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Phosphoinositide turnover in human platelets is stimulated by albumin
Life Sciences, Vol. 49, pp. 1707-1719 Printed in the U.S.A.
Pergamon Press
PHOSPHOINOSITIDE TURNOVER IN HUMAN PLATELETS IS STIMULATED BY ALBUMIN Riyad M. Amin, James H. Hays and K. M. Mohamed Shakir Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland. (Received in final form September 26, 1991)
Summary The effect of human serum albumin (HSA) on the hydrolysis of phosphatidylinositides in human platelets labeled with myo(q-I)inositol was studied. Incubation of platelets with HSA (4 gm/dl) for 10 seconds increased IP~ and IP~ by 169% and 217% respectively. 93% of IP~ accumulated within the first 10 seconds. This effect was also shared by bovine serum albumin, although no changes in IP~ levels occurred with ovalbumin. All albumin species used induced 'SCa*~release from platelets irrespective of its effect on IP~ accumulation. These findings indicate that albumin may function in biological systems by inducing intracellular signaling. Platelets from humans demonstrate rapid changes in membrane phospholipid content in response to physiologic stimuli (1-4). The stimulation of platelets is associated with rapid generation of inositol-l,4,5-trisphosphate (IP,) (5), and an increase of Ca 2÷ in the cytoplasm (6). This effect is in close association with the physiologic platelet response (7). Human serum albumin has been shown to bind specifically, reversibly and saturably to the cell surface (8,9), but the presence of an albumin receptor has been a subject of continuing controversy (10,11). In this study, we present the effect of human serum albumin on the hydrolysis of phosphoinositides in human platelets. Materials and Methods Materials L-Myo[2-SH(N)]inositol (myo(q-I)inositol) (16 Ci/mmol), 'H glycerol, phosphatidylinositol [myo-inositol-2-q-I(N)]-, radiolabeled inositol phosphate standards and '~Ca2÷ (12.5mCi/mg) were purchased from New England Nuclear Research Products, Dupont Company, Boston, Ma. Dowex AG-1-X8 (formate form, 100-200 mesh) was from Bio-Rad Laboratories, Richmond, Va. Fatty acid-free HSA fraction V, BSA, ovalbumin, phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid were purchased from Sigma Chemical Co., St. Louis, Mo. HSA fraction V was obtained from Calbiochem Corporation, San Diego, Ca. Phosphatidylethanol was prepared from egg lecithin (12). Address Correspondence to CAPT K.M.M. Shakir, MC, USN, Division of EndocrinologyMetabolism, Department of Medicine, National Naval Medical Center, Bethesda, Maryland 20889-5000. 0024-3205/91 $3.00 + .00
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49, No. 23, 1991
ACD anticoagulant was obtained from Baxter Corporation, Deerfield, I1. chemicals and reagents were of the highest commercially available grade.
All other
Preparation of Platelets Fresh platelet concentrates, suspended in ACD anticoagulant and obtained from the blood bank of the National Institutes of Health, Bethesda, Md. were centrifuged at 200xg for 15 minutes at 22°C to remove residual erythrocytes and leukocytes. All of the subsequent handling of the platelets were performed at 22 ° C unless otherwise mentioned. The platelet-rich plasma was then centrifuged at 2000xg for 20 minutes. The platelet pellet was washed three times with a modified Tyrode's-Hepes (N-2-hydroxyethyl-piperazine-N2-ethanesulfonic acid) buffer (134 mM NaC1, 12 mM NaHCO~, 2.9 mM KC1, 0.36 mM NaH:PO,, 1 mM MgCI:, 5 mM glucose; pH 7.4) containing 500 ng/ml prostacyclin and 0.6 ADPase U/ml apyrase. The platelet pellet was finally resuspended in the same buffer containing apyrase and adjusted to a final concentration of lxl0 ~ platelets/ml. Labelin~ of Platelets with Mvo('H)inositol Platelets were labeled as described previously (13). Briefly, platelets were incubated with 1.0 mCi/ml myo(q-I)inositol in the presence of 2 mM MnCI~ for four hours at 37°C under oxygen. The labeled platelets were then centrifuged, washed and resuspended in Tyrode's-Hepes buffer containing 10 mM LiCI. Delipidation of Albumin All albumin solutions including fatty acid-free albumin were delipidated as previously described (14). Delipidated albumin solutions were adjusted to the desired concentration using protein determination by the Lowry method (15). Fatty acids in delipidated albumin solutions were not detectable by HPLC. Modification of Albumin by Treatment with Pronase and NaOH Albumin was dissolved in Tyrode's-Hepes buffer and incubated at a ratio of 0.1 mg pronase to 10 mg albumin at 37 °C for 30 minutes. At the end of the incubation period enzyme activity was inactivated by heating the albumin solution at 55°C for 5 minutes. In control experiments albumin solution was incubated for an identical period and pronase was added at the end of incubation time and following this, pronase was inactivated. Experiments were performed to determine the quantity of protein hydrolyzed by pronase and for this purpose an aliquot of the pronase-treated and control albumin solution was treated with trichloroacetic acid (40%), and the protein precipitate was removed by centrifugation. The precipitate was dissolved in 0.05 N NaOH and protein was determined by the method of Lowry et al (15). The pronase treatment hydrolyzed almost 95% of albumin. Hydrolysis of albumin by NaOH was performed by dissolving in 6 N NaOH and incubating at room temperature for 16 hours. Following this, the pH was adjusted to 7.0 with concentrated HCI. The albumin was then dialyzed extensively against distilled water and subsequently lyophilized. In control experiments albumin was dissolved in distilled water and incubated at room temperature for 16 hours. At the end of incubation an equivalent amount of 6 N NaOH previously neutralized with HCI was added and subsequently dialyzed. The protein hydrolysis by NaOH was verified (15) as described for pronase-treatment and this method hydrolyzed albumin by 96%.
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49, No. 23,
1991
E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
1709
Evaluation of Phosphoinositid¢ Hvdrolvsis Samples (0.2 ml) of washed platelets at a concentration of 10° platelets/ml were incubated in 12x75 mm borosilicate glass tubes for 15 minutes at 37 °C in the absence of Ca ÷~and presence of 10 mM LiC1. Aliquots (0.2 ml) of the various albumin solutions were then added, and the incubation was continued at 37 °C while being gently stirred for the time intervals as indicated in each experiment. The reaction was terminated by adding 0.75 ml of acidic chloroform-methanol solution (1:2, v/v) followed by the addition of 0.25 ml 0.1N HCI and 0.25 ml chloroform. The tubes were subsequently mixed vigorously and centrifuged at 900xg for 20 minutes. The aqueous phase was separated and frozen at -70 ° C until inositol phosphates were determined within 48 hours. Separation of Inositol Phosphates The procedure for the separation of water soluble ['H]inositol phosphates was modified from that described by Berridge et al (16). Briefly, an aliquot (0.675 ml) of the upper aqueous phase of the chloroform-methanol extraction was applied to a glass column containing 0.5 ml of 1:1 (v/v) mixture of Dowex AG-1-X8 resin (100-200 mesh, formate form)/distilled water. The inositol phosphates were eluted by the stepwise addition of solutions containing increasing levels of formate. Free [q-I]inositol was eluted by the addition of 4 ml H~O. [q-I]Glycerophosphoinositol was eluted by the addition of 1.25 ml of 50 mM sodium formate, and 5 mM sodium tetraborate. [~H]inositol-l-phosphate was eluted by the addition of 7 ml of 200 mM ammonium formate, 100 mM formic acid. ['H]inositol bisphosphate was eluted by the addition of 6 ml of 400 mM ammonium formate, and 100 mM formic acid and [~H]inositol trisphosphate was eluted by the addition of 6 ml of 1.0 M ammonium formate, and 100 mM formic acid. Radioactivity was determined in the eluates collected directly into vials by liquid scintillation counting. The separation of inositol phosphates was verified by putting the known standards through the columns. Phospholipase C Assay Platelet suspension, in Tyrode's-Hepes buffer, containing prostacyclin and ADPase was incubated at 37 ° C with human serum albumin for 10 seconds, 15 minutes or 30 minutes. At the end of incubation, the reaction was terminated by freezing and the platelet suspension was subsequently homogenized by sonication. Phospholipase C assay, using PI as a substrate was carried out as described previously (17). Phospholipase C activity was determined by counting aliquots of the aqueous phase (17). Phospholipase D Assay Platelet-rich plasma was incubated with q-I glycerol (final concentration 100/~Ci/ml) at 37 ° C for two hours. The platelets were then isolated and suspended Ca2-free Tyrode's buffer as described previously (18). However bovine serum albumin was omitted from this buffer. Aliquots of platelet suspension were warmed to 37* C and human serum albumin was then added to give a final concentration of 4 gm/dl. Incubations were carried for 10 seconds, 15 minutes or 30 minutes. The reaction was terminated by freezing and the platelet suspension was subsequently homogenized by sonication. The extraction and determination of (q-l) phospholipids in the platelets were carried out as described previously (18).
