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Lysosomotropic Agents Selectively Potentiate Thrombin-Induced Acid

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Lysosomotropic Agents Selectively Potentiate Thrombin-Induced Acid Proc. Nat!. Acad. Sci. USA Vol. 82, pp. 2374-2378, April 1985 Cell Biology Lysosomotropic agents selectively potentiate thrombin-induced acid hydrolase secretion from platelets (lysosomal enzymes/ammonium chloride/lysosomes/methylamine/exocytosis) BERNARD A. VAN OOST*t, J. BRYAN SMITH**, HOLM HOLMSEN§, AND GEORGIRENE D. VLADUTIU¶ *Thrombosis Research Center of the Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140; §Department of Biochemistry, University of Bergen, 5000 Bergen, Norway; and ¶Department of Pediatrics, Children's Hospital of Buffalo, State University of New York at Buffalo, Buffalo, NY 14222 Communicated by Sidney Weinhouse, November 29, 1984 ABSTRACT Thrombin induces partial secretion (up to platelets have made it clear that the characteristics of dense 60%) of -N-acetyl-D-hexosaminidase (EC 3.2.1.52) from un- granule and a granule secretion are very similar, but that treated platelets. Preincubation of platelets with 10 mM lysosome secretion is quite different. Secretion from dense NH4Cl for up to 2 hr resulted in a time-dependent and marked granules and a granules is very rapid and occurs in response stimulation of thrombin-induced secretion of both this enzyme to low concentrations of most platelet agonists (15, 16). In and other acid glycosidases from platelets. The enhancement contrast, secretion of lysosomal enzymes is slow, never ofthe thrombin-induced secretion was not due to cell lysis, and reaches completion, and is induced only by certain platelet NH4Cl alone did not cause leakage of lysosomal enzymes into stimuli (15-17). Platelets resemble other lysosomal enzyme- the medium. The effect could be reversed by reincubating the secreting cells such a neutrophils, in that even at maximal platelets in NH4CI-free medium. Stimulation of thrombin-in- agonist concentrations, only a fraction of a given lysosomal duced secretion also was produced by a series of aliphatic pri- enzyme is secreted (18). mary amines from methylamine to butylamine, and by micro- Little is known about what distinguishes secretion from molar concentrations of chloroquine. The effect of weak bases lysosomes as compared to secretion from dense granules and on platelets appeared to be quite specific for enhancing lyso- a granules. Secretion of lysosomal enzymes from platelets in somal enzyme secretion. Thrombin-induced secretion of ade- vitro is always accompanied by secretion of dense granule nine nucleotides from dense granules and of 1&thromboglobu- and a granule components. However, despite the higher ago- fin from a granules was slightly enhanced by NH4CI but was nist requirement for secretion from lysosomes compared to slightly inhibited by methylamine. The only direct effect of the dense granule secretion, both platelet responses appear to weak bases on platelets was the displacement of serotonin from have the same sensitivity to cytosolic calcium (19). dense granules. Accumulation of weak bases in acidic pools in There is ample evidence that lysosomes are acidic inside, the platelets (e.g., lysosomes) might, therefore, be responsible which is essential for their function (20-25, 27, 42). It was, for the enhanced secretion of lysosomal enzymes. By using therefore, of interest to study the effect of perturbation of controlled digitonin-induced platelet lysis, it was found that the intralysosomal pH on the secretion of the lysosomal con- preincubation of platelets with NH4CI lowered the digitonin tents. It has been shown that weak bases such as ammonium concentration required for enzyme solubilization. We suggest chloride, methylamine, and chloroquine, are concentrated in that loading of lysosomes with weak bases dissociates already the lysosome (26), resulting in neutralization of its contents bound enzyme inside the lysosomes, resulting in a more effec- (27, 28). The accumulation of weak bases inside lysosomes tive discharge upon stimulation by thrombin. interferes with lysosomal functions, such as degradation of proteins and glycosaminoglycans, processing of endocy- Platelets are the richest known source of lysosomal enzymes tosed receptor complexes, and packaging of lysosomal en- (1-3), which have been demonstrated to be stored in primary zymes (for compilation of recent data, see ref. 29). lysosomes (4). Platelets secrete lysosomal enzymes from We report here new effects of weak bases on lysosomal their stores in response to stimuli such as thrombin or colla- function. We find that thrombin-induced secretion of the gen (5). The abundance of activated platelets at sites of vas- contents of lysosomes, but not of dense or a granules, is cular injury (6) and inflammation (7) makes platelets a poten- enhanced by weak bases. We also demonstrate that weak tially important source of lysosomal enzymes acting locally bases interfere with the intracellular binding of lysosomal in the vascular system. In addition to lysosomes, platelets enzymes and, therefore, hypothesize that intragranular bind- contain