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Thromboxane A2 (TXA2) formation by washed human platelets under the influence of low and high density lipoproteins from healthy donors
Prostaglandins,
Leukotrienes
and Medicine
23: 303-300, 1986
THROMBOXANE A2 (TXA2) FORMATIONBY WASHEOHUMANPLATELETS UNDER THE INFLUENCE OF LOWAN0 HIGH DENSITY LIPOPROTEINS FROM HEALTHYDONORS and H.-J. Mest J. Beitz Department of Pharmacology and Toxicology, Martin Luther University Halle-Wittenberg, 4020 Halle, GOR (reprint requests to HJM)
ABSTRACT Low density lipoprotein (LOL) stimulates TXA formation (measured as malondialdehyde) in washe human platelets with active cyclooxygenase, whereas ‘high density lipoprotein (HOL) does not show any influence on TXA2 formation in this system. These results’support the hypothesis that the proaggregatory action of a high plasma LOL concentration may be mediated by modification in the biosynthesis of eicosanoids. The mode of action of lipoproteins is discussed. INTRODUCTION The presence of high levels of low density lipoprotein (LOL) and low concentrations of high density lipoprotein (HOL) is generally accepted as a risk factor for the develppment of atherosclerosis (l)-. On the other hand the development of atherosclerosis may be associated with a modified formation of prostaglandin I (PGI > and TXA (2, 3). Recently it was suggested that betgeen these modif?cations in the metabolism of eicosanoids and of lipoproteins close LOL inhibited the formation of the connections may exist. it ;fti; gregatory PG12 (4, 51, whereas HOL stimulated LDL or HDL the question arises as to whether B. From this alGo influence the formation of the physiological antagonist the purpose of this the TXA Therefore of PGI study 8;s to invgstigate the influence of LDL and HOL on TXA formation from exogenous prostaglandin H2 (PGH2) by was z ed human platelets. 303
METHODS Lipoproteins were prepared from venous blood of healthy humans by ultracentrifugation as described earlier (4). All donors had normal lipid values, and drugs influencing platelet aggregation were not given for one week before taking blood. Immediately before the experiment the lipoproteins were ultrafiltrated redissolved in 0.9 % NaCl and brought again to the experimental concentration.
Venous blood was taken from healthy volunteers. The titrated blood was divided into two equal parts. The first concentrawas incubated with acetylsalicylic acid (final tion: 1 mmol/l) and allowed to stand at room temperature for 30 minutes. The second part also stood for 30 minutes at room temperature without addition of any effector. Thereafter the platelets were separated according to the method of Dkuma (7) and concentrated to 6 x 10 platelets per ml. PGH2 was prepared according to (8). Inbubation conditions: 330 ul of platelet solution x 10 platelets) and 330 pl 0.05 mol/l Tris-HCl buffer pH 7.4 were mixed with 330 ul of 0.9 % NaCl (controls), 330 ul of 0.9 % NaCl with fat-free human serum albumin (HSA) or 330 ul 0.9 % NaCl with lipoproteins, respectively, and preincubated for 15 min at 37 C. The reaction was started by addition of 160 ng PGH2 in 40 pl solution (4 pl absolute acetone containing the PGH and 36 pl of 0.9 % NaCl, mixed immediately before addin 8 to the incuAfter an incubation time of 3 min, the bation fluid). reaction was terminated by acidification with 0.8 ml of 20 % trichloroacetic acid containing 5 % (w/v> SnC12. The amount of malondialdehyde formed by thromboxane synthetase, without malondialdehyde (MDA) formed by thermic decomposition of hydroand endoperoxides during the reaction with thiobarbituric acid, was determined as described earlier (9). (2
t-test.
Statistics: Data were statistically A P value c 0.05 was considered
analysed by to be significant.
RESULTS The influence of LDL (0.5 - 2.0 mg LDL-cholesterol/ from ml) and HDL (0.25 - 1.0 mg HDL-cholesterol/ml) healthy volunteers and HSA (1.0 - 10.0 mg/ml> on the biotransformation of exogenous PGH2 into MDA by washed human platelets is summarized in Fig. 1. HSA inhibited rn a dose dependent manner MDA formation from exogenous PGH2 by But the washed platelets with inactive cyclooxygenase. inhibition seems not to be a specific action of I-ISA, because LDL and HDL (at the same concentration, measured
304
as protein in the lipoprotein fraction) inhibited MDA formation by nearly the same extent. The isolated lipoprolein fractions (not recentrifuged) con)ained (LDL fraction) or 10.6-2.0 mg 3.2-0.6 mg protein protein (HDL fraction) per mg lipoprotein cholesterol. This inhibition ma be caused by a nonspecific adsorption of substrate (PGH2 Y or product (MDA) on the proteins.
