ARCHIVES

OF BIOCHEMISTRY

Vol. 284, No. 1, January,

AND

BIOPHYSICS

pp. 47-52,

1991

Stimulation of Phospholipase A2 Activity in Human Platelets by Trypsin and Collagen Thomas

M. Chiang,l

Andrew

H. Kang,

and John

Veterans Administration Medical Center and Departments University of Tennessee, Memphis, Tennessee 38163

Received

May

25, 1990, and in revised

form

September

N. Fain of

Medicine

10, 1990

Type I collagen enhanced human platelet phospholipase A2 activity whether added to platelet-rich plasma or washed platelets. The stimulatory effect of type I collagen on platelet membrane phospholipase A2 activity was also observed in a cell-free system utilizing platelet membranes. The release of arachidonic acid was enhanced by types I and III but not by type V collagen. The activation of platelet phospholipase A2 by type I collagen was inhibited by soybean trypsin inhibitor and mimicked by trypsin. However, type I collagen addition was not associated with any detectable changes in platelet membrane proteins while trypsin altered many proteins. These results point to acid soluble phospholipase A2 activity of platelets as an enzyme activated by type I collagen. 8 1991 Academic Press, Inc.

Activation of human platelets by various agonists is associated with phospholipase A2 activation and the release of arachidonate from membrane phospholipid(s) (l5). The released arachidonate serves as substrate for the synthesis of prostaglandins and thromboxanes (6). However, arachidonic acid can also be released as a result of the combined activation of phospholipase C and diacylglycerol lipase in platelets (2, 3, 6-8). The relative importance of these pathways with regard to arachidonate release in vim remains to be determined. Collagen is a potent stimulant of platelet responses including platelet shape change, aggregation, secretion, phosphatidylinositide hydrolysis, and calcium translocation from intracellular stores (9). Collagen also stimulates the release of arachidonic acid (AA)2 from phos’ To whom correspondence should be addressed at Veterans Affairs Medical Center, Research Service (151), 1030 Jefferson Avenue, Memphis, TN 38104. * Abbreviations used: Tris-EDTA, 0.05 M Tris-HCl-0.145 M NaCl0.001 M EDTA, pH 7.4; PC, phosphatidylcholine; AA, arachidonic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 0003.9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

and Biochemistry,

pholipids and its subsequent conversion to eicosanoids (6). Several laboratories, on the basis of studies in intact platelets, have suggestedthis involved activation of phospholipase A2 (10-15). In the present investigation, we show that the activity of phospholipase A2 was enhanced by the addition of collagen (type I collagen was used in all studies except as specified) to washed intact human platelets or platelet membrane preparations. The effect of collagen on the activity of phospholipase A2 was not mimicked by albumin but was mimicked by trypsin. However, there is as yet no evidence that collagen activates endogenous platelet proteases. METHODS Preparation of washed human platelets. Blood was collected from normal volunteers who had fasted overnight. The blood was then placed in a polypropylene tube containing 0.1 vol of 3.8% sodium citrate. Platelet-rich plasma was prepared by centrifuging the titrated blood at room temperature for 15 min at 226g (16). Washed platelets were prepared by addition of an equal volume of 0.05 M Tris-HCl-0.145 M NaCl-0.001 M EDTA, pH 7.4 (Tris-EDTA) to platelet-rich plasma and then centrifuging the mixture for 15 min at 226g. The platelets were resuspended in the same buffer at the concentration of 300,000-400,000 per mma.

Preparation ofplatelet membranes. Human platelet membranes were isolated according to the method developed by Barber and Jamieson (17). Ten units of platelet-rich plasma (l-day-old) was obtained from a local blood bank. Platelets were sedimented by centrifugation and washed with Tris-EDTA as described above. The washed platelets were lysed by the addition of a buffer containing 0.01 M Tris-HCI (pH 7.5) and 0.25 M sucrose and sonicated for 10 s with a Branson sonifier. The membrane fraction was obtained by centrifuging the platelet lysate through a solution of 27% sucrose at 65,000g for 4 h. The interface containing the platelet membranes was collected, and the membranes were washed with Tris-EDTA buffer. The washed platelet membranes were suspended in 50 mM Tris-HCl, pH 7.5, and were either used directly or stored in test tubes at -2O’C. Preparation of type I collagen. Neutral salt soluble collagen (type I) was extracted from the skins of 3-week-old white Leghorn chicks which had been rendered lathyritic by the administration of p-amino propionitrile (Aldrich) for 2 weeks. The extracted collagen was purified by methods previously described (18). 47

Inc. reserved.