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49, No. 23, 1991
"Ca ~+ Study Ten ml of platelets previously washed with modified Tyrode's-Hepes buffer, were suspended at a concentration of 10' cells/ml in Tyrode's-Hepes buffer without phosphate (pH 7.4) and labeled with "Ca 2÷ (5 /~Ci/ml) for 60 minutes at 37°C. At the end of incubation the suspension was centrifuged at 1000xg for 15 minutes. The cell pellet was washed three times at 22°C by centrifugation and resuspension with 100 volumes of Tyrode's-Hepes buffer containing no calcium or phosphate and finally resuspended in the same buffer at a concentration of 10°/ml. 0.5 ml aliquots of the platelet suspension was then incubated with 0.1 ml of albumin solution (4 gm/dl - final concentration) for four minutes at 37" C. At the end of incubation 300/~1 of platelet suspension was added to 100 ~1 silicone oil in a microfuge tube, followed by centrifugation at 10,000xg for 60 seconds and the radioactivity in the supernatant was determined by counting 100/~1 aliquots in a liquid scintillation counter. '5Ca2* release was expressed as percent of total radioactivity present in platelets at the beginning of incubation. Data Analvsis Statistical analysis was performed using the Student's t-test for paired data. The level of significance was taken as p value less than 0.05. Results and Discussion Effect of Human Serum Albumin on Time-Dependent Accumulation of Inositol Phosphates in Platelets The addition of HSA 4 gm/dl (final concentration) to washed human platelets, prelabeled with myo(3H)inositol induced the accumulation of water soluble inositol phosphates as illustrated in Fig. 1. The accumulation of total [3H]inositol bisphosphate (IP2) and [~H]inositol trisphosphate (IP~) was rapid and reached its peak levels of 169% and 217% respectively, by 10 seconds compared to control values. Both [~H]inositol phosphate levels declined by 30 seconds followed by a gradual increase which continued up to 60 minutes. The data establish that there is biphasic accumulation of ['H]inositol bisphosphate and [q-I]trisphosphate in platelets after HSA stimulation, with a peak increase occurring within 10 seconds. Although this is in agreement with the typical cell stimulation by agonist (19) the reason for the peak increase in IP2 and IP3 after 30 minutes of agonist stimulation is not clear. It is possible that the second peak in IP2 and IPj levels might be caused by an increase in protein kinase C activity, due to endogenous metabolites formed after phosphatidylinositol hydrolysis. On the other hand, accumulation of total ['H]inositol monophosphate (IP~) was not detectable during the first 15 minutes after the addition of albumin. After this lag period, IP, showed a continuous and large accumulation (Fig. 1), which suggests the continuous hydrolysis of inositol phosphates for 60 minutes. However the data in Fig. 1 cannot exclude the possibility that some IP~ might be produced by direct action of specific phospholipase C on phosphatidylinositol phosphates. Unstimulated platelets contain phosphatidylinositol (PI), phosphatidylinositol-4-p (PIP) and phosphatidylinositol-4,5-p2 (PIP~) in a ratio of 13.8:2.3:1 respectively. When platelets are stimulated with agonist there is a 50% breakdown of PI, and no change in PIP and a transient fall in PIP2 occurs for 90 seconds (20). Because all these phosphatidylinositol phosphates are substrates for phospholipase C enzymes (21), there is a good possibility that the progressive accumulation of IP, is due to increased hydrolysis of PI. In agreement with this concept it was observed that HSA activated phospholipase C in platelet cytosol, using PI as the substrate (Table I). Incubation of platelets with HSA at a concentration of 4 gm/dl for 10 seconds increased phospholipase C activity by 254%. After 15 and 30 minutes of incubation these values were 292% and 414% respectively.
Vol.
49, No. 23, 1991
E f f e c t of A l b u m i n
on I n o s i t i d e
Turnover
171]
0.3
IP2 O.R
// ~
0.1
//
0
//
3
25 0 0 0v X
d
Q. U
IP1
20
Y
15 10 $
0 3OO
Inos~ol
200,
100,
0 10 -seconds-
60 // 5
30
60
TIME (rnin) Fig. 1
Effect of human serum albumin (4gm/dl) on the accumulation of inositol phosphates in human platelets. Values shown are mean + S.E.M. for three separate experiments. Control values remained unchanged over the experiment period.
Of particular interest is the rapid and large increase in ['H]inositol to 351% by 10 seconds compared to control value in the presence of Li ÷ which blocked the dephosphorylation of inositol phosphate. Accumulation of [q-I]inositol might arise from phospholipase D activation that results from the mobilization of intracellular [Ca']i stores due to accumulation of IP, (22,23) or by the direct effect of albumin on this enzyme. For this purpose the effect of albumin on phospholipase D activity in platelets was investigated (Table II). As shown in Table II the formation phosphatidic acid in the absence of added
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E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
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49, No. 23, 1991
TABLE I Effect of HSA on Phospholipase C Activity in Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Treatment of Platelets
Incubation Period
Phospholipase C Activity (pmoles/15 minutes/mg protein)
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Control
0 10 seconds 15 minutes 30 minutes
HSA (4gm/dl)
10 seconds 15 minutes 30 minutes
337 344 370 369
+ + + +
31 19 20 43
875 + 48* 1080 + 106" 1527 + 111"
Phospholipase C activity was measured in homogenates of platelets, previously incubated with or without HSA. The enzyme reaction was carried out as described previously (17). The values are mean + S.E.M. for four seperate experiments. * p value significantly different than control (p < 0.001). ATP, the diminution in phosphatidic acid on addition of ethanol and finally the formation of phosphatidylethanol in the presence of ethanol are all consistent with activation of phospholipase D. In control experiments the accumulation of phosphatidic acid decreased during 30 minutes of incubation whereas the formation of phosphatidylethanol increased. When platelets were incubated with albumin for 10 seconds phosphatidic acid increased by 224% whereas the accumulation of phosphatidylethanol rose by 182%. In the presence of ethanol the increases in phosphatidic acid and phosphatidylethanol were 159% and 250% respectively. These effects could be seen over the 30 minutes of incubation. These results confirm that phospholipase D activation takes place within seconds after adding albumin. Dose-Deoendencv of HSA-Stimulated Inositol Phosohates Accumulation in Human Platelets Dose response relationship of HSA to inositol phosphates accumulation is shown in Fig. 2. As indicated in Fig. 1, HSA concentration up to 4 gm/dl did not induce accumulation of IP~ in platelets incubated for 15 minutes (Fig. 2). While exposing platelets to 1 gm/dl HSA for 10 seconds did not induce a significant accumulation of IP2 and IP~, a maximum accumulation was observed at HSA concentration of 3 gm/dl. An incubation period of 15 minutes induced significant accumulation of IP2 and IP3 at all HSA concentrations used and gave similar accumulation data as to that obtained at 10 seconds. The percentage distribution of radioactivity between the inositol phosphates in washed prelabeled platelets and exposed to various concentrations of HSA is shown in Table III. Increasing the concentration of HSA changed the distribution of radioactivity between the inositol phosphates towards the production of IP,. Exposing washed platelets to HSA (4 grn/dl) for 10 seconds increased the accumulation ot IP3 3.16 fold and this reached 3.4 fold by 15 minutes. The data shown in Table III indicate that 93% of IP~ accumulated within the first 10 seconds.