HSA
HOI
lin nghll 1
+60
5
LDL
lin mg HDCChokStemVml) 10
025
0.5
(in mg LDL-Chdmefdlmll 10
0.5
l.0
2.0
% pFi
77
*
+30
Figure
1:
from healthy Influence of HDL and LDL taken humans and of HSA on the biotransformation prostaglandin H2 into MDA by washed human platelets
of
Open columns: Influence on platelets with inhibited cyclooxygenase. Hatched columns: Influence on platelets with active cyclooxygenase. a6 - significantly different in comparison to controls (P40.05)
305
A statistically not significant stimulation of MDA fornation was observed after incubation of 1 ng HSA with platelets with active cyclooxygenase, whereas all other investigated concentrations of HSA (5 and 10 mg/nl> inhibited MDA formation. The difference in the action of HSA on MDA formation by platelets with active versus those with inhibited cyclooxygenase is not statistically significant. The amount of MDA formed was higher in platelets with active cyclooxygenase compared to platelets with inhibited cyclooxygenase under the influence of HDL at all investigated concentrations, but the difference did not reach the level of significance. LDL (0.5 and 2.0 ng LDL-cholesterol/ml) stimulated MDA formation from exogenous with active cyclooxygenase, whereas at PGH2 by platelets 1 mg/nl only a tendency to stimulation was observed. This influence of LDL on MDA formation by platelets with active cyclooxygenase is statisticallv significantly of the same condifferent in comparison to the influence centration of LDL on MDA formation by aspirin inhibited platelets. DISCUSSION
In the literature it has been suggested that an elevated LDL concentration promotes the platelet aggregation, whereas HDL inhibits it (10, 11, 12). One important step in platelet aggregation is the formation of TXA2.With a possible modified method for the estimation of MDA it is MDA formed without to measure MDA formed by TXA synthetase and endoperoxides. by thermic decomposition of ifydroRecently this method demonstrated that LOL stimulated TXA2 formation by frozen-thawed platelets (9). In this paper we have demonstrated, that HSA, LOL or HOL at nearly the same,protein concentration decreased the detectable MDA concentration after 3 min incubation of platelets with inhibited cyclooxygenase and stimulation with PGH by nearly the same extent. It is possible that this diiinished level of MDA may be a result of a nonspecific adsorption of PGH . A different result was HSA demonstrated in platelets iith active cyclooxygenase: inhibited in a dose dependent nanner,HDL was without significant influence and LOL stimulated significantly (0.5 and 2.0 mg LDL-cholesterol/ml) MDA formation. The difference in the action of LOL on platelets with active versus inhibited cyclooxygenase is statistically significant at 1.0 and 2.0 mg LDL-cholesterol/ml, whereas the effect was not statistically diffeof HSA or HDL, respectively, rent in the two systems. This enhanced MDA formation in platelets with active cyclooxygenase may therefore not be a result of a direct influence of LDL on the TXA2 synthetase, but nay rather be a result of an activation of other steps But it is not possible to investigate in TXA formation. 2
306
which step of the arachidonic acid cascade is activated because HSA (5 or 10 mg/ml> under the influence of LDL, inhibited almost completely arachidonic acid (1.25 or 25 pmol/l)as well as thrombin (2.5 or 5 U/ml>- induced MDA formation in our systems. The influence of LDL did not depend on the sex of the donors. This is qualitatively different from the in the microsomal action of LDL on the PGI formation fraction of pig aorta (13). The mode of action of LDL on TXA2 formation is unknown, because the effect of LDL may be caused in different ways. It is known that an increased platelet cholesterol content gives rise to hypersensitive platelets in terms of response to aggregating agents (14) and, in addition these platelets produce significantly more TXA (15). Aviram and Brook (10) suggest that physiological concentrations of LDL and HDL were bound by platelets in a low affinity and calcium-independent way and enhance the platelet cholesterol content. But after our short incubation time it seems unlikely that the cholesterol from lipoproteins is transferred into the platelets. Additionally these effects of LDL were independent of a receptor mediated uptake because LDL stimulated TXA2 formation also in frozen-t.hawed possibility may be’that LDL platelets (9). Another was incorporated into the membrane and affects the membrane integrity and fluidity. This process may induce a modification of the microenvironment of membrane-associated and in this way their activity (16). A third enzymes, possible mode of action of LDL on the stimulation of TXA2 formation is the direct release of arachidonic acid from membranes. Considering this stimulation of TXA2 formation by PG12 formation LDL together with the inhibition of the concept by LDL (4, 51, the current data support the of close connections between modifications in the metabolism of lipoproteins during the development of atherosclerosis and modifications in the metabolism of This hypothesis may be supported by the fact eicosanoids. that HDL, the physiological antagonist of LDL, had an opposite effect on the metabolism of eicosanoids. HDL stimulated PGI formation (5, 6) and was without significant influen 8 e on TXA2 formation under our conditions. ACKNOWLEDGEMENT Ne gratefully support provided
acknowledge the excellent technical by Ms. I. Adler and Ms. A. Seydlitz.