48

CHIANG,

KANG,

Preparation of types III and V collagen. Types III and V collagens were isolated and purified from human placentas by limited pepsin digestion according to the procedure of Rhodes and Miller (19). Preparation of platelet phospholipase A2 extract. Intact washed platelets or platelet membrane-associated phospholipase A2 was stimulated with collagen or other stimuli for various time intervals as described under Results. Phospholipase A2 was extracted with 0.18 N H&II as described by Chang et al. (20). Briefly, the agonist-stimulated platelets or platelet membranes were mixed with equal volumes of cold 0.18 N H&SO,. The mixture was frozen and thawed for 10 cycles and kept at 4°C for 1 h, vortexed, and centrifuged for 15 min at 10,OOOg. The amount of protein in the supernatant fluids enriched with phospholipase A2 was determined according to the method of Lowry et al. (21) using bovine serum albumin as a standard. The supernatants were used to assay the activity of phospholipase A2 or kept at -20°C until used. Assay of phospholipase A2 activity. Phospholipase A2 activity was measured routinely by the hydrolysis of [3H]arachidonic acid-labeled phosphatidylcholine (45,000 cpm). Incubation mixtures contained: 100 mM Tris buffer, pH 7.5, 5 mM CaCl,, 50 ~1 of 100 pM [3H]arachidonic acid-labeled phosphatidylcholine dissolved in chloroform:methanol: HZ0 (870:1100:30), and 100 ~1 of test sample in a total volume of 0.5 ml (20). Incubations were carried out at 37°C in a shaking water bath for 1 h. The reaction was terminated by the addition of 1.5 ml of Dole’s reagent, 0.75 ml of heptane, and 100 ~1 of water. After vortexing the mixture was placed in a shaker for 15 min and centrifuged at 2OOOg for 10 min at 4°C. The upper phase containing liberated arachidonic acid (0.5 ml) was withdrawn and mixed with 10 ml Scintiverse II for counting in a Packard scintillation counter (Model A2000). For comparison, the substrate was also dissolved in 100% dimethyl sulfoxide. Results obtained were similar. Therefore, all experiments were performed with arachidonic acid dissolved in the chloroform:methanol:H,O mixture. The same concentration of phosphate buffer can also be used to replace the TrisHCl buffer. An addition of physiological salt to the extraction or reaction mixtures did not interfere with the assay of phospholipase A2 activity. The phospholipase A2 activity required the addition of Ca*+. Addition of EDTA abolished soluble phospholipase A2 activity. Other divalent cations were tested. The relative activity to Ca*+ was M$+ (56%), Zn*+ (45%), Mn*+ (39%), Co’+ (27%), Cu*+ (O%), and Ba2+ (0%) at 5 mM concentration. Nondenaturing gel electrophoresis. Nondenaturing gels (7.5%) contained 10 ml of monomer solution (40% acrylamide, 5% bisacrylamide), 10 ml of Tris-buffer (947 mM Tris-HCl, pH 8.48), 5 ml of catalyst (0.06% ammonium persulfate, 0.002% riboflavin phosphate), 15 ml of water, and 0.08 ml of N,N,N’,N’-tetramethylethylenediamine. The gels were electrophoresed for 4 h before samples were loaded with lower tank buffer containing 63 mM Tris, 50 mM HCl, pH 7.47, and upper tank buffer containing 37.6 mM Tris, 40 mM glycine, pH 8.89. After the samples were loaded, the gels were electrophoresed overnight at 40 V. At the end of electrophoresis, gels were sliced into 0.5.mm slices, broken into small pieces, and assayed for the release of [sH]arachidonic acid as described in the above section. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) of samples. Samples from buffer control, and various types of collagen-treated platelet membrane extracts were dissolved in sample buffer (containing 1.5% /!I-mercaptoethanol, 1.5% SDS, 1.5% glycerol, and 0.05% bromphenyl blue in 50 mM Tris-HCl, pH 6.8) boiled for 2 min. The boiled samples were analyzed on a 7.5% SDS-PAGE slab gel containing 8.25 ml (1.5 M TrisHCl, pH 8.8), 8.25 ml acrylamide (30 g acrylamide 0.8 g bisacrylamide in 100 ml H,O), 0.33 ml (10%) SDS, 0.011 ml N,N,N’,iV-tetramethylethylenediamine, 1.1 ml (1% ammonium persulfate and 15 ml H,O) (22). After electrophoresis at 40 V in Triss glycine buffer, pH 8.3, overnight, the gel was stained with Coomassie brilliant blue (Eastman Kodak Co., Rochester, NY), destained with methanol/acetic acid (10/7%) solution, and photographed. For the radioactive samples, the destained gel was dried under vacuum. The dried gel was radioautographed using a Kodak XR-5 film (Picker International, Norcross, GA).