Vol.
49, No. 23,
1991
E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
1713
TABLE II Effect of HSA on Phospholipase D Activity in (q-I) Glycerol-Labeled Platelets .
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Incubation Treatment Period .
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Control
HSA 4 gm/dl (Final Concentration)
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0 10 10 15 15 30 30 10 10 15 15 30 30
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10'x(~H) PA d.p.m./mg of Protein
Incubation Conditions .
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seconds seconds minutes minutes minutes minutes
None 0 Ethanol 0 Ethanol 0 Ethanol
7.7 8.5 6.6 6.1 5.0 3.5 2.9
seconds seconds minutes minutes minutes minutes
0 Ethanol 0 Ethanol 0 Ethanol
19.1 10.5 16.0 11.1 20.1 15.2
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10~x(q-I)PEt d.p.m./mg of Protein .
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_+ 0.98 + 0.45 + 0.80 _+ 0.84 + 0.72 + 0.58 + 0.43 + 1.21• _+ 1.13" + 1.22"* _+ 0.92, _+ 2.19" + 1.96"*
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0.64 0.67 1.04 1.36 1.94 2.28 3.23
_+ 0.09 + 0.07 + 0.06 + 0.14 + 0.31 + 0.21 + 0.31
1.22 2.60 1.67 3.58 3.13 5.55
+ _+ _+ _+ + +
0.11" (129'* 018"* 056" 0.58 0.33*
Values are mean + S.E.M. for four separate experiments. Platelets were labeled with (3H) glycerol and incubated with or without HSA as described in Methods. The final concentration of ethanol was 300 mM. ~(H) phosphatidic acid (PA) and "(H) phosphatidylethanol (PEt) were isolated and counted for radioactivity as described previously (18). * significantly different from corresponding control values (p < 0.05) ** significantly different from corresponding control values (p < 0.01) • significantly different from corresponding control values (p < 0.002)
Soecificitv of HSA Effect in Accumulating In0sitol Phosphates in Human Platelets Albumins obtained from various sources were used to examine the specificity of phosphatidylinositide hydrolysis in platelets by HSA. As shown in Table IV, HSA prepared by Sigma or Calbiochem, significantly elevated inositol and inositol phosphates (IP2 and IP,) in human platelets when exposed to albumin solutions for 10 seconds or 15 minutes. As expected IPj levels did not change significantly. Upon the addition of ovalbumin there was no significant increase in the levels of [~H]inositol trisphosphate. However, a slight increase in [q-I]inositol bisphosphate was observed. The data in Table IV is in agreement with the effect of albumin on neonatal rat pancreatic islets cell culture. HSA and BSA but not ovalbumin induced a significant accumulation of the inositol phosphates (24). ~'Ca ÷~ Studv Ca ÷2 release from intracellular stores appears to be mediated by inositol trisphosphate (23,25). It has been shown that IP~ releases Ca ÷2 from membrane vesicles enriched in plasma membranes of platelets (26). This indicates that IP~ generates Ca ÷2 influx in platelets which in turn results in an increase in free cytosolic ([Ca+2],) and subsequent efflux of Ca ÷2 from the cell (27,28). HSA and BSA which increase the accumulation of IP~ also
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E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
Vol.
49, No. 23, 199]
IP3 ,ix
' T
•
O v
d O
01
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150
IP1 lr .11,
100
60
0
o.o
,'.o
21o
31o
2.0
HSA (gm/dl)
Fig. 2 Dose-dependency of HSA-stimulated inositol phosphates accumulation in human platelets. Cells were exposed to different HSA concentrations for 10 seconds (o---n) and 15 minutes (o---e). Values shown are mean + S.E.M. for three separate experiments. * Significantly different from control (p < 0.05).
Vol.
49, No. 23, 1991
E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
1715
TABLE III Distribution of Myo(~H)inositol Within Inositol Phosphates in Human Platelets in Response to Different Concentrations of HSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
HSA gm/dl
10 Seconds IP~ .
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IP2 .
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IP~
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None
98.34+1.97
1.16+0.02
0.49+0.03
1
98.09+0.98
1.36+0.04"
0.62+0.03"
2
97.57+0.98
1.39+0.03"
1.04+0.10"
3
96.81+0.97
1.70+0.11"
1.48+0.10"
4
96.77+0.97
1.67+0.03"
1.55+0.10"
HSA gm/dl .
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15 Minutes .
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IPI .
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IP~ .
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IP3 .
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None
97.83+0.98
1.64+0.03
0.48+0.03
1
96.32+0.96
2.95+0.20"
0.73+0.03"
2
95.45+0.95
3.25+0.20*
1.30+0.05"
3
94.49+0.94"
4.04+0.30"
1.47+0.066"
4
94.56+0.95"
3.81+0.35"
1.63+0.08"
The data are presented as percentage of total inositol phosphates and the values are mean + S.E.M. for three separate experiments. Platelets were labeled with myo(~H) inositol, washed and resuspended in Tyrode's-Hepes buffer and exposed to various concentrations of HSA. The amount of label incorporated into inositol phosphates was determined as described under "Materials and Methods". * Significantly different from control (p < 0.05).
induced a significant increase in the release of OCa÷2from platelets as shown in Fig. 3. The fact that ovalbumin increased '~Ca÷2 release in platelets without affecting IPs levels emphasizes the fact that alterations in IP~ concentrations cannot explain all features of Ca+~-dependent signalling (29). However, it must be noted that we have not determined intracellular pools of calcium and free cytosolic calcium.
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Vol.
49, No. 23, 1991
TABLE IV Effect of Albumin from Various Sources on the Accumulation of Inositol and Inositol Phosphates in Human Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Addition
Levels of Inositol Metabolites (c.p.m. x 1000)
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Inositol
IP~
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10" .
None
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15' .
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61.1 +__3.3
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10"
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15' .
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66.8+2.1
9.7+0.1
9.9+0.2
HSA(Sigma)
214.3+5.7"
227.5+1.4"
9.8+0.8
10.2+0.2
HSA(Calbio)
225.7+13.9"
228.4+14.1"
9.7+0.3
9.2+0.3
BSA
252.2+0.5*
237.3+1.1"
10.2+0.3
10.1+0.1
45.0+1.4"
45.5+0.7*
9.3+0.3
10.8+0.1
Ovalb Addition
IP2
IP3
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10" .
.
.
.
.
15' .
.
.
.
.
.
.
.
.
.
.
.
.
10" .
None
143+8
153+7
HSA(Sigma)
194+__20"
HSA(Calbio)
.
.
.
.
.
.
.
.
.
.
.
.
15'
.
46+1
54+6
225+17"
100+4"
110+9"
221+24"
252 +___25"
126+6"
122+4"
BSA
232+10"
291+17"
107+17"
94+2*
Ovalb
188+19"
220+11"
57+10
45+3
Values are mean + S.E.M. for three separate experiments. Platelets were labeled with myo(q-I)inositol, washed and resuspended in Tyrode's-Hepes buffer and exposed to albumin (4 gm/dl) from various sources. The amount of label incorporated into inositol phosphates was determined as described under "Materials and Methods". * Significantly different from control (p <0.05).