two other types of granules whose contents are se- ing of lysosomal enzymes regulates the extent of lysosomal creted upon activation: dense granules and a granules (8). enzyme secretion. Secretion from these different granules has different physio- logical effects. Thus, (i) secretion of adenosine diphosphate MATERIALS AND METHODS from granules is important for the propagation of platelet ag- gregation and aids hemostasis (9, 10), (ii) secretion of plate- Platelet Isolation and Exposure to Lysosomotropic Agents. let-derived growth factor from a granules initiates wound re- Platelet-rich plasma was obtained from human blood anti- pair (11, 12), and (iii) secretion of acid hydrolases from lyso- coagulated with citrate (30) and, in some experiments, was somes promotes clearing of platelet aggregates (13, 14). incubated at room temperature for 15 min with 1 ,M Because secretion from these different granules may not be [14C]serotonin (side chain 2-14C; Amersham), which is incor- appropriate at the same time and at the same site, it is impor- porated into the dense granules. The platelets in platelet-rich tant to learn more about the regulatory mechanisms of plate- plasma were transferred to a Ca-free Tyrode's solution con- let secretion. Studies of the in vitro secretion response of taining 0.2% human albumin/5 mM glucose by filtration The publication costs of this article were defrayed in part by page charge tPresent address: Department of Biochemistry, University of Brit- payment. This article must therefore be hereby marked "advertisement" ish Columbia, Vancouver, BC, Canada V6T 1W5. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2374 Downloaded by guest on October 2, 2021 Cell Biology: van Oost et aL Proc. NatL. Acad. Sci. USA 82 (1985) 2375 through Sepharose CL-2B (31). Ammonium chloride, chlo- granular components in hepatocytes (35) and in platelets roquine, methylamine, ethylamine, propylamine, and buty- (36). Ten-fold concentrated digitonin (Calbiochem) stock so- lamine were obtained from Sigma, and were added to plate- lutions were prepared in calcium-free Tyrode's solution. let-rich plasma or to platelet suspensions from 100-fold con- Gel-filtered platelet suspensions (0.9 ml) were preincubated centrated stock solutions that were adjusted to pH 7.3 with for 5 min at 37°C and subsequently incubated with digitonin concentrated HCl. (0.1 ml; for final concentrations see Table 4) for 1 min at Platelet Secretion. For measurements of secretion of lyso- 37°C. The suspensions were centrifuged for 2 min at 10,000 somal enzymes and P-thromboglobulin, 1-ml samples of x g and 0.5 ml of supernatant was collected for assay of platelet suspension were preincubated at 370C for 5 min. solubilized enzymes as described above. Then, 50 ,u1 of thrombin (Parke, Davis) in saline was added Other Methods. The amounts of ATP and ADP secreted to give final concentrations from 0.01 to 100 units/ml. The from platelets (dense granule constituents) were determined samples were mixed once on a Vortex, and the incubation in ethanolic extracts by a luciferase method (37). ,B-Throm- was continued at 37TC for 10 min without stirring. The sam- boglobulin (low affinity platelet factor 4, an a granule con- ples were centrifuged at room temperature in an Eppendorf stituent) was measured by radioimmunoassay (38). centrifuge for 2 min at 10,000 x g. Portions of the superna- tant (200 ,1) or total noncentrifuged suspension (200 ,ul) were RESULTS mixed with 200 1.l of 0.6% Triton X-100, and were frozen and thawed 3 times. Incubation of gel-filtered platelets with 10 mM NH4Cl for 2 For measurement of adenine nucleotide secretion, por- hr did not cause acid hydrolase secretion by itself. However, tions (400 /l) were taken after a 3-min incubation with thrombin-induced 3-hexosaminidase secretion was greatly thrombin, mixed with 100 ,4 of 38 mM EDTA/0.65 M for- enhanced (Fig. 1). While thrombin (2 units/ml) induced malin, and centrifuged as described above. The supernatant 509-60%o secretion in control platelet suspensions, virtually (200 ,l) was mixed with 200 ,u1 of freshly prepared ice-cold complete secretion of 3hexosaminidase was obtained after 10 mM EDTA/87.4% ethanol as described (32). This proce- the platelets had been incubated with 10 mM NH4Cl for 2 hr. dure prevented the artefactual centrifugation-induced ade- Because the effect of NH4Cl was most pronounced at inter- nine nucleotide secretion. mediary thrombin concentrations, experiments to further The extent of adenine nucleotide and ,3-thromboglobulin study the observations in Fig. 1 were carried out with 1-2 secretion was expressed as percentage of maximal secreta- units of thrombin per ml. ble amount with thrombin at 5 units/ml. It was verified that The time course of the effect of NH4Cl is shown in Fig. 2. the thrombin incubation periods were sufficiently long for At zero time, NH4Cl had no discernible effect on thrombin- maximal secretion at any given thrombin concentration. The induced 3-hexosaminidase secretion. However, the secre- percentage secretion was calculated using the formula tion was progressively enhanced, up to 2-fold, over 90 min.
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