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. On the other hand, in the absence of NH4Cl, thrombin-in- - Stest Scontrol x 100 = % secretion, duced lysosomal enzyme secretion decreased. Thus, the true Tcontrol - Scontrol enhancing effect of NH4Cl was 3-fold at the end of the incu- bation. where S designates the enzyme activity in the supernatants The effect of NH4Cl was found to be reversible. As shown and T designates the enzyme activity in the total uncentri- in Table 1, reincubation of NH4Cl-treated platelets in fuged platelet suspension. NH4CI-free medium restored the level of secretion to that of Acid Hydrolase Assay. f-N-Acetyl-D-hexosaminidase (/3 untreated platelets. hexosaminidase; EC 3.2.1.52) was measured fluorimetrically Potentiation of ,3-hexosaminidase secretion also was ob- by a modification of the method described (33). Namely, 75 served when platelets were incubated with short-chain pri- ,ul of 4 mM 4-methylumbelliferyl 2-acetamido-2-deoxy-3-D- mary aliphatic amines at a 10 mM concentration (Table 2). glucopyranoside in 0.1 M sodium phosphate/sodium citrate buffer (pH 4.5) containing 0.2% human serum albumin (Sig- 100r- ma) was mixed at 37°C with 50 ,ud of sample. After a 12-min incubation at 37°C, the reaction was stopped by addition of 2.5 ml of 0.1 M glycine/NaOH, pH 10.7. The fluorescence 80k was read at 450 nm with excitation at 365 nm in a Farrand 0-I Mark I Spectrofluorimeter. The enzymes -D-glucuronidase (EC 3.2.1.31), ,B-D-galactosidase (EC 3.2.1.23), a-D-manno- z 60k sidase (EC 3.2.1.24), and acid phosphatase (EC 3.1.3.2) were 0 control measured as described for -hexosaminidase with the fol- 40k lowing substrate solutions, respectively: 10 mM 4-methyl- w umbelliferyl ,B-D-glucuronide (pH 5.0), 1 mM 4-methylum- C/) belliferyl ,B-D-galactoside (pH 4.5), 4 mM 4-methylumbelli- feryl a-D-mannopyranoside (pH 5.5), and 0.6 mM 4- 20 methylumbelliferyl phosphate (pH 5.0). All substrates were obtained from Sigma. Lactate Dehydrogenase Assay. Lactate dehydrogenase was 0 K assayed according to a micro-modification of the method of U/mi THROMBIN Wroblewski and LaDue (34). Thus, 260 Al of 0.2 M sodium phosphate buffer (pH 7.3) was mixed with a 200-Al sample FIG. 1. Effect of thrombin concentration on secretion of 3-hexo- and 20 Al of NADH (Boehringer Mannheim; 2.6 mg/ml, saminidase in untreated platelets or in platelets treated with NH4CI. freshly prepared) and the absorbance at A280 was measured Platelet-rich plasma was incubated at room temperature with 10 mM NH4Cl. Gel-filtered platelets (200,000 cells per IlI) were prepared in at room temperature. Then, 20 Al of sodium pyruvate (Sig- calcium-free Tyrode's solution supplemented with 10 mM NH4Cl. ma; 2.5 mg/ml) was added and the rate of the decrease in Thrombin-induced ,3-hexosaminidase secretion was performed after optical density was taken as the enzymatic activity. 2 hr exposure of the platelets to NH4Cl as described. Data shown Digitonin-Induced Cell Lysis. Controlled digiton-induced are for one platelet suspension and are representative of two other cell lysis is a rapid technique for separating soluble from experiments using different platelet suspensions. Downloaded by guest on October 2, 2021 2376 Cell Biology: van Oost et aL Proc. NatL Acad ScL USA 82 (1985)

60r Table 2. Potentiation of f-hexosaminidase secretion by aliphatic primary amnes Addition % secretion (10 mM) (mean ± SD)

40 None 18 1 CH3NH2 43 ± 2 z C2H5NH2 52 ± 4 C3H7NH2 48 ± 3 w C4Hj0NH2 47 ± 2 w 20F- NH4Cl 37 ± 1 CI) Gel-filtered platelets (4 ml; 160,000 cells per jAl) were incubated with the, amines for 60 min at room temperature. 13-Hexosaminidase secretion was induced with thrombin (final concentration, 1 unit/ ml). Data represent means ± SD for secretion experiments carried 0 60 out in triplicate. PREINCUBATION TIME (MIN) age of adenine nucleotides, the latter were used as markers FIG. 2. Effect of time of incubating platelets with NH4Cl on for secretioni from dense granules. While NH4Cl slightly en- thrombin-induced secretion. Gel-filtered platelets in calcium-free hanced thrombin-induced secretion of dense granule and a Tyrode's solution were prepared as described. Gel-filtered platelets methylamine was slightly inhibitory per were with 10 mM NH4Cl at granule constituents, (25 ml; 130,000 cells j1) incubated Both agents, however, stimulated f3hexosamini- room temperature for up to 120 min. At indicated times, 0.5-ml sam- (Fig. 3). ples were taken, ,3-hexosaminidase secretion was induced with