AND

FAIN

RESULTS Since the stimulation by collagen of platelet aggregation is associated with arachidonic acid release in intact platelets, we examined the effect of collagen on the acid soluble phospholipase A2 activity of platelets. It has been known for some time that phospholipase A2 activity can be measured in sulfuric acid extracts of platelets after neutralization (20, 23). This has been used in purification of phospholipase A2 since the bulk of platelet proteins are precipitated by acid treatment. The deacylation of phosphyatidylcholine (PC) by phospholipase A2 represents a major route for the release of AA in collagen-stimulated platelets (7,8,24). We have used [3H]arachidonate-PC as the substrate for assaying the activity of phospholipase A2. Our results indicate that the acid-soluble phospholipase A2 activity was enhanced two- to fourfold by the addition of collagen to plateletrich plasma (Table I). The activation of acid-soluble phospholipase A2 activity was time dependent and reached maximum at 2 min (Table I). Furthermore, isolated platelet membrane phospholipase A2 activity was stimulated by collagen (Table II). Maximum activity of phospholipase A2 required 3-6 pg of collagen in the presence of 250 pg of platelet membrane protein. The addition of collagen in excess of 30 pg was inhibitory (data not shown). The activation by collagen of phospholipase A2 activity was seen within 15 s after collagen was added to platelet membranes and was maximal by 60 s (data not shown). Bovine serum albumin addition to platelet membranes was less effective (30%) in the activation of phospholipase A2 than was collagen (Table III). Furthermore, in the presence of albumin collagen was unable to further stim-

TABLE Time Dependence for Type Phospholipase A2 Activity Incubation (min) 0 0.5 1 2 4 6 8

time

Arachidonic (cpm) 1252 2565 3157 4515 5227 5632 6105

I I Collagen Activation of in Platelet-Rich Plasma acid release + S.D. +k t + + f f

306 329 207 364 355 253 229

% Control 100 204 252 360 417 450 480

Note. One milliliter of platelet-rich plasma (3 X 10’ platelets) was incubated with 20 /Lg of type I collagen (with stirring) for various time points as listed in the table. The mixture was spun in an Eppendorf centrifuge for 1 min. The platelet pellets were suspended in 0.3 ml of 0.18 N H2S04 then frozen and thawed for 10 cycles. Samples were centrifuged at 10,OOOg for 15 min at 4°C. The supernatant was assayed for phospholipase A2 activity using [3H]PC. The data shown are means + S.D. of two duplicate experiments.

COLLAGEN-PLATELET TABLE

TABLE

II

Dose-Dependent Stimulation of Isolated Platelet Membrane PhospholipaseA2 Activity by Type I Collagen Amount

of type (/.G) Buffer

I collagen

Arachidonic (cpm) 1365 1385 1570 2060 2252 2685

control 0.45 0.9 1.8 3.7 7.5

acid release f S.D. f k +f + k

12 68 104 89 40 131

Effect of Various Platelet

Types Buffer

Note. Platelet membranes (200 pg) were incubated with various amounts of type I collagen for 4 min. At the end of the incubation, an equal volume of 0.18 N H,SO* was added and kept at 4°C for 1 h. The mixture was centrifuged and the supernatant was assayed for phospholipase A2 activity. The data shown are means + S.D. of one triplicate experiment.

