuffered saline. Platelets prepared in this fashion do not aggregate well in response to ADP 171S" and their use decreases the likelihood of aggregation-induced secretion " contributing to the observed serotonin release. PRP from blood anticoagulated with 7.7 mM EDTA was incubated at 37 C for 30 minutes with 10 ;Ci 'H-serotonin.'2 The PRP was then sedimented at lOOOg at 8 C for 15 minutes, resuspended in TBS, pH 7.4, recentrifuged, and resuspended twice more before being suspended in TBS, pH 7.4. Morphologic Studies
Platelets were prewarmed at 37 C for 10 minutes before addition of urate crystals to a final concentration of 2 mg/ml. Incubations were performed with horizontal shaking on a Dubnoff metabolic incubator and were terminated by addition of 6X volume of prewarmed (37 C) 2% glutaraldehyde in isotonic buffered saline, approximately 500 mOsm/ liter." The mixture was incubated at 37 C for 30 minutes and at 4 C for 1 hour. Washed cell pellets were then postfixed at 4 C in 1% OsO in isotonic buffered saline for 30 minutes, dehydrated in graded alcohol, embedded in Epon, and sectioned with an LKB ultramicrotome (LKB Instruments Inc., Rockville, Md). Grids stained with uranyl acetate and lead citrate were examined in a Hitachi Hu 11E electron microscope. Measurement of Constituent Release
Labeled platelet suspensions (0.8 ml) in polyethylene tubes were preincubated at 37 C for 10 minutes; then 0.2 ml of buffer or crystals was added and the mixture was incubated with continuous shaking at 37 C in a Dubnoff metabolic incubator. At appropriate times, tubes were placed into iced methanol for 2 to 5 minutes and subsequently centrifuged at 12,500g at 0 C for 20 minutes. Supernates were removed for assays and 1 ml of buffer was added to the pellets, which were then sonicated at settings "7", 4 s, four times in a Sonifier (Bronwill Scientific, Rochester, NY: model 1850) cell disrupter. The sonicate was centrifuged at 12,500g for 20 minutes and the platelet extract was taken for assays. Percent release of any component was defined using the following equation: Activity in crystal-treated supernate - Activity in buffer-treated supemate x 100 Activity in buffer-treated pellet Less than 10% of total platelet radioactivity was noted in control supernates. Assays
Radioactivity was assayed by mixing 100 gl of sample with 900 Al of water and adding 10 ml of Bray's solution before counting in a Packard Tri-Carb liquid scintillation counter (Packard Instrument Co., Inc., Downers Grove, Ill). Lactic dehydrogenase (LDH) was assayed by a standard technique, and 0-glucuronidase was assayed by hydrolysis of phenolphthalein glucuronic acid as previously described.One hundred microliters of ethanol-EDTA extracts of supernatants and platelet pellets were prepared for assay of ATP and ADP as recommended by Holmsen et al.2' Light emission after addition of 500 Mg of firefly lantern extract was measured in a Packard TnCarb liquid scintillation counter as suggested by Stanley and Williams.