dase secretion to the same extent. In view of the opposite thrombin at 1 unit/ml (final concentration). At zero time, 3-hexosa- effects of NH4Cl and methylamine on adenine nucteotide se- miniddse secretion in throthbin-incubated platelets was 34% ± 2% in cretion, the amine-induced leakage of preabsorbed ['4C]ser- the presence, and 34% ± 4% in the absence of NH4Cl, respectively. otonin was studied. Both NH4Cl and Inethylamine caused Indicated are means ± SD (vertical bars) for secretion experiments leakage of 20% of preabsorbed [14C]serotonin over a 4-hr carried out in triplicate. incubation period at room temperature, and they did so in an approximately linear and superimposable fashion. The aliphatic amines were slightly more potent than NH4Cl The effect of NH4C1 preincubation on the solubilization of in stimulating ,-hexosaminidase secretion. Chloroquine, at a P-hexosaminidase caused by the cell disruptive agent, digito- concentration of 30 AM, potentiated 3-hexosaminidase se- nin, also was studied. Incubation of platelets for 1 min with cretion induced by 1 unit of thrombin per ml from 51% + 1% digitonin at several concentrations revealed that 3-hexosa- up to 72% 2% (mean SD, experiments in triplicate). minidase was more easily solubilized when the platelets Electron microscopical.inspection of platelets exposed to were preincubated for 2 hr with 10 mM NH4C1 (Table 4). 10 mM NH4Cl for 2 hr showed some pseudopod formation, Prolonged exposure ofplatelets to 10 mM NH4C1 was clearly but no sign of platelet activation, such as granule centraliza- essential because when NH4Cl was added together with diki- tion, vacuolization, or swelling. After addition of thrombin tonin to the platelet suspension and incubated for 1 min, no to platelets that had been preinicubated with NH4Cl, identi- effect of NH4C1 could be demonstrated whichever digitonin cal ultra~tructural changes took place compared to those ob- concentration was used (data from two separate experi- served in normal control platelets. ments, not shown). The effect' of NH4Cl on the thrombin-induced secretion of platelet enzymes other than /3-hexosaminidase is shown in Table 3. As with P3-he.Nosaminidase, secretion of 3glucuroni- DISCUSSION idase and f-galactosidase was enhanced. In contrast, a-man- This work shows that weak bases specifically enhance nosidase secretion wasnoot enhanced. Acid phosphatase and thrombin-induced lysosomal enzyme secretion from plate- lactate dehydrogenase were not secreted by thrombin, been lets. Using this approach, we demonstrate here that P-hexo- regardless of whether the platelets had preincubated saminidase can be completely secreted from platelets. This with NH4Cl. rules out at least one explanation for the nordially incom- The effect of weak bases on dense granule and a granule plete secretion of,hexosAminidase-i.e., partial localiza- secretion from platelets wds assessed using either NH4Cl or methylamine. Since the lysosomotropic agents caused leak- age of serotonin from the platelets (see below) but not leak- Table 3. Stimulation of lysosomal enzyme secretion by NH4CI % secretion Table 1. Reversibility of NH4Cl effect on f-hexosaminidase NH4CI secretion (10 inM) Control Platelet-rich Gel-filtration t f3-Hexosaminidase 72 30 plasma buffer secretion 1-Glucuronidase 51 24 -- 32 ± 2 /3-Galactosidase 28 9 + + 61 ± 2 a-Mannosidase 38 38 + 55 ± 2 Acid phosphatase 0 0 + - 35±1 Lactate dehydrogenase 0 0 Platelet-rich.plasma (7 ml) was incubated for 1 hr at room Gel-filtered platelets (150,000 cells per il) in calcium-free Ty- with or without (-) 10 MM NH4Cl. Gel-filtered rode's solution were exposed for 2 hr to 10 mM NH4Cl. Platelet temperature (+) 2 platelets were prepared in Tyrode's buffer with (+) or without (-) secretion was induced with thrombin (final concentration, units/ a total of 2 hr incubation with or without ml), and secretion was calculated. Control represents platelets that 10 mM NH4Cl. After obtained NH4Cl, secretion was induced in 1-ml samples (185,000 cells per 1ul) were not exposed to NH4Cl. Data shown are mean values with thrombin (final concentration, 2 units/ml). Data represent with one platelet suspension and are representative of two other different means ± SD for secretion experiments carried out in triplicate. experiments using platelet suspensions. Downloaded by guest on October 2, 2021 Cell Biology: van Oost et aL Proc. NatL. Acad. Sci USA 82 (1985) 2377