307
REFERENCES
1.
Scanu AM, ilissler RN, Getz GS (Eds.). The Biochemistry of Atherosclerosis. Marcel Dekker, Inc., 1379. New York and Basel,
2.
Gryglewski RJ, Dembinska-Kiec A, Zmuda M, Gryglewska Prostacyclin and thromboxane A biosynthesis capacities of heart, arteries 2 nd platelets of various stages of experimental atherosclerosis. Atherosclerosis 31: 385, 1978.
3.
Sinzinger H, Clopath P, Silberbauer K, Auerswald W. Prostacyclin synthesis and experimental atherosclerosis. Atherosclerosis 34: 345, 1978.
4.
Beitz
5.
Nordrdy A, Svensson and inhibitory on the platelet 1978.
6.
LN, Tall AR, Witte LD, Miller RW, Cannon PJ. Fleisher Stimulation of arterial endothelial cell prostacyclin synthesis by high density lipoproteins. J. Biol. Chem. 257: 6653, 1982.
7.
Okuma H, Uchina H. Altered arachidonate metabolism by platelets in patients with myeloproliferative disorders. Blood 54: 11258, 1979.
8.
Beitz
J, Block HU, Hoffmann P, Fijrster W. Influence from biological of fatty acids and other lipids sources on the in vitro biosynthesis of prostaglandin I2 (PG12). Pharmacol. Res. Commun. 13: 363, 1981.
9.
Beitz
J, Panse M, Farster W. Low density lipoprotein (LDL) from male donors stimulated the thromboxane formation by human platelets. Prostaglandins, Leukotrienes and Medicine 10: 443, 1983.
10.
11.
J, Ftirster W. Influence of human low density and high density lipoprotein cholesterol on the in vitro prostaglandin I synthetase activity. Biochim. Biophys. Acta 6?0r 352, 1980.
Aviram M, Brook JC. and low density 259, lg83.
B, Wieber effect of function.
Cl, Noack JC. Lipoproteins human endothelial cells Circ. Res. 43: 527,
Platelet interaction with lipoproteins. Atherosclerosis
Hassel DG, Owen JS, Bruckdorfer of isolated human platelets lipoproteins and prostacyclin. 43, 1983.
308
KR.
The
high 46:
8ggregatiOn
in the presence Biochem. J.
of 216:
T
12.
Fujetani 8, Tsubou T, Yoshida K,Shimizu Aggregation of gel-filtrated guinea by lipoprotein. Thromb. Haemostasis
13.
Seitz J, Fijrster W. Differential influence of lipoproteins isolated from women and men on the synthetase. activity of PGI Prostaglandins and Medicine 6: 515: 1981.
14.
Shatil SJ, Anaya-Gaklindo R, Bennett J, Colman RW, Coppen RA. Platelet hypersensitivity induced by cholesterol incorporation. J. Clin. Invest. 55: 636, 1975.
15.
Stuart MJ, Gerrard JM, White JG. Effect of cholesterol on the production of thromhoxane 82 by platelets 1982. in vitro. N. Engl. J. Med. 302: 6,
16.
VA, Gerasimova EN. Interaction of lipoTverdislov proteins and apolipoproteins with bilayer lipid membrane. In: Vessel Wall in Atheroand Thromhogenesis (EI Chazov,VN Smirnov eds.), pp 107-117, Springer Verlag Berlin, Heidelberg, New York, 1982.