Results Following addition of urate crystals to washed platelet suspensions there is rapid, active release of serotonin. After 10 minutes of incubation,
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lysosomal /3-glucuronidase and cytoplasmic lactic dehydrogenase loss begins.12 This suggests that platelet lysis begins at this time. To further analyze these phases of platelet-urate-crystal interactions, ultrastructural studies were performed. Morphology of Platelet-Urate-Crystal Interaction
The unstimulated platelets (Figure 1) were predominantly discoid, had undilated to slightly dilated canaliculi, and contained both dense bodies and a-granules. One minute after addition of urate crystals, the cells had changed shape, becoming spherical with multiple pseudopodia (Figures 2 and 3). Strikingly, pseudopodia (Figure 3) appeared to surround crystal artifacts which appeared as electron-lucent areas or as needle-shaped electron-dense structures with lucent cores. In some planes of section, the crystals appeared to lie in membrane-lined compartments within the platelet cytoplasm, but this may merely reflect a tangential section through a partially "surrounded" crystal. Centralization of platelet granules was observed, and in some cells this had progressed to fusion of some granules near the center of the cell (Figure 2). White 23 has termed this the "contractile wave" and demonstrated it in association with the platelet "release reaction." At 5 minutes of incubation, platelet aggregates were observed (Figure 4). Crystal artifacts appeared to lie within these aggregates. In some cases the artifacts were obviously in the open canalicular system. In other cases, perhaps due to the plane of section, the crystals appeared to be within membrane-lined compartments in the platelet cytoplasm. At 30 minutes of incubation, when substantial loss of cytoplasmic enzymes has occurred, there was striking evidence of loss of platelet internal structure and of platelet plasma membrane integrity, suggesting cell death (Figure 5). Within these cells, crystal artifacts often appeared to lie free in the platelet cytoplasm. Additional evidence of cell lysis was observed in platelet aggregates in which membrane fragments and free mitochondria were observed along with intact cells and trapped urate crystals (Figure 6). These morphologic data indicated that the urate crystal may stimulate platelets in the usual sense, ie, produce shape change, aggregation, and "contracile wave" in addition to provoking platelet lysis. To further explore the role of urate-induced platelet lysis in serotonin release, the effects of cytochalasin B,24'25 an agent which inhibits urate crystal-induced neutrophil lysis, on the urate-platelet interaction were studied.
_
Vol. 94, No. 3 March 1979
_
553
MONOSODIUM URATE CRYSTALS
by Urate Crystals
Effects of Cyhaasin B on Reease of Platet Costue
Biochemical Studies
When platelets were preincubated with cytochalasin B prior to addition of urate crystals, serotonin release at 10 minutes of incubation was unaltered (Text-figure 1). This serotonin release occurs during the initial "'secretory phase" of urate stimulation of platelets. Serotonin release at 30 minutes of incubation, however, was partially inhibited. The amount of inhibition appeared to correspond to the increment in release from 10 to 30 minutes, ie, during the so-called lytic phase. Our explanation of this is that the cytochalasin B inhibited the lytic component of serotonin release. This possibilitv was substantiated by the concurrent abolition of urateinduced LDH loss by cytochalasin B. Thus, cytochalasin B inhibited crvstal-induced platelet lysis but did not inhibit the "secretory phase" of urate-crystal-induced serotonin release. Morphologic Studies
The influence of cytochalasin B on the morphologic aspects of uratecrystal-induced serotonin release was also assessed. Cytochalasin-Btreated unstimulated platelets were somewhat "'fatter" disks than un-
TE.XT-FIGLRE 1-Effect of cvtochalasin B on platelet response to urate crvstals. Cytochalasin B was preincubated w%ith platelets at the indicated final concentration. After 10 minutes, 0.8 mg/ml urate crystals was added, and the percent release was determined after 10 (lower panel) and 30 (upper panel) minutes. Solid circles, serotonin, open circles, ,-glucuronidase; triangles, lactic dehvdrogenase. Error bars indicate ± 1 standard error of triplicate determinations. Where no bars appear, the error is within the point.
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treated cells and showed a dilated open canalicular system (Figure 7). One minute after addition of crystals to cytochalasin-B-treated platelets, crystal artifacts were observed in relation to the platelet plasma membranes. The cells, however, remained discoid and did not show centralization of granules in spite of intact microtubules (Figures 8 and 9). In 15 of 75 untreated platelets examined, crystals were observed in membrane-lined structures apparently within the platelet cytoplasm. In contrast, this was observed in only 3 of 75 cytochalasin-B-treated cells. At 30 minutes of incubation, discrete, intact, discoid platelets in contact with urate crystals were observed. Many of these cells were completely degranulated (Figure 10). Thus, cytochalasin B inhibited urate-crystal-induced shape change, aggregation, and lysis but did not inhibit urate-induced platelet degranulation. Release of ATP and ADP
To determine whether urate crystals induced an active secretion of dense body constituents or merely caused selective exchange of serotonin, the release of two other dense body constituents was assayed. As shown in Text-figure 2, ADP and ATP release occurred concurrently with serotonin release. This indicates that urate crystals provoke release of a number of dense granule constituents, including two for which no reuptake mechanism is known. Discussion
The data provided in this report indicate that urate crystals release platelet constituents by at least two mechanisms, ie, secretion and lysis.
