Published online before print January 17, 2008, doi: 10.1161/ATVBAHA.107.151837
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© 2008 American Heart Association, Inc.
Clinical and Population Studies |
Individual Heterogeneity in Platelet Response to Lysophosphatidic Acid
Evidence for a Novel Inhibitory Pathway
From the Division of Cardiovascular Medicine (Z.P., H.-Y.C., M.P., S.S., A.J.M., R.C., S.S.), The Gill Heart Institute, University of Kentucky, Lexington; the School of Medicine (J.S.L.), The University of North Carolina at Chapel Hill; and the Department of Biostatistics (R.C.), College of Public Health, University of Kentucky, Lexington and the Department of Veterans Affairs Medical Center, Lexington, KY (S.S.S.).
Correspondence to Susan S. Smyth, MD PhD, The Gill Heart Institute, 326 Charles T Wethington Building, 900 S. Limestone Street, Lexington, KY 40536. E-mail susansmyth{at}uky.edu
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Abstract |
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Objective— The bioactive lipid lysophosphatidic acid (LPA) stimulates platelet actin reorganization, adhesion, shape change, and aggregation. LPA is present in blood and exposure or release of LPA after atherosclerotic plaque rupture has been proposed to trigger platelet thrombus formation.
Methods and Results— In this report, we characterize heterogeneity in LPA responses among individuals. Platelets isolated from approximately 20% of healthy donors consistently failed to aggregate in response to LPA. Our studies indicate that, rather than lacking stimulatory pathways, platelets from "nonresponsive" donors respond to LPA by triggering inhibitory pathway(s) to block platelet aggregation. Consistent with this observation, LPA-induced aggregation could be partially restored to "nonresponsive" platelets by pharmacological inhibition of cAMP generation. LPA "nonresponsiveness" may be related to heightened platelet expression of LPA receptor 4 and PPAR
. Among 70 patients with stable coronary artery disease (CAD), only 1 (1.4%) was identified as an LPA nonresponder. Moreover, in 33 patients presenting for diagnostic catheterization, CAD was identified as having a bivariate association with platelet LPA responder/nonresponder status.
Conclusions— Platelet LPA signaling may involve stimulatory and inhibitory pathways, with the inhibitory pathway predominating in 
20% of individuals. Diseases such as CAD that affect platelet reactivity may attenuate this inhibitory pathway in platelets.
The bioactive lipid mediator lysophosphatidic acid stimulates actin reorganization and aggregation of human platelets. We report that LPA may trigger an inhibitory response in platelets from 20% of healthy individuals. Furthermore, we provide evidence that the presence of coronary artery disease may attenuate this inhibitory pathway.
Key Words: platelet aggregation • platelet activation • lysophosphatidic acid • coronary artery disease
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Introduction |
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Lysophosphatidic acid (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate; LPA), the simplest glycerophospholipid, exerts growth factor–like effects on a variety of cells thereby controlling proliferation, differentiation, apoptosis, and development.1–3 Some of the earliest observations of receptor-mediated actions of LPA came from studies in platelets.4–7 LPA stimulates platelet shape change,6,8 fibronectin matrix assembly,9 and platelet-monocyte coaggregate formation.10 LPA induces partial platelet aggregation and also functions synergistically to enhance platelet aggregation by epinephrine and adenosine triphosphate (ADP). LPA is present in physiologically relevant levels (1 to 5 µmol/L) in serum, but is found at 10- to 50-fold lower levels in whole blood and platelet-poor plasma. Evidence suggests that platelets may be necessary for production of serum LPA.11–16 LPA also accumulates in mildly-oxidized low-density lipoprotein (LDL) and is abundant in atherosclerotic plaques, with highest levels in the lipid-rich core.17 Mildly-oxidized LDL stimulates platelet function in an LPA dependent manner,17 and platelet aggregation induced by components of atherosclerotic plaque lipid-rich cores can be inhibited by LPA receptor antagonists.18 Thus, after plaque rupture or erosion, exposure of LPA may serve a key role in triggering or potentiating platelet responses during acute thrombosis.