ulate phospholipase A2 activity (Table III). It is possible that albumin inhibited the proteolytic enzyme(s) to activate phospholipase A2. We compared the effects of soluble types I, III, and V collagen on activation of phospholipase A2. At the same concentration of collagen (5 pg), types I and III but not V collagen stimulated arachidonic acid release (Table IV). Reports from other laboratories have shown that platelets possessmembrane-bound phospholipase C (25, 26). This raised the possibility that we might be observing the results of sequential actions of a G-protein-activated phospholipase C followed by a diacylglycerol lipase. We, therefore, used neomycin, a phospholipase C inhibitor (14), to determine a possible contribution to the arachi-

TABLE

49

INTERACTION

III

Effect of Type I Collagen on the Activity of Platelet Membrane PhospholipaseA2 RequiresIncubation

IV

Types of Collagen on the Activation Membrane Phospholipase A2

of collagen

Arachidonic (cpm) 1440 2610 2370 1620

control I III V

acid release -C S.D. ?I f f +

of

% Control

59 67 261 251

100 180 165 110

Note. Platelet membranes (250 pg) were incubated with various types of collagen (5 rg) for 1 min at 37°C. The incubations were stopped by adding an equal volume of cold 0.18 N H,SO1 and the neutralized supernatants analyzed for phospholipase A2 activity. The data shown are means + S.D. of two duplicate experiments.

donic acid release by phospholipase C/diacylglycerol lipase pathway. The stimulatory effect of collagen on phospholipase A2 was not inhibited by neomycin (Table V). We examined the proteins in the acid extract using nonreducing gel electrophoresis and compared the phospholipase A2 activity in the bands eluted from control versus collagen-treated platelet membranes (Fig. 1). There was no visible effect of collagen on the protein pattern after electrophoresis of the acid extract (Figs. 1 and 2). None of the major protein bands correlated with phospholipase A2 activity, which was in gel slice 19 (from the top of the gel), while the major protein band was in gel slice 18 (Fig. 1). In controls the detectable phospholipase A2 activity was also in slice 19. In collagen-treated membranes the amount of activity in this region increased about two-fold. The migration of phospholipase A2 upon gel electrophoresis was not altered by treatment with collagen. This result suggests that the activation of phospholipase A2 is not accompanied by any major change in the size of the enzyme.

with Membranes TABLE Arachidonic (cpm)

0 time control

Treatment Buffer control Type I collagen, Albumin, 5 pg Type I collagen,

5 pg 5 pg + albumin,

5 pg

1435 2485 2345 2940

& 48 f 388 + 238 * 357

acid release f S.D. A Due to incubation for 4 min 1890 6820 3395 3010

I 115 I 1,475 + 180 + 417

Note. Platelet membranes (250 gg) were incubated with type I collagen or albumin for 0 or 4 min. The samples were extracted using 0.18 N H,SOI with brief sonication (10 s) and then centrifuged at 10,OOOg for 15 min at 4°C. The supernatants (100 ~1 each) were assayed for phospholipase A2 activity. The data are means -t S.D. of two duplicate experiments.

V

Effect of Neomycin on Type I Collagen Activation Membrane Phospholipase A2 Arachidonic (cpm)

Treatment Collagen Collagen Collagen

plus neomycin, plus neomycin,

0.3 mM 0.9 mM

of Platelet

acid release t SD.

1980? 47 1829 + 94 2199 f 284

Note. Platelet membranes (200 pg) were incubated with neomycin as listed in the table for 10 min before adding type I collagen. The incubation was continued for 4 min in the presence of type I collagen. The samples were extracted with 0.18 N H,SO, and the supernatants were assayed for phospholipase A2 activity as described in the text. The data shown are means f SD. of one triplicate experiment. Basal arachidonic acid release in the absence of collagen was 1201 f 35 cpm.

50

CHIANG,

KANG.

AND

FAIN a

-b -C

1600 :

0 :z-

2 E '0 x : G

P

Top of gel

-d

1400-

2 1200low600-l

0

I

1

5

10

.