Effect of Hydrolysis of HSA on the Acc0mulation of Inositol Phosohates in Human Platelets To gain insight into the nature of the effect of albumin on the induction of phosphatidylinositide hydrolysis, HSA was subjected to chemical and enzymatic hydrolysis prior to its addition to platelets. As shown in Table V, chemical or enzymatic hydrolysis
Vol.
49, No. 23,
1991
E f f e c t of A l b u m i n
on I n o s i t i d e
Turnover
1717
100
2o 0
.
.
.
.
.
.
°
U.
~
"~" Fig. 3
Effect of various types of albumin on "Ca 2+ release from prelabeled platelets. Values shown are mean _+S.E.M. for four separate experiments. * Significantly different from control (p <0.05).
abolished the effect of HSA on inositol phosphate accumulation in human platelets. Data in Table V indicates that the intact polypeptide structure is needed for HSA to exert its effect in platelets and eliminates the possibility that this effect may be mediated through fatty acids bound to albumin molecule (30). It is possible that polypeptide contaminants in albumin may mediate this effect in platelets (31). But in another study, performed in this laboratory, albumin, dialyzed against acetic acid reproduced effects similar to the present study on phosphatidylinositide hydrolysis in neonatal rat pancreatic islet cultures (24). It is important to consider the fact that the platelets circulating in blood are constantly exposed to albumin. Walsh et al (32) demonstrated that albumin density-gradient washing procedure had a beneficial effect on platelet morphology. These authors suggested that albumin gradient protected the cells from disruption by centrifugal forces. It is possible that platelets when suspended in albumin-free buffer for a period of time might demonstrate the effects on phosphoinositide hydrolysis upon reexposure to this protein. Although our studies indicate that albumin may affect phosphatidylinositide hydrolysis, further confirmation awaits the demonstration of a specific receptor for albumin in these cells and investigation of albumin effect on various aspects of platelet functions.
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E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
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TABLE V Effect of Hydrolyzed HSA on the Accumulation of Inositol Phosphates in Human Platelets Addition ................................................................................................ IPl IP2 IP~ .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
None
8120+132
168+7
HSA (4 gm/dl)
8090+179
273+16"
HSA + PRONASE (4 gm/dl)
8254+279
159+8
HSA + N a O H .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
8211+251 .
.
.
.
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.
.
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.
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.
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.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
51+5 132+12" 62+9
151+9 .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
54+3 .
.
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.
.
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.
.
.
.
.
Values are mean + S.E.M. for three separate experiments. Platelets were labeled with myo['H]inositol, washed and resuspended in Tyrode's-Hepes buffer and exposed to albumin. Pronase was incubated with albumin solution at 37 ° C for 30 minutes as described in Methods. Pronase added to albumin solution at the end of 30 minute incubation as described in Methods was used as control. The amount of label was determined as described under "Materials and Methods". * Significantly different from control (p < 0.05).
Acknowledgements We are grateful to Ms. Pat Rattal for the expert editorial assistance. Dr. R.M. Amin was a Fulbright Scholar from the Department of Biology and Biochemistry, Birzeit University, Birzeit, West Bank, Israel. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Navy Department or the Naval Service at large. References .
2. 3. 4. 5. 6.
E.G. L A P E T I N A and P. CUATRECASAS, Biochim. Biophys. Acta. 573 394-402 (1979). S. R r I T E N H O U S E - S I M M O N S , J. Clin. Invest. 63 580-587 (1979). M.J. BROEKMAN, J.W. WARD and A.J. MARCUS, J. Clin. Invest. 66 275-283 (1980). W. SIESS and H. BINDER, FEBS Lett. 180 107-112 (1985). J.D. VICKERS, R.L. K I N L O U G H - R A T H B O N E and J.F. MUSTARD, Biochem. J. 219 25-31 (1984). A. KISHIMOTO, Y. TAKAI, T. MORI, U. KIKKAWA and Y. NISHIZUKA, J. Biol. Chem. 255 2273-2276 (1980).
Vo1.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
49, No. 23, 1991
E f f e c t of A l b u m i n
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P.W. MAJERUS, The Molecular Basis of Blood Diseases, G. Stamatoyannopoulos, A.W. Nienhuis, P. l_~der and P.W. Majerus (eds), 689, W.B. Saunders Co. (1987). R.K. OCKNER, R.A. WEISIGER and J.L. GOLLAN, Amer. J. Physiol. 245 G13G18 (1983). R. BRANDES, R.K. OCKNER, R.A. WEISIGER and N. LYSENKO, Biochem. Biophys. Res. Commun. 105 821-827 (1982). A.W. WOLKOFF, Hepatology. 7 777-779 (1987). R.G. REED and C.M. BURRINGTON, J. Biol. Chem. 264 9867-9872 (1989). M. KOBAYASHI and J.N. KANFER, J. Neurochem. 48 1597-1603 (1987). E.G. LAPETINA and W. SIESS, Methods in Enzymol. 141 176-192 (1987). R.F. CHEN, J. Biol. Chem. 242 173-181 (1967). O.H. LOWRY, N.J. ROSENBROUGH, A.L. FARR and R.J. RANDAL Methods in Enzymology, S.P. Colowick and N.O. Kaplan (eds), Vol 111 448, Academic Press, New York (1957). M.J. BERRIDGE, C.P. DOWNES and M.R. HANLEY, Biochem. J. 206 587-595 (1982). Y. BANNO, A. YU, T. NAKASHIMA, Y. HOMMA, T. TAKENAWA, and Y. NOZAWA, Biochem. Biophys. Res. Commun. 167 396-401 (1990). J. VAN DER MEULEN and R.J. HASLAM, Biochem. J. 271 693-700 (1990). S.B. CHAHWALA, L.F. FLEISCHMAN and L. CANTLEY, Biochemistry. 26 612622 (1987). P.W. MAJERUS, D.B. WILSON, T.M. CONNOLLY, T.E. BROSS and E.J. NEUFELD, Adv. Prostaglandin Thromboxane Leukotriene Res. 15 109-112 (1985). D.B. WILSON, T.E. BROSS, S.L. HOFMANN and P.W. MAJERUS, J. Biol. Chem. 259 11718-11724 (1984). J.H. EXTON, J. Biol. Chem. 265 1-4 (1990) M.J. BERRIDGE and R.F. IRVINE, Nature. 312 315-321 (1984). J.H. HAYS, M. FLETCHER, R. AMIN and K.M.M. SHAKIR, Diabetes. 40(Suppl) 237A (1991). J.R. WlLLIAMSON, R.H. COOPER, S.K. JOSEPH and A.P. THOMAS, Am. J. Physiol. 248 C203-C216 (1985). A. RENGASAMY and H. FEINERG, Biochem. Biophys. Res. Commun. 150 10211026 (1988). S.J. PANDOL, M.S. SCHOEFFIELD, G. SACHS and S. MUALLEM, J. Biol. Chem. 260 10081-10086 (1985). D.L. OCHS, J.I. KORENBROT and J.A. WILLIAMS, Am. J. Physiol. 249 G389G398 (1985). O.H. PETERSEN, Cell. Calcium. 10 375-383 (1989). R.F. IRVINE, A.J. LETCHER and R.M.C. DAWSON, Biochem J. 178 497-500 (1979). A.L. RUBIN, G.D. LUBASH, R.F. ARONSON and P.F. DAVISON, Nature. 197 1009-1010 (1963). P.N. WALSH, D.C.B. MILLS and J.G. WHITE, Br. J. Haemato.1 36 281-296 (1977).