Table 4. Effect of platelet incubation with NH4Cl on A /3-hexosaminidase solubilization by digitonin % solubilization Digitonin, mM NH4Cl (10 mM) Control %- 0 1 1 z 60 0 0.1 60 24 0.2 74 58 w 40 0.4 90 87 w 1 93 95 cn Gel-filtered platelets (400,000 cells per ul) were either untreated 20 (control) or exposed for 2 hr to 10 mM NH4Cl at room temperature. Samples of the suspension were exposed to a series of digitonin concentrations and the solubilized B3-hexosaminidase was assayed. The extent of enzyme solubilization is expressed as % of total en- zyme activity, as measured in an uncentrifuged platelet suspension U/ml THROMBIN exposed to 1 mM digitonin. The data shown are mean values ob- tained using one platelet suspension and are representative of two other experiments using different platelet suspensions. and a consistent marked stimulation was observed only for lysosomal enzyme secretion. This observed specificity of the effect of weak bases on platelet secretion and the well-known concentration of weak Z 60- ; the in- =F that of bases inside lysosomes suggests modification 0 tralysosomal milieu is the mechanism by which the weak bases work. This was tested by examination of the intracel- 40 lular binding of lysosomal enzymes in intact platelets ex- CO) posed to weak bases using controlled digitonin-induced cell 20 lysis. Digitonin is a glycoside exhibiting detergent-like prop- erties by complexing with free cholesterol in cell membranes (35). f3Hexosaminidase was rendered soluble at lower digi- 0 L tonin concentrations when the platelets were incubated for 2 0 0.02 0.04 0.06 0.08 0.1 1 hr with 10 mM NH4CI, indicating that less membrane disrup- U/mi THROMBIN tion is required for ,-hexosaminidase solubilization. Without FIG. 3. Effect of platelet incubation with NH4Cl or methylamine preincubation, the effect of NH4Cl on enzyme solubilization on adenine nucleotide secretion (A) and /3-thromboglobulin secre- was negligible. This indicates that prolonged exposure of tion (B). Gel-filtered platelets (150,000 cells per /.d) were exposed for platelets to NH4Cl dissociates lysosomal enzymes from 2 hr to 10 mM NH4Cl or to 10 mM methylamine. /3-Hexosaminidase binding sites inside the platelet and is in keeping with the secretion induced by thrombin (0.1 unit/ml) was stimulated from time dependence of the effect of NH4Cl on thrombin-in- 9%o to 40% by NH4Cl and to 56% by methylamine. Maximal secre- duced,-hexosaminidase secretion. table adenine nucleotide was 43% of the total adenine nucleotide It has been shown by Ohkuma and Poole (27) that the in- content in platelets. Maximal secretable 3-thromboglobulin was 89%o of the total 3-thromboglobulin content in platelets. Superna- tralysosomal pH in intact cells increases within a few min- tants of control platelets incubated with or without weak bases con- utes after addition of the weak bases, then the accumulation tained 1% of the total adenine nucleotide content and 3.5% or less of of weak bases continues, presumably reflecting the action of the total ,B-thromboglobulin content. Data shown are for one platelet an ATP-dependent proton pump (42-44). The kinetics of the suspension and are representative of two other experiments using effect of NH4Cl on f3-hexosaminidase secretion from plate- different platelet suspensions. A, Platelets incubated with 10 mM lets are thus more consistent with accumulation of NHW in- methylamine; o, control platelets; z, platelets incubated with 10 mM side the lysosomes rather than an effect only on the intraly- NH4Cl. sosomal pH. The stimulation of secretion by weak bases appeared to be tion of P-hexosaminidase in the cytoplasm or in nonsecre- specific for lysosomal enzymes. While NH4Cl stimulated tory vesicles. ,B3hexosaminidase, 3-galactosidase, and 3-glucuronidase se- The effect of weak bases was dependent on the duration of cretion 2- to 3-fold, a-mannosidase secretion was not affect- their incubation with the platelets and was fully reversible, ed. This is in line with findings of others (45) that the secre- consistent with the idea that intracellular accumulation of tion of a-mannosidase is kinetically distinct from that of /3- weak bases is required to cause enhancement of lysosomal hexosaminidase. Thus, the effect of weak bases may be of enzyme secretion. All three secretory granules in platelets help in distinguishing lysosomal enzyme secretion from se- are possible target sites for weak base action. Direct evi- cretion of enzymes from other cell compartments. dence for penetration of weak bases in dense granules is pro- To our knowledge, weak