The effects of LPA are mediated, at least in part, by specific cell surface G protein–coupled receptors (GPCR).19 To date, at least 5 GPCRs for LPA (LPA1–5; formerly Edg2, 4, 7, GPR23, and GPR92) have been identified that couple to intracellular signaling pathways to activate Rho-family GTPases, phospholipase D, phosphatidylinositol 3-kinase, and mitogen activated protein kinases.20 Human platelets contain messenger RNA for 3 of the known G protein–coupled LPA receptors, LPA 1 to 3,21 but the receptor systems mediating LPA responses in platelets have yet to be identified. Moreover, although LPA appears to be well positioned to serve as a physiologically-relevant mediator of platelet function, attempts to define physiological or pathophysiologic roles for platelet LPA signaling systems have been hampered, in part, by a lack of specific pharmacological tools for probing receptor function. Additionally, although LPA is a well-documented activator of human platelets, it does not appear to induce aggregation in rodent platelets, which limits the insight that can be gained about its pathophysiologic role in rodent thrombosis models. There is increasing awareness of heterogeneity among individuals in terms of baseline platelet function and responses to antiplatelet therapy,22–24 which may impact clinical manifestation of atherothrombotic disease.25,26 Early reports identified individuals whose platelets fail to respond to LPA,27 but the frequency, mechanism, and significance of these observations has not been investigated. Herein, we report differences in platelet responses to LPA among normal individuals and use this heterogeneity to obtain insight into LPA signaling in platelets. Comparisons of the expression of signaling proteins and downstream signaling responses identify an LPA-regulated inhibitory pathway in nonresponsive platelets. Our results indicate that the balance between stimulatory and this newly described inhibitory LPA signaling systems may account for differences in LPA responsiveness between individuals. Additionally, we report that individuals with atherosclerotic coronary artery disease (CAD) are less likely to be nonresponders, suggesting that systemic conditions that are associated with platelet hyperreactivity may attenuate inhibitory signaling through LPA. These findings provide additional support for the notion that LPA may be a pathophysiologically relevant mediator of cardiovascular disease in humans and a viable target for novel antithrombotic strategies.
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Methods |
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All donors and patients provided written consent, and blood was collected according to protocols approved by the Institutional Review Boards of both the University of North Carolina at Chapel Hill (UNC) and the University of Kentucky. Platelets were isolated as previously described.28,29 Please see supplemental methods for details of the platelet function assays, including aggregation, Rho activation, and VASP phosphorylation.30 For analysis of LPA receptors by polymerase chain reaction (PCR), platelets were depleted of leukocytes31 and PCR performed as described in supplemental methods (available online at http://atvb.ahajournals.org) using the 2–
CT method32 to express fold change. Data analyses were carried out in SAS Version 8.2 (SAS Institute, Cary, NC). Please see supplemental Methods for details. |
Results |
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In our ongoing studies of agonist activation of platelets from healthy volunteers, we occasionally identified individuals whose platelets failed to aggregate in response to LPA. To investigate the frequency of this phenotype, we examined aggregation responses to 1 and 5 µmol/L LPA in platelets isolated from 45 consecutive healthy donors (60% male). We found that platelet aggregation in response to LPA occurred in only 36 of these donors (Figure 1A). Platelets isolated from the remaining 9 donors consistently failed to aggregate to LPA (95% CI 0.096 to 0.346 for the nonresponse rate), although responses to a range of other agonists, including thrombin, collagen, and ADP, were normal (Figure 1B). Aggregation was not observed with either prolonged exposure to LPA or with increased concentrations of LPA (not shown). Blood was collected on LPA nonresponsive donors up to 10 times over 2 to 3 years with similar results. On the basis of an absence of LPA-induced aggregation (no change in light transmission), these donors were classified as being LPA "nonresponders". Of the 9 nonresponsive donors, 5 were males.