Pergamon Press
PHOSPHOINOSITIDE TURNOVER IN HUMAN PLATELETS IS STIMULATED BY ALBUMIN Riyad M. Amin, James H. Hays and K. M. Mohamed Shakir Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland. (Received in final form September 26, 1991)
Summary The effect of human serum albumin (HSA) on the hydrolysis of phosphatidylinositides in human platelets labeled with myo(q-I)inositol was studied. Incubation of platelets with HSA (4 gm/dl) for 10 seconds increased IP~ and IP~ by 169% and 217% respectively. 93% of IP~ accumulated within the first 10 seconds. This effect was also shared by bovine serum albumin, although no changes in IP~ levels occurred with ovalbumin. All albumin species used induced 'SCa*~release from platelets irrespective of its effect on IP~ accumulation. These findings indicate that albumin may function in biological systems by inducing intracellular signaling. Platelets from humans demonstrate rapid changes in membrane phospholipid content in response to physiologic stimuli (1-4). The stimulation of platelets is associated with rapid generation of inositol-l,4,5-trisphosphate (IP,) (5), and an increase of Ca 2÷ in the cytoplasm (6). This effect is in close association with the physiologic platelet response (7). Human serum albumin has been shown to bind specifically, reversibly and saturably to the cell surface (8,9), but the presence of an albumin receptor has been a subject of continuing controversy (10,11). In this study, we present the effect of human serum albumin on the hydrolysis of phosphoinositides in human platelets. Materials and Methods Materials L-Myo[2-SH(N)]inositol (myo(q-I)inositol) (16 Ci/mmol), 'H glycerol, phosphatidylinositol [myo-inositol-2-q-I(N)]-, radiolabeled inositol phosphate standards and '~Ca2÷ (12.5mCi/mg) were purchased from New England Nuclear Research Products, Dupont Company, Boston, Ma. Dowex AG-1-X8 (formate form, 100-200 mesh) was from Bio-Rad Laboratories, Richmond, Va. Fatty acid-free HSA fraction V, BSA, ovalbumin, phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid were purchased from Sigma Chemical Co., St. Louis, Mo. HSA fraction V was obtained from Calbiochem Corporation, San Diego, Ca. Phosphatidylethanol was prepared from egg lecithin (12). Address Correspondence to CAPT K.M.M. Shakir, MC, USN, Division of EndocrinologyMetabolism, Department of Medicine, National Naval Medical Center, Bethesda, Maryland 20889-5000. 0024-3205/91 $3.00 + .00
1708
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ACD anticoagulant was obtained from Baxter Corporation, Deerfield, I1. chemicals and reagents were of the highest commercially available grade.
All other
Preparation of Platelets Fresh platelet concentrates, suspended in ACD anticoagulant and obtained from the blood bank of the National Institutes of Health, Bethesda, Md. were centrifuged at 200xg for 15 minutes at 22°C to remove residual erythrocytes and leukocytes. All of the subsequent handling of the platelets were performed at 22 ° C unless otherwise mentioned. The platelet-rich plasma was then centrifuged at 2000xg for 20 minutes. The platelet pellet was washed three times with a modified Tyrode's-Hepes (N-2-hydroxyethyl-piperazine-N2-ethanesulfonic acid) buffer (134 mM NaC1, 12 mM NaHCO~, 2.9 mM KC1, 0.36 mM NaH:PO,, 1 mM MgCI:, 5 mM glucose; pH 7.4) containing 500 ng/ml prostacyclin and 0.6 ADPase U/ml apyrase. The platelet pellet was finally resuspended in the same buffer containing apyrase and adjusted to a final concentration of lxl0 ~ platelets/ml. Labelin~ of Platelets with Mvo('H)inositol Platelets were labeled as described previously (13). Briefly, platelets were incubated with 1.0 mCi/ml myo(q-I)inositol in the presence of 2 mM MnCI~ for four hours at 37°C under oxygen. The labeled platelets were then centrifuged, washed and resuspended in Tyrode's-Hepes buffer containing 10 mM LiCI. Delipidation of Albumin All albumin solutions including fatty acid-free albumin were delipidated as previously described (14). Delipidated albumin solutions were adjusted to the desired concentration using protein determination by the Lowry method (15). Fatty acids in delipidated albumin solutions were not detectable by HPLC. Modification of Albumin by Treatment with Pronase and NaOH Albumin was dissolved in Tyrode's-Hepes buffer and incubated at a ratio of 0.1 mg pronase to 10 mg albumin at 37 °C for 30 minutes. At the end of the incubation period enzyme activity was inactivated by heating the albumin solution at 55°C for 5 minutes. In control experiments albumin solution was incubated for an identical period and pronase was added at the end of incubation time and following this, pronase was inactivated. Experiments were performed to determine the quantity of protein hydrolyzed by pronase and for this purpose an aliquot of the pronase-treated and control albumin solution was treated with trichloroacetic acid (40%), and the protein precipitate was removed by centrifugation. The precipitate was dissolved in 0.05 N NaOH and protein was determined by the method of Lowry et al (15). The pronase treatment hydrolyzed almost 95% of albumin. Hydrolysis of albumin by NaOH was performed by dissolving in 6 N NaOH and incubating at room temperature for 16 hours. Following this, the pH was adjusted to 7.0 with concentrated HCI. The albumin was then dialyzed extensively against distilled water and subsequently lyophilized. In control experiments albumin was dissolved in distilled water and incubated at room temperature for 16 hours. At the end of incubation an equivalent amount of 6 N NaOH previously neutralized with HCI was added and subsequently dialyzed. The protein hydrolysis by NaOH was verified (15) as described for pronase-treatment and this method hydrolyzed albumin by 96%.
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E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
1709
Evaluation of Phosphoinositid¢ Hvdrolvsis Samples (0.2 ml) of washed platelets at a concentration of 10° platelets/ml were incubated in 12x75 mm borosilicate glass tubes for 15 minutes at 37 °C in the absence of Ca ÷~and presence of 10 mM LiC1. Aliquots (0.2 ml) of the various albumin solutions were then added, and the incubation was continued at 37 °C while being gently stirred for the time intervals as indicated in each experiment. The reaction was terminated by adding 0.75 ml of acidic chloroform-methanol solution (1:2, v/v) followed by the addition of 0.25 ml 0.1N HCI and 0.25 ml chloroform. The tubes were subsequently mixed vigorously and centrifuged at 900xg for 20 minutes. The aqueous phase was separated and frozen at -70 ° C until inositol phosphates were determined within 48 hours. Separation of Inositol Phosphates The procedure for the separation of water soluble ['H]inositol phosphates was modified from that described by Berridge et al (16). Briefly, an aliquot (0.675 ml) of the upper aqueous phase of the chloroform-methanol extraction was applied to a glass column containing 0.5 ml of 1:1 (v/v) mixture of Dowex AG-1-X8 resin (100-200 mesh, formate form)/distilled water. The inositol phosphates were eluted by the stepwise addition of solutions containing increasing levels of formate. Free [q-I]inositol was eluted by the addition of 4 ml H~O. [q-I]Glycerophosphoinositol was eluted by the addition of 1.25 ml of 50 mM sodium formate, and 5 mM sodium tetraborate. [~H]inositol-l-phosphate was eluted by the addition of 7 ml of 200 mM ammonium formate, 100 mM formic acid. ['H]inositol bisphosphate was eluted by the addition of 6 ml of 400 mM ammonium formate, and 100 mM formic acid and [~H]inositol trisphosphate was eluted by the addition of 6 ml of 1.0 M ammonium formate, and 100 mM formic acid. Radioactivity was determined in the eluates collected directly into vials by liquid scintillation counting. The separation of inositol phosphates was verified by putting the known standards through the columns. Phospholipase C Assay Platelet suspension, in Tyrode's-Hepes buffer, containing prostacyclin and ADPase was incubated at 37 ° C with human serum albumin for 10 seconds, 15 minutes or 30 minutes. At the end of incubation, the reaction was terminated by freezing and the platelet suspension was subsequently homogenized by sonication. Phospholipase C assay, using PI as a substrate was carried out as described previously (17). Phospholipase C activity was determined by counting aliquots of the aqueous phase (17). Phospholipase D Assay Platelet-rich plasma was incubated with q-I glycerol (final concentration 100/~Ci/ml) at 37 ° C for two hours. The platelets were then isolated and suspended Ca2-free Tyrode's buffer as described previously (18). However bovine serum albumin was omitted from this buffer. Aliquots of platelet suspension were warmed to 37* C and human serum albumin was then added to give a final concentration of 4 gm/dl. Incubations were carried for 10 seconds, 15 minutes or 30 minutes. The reaction was terminated by freezing and the platelet suspension was subsequently homogenized by sonication. The extraction and determination of (q-l) phospholipids in the platelets were carried out as described previously (18).
