Lipid fluidity modulates platelet aggregation and agglutination in vitro

Life Sciences, Vol. 53, pp. 1053-1060 Printed in the USA

LIPID

FLUIDITY

Pergamon Press

MODULATES

PLATELET AGGREGATION IN VITRO

AND

AGGLUTINATION

Niksa Vlasic, Marvin S. Medow ~, Steven M. Schwarz ~ Kirkwood A. Pritchard Jr. and Michael B. Stemerman Departments of Experimental New York Medical College,

Pathology Valhalla,

and Pediatrics ~, New York, USA

(Received in final form July 14, 1993)

Summary To determine the effect of altered membrane fluidity on platelet aggregation/agglutination, fresh, washed human platelets were treated with A2C, a cyclopropyl fatty acid ester which is known to enhance mobility of intrinsic membrane bilayer constituents and increase membrane fluidity. Fluorescence polarization studies demonstrated A2C incubation time- and concentrationdependent increases in platelet membrane fluidity (decreased fluorescence anisotropy). Preincubation with A2C was associated with diminished collagen, thrombin and ristocetin-induced platelet aggregation/ agglutination. A g g r e g a t i o n / a g g l u t i n a t i o n was diminished by 93 ± 5% for collagen (0.2 mg/ml), 53 ± 3% for thrombin (i.0 U/ml) and 85 ± 9% for ristocetin (i.i mg/ml). These data suggest that membrane fluidity is involved in the regulation of platelet function. Available evidence suggests that specific functions of human platelets are regulated, in part, by membrane lipid dynamics (6,10,25). For example, alterations in platelet cholesterol content and the cholesterol to phospholipid molar ratio (major determinants of membrane fluidity) are associated with changes in platelet aggregation (1,7,23,24). Incubation of platelets with cis-polyunsaturated fatty acids, which results in increased lipid fluidity, is also associated with inhibition of thrombin and ADP-induced aggregation. To further define the role of membrane fluidity in the modulation of platelet activity, various amphiphilic molecules (soaps, alcohols, detergents) have been employed to alter membrane physicochemical properties (8,10). Few studies, however, have addressed the importance of fluidity per s_ee (in the absence of significant perturbations in bilayer lipid composition and/or integrity) in controlling specific platelet functions. Accordingly, the experiments presented herein employ the cyclopropane fatty acid ester 2(2-methoxyethoxy)ethyl-8-(cis-2-octyl-cyclo-propyl)-octanoate, C o r r e s p o n d i n g Author: Marvin S. Medow, Ph.D, Dept of Pediatrics New York Medical College, Valhalla, New York, 10595 ~2~3205/93 ~ . ~ + . ~ Cop~ight©1993PergamonPressLtd Allrightsrese~ed.

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(A2C), an agent known to enhance m o b i l i t y of intrinsic plasma m e m b r a n e c o n s t i t u e n t s (3,12,13,17) without s i g n i f i c a n t l y altering bilayer lipid composition, to effect increases in platelet membrane fluidity (assessed by fluorescence p o l a r i z a t i o n spectrophotometry). Functional consequences of A2C-induced fluidity changes were determined by measuring platelet a g g r e g a t i o n / a g g l u t i n a t i o n responses to several platelet agonists.