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TEXT-FIGURE 2-Kinetics
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Vol. 94, No. 3 March 1979
MONOSODIUM URATE CRYSTALS
555
The evidence that urate crystals induce platelet secretion is as follows: 1. Urate crystals induced a platelet morphologic response typical of the reaction to the other secretion-inducing agents. 2. Urate-induced platelet lysis could be abrogated by cytochalasin B as measured morphologically and biochemically, without abrogation of serotonin release. 3. The initial (secretory) phase of platelet serotonin release occurred without evident loss in platelet membrane integrity as assessed morphologically or biochemically. 4. Other dense body constituents were released by urates concurrently with serotinin. This rules out the possibility that urates act merely to reverse serotonin reuptake. 5. Agents which deplete cellular energy inhibited the early secretory phase of urate-induced serotonin release.12 Urate crystals avidly bind IgG,ft and IgG-coated urate crystals are also potent platelet stimuli. This raises the possibility that IgG in the platelet suspensions may mediate urate-induced secretion. Recent work," has established that this is not the case and has suggested that the urate crystal surface provides a direct trigger for platelet secretion. Thus, urate crystals are polyanions which apparently directly trigger platelet secretion. The mechanism by which they do so is under study. By both morphologic and biochemical criteria, prolonged incubation of urate crystals with platelets leads to cell lysis. We previously noted 12 that metabolic inhibitors decreased urate-induced platelet lysis. The present studies demonstrate that cytochalasin B, an agent which disrupts microfilaments, also abrogates crystal-induced platelet lysis. This suggests a role for cellular contractile elements in this phenomenon. Cytochalasin B is known to inhibit platelet aggregation," shape change," and apparent particle uptake.' These three effects were observed on platelet response to urate crystals. Thus, it is not possible to ascertain which, if any, of these platelet responses is critical for lysis. What is clear is that some active response of the platelet is required for urate-induced lysis. Since cytochalasin B did not inhibit urate-induced platelet secretion, secretion is not the active response which leads to urate-induced platelet lysis. Thus, urate crystals release platelet constituents by at least two mechanisms: 1) By active secretion of dense body constituents. Since this is followed by, but not dependent on, lysis, it appears to be a true case of secretion. 2) Urate crystals also induce platelet lysis. This requires an active platelet response, possibly involving cellular contractile mechanisms. The relative roles of each mechanism of release of platelet constituents by urate crystals in the gouty patient remains to be assessed.