1710
E f f e c t of A l b u m i n o n I n o s i t i d e T u r n o v e r
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"Ca ~+ Study Ten ml of platelets previously washed with modified Tyrode's-Hepes buffer, were suspended at a concentration of 10' cells/ml in Tyrode's-Hepes buffer without phosphate (pH 7.4) and labeled with "Ca 2÷ (5 /~Ci/ml) for 60 minutes at 37°C. At the end of incubation the suspension was centrifuged at 1000xg for 15 minutes. The cell pellet was washed three times at 22°C by centrifugation and resuspension with 100 volumes of Tyrode's-Hepes buffer containing no calcium or phosphate and finally resuspended in the same buffer at a concentration of 10°/ml. 0.5 ml aliquots of the platelet suspension was then incubated with 0.1 ml of albumin solution (4 gm/dl - final concentration) for four minutes at 37" C. At the end of incubation 300/~1 of platelet suspension was added to 100 ~1 silicone oil in a microfuge tube, followed by centrifugation at 10,000xg for 60 seconds and the radioactivity in the supernatant was determined by counting 100/~1 aliquots in a liquid scintillation counter. '5Ca2* release was expressed as percent of total radioactivity present in platelets at the beginning of incubation. Data Analvsis Statistical analysis was performed using the Student's t-test for paired data. The level of significance was taken as p value less than 0.05. Results and Discussion Effect of Human Serum Albumin on Time-Dependent Accumulation of Inositol Phosphates in Platelets The addition of HSA 4 gm/dl (final concentration) to washed human platelets, prelabeled with myo(3H)inositol induced the accumulation of water soluble inositol phosphates as illustrated in Fig. 1. The accumulation of total [3H]inositol bisphosphate (IP2) and [~H]inositol trisphosphate (IP~) was rapid and reached its peak levels of 169% and 217% respectively, by 10 seconds compared to control values. Both [~H]inositol phosphate levels declined by 30 seconds followed by a gradual increase which continued up to 60 minutes. The data establish that there is biphasic accumulation of ['H]inositol bisphosphate and [q-I]trisphosphate in platelets after HSA stimulation, with a peak increase occurring within 10 seconds. Although this is in agreement with the typical cell stimulation by agonist (19) the reason for the peak increase in IP2 and IP3 after 30 minutes of agonist stimulation is not clear. It is possible that the second peak in IP2 and IPj levels might be caused by an increase in protein kinase C activity, due to endogenous metabolites formed after phosphatidylinositol hydrolysis. On the other hand, accumulation of total ['H]inositol monophosphate (IP~) was not detectable during the first 15 minutes after the addition of albumin. After this lag period, IP, showed a continuous and large accumulation (Fig. 1), which suggests the continuous hydrolysis of inositol phosphates for 60 minutes. However the data in Fig. 1 cannot exclude the possibility that some IP~ might be produced by direct action of specific phospholipase C on phosphatidylinositol phosphates. Unstimulated platelets contain phosphatidylinositol (PI), phosphatidylinositol-4-p (PIP) and phosphatidylinositol-4,5-p2 (PIP~) in a ratio of 13.8:2.3:1 respectively. When platelets are stimulated with agonist there is a 50% breakdown of PI, and no change in PIP and a transient fall in PIP2 occurs for 90 seconds (20). Because all these phosphatidylinositol phosphates are substrates for phospholipase C enzymes (21), there is a good possibility that the progressive accumulation of IP, is due to increased hydrolysis of PI. In agreement with this concept it was observed that HSA activated phospholipase C in platelet cytosol, using PI as the substrate (Table I). Incubation of platelets with HSA at a concentration of 4 gm/dl for 10 seconds increased phospholipase C activity by 254%. After 15 and 30 minutes of incubation these values were 292% and 414% respectively.
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E f f e c t of A l b u m i n
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171]
0.3
IP2 O.R
// ~
0.1
//
0
//
3
25 0 0 0v X
d
Q. U
IP1
20
Y
15 10 $
0 3OO
Inos~ol
200,
100,
0 10 -seconds-
60 // 5
30
60
TIME (rnin) Fig. 1
Effect of human serum albumin (4gm/dl) on the accumulation of inositol phosphates in human platelets. Values shown are mean + S.E.M. for three separate experiments. Control values remained unchanged over the experiment period.
Of particular interest is the rapid and large increase in ['H]inositol to 351% by 10 seconds compared to control value in the presence of Li ÷ which blocked the dephosphorylation of inositol phosphate. Accumulation of [q-I]inositol might arise from phospholipase D activation that results from the mobilization of intracellular [Ca']i stores due to accumulation of IP, (22,23) or by the direct effect of albumin on this enzyme. For this purpose the effect of albumin on phospholipase D activity in platelets was investigated (Table II). As shown in Table II the formation phosphatidic acid in the absence of added
1712
E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
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49, No. 23, 1991
TABLE I Effect of HSA on Phospholipase C Activity in Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Treatment of Platelets
Incubation Period
Phospholipase C Activity (pmoles/15 minutes/mg protein)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Control
0 10 seconds 15 minutes 30 minutes
HSA (4gm/dl)
10 seconds 15 minutes 30 minutes
337 344 370 369
+ + + +
31 19 20 43
875 + 48* 1080 + 106" 1527 + 111"
Phospholipase C activity was measured in homogenates of platelets, previously incubated with or without HSA. The enzyme reaction was carried out as described previously (17). The values are mean + S.E.M. for four seperate experiments. * p value significantly different than control (p < 0.001). ATP, the diminution in phosphatidic acid on addition of ethanol and finally the formation of phosphatidylethanol in the presence of ethanol are all consistent with activation of phospholipase D. In control experiments the accumulation of phosphatidic acid decreased during 30 minutes of incubation whereas the formation of phosphatidylethanol increased. When platelets were incubated with albumin for 10 seconds phosphatidic acid increased by 224% whereas the accumulation of phosphatidylethanol rose by 182%. In the presence of ethanol the increases in phosphatidic acid and phosphatidylethanol were 159% and 250% respectively. These effects could be seen over the 30 minutes of incubation. These results confirm that phospholipase D activation takes place within seconds after adding albumin. Dose-Deoendencv of HSA-Stimulated Inositol Phosohates Accumulation in Human Platelets Dose response relationship of HSA to inositol phosphates accumulation is shown in Fig. 2. As indicated in Fig. 1, HSA concentration up to 4 gm/dl did not induce accumulation of IP~ in platelets incubated for 15 minutes (Fig. 2). While exposing platelets to 1 gm/dl HSA for 10 seconds did not induce a significant accumulation of IP2 and IP~, a maximum accumulation was observed at HSA concentration of 3 gm/dl. An incubation period of 15 minutes induced significant accumulation of IP2 and IP3 at all HSA concentrations used and gave similar accumulation data as to that obtained at 10 seconds. The percentage distribution of radioactivity between the inositol phosphates in washed prelabeled platelets and exposed to various concentrations of HSA is shown in Table III. Increasing the concentration of HSA changed the distribution of radioactivity between the inositol phosphates towards the production of IP,. Exposing washed platelets to HSA (4 grn/dl) for 10 seconds increased the accumulation ot IP3 3.16 fold and this reached 3.4 fold by 15 minutes. The data shown in Table III indicate that 93% of IP~ accumulated within the first 10 seconds.