309
M. pig platelets 41: 416, 1979.
Leukotrienes
and Medicine
23: 303-300, 1986
THROMBOXANE A2 (TXA2) FORMATIONBY WASHEOHUMANPLATELETS UNDER THE INFLUENCE OF LOWAN0 HIGH DENSITY LIPOPROTEINS FROM HEALTHYDONORS and H.-J. Mest J. Beitz Department of Pharmacology and Toxicology, Martin Luther University Halle-Wittenberg, 4020 Halle, GOR (reprint requests to HJM)
ABSTRACT Low density lipoprotein (LOL) stimulates TXA formation (measured as malondialdehyde) in washe human platelets with active cyclooxygenase, whereas ‘high density lipoprotein (HOL) does not show any influence on TXA2 formation in this system. These results’support the hypothesis that the proaggregatory action of a high plasma LOL concentration may be mediated by modification in the biosynthesis of eicosanoids. The mode of action of lipoproteins is discussed. INTRODUCTION The presence of high levels of low density lipoprotein (LOL) and low concentrations of high density lipoprotein (HOL) is generally accepted as a risk factor for the develppment of atherosclerosis (l)-. On the other hand the development of atherosclerosis may be associated with a modified formation of prostaglandin I (PGI > and TXA (2, 3). Recently it was suggested that betgeen these modif?cations in the metabolism of eicosanoids and of lipoproteins close LOL inhibited the formation of the connections may exist. it ;fti; gregatory PG12 (4, 51, whereas HOL stimulated LDL or HDL the question arises as to whether B. From this alGo influence the formation of the physiological antagonist the purpose of this the TXA Therefore of PGI study 8;s to invgstigate the influence of LDL and HOL on TXA formation from exogenous prostaglandin H2 (PGH2) by was z ed human platelets. 303
METHODS Lipoproteins were prepared from venous blood of healthy humans by ultracentrifugation as described earlier (4). All donors had normal lipid values, and drugs influencing platelet aggregation were not given for one week before taking blood. Immediately before the experiment the lipoproteins were ultrafiltrated redissolved in 0.9 % NaCl and brought again to the experimental concentration.
Venous blood was taken from healthy volunteers. The titrated blood was divided into two equal parts. The first concentrawas incubated with acetylsalicylic acid (final tion: 1 mmol/l) and allowed to stand at room temperature for 30 minutes. The second part also stood for 30 minutes at room temperature without addition of any effector. Thereafter the platelets were separated according to the method of Dkuma (7) and concentrated to 6 x 10 platelets per ml. PGH2 was prepared according to (8). Inbubation conditions: 330 ul of platelet solution x 10 platelets) and 330 pl 0.05 mol/l Tris-HCl buffer pH 7.4 were mixed with 330 ul of 0.9 % NaCl (controls), 330 ul of 0.9 % NaCl with fat-free human serum albumin (HSA) or 330 ul 0.9 % NaCl with lipoproteins, respectively, and preincubated for 15 min at 37 C. The reaction was started by addition of 160 ng PGH2 in 40 pl solution (4 pl absolute acetone containing the PGH and 36 pl of 0.9 % NaCl, mixed immediately before addin 8 to the incuAfter an incubation time of 3 min, the bation fluid). reaction was terminated by acidification with 0.8 ml of 20 % trichloroacetic acid containing 5 % (w/v> SnC12. The amount of malondialdehyde formed by thromboxane synthetase, without malondialdehyde (MDA) formed by thermic decomposition of hydroand endoperoxides during the reaction with thiobarbituric acid, was determined as described earlier (9). (2
t-test.
Statistics: Data were statistically A P value c 0.05 was considered
analysed by to be significant.
RESULTS The influence of LDL (0.5 - 2.0 mg LDL-cholesterol/ from ml) and HDL (0.25 - 1.0 mg HDL-cholesterol/ml) healthy volunteers and HSA (1.0 - 10.0 mg/ml> on the biotransformation of exogenous PGH2 into MDA by washed human platelets is summarized in Fig. 1. HSA inhibited rn a dose dependent manner MDA formation from exogenous PGH2 by But the washed platelets with inactive cyclooxygenase. inhibition seems not to be a specific action of I-ISA, because LDL and HDL (at the same concentration, measured
304
as protein in the lipoprotein fraction) inhibited MDA formation by nearly the same extent. The isolated lipoprolein fractions (not recentrifuged) con)ained (LDL fraction) or 10.6-2.0 mg 3.2-0.6 mg protein protein (HDL fraction) per mg lipoprotein cholesterol. This inhibition ma be caused by a nonspecific adsorption of substrate (PGH2 Y or product (MDA) on the proteins.