Methods Platelet preparation. Platelet suspensions were prepared from whole blood collected from healthy volunteers by antecubital venipuncture. Blood was diluted (1:9) in 3.8% trisodium citrate buffer, and p l a t e l e t - r i c h plasma (PRP) was prepared by c e n t r i f u g a t i o n of the citrate diluted blood at 150 x g for I0 min at 25°C. Platelets were separated from the PRP by c e n t r i f u g a t i o n at 200 x g for 15 min. The pelleted platelets were w a s h e d once with Ca 2. and Mg2÷-free H E P E S / T y r o d e ' s / A l b u m i n buffer (HTA), c o n t a i n i n g 5 ram N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 ethanesulphonic acid (HEPES), 149 mM NaCl, 2.7 mM KCI, 0.45 mM Na2HPO4, 5.5 mM ~-D(+)glucose, 12 mM NaHCO3, 2 mM ethylene diaminetetraacetic acid (EDTA), and 0.2%(w/v) bovine serum albumin (BSA), pH 7.4 (2). Platelets were r e s u s p e n d e d to a final c o n c e n t r a t i o n of 2x10 s platelets per ml, in EDTA-free HTA buffer. F l u o r e s c e n c e p o l a r i z a t i o n studies. Platelet membrane fluidity was d e t e r m i n e d by fluorescence polarization spectrophotometry, employing the lipid soluble fluorescent probe 1 , 6 - d i p h e n y l - l , 3 , 5 - h e x a t r i e n e (DPH), which was prepared and stored according to established techniques (22). Estimates of membrane fluidity were calculated following f l u o r e s c e n c e polarization measurements, using a Shimadzu RF-540 s p e c t r o f l u o r o p h o t o m e t e r (shimadzu Inc., Columbia, MD), equipped with rotating polarizers and a t h e r m o r e g u l a t e d sample chamber. P l a t e l e t a q q r e q a t i o n / a g q l u t i n a t i o n studies. Washed platelets were p r e i n c u b a t e d for 3 min at 37°C prior to the addition of agonist, and aggregation studies were performed by the turbidometric method of Born (22) using a Chrono-log dual channel aggregometer (Chrono-Log Co., Havertown, PA). The a g g r e g a t i o n response to collagen (0.2 mg/ml) and t h r o m b i n (i.O U/ml) was d e t e r m i n e d in plasma free HTA (pH 7.4). Ristocetin (i.i mg/ml) was added to platelets suspended in HTA buffer with 2.5% platelet poor plasma. Platelet a g g r e g a t i o n was m e a s u r e d 5 min after agonist addition. To evaluate the effects of m e m b r a n e fluidization on platelet function, agonists were added to p l a t e l e t s following a 1 min preincubation with A2C. I n h i b i t i o n of the a g o n i s t - s t i m u l a t e d a g g r e g a t i o n response following A2C treatment was expressed as a percentage of a g o n i s t - i n d u c e d aggregation, in the absence of A2C. All data presented are triplicate determinations from at least 3 d i f f e r e n t p l a t e l e t preparations. Data are expressed as means ± SD unless otherwise stated. Results were compared using analysis of variance (ANOVA) or Student's t-test for paired samples (30); d i f f e r e n c e s of pS 0.05 were considered significant.

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Reagents. Calf skin collagen, h u m a n thrombin, ristocetin, bovine serum a l b u m i n (BSA F r a c t i o n V), and A2C were obtained from Sigma Chemical Co. (St. Louis, MO). 1 , 6 - d i p h e n y l h e x a - l , 3 , 5 - t r i e n e (DPH) was p u r c h a s e d from A l d r i c h Chemical (Milwaukee, WI) and lactated R i n g e r ' s from Travenol (Deerfield, IL). All other c h e m i c a l s were obtained from Fisher Scientific (Fairlawn, NJ).

Results

F l u o r e s c e n c e P o l a r i z a t i o n Studies. To d e t e r m i n e the effects of the membrane m o b i l i t y agent A2C on platelet fluidity, f l u o r e s c e n c e p o l a r i z a t i o n studies were performed. F o l l o w i n g a 1 min p r e i n c u b a t i o n with A2C (Table I), anisotropy measurements demonstrated significant decreases in the p a r a m e t e r s r and r® at both 25 and 37°C, indicating increased fluidity. Platelet fluidity showed a linear timed e p e n d e n t increase when incubated with A2C for various times up to i0 min. Additional studies indicated an inverse, linear relationship between fluorescence anisotropy and A2C concentration, with r and r. decreasing significantly (p < 0.03), c o m p a r e d to control, after a 1 min preincubation with i0 uM A2C. All subsequent evaluations of p l a t e l e t function were c o m p a r e d to p l a t e l e t s p r e i n c u b a t e d for 1 min with 2 ~M A2C, as this was the lowest concentration which resulted in a s i g n i f i c a n t increase in m e m b r a n e fluidity.