1

1

15

20

.

1

25

Gel Slices

0

10

5

2.5

COLLAGEN,

TRYPSIN,“g

1.2

ug

of proteins in acid extract of platelet membranes. FIG. 2. Detection Platelet membranes were incubated with buffer (0) and various amounts of trypsin (left) or type I collagen (right), as indicated for 4 min then extracted with 0.18 N H,SO,. The supernatants were neutralized and subjected to 10% SDS-PAGE. Protein bands were located with Coomassie brilliant blue stain. Molecular weight marker proteins are: a, myosin, 200,000; b, P-galactosidase, 130,000; c, phosphorylase b, 97,400; d, bovine serum albumin, 66,000 and; e, ovalbumin, 42,699, respectively.

1 2 12

3

3

FIG. 1. Localization of phospholipase A2 activity by nonreducing gel electrophoresis. Platelet membranes were incubated without (m) or with type I collagen (B) for 4 min then extracted with 0.18 N H,SOI. The supernatants were neutralized and subjected to 7.5% nonreducing polyacrylamide gel electrophoresis. Each gel was cut into 24 pieces, transferred into tubes, broken into small pieces, extracted with 0.2 ml 0.18 N H,SO,, and then assayed for the release of [3H]AA from [3H]PC (top). The stained gels after electrophoresis under nonreducing conditions in the absence of SDS are shown in A. For comparison, the gel (7.5%) separations in the presence of SDS and P-mercaptoethanol are shown in B. Lanes 1, 2, and 3 are, respectively, samples of buffer control, type I collagen treated, and molecular weight marker proteins (phosphorylase b, 97,400, bovine serum albumin, 66,200; ovalbumin, 42,699; and carbonic anhydrase, 31,000). The peak of phospholipase A2 activity (slice 19 from the top of the gel) is shown by the arrow in the lower panel and was below the ovalbumin standard as well as the major protein band in the platelet extract which was in slice 18. The data represent one of three independent experiments.

pepstatin and leupeptin inhibited the basal phospholipase A2 activity but not the increase due to collagen (Table VI). This stimulated us to compare trypsin with collagen as an activator of phospholipase A2. We found that as little as 25 ng of trypsin increased acid-soluble phospholipase A2 activity (Table VII). Addition of 5 pg trypsin to the

TABLE

Effect of ProteaseInhibitors on Type I CollagenActivation of Platelet Membrane PhospholipaseA2 Arachidonic (cpm) Treatment

The stimulatory effect of collagen on phospholipase A2 was the same at different phosphatidylcholine concentrations (1 PM, 150%; 10 PM, 175%; and 100 PM, 150%). An attempt was made to see whether protease inhibitors inhibited the activation of platelet membrane phospholipase A2 by collagen. Among the four inhibitors we examined, only the soybean trypsin inhibitor inhibited the collagen activation of membrane phospholipase A2 activity (Table VI). However, this may be a nonspecific effect of the soybean trypsin inhibitor preparation. In contrast,

VI

Buffer control Phenylmethylsulfonyl fluoride, 1 mM Soybean trypsin inhibitor, 5 pg Pepstatin, 5 pg Leupeptin, 5 pg

-Collagen 1414 1130 1440 730 530

* f f -r*

acid release * S.D. A Due to collagen

44 37 30 31 10

742 720 llO-+ 880 790

f 29 k 18 2 f 67 f 7

Note. Platelet membranes (200 fig) were incubated with protease inhibitors as listed in the table for 10 min before addition of type I collagen. The incubation was continued for 4 min in the presence of type I collagen. The samples were extracted with 0.18 N H,SO, and the supernatants were assayed for phospholipase A2 activity as described in the text. The data shown are means f SD. of one triplicate experiment.

COLLAGEN-PLATELET

TABLE Activation

of

5% 0.5 Fcg 0.05 pg 0.025 fig 0.005 pg

Platelet Membrane PhospholipaseA2 by Trypsin Arachidonic (cpm)

Treatment Buffer control Type I collagen, Trypsin

VII

acid release + SD.