HSA
HOI
lin nghll 1
+60
5
LDL
lin mg HDCChokStemVml) 10
025
0.5
(in mg LDL-Chdmefdlmll 10
0.5
l.0
2.0
% pFi
77
*
+30
Figure
1:
from healthy Influence of HDL and LDL taken humans and of HSA on the biotransformation prostaglandin H2 into MDA by washed human platelets
of
Open columns: Influence on platelets with inhibited cyclooxygenase. Hatched columns: Influence on platelets with active cyclooxygenase. a6 - significantly different in comparison to controls (P40.05)
305
A statistically not significant stimulation of MDA fornation was observed after incubation of 1 ng HSA with platelets with active cyclooxygenase, whereas all other investigated concentrations of HSA (5 and 10 mg/nl> inhibited MDA formation. The difference in the action of HSA on MDA formation by platelets with active versus those with inhibited cyclooxygenase is not statistically significant. The amount of MDA formed was higher in platelets with active cyclooxygenase compared to platelets with inhibited cyclooxygenase under the influence of HDL at all investigated concentrations, but the difference did not reach the level of significance. LDL (0.5 and 2.0 ng LDL-cholesterol/ml) stimulated MDA formation from exogenous with active cyclooxygenase, whereas at PGH2 by platelets 1 mg/nl only a tendency to stimulation was observed. This influence of LDL on MDA formation by platelets with active cyclooxygenase is statisticallv significantly of the same condifferent in comparison to the influence centration of LDL on MDA formation by aspirin inhibited platelets. DISCUSSION
In the literature it has been suggested that an elevated LDL concentration promotes the platelet aggregation, whereas HDL inhibits it (10, 11, 12). One important step in platelet aggregation is the formation of TXA2.With a possible modified method for the estimation of MDA it is MDA formed without to measure MDA formed by TXA synthetase and endoperoxides. by thermic decomposition of ifydroRecently this method demonstrated that LOL stimulated TXA2 formation by frozen-thawed platelets (9). In this paper we have demonstrated, that HSA, LOL or HOL at nearly the same,protein concentration decreased the detectable MDA concentration after 3 min incubation of platelets with inhibited cyclooxygenase and stimulation with PGH by nearly the same extent. It is possible that this diiinished level of MDA may be a result of a nonspecific adsorption of PGH . A different result was HSA demonstrated in platelets iith active cyclooxygenase: inhibited in a dose dependent nanner,HDL was without significant influence and LOL stimulated significantly (0.5 and 2.0 mg LDL-cholesterol/ml) MDA formation. The difference in the action of LOL on platelets with active versus inhibited cyclooxygenase is statistically significant at 1.0 and 2.0 mg LDL-cholesterol/ml, whereas the effect was not statistically diffeof HSA or HDL, respectively, rent in the two systems. This enhanced MDA formation in platelets with active cyclooxygenase may therefore not be a result of a direct influence of LDL on the TXA2 synthetase, but nay rather be a result of an activation of other steps But it is not possible to investigate in TXA formation. 2
306
which step of the arachidonic acid cascade is activated because HSA (5 or 10 mg/ml> under the influence of LDL, inhibited almost completely arachidonic acid (1.25 or 25 pmol/l)as well as thrombin (2.5 or 5 U/ml>- induced MDA formation in our systems. The influence of LDL did not depend on the sex of the donors. This is qualitatively different from the in the microsomal action of LDL on the PGI formation fraction of pig aorta (13). The mode of action of LDL on TXA2 formation is unknown, because the effect of LDL may be caused in different ways. It is known that an increased platelet cholesterol content gives rise to hypersensitive platelets in terms of response to aggregating agents (14) and, in addition these platelets produce significantly more TXA (15). Aviram and Brook (10) suggest that physiological concentrations of LDL and HDL were bound by platelets in a low affinity and calcium-independent way and enhance the platelet cholesterol content. But after our short incubation time it seems unlikely that the cholesterol from lipoproteins is transferred into the platelets. Additionally these effects of LDL were independent of a receptor mediated uptake because LDL stimulated TXA2 formation also in frozen-t.hawed possibility may be’that LDL platelets (9). Another was incorporated into the membrane and affects the membrane integrity and fluidity. This process may induce a modification of the microenvironment of membrane-associated and in this way their activity (16). A third enzymes, possible mode of action of LDL on the stimulation of TXA2 formation is the direct release of arachidonic acid from membranes. Considering this stimulation of TXA2 formation by PG12 formation LDL together with the inhibition of the concept by LDL (4, 51, the current data support the of close connections between modifications in the metabolism of lipoproteins during the development of atherosclerosis and modifications in the metabolism of This hypothesis may be supported by the fact eicosanoids. that HDL, the physiological antagonist of LDL, had an opposite effect on the metabolism of eicosanoids. HDL stimulated PGI formation (5, 6) and was without significant influen 8 e on TXA2 formation under our conditions. ACKNOWLEDGEMENT Ne gratefully support provided
acknowledge the excellent technical by Ms. I. Adler and Ms. A. Seydlitz.