TABLE I Effects of 2 ~M A2C on F l u o r e s c e n c e A n i s o t r o p y V a l u e s (DPH) of Washed, Human Platelets at 25 and 37°C. Anisotropy

K

parameter

r_~

B u f f e r at 2 5 : C PBS (control) PBS + 2 ~M A2C

0.198 ± 0.002 0.177 ± 0.003"

0.165 ± 0.002 0.136 ± 0.004"

B u f f e r at 3 7 * c PBS (control) PBS + 2 ~M A2C

0.172 ± 0.004 0.154 ± 0.005"

0.130 ± 0.005 0.106 ± 0.007"

R e s u l t s p r e s e n t e d as means ± SEM for 3 d e t e r m i n a t i o n s on each of 5 p l a t e l e t s preparations. Buffer is p h o s p h a t e buffered saline (PBS). "Significantly different from control, p < 0.01, Student's t-test for paired observations.

A g g r e g a t i o n Studies. To evaluate the effects of A2C on p l a t e l e t function, the aggregation/agglutination of w a s h e d platelets, with and w i t h o u t added A2C, was d e t e r m i n e d following incubation with known p l a t e l e t agonists. F o l l o w i n g a 1 min p r e i n c u b a t i o n with 2 ~M A2C, the r e s p o n s e s of p l a t e l e t s to c o l l a g e n (Figure IA), t h r o m b i n (Figure IB) and r i s t o c e t i n (Figure IC) were inhibited by 92.5 ± 5.0%, 59.0

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± 2.8%, and 85.0 ± 9.0%, respectively, inhibition w i t h o u t addition of A2C).

compared

to control

(0%

To study r i s t o c e t i n - i n d u c e d agglutination, experiments were p e r f o r m e d in the presence of platelet poor plasma (PPP). When w a s h e d human p l a t e l e t s were p r e i n c u b a t e d with 2 ~M A2C, ristocetin-induced platelet agglutination was markedly diminished. Inhibition was dependent on the c o n c e n t r a t i o n of plasma used; thus, 2.5% PPP resulted in 64% inhibition, and 5.0% PPP r e s u l t e d in an 85% inhibition, compared to ristocetininduced p l a t e l e t a g g r e g a t i o n in the absence of A2C (Figure IC).

Figure 1A =7 m

5 Time (min)

Figure 1 C b)

'~

b)

=2_ c as

lL3

V. 0

0

5 T i m e (min)

Time (min)

Fig.

1

R e p r e s e n t a t i v e tracing of collagen (Fig. IA, 0.2 mg/ml), t h r o m b i n (Fig. IB, 1 U/ml) and r i s t o c e t i n (Fig IC, i.I m g / m l ) - i n d u c e d platelet aggregation inhibition by 2 ~M A2C: a) = Control: agonist-induced platelet a g g r e g a t i o n without A2C; b) = platelets p r e i n c u b a t e d with 2 ~M A2C for 1 min prior to addition of agonist.