Vol.
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1991
E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
1713
TABLE II Effect of HSA on Phospholipase D Activity in (q-I) Glycerol-Labeled Platelets .
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Incubation Treatment Period .
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Control
HSA 4 gm/dl (Final Concentration)
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0 10 10 15 15 30 30 10 10 15 15 30 30
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10'x(~H) PA d.p.m./mg of Protein
Incubation Conditions .
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seconds seconds minutes minutes minutes minutes
None 0 Ethanol 0 Ethanol 0 Ethanol
7.7 8.5 6.6 6.1 5.0 3.5 2.9
seconds seconds minutes minutes minutes minutes
0 Ethanol 0 Ethanol 0 Ethanol
19.1 10.5 16.0 11.1 20.1 15.2
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.
10~x(q-I)PEt d.p.m./mg of Protein .
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.
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.
_+ 0.98 + 0.45 + 0.80 _+ 0.84 + 0.72 + 0.58 + 0.43 + 1.21• _+ 1.13" + 1.22"* _+ 0.92, _+ 2.19" + 1.96"*
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.
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.
0.64 0.67 1.04 1.36 1.94 2.28 3.23
_+ 0.09 + 0.07 + 0.06 + 0.14 + 0.31 + 0.21 + 0.31
1.22 2.60 1.67 3.58 3.13 5.55
+ _+ _+ _+ + +
0.11" (129'* 018"* 056" 0.58 0.33*
Values are mean + S.E.M. for four separate experiments. Platelets were labeled with (3H) glycerol and incubated with or without HSA as described in Methods. The final concentration of ethanol was 300 mM. ~(H) phosphatidic acid (PA) and "(H) phosphatidylethanol (PEt) were isolated and counted for radioactivity as described previously (18). * significantly different from corresponding control values (p < 0.05) ** significantly different from corresponding control values (p < 0.01) • significantly different from corresponding control values (p < 0.002)
Soecificitv of HSA Effect in Accumulating In0sitol Phosphates in Human Platelets Albumins obtained from various sources were used to examine the specificity of phosphatidylinositide hydrolysis in platelets by HSA. As shown in Table IV, HSA prepared by Sigma or Calbiochem, significantly elevated inositol and inositol phosphates (IP2 and IP,) in human platelets when exposed to albumin solutions for 10 seconds or 15 minutes. As expected IPj levels did not change significantly. Upon the addition of ovalbumin there was no significant increase in the levels of [~H]inositol trisphosphate. However, a slight increase in [q-I]inositol bisphosphate was observed. The data in Table IV is in agreement with the effect of albumin on neonatal rat pancreatic islets cell culture. HSA and BSA but not ovalbumin induced a significant accumulation of the inositol phosphates (24). ~'Ca ÷~ Studv Ca ÷2 release from intracellular stores appears to be mediated by inositol trisphosphate (23,25). It has been shown that IP~ releases Ca ÷2 from membrane vesicles enriched in plasma membranes of platelets (26). This indicates that IP~ generates Ca ÷2 influx in platelets which in turn results in an increase in free cytosolic ([Ca+2],) and subsequent efflux of Ca ÷2 from the cell (27,28). HSA and BSA which increase the accumulation of IP~ also
1714
E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
Vol.
49, No. 23, 199]
IP3 ,ix
' T
•
O v
d O
01
.
.
.
.
150
IP1 lr .11,
100
60
0
o.o
,'.o
21o
31o
2.0
HSA (gm/dl)
Fig. 2 Dose-dependency of HSA-stimulated inositol phosphates accumulation in human platelets. Cells were exposed to different HSA concentrations for 10 seconds (o---n) and 15 minutes (o---e). Values shown are mean + S.E.M. for three separate experiments. * Significantly different from control (p < 0.05).
Vol.
49, No. 23, 1991
E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
1715
TABLE III Distribution of Myo(~H)inositol Within Inositol Phosphates in Human Platelets in Response to Different Concentrations of HSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
HSA gm/dl
10 Seconds IP~ .
.
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.
.
IP2 .
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.
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.
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.
IP~
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.
.
.
.
.
.
None
98.34+1.97
1.16+0.02
0.49+0.03
1
98.09+0.98
1.36+0.04"
0.62+0.03"
2
97.57+0.98
1.39+0.03"
1.04+0.10"
3
96.81+0.97
1.70+0.11"
1.48+0.10"
4
96.77+0.97
1.67+0.03"
1.55+0.10"
HSA gm/dl .
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15 Minutes .
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IPI .
.
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.
IP~ .
.
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.
.
.
.
.
.
.
.
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.
.
IP3 .
.
.
.
.
.
.
.
.
None
97.83+0.98
1.64+0.03
0.48+0.03
1
96.32+0.96
2.95+0.20"
0.73+0.03"
2
95.45+0.95
3.25+0.20*
1.30+0.05"
3
94.49+0.94"
4.04+0.30"
1.47+0.066"
4
94.56+0.95"
3.81+0.35"
1.63+0.08"
The data are presented as percentage of total inositol phosphates and the values are mean + S.E.M. for three separate experiments. Platelets were labeled with myo(~H) inositol, washed and resuspended in Tyrode's-Hepes buffer and exposed to various concentrations of HSA. The amount of label incorporated into inositol phosphates was determined as described under "Materials and Methods". * Significantly different from control (p < 0.05).
induced a significant increase in the release of OCa÷2from platelets as shown in Fig. 3. The fact that ovalbumin increased '~Ca÷2 release in platelets without affecting IPs levels emphasizes the fact that alterations in IP~ concentrations cannot explain all features of Ca+~-dependent signalling (29). However, it must be noted that we have not determined intracellular pools of calcium and free cytosolic calcium.
1716
E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
Vol.
49, No. 23, 1991
TABLE IV Effect of Albumin from Various Sources on the Accumulation of Inositol and Inositol Phosphates in Human Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Addition
Levels of Inositol Metabolites (c.p.m. x 1000)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Inositol
IP~
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10" .
None
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15' .
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61.1 +__3.3
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10"
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15' .
.
66.8+2.1
9.7+0.1
9.9+0.2
HSA(Sigma)
214.3+5.7"
227.5+1.4"
9.8+0.8
10.2+0.2
HSA(Calbio)
225.7+13.9"
228.4+14.1"
9.7+0.3
9.2+0.3
BSA
252.2+0.5*
237.3+1.1"
10.2+0.3
10.1+0.1
45.0+1.4"
45.5+0.7*
9.3+0.3
10.8+0.1
Ovalb Addition
IP2
IP3
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10" .
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15' .
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10" .
None
143+8
153+7
HSA(Sigma)
194+__20"
HSA(Calbio)
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.
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.
.
15'
.
46+1
54+6
225+17"
100+4"
110+9"
221+24"
252 +___25"
126+6"
122+4"
BSA
232+10"
291+17"
107+17"
94+2*
Ovalb
188+19"
220+11"
57+10
45+3
Values are mean + S.E.M. for three separate experiments. Platelets were labeled with myo(q-I)inositol, washed and resuspended in Tyrode's-Hepes buffer and exposed to albumin (4 gm/dl) from various sources. The amount of label incorporated into inositol phosphates was determined as described under "Materials and Methods". * Significantly different from control (p <0.05).