307
REFERENCES
1.
Scanu AM, ilissler RN, Getz GS (Eds.). The Biochemistry of Atherosclerosis. Marcel Dekker, Inc., 1379. New York and Basel,
2.
Gryglewski RJ, Dembinska-Kiec A, Zmuda M, Gryglewska Prostacyclin and thromboxane A biosynthesis capacities of heart, arteries 2 nd platelets of various stages of experimental atherosclerosis. Atherosclerosis 31: 385, 1978.
3.
Sinzinger H, Clopath P, Silberbauer K, Auerswald W. Prostacyclin synthesis and experimental atherosclerosis. Atherosclerosis 34: 345, 1978.
4.
Beitz
5.
Nordrdy A, Svensson and inhibitory on the platelet 1978.
6.
LN, Tall AR, Witte LD, Miller RW, Cannon PJ. Fleisher Stimulation of arterial endothelial cell prostacyclin synthesis by high density lipoproteins. J. Biol. Chem. 257: 6653, 1982.
7.
Okuma H, Uchina H. Altered arachidonate metabolism by platelets in patients with myeloproliferative disorders. Blood 54: 11258, 1979.
8.
Beitz
J, Block HU, Hoffmann P, Fijrster W. Influence from biological of fatty acids and other lipids sources on the in vitro biosynthesis of prostaglandin I2 (PG12). Pharmacol. Res. Commun. 13: 363, 1981.
9.
Beitz
J, Panse M, Farster W. Low density lipoprotein (LDL) from male donors stimulated the thromboxane formation by human platelets. Prostaglandins, Leukotrienes and Medicine 10: 443, 1983.
10.
11.
J, Ftirster W. Influence of human low density and high density lipoprotein cholesterol on the in vitro prostaglandin I synthetase activity. Biochim. Biophys. Acta 6?0r 352, 1980.
Aviram M, Brook JC. and low density 259, lg83.
B, Wieber effect of function.
Cl, Noack JC. Lipoproteins human endothelial cells Circ. Res. 43: 527,
Platelet interaction with lipoproteins. Atherosclerosis
Hassel DG, Owen JS, Bruckdorfer of isolated human platelets lipoproteins and prostacyclin. 43, 1983.
308
KR.
The
high 46:
8ggregatiOn
in the presence Biochem. J.
of 216:
T
12.
Fujetani 8, Tsubou T, Yoshida K,Shimizu Aggregation of gel-filtrated guinea by lipoprotein. Thromb. Haemostasis
13.
Seitz J, Fijrster W. Differential influence of lipoproteins isolated from women and men on the synthetase. activity of PGI Prostaglandins and Medicine 6: 515: 1981.
14.
Shatil SJ, Anaya-Gaklindo R, Bennett J, Colman RW, Coppen RA. Platelet hypersensitivity induced by cholesterol incorporation. J. Clin. Invest. 55: 636, 1975.
15.
Stuart MJ, Gerrard JM, White JG. Effect of cholesterol on the production of thromhoxane 82 by platelets 1982. in vitro. N. Engl. J. Med. 302: 6,
16.
VA, Gerasimova EN. Interaction of lipoTverdislov proteins and apolipoproteins with bilayer lipid membrane. In: Vessel Wall in Atheroand Thromhogenesis (EI Chazov,VN Smirnov eds.), pp 107-117, Springer Verlag Berlin, Heidelberg, New York, 1982.
309
M. pig platelets 41: 416, 1979.