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Washed human platelets preincubated for 1 min with 2 ~M A2C, exhibited 93% aggregation inhibition, compared to platelets without A2C. Studies examining the time-dependence of A2C's effect on collagen-induced platelet aggregation were performed using 2 ~M A2C to achieve maximal inhibition (Figure 2). A p p r o x i m a t e l y 80% aggregation inhibition was achieved following both a 1 and 3 min incubation with 2 ~M A2C. Incubations of 5 and I0 min, inhibited aggregation by 17 and 20%, respectively. Platelets treated with 2 ~M A2C showed a 59% inhibition of platelet aggregation induced by thrombin (i.0 U/ml), which was similar to that seen with collagen. Time-dependence experiments (Figure 2) demonstrated that maximal A2C inhibition was achieved after a 1 min preincubation with 2 ~M A2C. Increased time of incubation with the ester, prior to thrombin addition, resulted in decreased inhibition of aggregation, similar to that measured using collagen. I00-

[~1 Thrombin ~\Xl Collagen

Oc

80-

x~ E E .2 ..l_a "13n <

60-

p ( 0.01 \\\ \\\

4-020-

~N

T ~ k~ k~ kA k~

- -

\\\ \\\ \\N

5

5

10

Time (rain) Fig.

2

Time-dependence of A2C inhibition of collagen and thrombin induced platelet aggregation. Human washed platelets were preincubated at 37°C with A2C for i-i0 minutes, prior to addition of collagen ( ~ . ! .(0.2 mg/ml) or thrombin ([--7) (i U/ml). Percent of innlDltlOn of platelet aggregation was calculated as described in Materials and Methods; n=3 experiments (* p< 0.01, compared with shorter incubation times). Discussion The present studies demonstrate that the membrane fluidity of washed, human platelets is increased in a concentrationdependent manner following incubation with the cyclopropyl fatty acid ester A2C. Fluidization of platelet membranes is

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associated with significant inhibition of the platelet aggregation or agglutination response to diverse stimuli. In contrast to prior reports, our data suggest that alterations in fluidity, in the absence of significant changes in lipid composition or disruption of the plasma membrane by detergent solubilization, directly influence platelet function. Previous investigations suggested direct relationships between platelet lipid physicochemical properties and cellular functions, by showing that the number and affinity of platelet thrombin receptors are controlled in part by cellular cholesterol content (15,29). Incubation of platelets with A2C did not alter cellular cholesterol content. Although the precise relationships between cholesterol levels and platelet aggregation, in response to other agonists such as collagen and ristocetin, are less well understood, available evidence supports a role for lipidrelated fluidity changes in the modulation of agonist-stimulated activity (7,9,20,23,26,29). The data presented herein also describe the inhibition of platelet aggregation and agglutination by A2C, which appears to exert its physicochemical and functional effects by enhancing the mobility of intrinsic bilayer components, thus perturbing bilayer lipid dynamics (5). This inhibition is observed using several agonists (thrombin, collagen, ristocetin), which are known to promote platelet activation by different pathways (1,16,20). Effects on platelet aggregation/agglutination are, to an extent, dependent upon the incubation medium, since we have also found that A2C has no effect on platelet activity when aggregation studies are performed in platelet rich plasma (data not shown). This lack of effect on aggregation may be a consequence of fatty acid ester hydrolysis in the incubating medium; or, alternatively, this may involve binding of A2C to plasma proteins, making it unavailable for incorporation into platelet membranes. The aggregation response of platelets to collagen preincubated with A2C was similar to that using collagen alone. Previous studies have shown that, when BSA is present in the incubation buffer, 5 to 10-fold higher levels of A2C are required to fluidize biological membranes, compared to effective A2C concentrations in the absence of BSA (14). In other biological membranes, fluidity exerts a regulatory influence on numerous intrinsic (lipid-dependent) bilayer functions. For example, hyperoxia-induced changes in fluorescence anisotropy-determined fluidity of cultured pulmonary endothelial cell membranes are associated with alterations in transport of 5-hydroxytryptamine and specific activity of (Na+-K÷)-dependent adenosine triphosphatase (22). Recent studies from our laboratory, utilizing cultured human umbilical vein endothelial cells, demonstrated that low-density lipoprotein-induced decreases in membrane fluidity were associated with enhancement of receptor-mediated mononuclear cell attachment. Restoration of fluidity to control levels, following incubation with i0 ~M A2C, reversed the LDL-increased monocyte binding (21). We have also demonstrated that agonist-stimulated eicosanoid release by cultured rabbit coronary microvessel endothelial cells is regulated, at least in part, by membrane fluidity (4,19).