1158 f 31 5 pg

1842 f 55

% Control 100 159

1527 f 20

132

1493 + 18

129

1449 k 16 1414 IL 43 1162 f 43

125 122 100

Note. Platelet membranes (200 pg) were incubated with buffer, type I collagen (5 pg), and various amounts of trypsin for 4 min. The samples were extracted with 0.18 N HzS04 and the supernatants were assayed for phospholipase A2 as described in the text. The data shown are means f SD. of one triplicate experiment.

control extracts increased acid soluble phospholipase A2 activity by 130%, which is similar to the data shown in Table VII. However, addition of 5 pg trypsin to neutralized collagen-treated extract did not further increase acid-soluble phospholipase A2 activity (data not shown). We compared the effects of collagen and trypsin on acid-soluble platelet membrane proteins. Collagen did not change the protein pattern of the stained gel (Fig. 2). Trypsin, unlike collagen, resulted in the formation of many new protein bands of lower molecular weights presumably as the result of protease action (Fig. 2). DISCUSSION

This report demonstrates that collagen and trypsin activate phospholipase A2 activity in platelet membranes. Our finding that collagen stimulates phospholipase A2 activity is consistent with reports from other laboratories indicating an increased release of arachidonic acid following the exposure of platelets to agonists (6, 7, 24). Collagen did not have a detectable effect on the molecular weight of the phospholipase A2 activity. However, activation of pancreatic phospholipase A2 activity has been reported after removal of a small surface loop on the molecule (27). We could not examine this since the change in M, of the enzyme might be so small that an effect of collagen under reducing conditions could not have been observed. Collagen, unlike trypsin, did not cause any detectable change in any of the protein bands of acid extracts from platelet membranes (Fig. 2). It has been reported that a small molecular weight (14.4 KDa) phospholipase A2 enzyme is present in platelets (28) and in rheumatoid arthritic synovial fluid (29). Whether the larger apparent iU, for the phospholipase A2 activity is due to aggregation of a low M, enzyme could not be examined because the phospholipase A2 activity

51

INTERACTION

was lost after SDS gel electrophoresis in reducing conditions. Type I as well as type III collagen either in soluble or in fibrillar form aggregates human platelets (30-32). However, the ability of type V collagen to induce platelet aggregation depends on the physical state of collagen, i.e., the fibrillar form but not the soluble form aggregates platelets (33, 34). The effect of collagen on the activity of phospholipase A2 correlated with the platelet aggregability of collagens. Types I and III but not V collagen stimulated arachidonic acid release (Table IV). Imai et al. (35) have found an inhibitor of phospholipase A2 in human platelets. An increase in phospholipase A2 activity was also seen during the course of enzyme purification (36). The effect of collagen on phospholipase A2 activation probably does not involve the removal of inhibitor(s) that bind to substrate such as lipocortin. Lipocortin effects were reversed by increasing the concentration of substrate (15) while those of collagen were unaffected (Table V). In summary, the activity of platelet membrane acidstable phospholipase A2 activity was enhanced by collagen. The increase in phospholipase A2 activity due to collagen was through a mechanism that was unaltered by extraction of the enzyme with 0.18 N sulfuric acid. Our findings indicate that types I and III collagens, but not type V collagen, produce a rapid increase in the acid-stable phospholipase A2 activity of isolated platelet membranes. Since trypsin mimicked the effect of collagen and gave no further activation in extracts from collagen-treated membranes, the collagen effect may involve protease activation. However, if collagen does activate a protease, it must be one with narrow specificty. Pancreatic phospholipase A2 is activated by proteolytic cleavage resulting in removal of only a small surface loop (27). Further studies are required to see if a similar mechanism is involved in collagen activation. ACKNOWLEDGMENTS We thank Ms. assistance. Sincere manuscript. This Veterans Affairs, DK-37004 and by Tennessee Affiliate.

V. Woo and Mr. D. Orwig for their expert technical thanks are given to Ms. K. Pearson for typing the research was supported in part by the Department of by U.S. Public Health Service Grants AM-16506 and a grant-in aid from the American Heart Association-

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Stimulation of phospholipase A2 activity in human platelets by trypsin and collagen.

Type I collagen enhanced human platelet phospholipase A2 activity whether added to platelet-rich plasma or washed platelets. The stimulatory effect of...
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