bases are unique in stimulating vided by our finding that preabsorbed [14C]serotonin leaks agonist-induced acid hydrolase secretion specifically. Cyto- from the platelets during incubation with weak bases. This is chalasin B also stimulates acid hydrolase secretion from in agreement with previous findings that dense granules are platelets (46). Cytochalasin B is thought to function by dis- acidic inside (39) and that ammonia liberates serotonin from rupting microfilaments, which facilitates the movement of intact isolated granules (40). Weak bases also may accumu- granules to the plasma membrane. As would be expected late in a granules. It recently was demonstrated that these with such a general mechanism, cytochalasin B stimulates granules may contain an ATP-dependent proton pump (41) not only acid hydrolase secretion, but also dense granule se- and, therefore, should be able to trap weak bases. However, cretion from platelets (46). in our studies, thrombin-induced secretion from dense gran- A number of other possible explanations of the observed ules and a granules was only slightly affected by weak bases effects of weak bases were explored. Exposure to weak Downloaded by guest on October 2, 2021 2378 Cell Biology: van Oost et aL Proc. NatL Acad ScL USA 82 (1985) bases had no effect on resting platelets, except for the leak- 14. Taylor, F. B. & Muller-Eberhard, H. J. (1970) J. Clin. Invest. age of serotonin. Electron microscopy revealed that the plas- 49, 2068-2085. ma membrane and granules in NH4Cl-treated platelets were 15. Holmsen, H. & Day, H. J. (1970) J. Lab. Clin. Med. 75, 840- 855. intact. The levels of adenine nucleotides, /3-thromboglobu- 16. Holmsen, H., Setkowsky, C. A., Lages, B., Day, H. J., lin, lactate dehydrogenase, and a variety of acid hydrolases Weiss, H. J. & Scrutton, M. C. (1975) Blood 46, 131-142. in unstimulated platelets were not affected by the weak 17. Holmsen, H., Kaplan, K. L. & Dangelmaier, C. A. (1982) Bio- bases. As demonstrated here, dense granule and a granule chem. J. 208, 9-18. secretion were only slightly affected. Also, we did not find 18. Henson, P. M. (1976) in Lysosomes in Biology and Pathology, any effect of NH4Cl on thrombin-induced platelet aggrega- eds. Dingle, J. T. & Dean, R. T. (Elsevier, Amsterdam), Vol. tion or thrombin binding to platelets, and the adenylate ener- 5, pp. 99-126. gy charge was unchanged (unpublished observations). 19. Knight, D. E., Hallam, T. J. & Scrutton, M. C. (1982) Nature that exposure of cells to high con- (London) 2%, 256-257. It is known prolonged 20. Jensen, M. S. & Bainton, D. F. (1973) J. Cell Biol. 56, 379- centrations of weak bases leads to extensive vacuolization 388. (47, 48), a decrease in cellular ATP levels (49), and loss of 21. Goldman, R. & Rottenberg, H. (1973) FEBS Lett. 33, 233-238. lysosomal enzyme contents (50, 51). Under these conditions, 22. Reijngoud, D. J. & Tager, J. M. (1973) Biochim. Biophys. Acta the effect of weak bases on cellular reactions probably ex- 297, 174-178. tends beyond their selective effect on lysosomes. Since this 23. Jacques, Y. V. & Bainton, D. F. (1978) Lab. Invest. 39, 179- paper was submitted, a report of the effect of weak bases on 185. neutrophil degranulation has appeared (52). In this study, 24. Styrt, B. & Klempner, M. S. (1982) FEBS Lett. 149, 113-116. weak bases were found to suppress lysozyme secretion from 25. Geisow, M. J., D'Arcy Hart, P. & Young, M. R. (1981) J. Cell stimulated neutrophils. Whether this represents a diminished Biol. 89, 645-652. re- 26. De Duve, C., de Barsy, T., Poole, B., Trouet, A., Tulkens, P. interaction of the cell stimulants with their target sites, a & van Hoof, F. (1974) Biochem. Pharmacol. 23, 2495-2531. versal of the known secretion-enhancing effect of cytochala- 27. Ohkuma, S. & Poole, B. (1978) Proc. Natl. Acad. Sci. USA 75, sin B, a reduction in cellular ATP available for secretion, or 3327-3331. an intralysosomal effect cannot be decided because all of the 28. Poole, B. & Ohkuma, S. (1981) J. Cell Biol. 90, 665-669. secretion reactions studied were depressed by weak bases 29. Dean, R. T., Jessup, W. & Roberts, C. R. (1984) Biochem. J. (52). 217, 27-40. In conclusion, we show here that thrombin can induce 30. Holmsen, H., Day, H. J. & Setkowsky, C. A. (1972) Biochem. complete secretion of fhexosaminidase if the platelets are J. 129, 67-82. with An attractive for the en- 31. Lages, B., Scrutton, M. C. & Holmsen, H. (1977) J. Lab. 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