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A2C (in ~molar amounts) increases m e m b r a n e fluidity in a variety of cell membranes (3,12,13,28). Following brief p r e i n c u b a t i o n s of A2C with isolated cells, the ester has been indirectly shown to segregate t r a n s i e n t l y to the plasma m e m b r a n e by the inability to detect a fluorescent labeled analogue in the cytosol unless cellular damage is induced (12). Although A2C's mechanism(s) of action are not well understood, f l u i d i z a t i o n effects are likely due to it's m o l e c u l a r structure, which contains both a h y d r o p h i l i c moiety anchoring the m o l e c u l e at the outer membrane surface, and a h y d r o p h o b i c chain e x t e n d i n g into the bilayer h y d r o c a r b o n core and p r o m o t i n g m o l e c u l a r disorder (14). A2C appears to influence bilayer physical p r o p e r t i e s by expanding the packing order of bilayer lipids, thus enhancing the lateral mobility of intrinsic membrane constituents (3,12,13,28). Resulting effects on m e m b r a n e lipid dynamics have been shown to m o d u l a t e l i p i d - d e p e n d e n t enzymes (22,28), second messenger (cyclic adenosine monophosphate, i n t r a c e l l u l a r Ca 2. (5)), cap formation (17), and cellular lipid m e t a b o l i s m (4,19). The present study confirms these prior o b s e r v a t i o n s of A2C's role in m o d i f y i n g membrane function, and demonstrates that relatively modest changes in bilayer lipid fluidity are associated with significant p e r t u r b a t i o n s in m e m b r a n e function. The t i m e - d e p e n d e n t decrease of a g g r e g a t i o n inhibition seen here is likely the c o n s e q u e n c e of h y d r o l y s i s of the ester, as well as it's d i s s i p a t i o n from the m e m b r a n e lipid bilayer into the cytoplasm and other i n t r a c e l l u l a r organelles. Under p h y s i o l o g i c a l conditions, plasma m e m b r a n e fluidity is maintained within a narrow range and resists m o d u l a t i o n s induced by lipid c o m p o s i t i o n a l changes, a p h e n o m e n o n known as h o m e o v i s c o u s a d a p t a t i o n (27). Accordingly, alterations in f l u o r e s c e n c e a n i s o t r o p i e s in the range of only 10% have been a s s o c i a t e d with s i g n i f i c a n t changes in l i p i d - d e p e n d e n t (i.e. intrinsic) membrane functions, as d e s c r i b e d above. In this regard, our results indicate that A 2 C - r e l a t e d fluidity changes of similar magnitude are associated with a greater than 50% inhibition of the a g g r e g a t i o n response to both collagen and thrombin. Our findings are similar to earlier studies, which employed other compounds known to influence m e m b r a n e dynamics, such as c i s - p o l y u n s a t u r a t e d fatty acids (7,11,18), a n e s t h e t i c alcohols (I0), and p h o s p h o l i p i d s (8). However, these compounds would be expected to significantly alter plasma membrane composition, either directly or by detergent action. In contrast, we have d e m o n s t r a t e d the absence of any substantial effect on cell v i a b i l i t y or activity following A2C incubation with cultured endothelial cells (19,21), suggesting a direct effect on m e m b r a n e receptor function. Acknowledqements The authors g r a t e f u l l y acknowledge the c o n t r i b u t i o n s of their c o l l e a g u e s Drs. Erica Asch, Pal Czobor, Kenneth Lerea and Julie Scheurer. The authors also wish to thank Ann Lambert for her technical assistance. This study was supported by NIH grants HL-21249, HL-33742, HL-43023 and American Heart Assoc. Grants 90-082G and 91-020G, (New York State Affiliate).

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