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References 1. Wyngaarden JB: The etiology and pathogenesis of gout. Arthritis and Allied Conditions, Eighth edition. Edited by JL Hollander, DJ McCarty. Philadelphia, Lea & Febiger, 1972, p 1079 2. Glynn MF, Movat HZ, Murphy EA, Mustard JF: Study of platelet adhesiveness and aggregation, with latex particles. J Lab Clin Med 65:179-201, 1965 3. Hardisty RM, Hutton RA: The kaolin clotting time of platelet rich plasma. Br J Haematol 11:258-268, 1965 4. Movat HZ, Weiser WJ, Glynn MF, Mustard JF: Platelet phagocytosis and aggregation. J Cell Biol 27:531-543, 1965 5. Spaet TH, Cintron J: Studies on platelet factor-3 availability. Br J Haematol 11:269-275, 1965 6. Mustard JF, Glynn MF, Nishizawa EE, Packham MA: Platelet-surface interactions: Relationship to thrombosis and hemostasis. Fed Proc 26:106-114, 1967 7. Mueller-Eckhardt C, Lulscher EF: Immune reactions of human blood platelets. II. The effect of latex partieles coated with gamma globulin in relation to complement activation. Thromb Diath Haemorrh 20:168-179, 1968 8. Morris CDW: Observations of the effect of glass beads on platelet aggregation and its relationship to platelet stickiness. Thromb Diath Haemorrh 20:345-353, 1968 9. Jobin F, Lapointe F, Gagnon F: Platelet reactions and immune processes. VI. The effect of immunoglobulins and other plasma proteins on platelet surface interactions. Thromb Diath Haemorrh 25:86-97, 1971 10. Pfueller SL, Lulscher EF: Studies of the mechanisms of the human platelet release reaction induced by immunologic stimuli. II. The effect of Zymosan. J Immunol 112:1211-1218, 1974 11. Zucker MB, Grant RA: Aggregation and release reaction induced in human blood platelets by zymosan. J Immunol 112:1219-1230, 1974 12. Ginsberg MH, Kozin F, O'Malley M, McCarty DJ: Release of platelet constituents by monosodium urate crystals. J Clin Invest 60:999-1007, 1977 13. Henson PM, Ginsberg MH, Morrison DC: Mechanisms of mediator release by inflammatory cells. Membrane Fusion. Edited by G. Poste, GL Nicholson. New York, Elsevier North-Holland, 1978, pp 407-508 14. McCarty DJ, Faires JS: A comparison of the duration of local anti-inflammatory effect of several adrenocorticosteroid esters: A bioassay technique. Curr Ther Res 5:284-290, 1963 15. Aster RH, Jandl JH: Platelet sequestration in man. J Clin Invest 43:843-855, 1964 16. Walsh PN: Albumin density gradient separation and washing of platelets and the study of platelet coagulant activities. Br J Haematol 22:205-217, 1972 17. Lages B, Scrutton MC, Holmsen H: Studies on gel-filtered human platelets: Isolation and characterization in a medium containing no added Ca2+, MG2+, or K+ J Lab Clin Med 85:811-825, 1975 18. Ginsberg MH: Unpublished observations 19. Charo IF, Feinman RD, Detwiler TC: Interrelationships of platelet aggregation and secretion. J Clin Invest 60:866-873, 1977 20. Morel FM, Baker RF, Wayland H: Quantitation of human red blood cell fixation by glutaraldehyde. J Cell Biol 48:91-100, 1971 21. Holmsen H, Storm E, Day HJ: Determination of ATP and ADP in blood platelets: A modification of the firefly luciferase assay for plasma. Anal Biochem 46:489-501, 1972 22. Stanley PE, Williams SG: Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal Biochem 29:381-392, 1969
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23. White JG: Electron microscopic studies of platelet secretion. Prog Hemost Thromb 2:49-98, 1974 24. Hoffstein S, Weissmann G: Mechanisms of lysosomal enzyme release from leukocytes. IV. Interaction of monosodium urate crystals with dogfish and human leukocytes. Arthritis Rheum 18:153-165, 1975 25. Spilberg I, Gallacher A, Mendell B: Studies on crystal-induced chemotactic factor II role of phagocytosis. J Lab Clin Med 85:631-636, 1975 26. Kozin F, McCarty DJ: Protein binding to monosodium urate monohydrate, calcium pyrophosphate dihydrate, and silicon dioxide crystals. I. Physical characteristics. J Lab Clin Med 89:1314-1325, 1977 27. Ginsberg MH, Kozin F: Mechanisms of cellular interaction with monosodium urate crystals: IgG-dependent and IgG-independent platelet stimulation. Arthritis Rheum (In press) 28. Haslam RJ, Davidson MML McClenagan MD: Cytochalasin B, the blood platelet release reaction and cyclic GMP. Nature 253:455-457, 1975 29. White JG, Krumweide M: Influence of cytochalasin B on the shape change induced in platelets in the cold. Blood 41:823-832, 1973 30. White JG: Uptake of latex particles by blood platelets: Phagocytosis or sequestration? Am J Pathol 69:439-458, 1972
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[Illustrations follow]
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