Effect of Hydrolysis of HSA on the Acc0mulation of Inositol Phosohates in Human Platelets To gain insight into the nature of the effect of albumin on the induction of phosphatidylinositide hydrolysis, HSA was subjected to chemical and enzymatic hydrolysis prior to its addition to platelets. As shown in Table V, chemical or enzymatic hydrolysis
Vol.
49, No. 23,
1991
E f f e c t of A l b u m i n
on I n o s i t i d e
Turnover
1717
100
2o 0
.
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.
.
.
.
°
U.
~
"~" Fig. 3
Effect of various types of albumin on "Ca 2+ release from prelabeled platelets. Values shown are mean _+S.E.M. for four separate experiments. * Significantly different from control (p <0.05).
abolished the effect of HSA on inositol phosphate accumulation in human platelets. Data in Table V indicates that the intact polypeptide structure is needed for HSA to exert its effect in platelets and eliminates the possibility that this effect may be mediated through fatty acids bound to albumin molecule (30). It is possible that polypeptide contaminants in albumin may mediate this effect in platelets (31). But in another study, performed in this laboratory, albumin, dialyzed against acetic acid reproduced effects similar to the present study on phosphatidylinositide hydrolysis in neonatal rat pancreatic islet cultures (24). It is important to consider the fact that the platelets circulating in blood are constantly exposed to albumin. Walsh et al (32) demonstrated that albumin density-gradient washing procedure had a beneficial effect on platelet morphology. These authors suggested that albumin gradient protected the cells from disruption by centrifugal forces. It is possible that platelets when suspended in albumin-free buffer for a period of time might demonstrate the effects on phosphoinositide hydrolysis upon reexposure to this protein. Although our studies indicate that albumin may affect phosphatidylinositide hydrolysis, further confirmation awaits the demonstration of a specific receptor for albumin in these cells and investigation of albumin effect on various aspects of platelet functions.
1718
E f f e c t of A l b u m i n on I n o s i t i d e T u r n o v e r
Vol.
49, No. 23, 1991
TABLE V Effect of Hydrolyzed HSA on the Accumulation of Inositol Phosphates in Human Platelets Addition ................................................................................................ IPl IP2 IP~ .
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None
8120+132
168+7
HSA (4 gm/dl)
8090+179
273+16"
HSA + PRONASE (4 gm/dl)
8254+279
159+8
HSA + N a O H .
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8211+251 .
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51+5 132+12" 62+9
151+9 .
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54+3 .
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.
Values are mean + S.E.M. for three separate experiments. Platelets were labeled with myo['H]inositol, washed and resuspended in Tyrode's-Hepes buffer and exposed to albumin. Pronase was incubated with albumin solution at 37 ° C for 30 minutes as described in Methods. Pronase added to albumin solution at the end of 30 minute incubation as described in Methods was used as control. The amount of label was determined as described under "Materials and Methods". * Significantly different from control (p < 0.05).
Acknowledgements We are grateful to Ms. Pat Rattal for the expert editorial assistance. Dr. R.M. Amin was a Fulbright Scholar from the Department of Biology and Biochemistry, Birzeit University, Birzeit, West Bank, Israel. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Navy Department or the Naval Service at large. References .
2. 3. 4. 5. 6.
E.G. L A P E T I N A and P. CUATRECASAS, Biochim. Biophys. Acta. 573 394-402 (1979). S. R r I T E N H O U S E - S I M M O N S , J. Clin. Invest. 63 580-587 (1979). M.J. BROEKMAN, J.W. WARD and A.J. MARCUS, J. Clin. Invest. 66 275-283 (1980). W. SIESS and H. BINDER, FEBS Lett. 180 107-112 (1985). J.D. VICKERS, R.L. K I N L O U G H - R A T H B O N E and J.F. MUSTARD, Biochem. J. 219 25-31 (1984). A. KISHIMOTO, Y. TAKAI, T. MORI, U. KIKKAWA and Y. NISHIZUKA, J. Biol. Chem. 255 2273-2276 (1980).
Vo1.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
49, No. 23, 1991
E f f e c t of A l b u m i n
on I n o s i t i d e
Turnover
1719
P.W. MAJERUS, The Molecular Basis of Blood Diseases, G. Stamatoyannopoulos, A.W. Nienhuis, P. l_~der and P.W. Majerus (eds), 689, W.B. Saunders Co. (1987). R.K. OCKNER, R.A. WEISIGER and J.L. GOLLAN, Amer. J. Physiol. 245 G13G18 (1983). R. BRANDES, R.K. OCKNER, R.A. WEISIGER and N. LYSENKO, Biochem. Biophys. Res. Commun. 105 821-827 (1982). A.W. WOLKOFF, Hepatology. 7 777-779 (1987). R.G. REED and C.M. BURRINGTON, J. Biol. Chem. 264 9867-9872 (1989). M. KOBAYASHI and J.N. KANFER, J. Neurochem. 48 1597-1603 (1987). E.G. LAPETINA and W. SIESS, Methods in Enzymol. 141 176-192 (1987). R.F. CHEN, J. Biol. Chem. 242 173-181 (1967). O.H. LOWRY, N.J. ROSENBROUGH, A.L. FARR and R.J. RANDAL Methods in Enzymology, S.P. Colowick and N.O. Kaplan (eds), Vol 111 448, Academic Press, New York (1957). M.J. BERRIDGE, C.P. DOWNES and M.R. HANLEY, Biochem. J. 206 587-595 (1982). Y. BANNO, A. YU, T. NAKASHIMA, Y. HOMMA, T. TAKENAWA, and Y. NOZAWA, Biochem. Biophys. Res. Commun. 167 396-401 (1990). J. VAN DER MEULEN and R.J. HASLAM, Biochem. J. 271 693-700 (1990). S.B. CHAHWALA, L.F. FLEISCHMAN and L. CANTLEY, Biochemistry. 26 612622 (1987). P.W. MAJERUS, D.B. WILSON, T.M. CONNOLLY, T.E. BROSS and E.J. NEUFELD, Adv. Prostaglandin Thromboxane Leukotriene Res. 15 109-112 (1985). D.B. WILSON, T.E. BROSS, S.L. HOFMANN and P.W. MAJERUS, J. Biol. Chem. 259 11718-11724 (1984). J.H. EXTON, J. Biol. Chem. 265 1-4 (1990) M.J. BERRIDGE and R.F. IRVINE, Nature. 312 315-321 (1984). J.H. HAYS, M. FLETCHER, R. AMIN and K.M.M. SHAKIR, Diabetes. 40(Suppl) 237A (1991). J.R. WlLLIAMSON, R.H. COOPER, S.K. JOSEPH and A.P. THOMAS, Am. J. Physiol. 248 C203-C216 (1985). A. RENGASAMY and H. FEINERG, Biochem. Biophys. Res. Commun. 150 10211026 (1988). S.J. PANDOL, M.S. SCHOEFFIELD, G. SACHS and S. MUALLEM, J. Biol. Chem. 260 10081-10086 (1985). D.L. OCHS, J.I. KORENBROT and J.A. WILLIAMS, Am. J. Physiol. 249 G389G398 (1985). O.H. PETERSEN, Cell. Calcium. 10 375-383 (1989). R.F. IRVINE, A.J. LETCHER and R.M.C. DAWSON, Biochem J. 178 497-500 (1979). A.L. RUBIN, G.D. LUBASH, R.F. ARONSON and P.F. DAVISON, Nature. 197 1009-1010 (1963). P.N. WALSH, D.C.B. MILLS and J.G. WHITE, Br. J. Haemato.1 36 281-296 (1977).