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To further investigate the heterogeneity in LPA responses, we first excluded that released ADP was mediating platelet aggregation to LPA under the conditions used by documenting that P2Y1 (A3P5PS) or P21Y12 (2-MeS-ADP) receptor antagonists alone or in combination lacked an effect on LPA-induced aggregation (data not shown). In addition, no rise in intraplatelet Ca2+ was observed in response to 1 µmol/L LPA in either population (data not shown), a finding that is consistent with previous observations that high concentrations of LPA induce very modest increases in platelet cytoplasmic Ca2+.5 Rho-GTPases are well-established mediators of platelet activation and previously have been proposed to be involved in platelet LPA responses.5,8,33 Interestingly, we found that LPA increased Rho activation to similar levels in platelets from both LPA responsive and "nonresponsive" donors (Figure 1C and 1D). This result suggests that the lack of LPA-induced aggregation in "nonresponsive" donors might be a consequence of LPA-promoted inhibitory signaling pathways rather than a selective loss of LPA-responsive signaling components of pathways upstream of Rho activation.
One prominent mechanism for inhibition of platelet function involves activation of protein kinase A and resultant phosphorylation of vasodilator stimulatory protein (VASP), which in turns leads to an attenuation of platelet responses by effects on actin dynamics. The effects of LPA on VASP phosphorylation were measured in platelets from both responsive and "nonresponsive" donors using flow cytometric analysis. In platelets from both populations, a maximally effective concentration of ADP significantly attenuated the ability of PGI2 to induce VASP phosphorylation (Figure 2A and 2B). 1 µmol/L LPA modestly reduced PGI2-mediated VASP phosphorylation in platelets from LPA responsive donors (Figure 2A). In contrast, LPA increased VASP-phosporylation in samples from nonresponders at the higher concentrations of PGI2 tested (Figure 2B). Consistent with this finding, 1 µmol/L LPA induced increases in cAMP levels in "nonresponsive" platelets greater than those observed in LPA responsive platelets (0.13±0.03 versus <0.05 pmoles with 1 µmol/L LPA and 0.95±0.05 versus 0.16±0.05 pmoles with 1 µmol/L LPA +0.3 nM PGI2; P<0.01 by t test for the latter). Taken together, these results suggest that in addition to its well-documented actions as a platelet activator, LPA may also be able to inhibit platelet function in certain individuals. These observations suggest the possibility that an imbalance between stimulatory and inhibitory pathways may account for the failure of LPA to promote aggregation in nonresponders. To test this hypothesis, we surmised that inhibition of the enzyme responsible for cAMP accumulation, adenylyl cyclase, should restore the ability of LPA to elicit aggregation of platelets from "nonresponsive" donors. Therefore, platelets from 3 responders and 3 nonresponders were preincubated with the adenylyl cylcase inhibitor SQ22536 (SQ) before being challenged with LPA. Preincubation with SQ had no effect on LPA-induced aggregation of platelets from responsive donors (maximum light transmission of 30±15% versus 29±5.6% [mean±SD; n=3], without and with SQ, respectively; Figure 2C). However, platelets from 3 "nonresponsive" donors exhibited a partial and transient restoration of LPA-induced aggregation following preincubation with SQ (0% aggregation versus 11±1.7% [mean±SD; n=3] maximal aggregation without and with SQ, respectively; Figure 2D). Similar results were obtained with the adenyly cyclase inhibitor MLD-12330A (data not shown).
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To determine whether changes in LPA receptors or G proteins might account for the differences in platelet responses among donors, and, in particular, the apparent exaggeration of inhibitory responses to LPA in nonresponders, we measured expression of both by immunoblot analysis. Only limited antibodies are available for LPA receptors; using ones specific for LPA1 and LPA2, we did not detect differences in levels of immunoreactive proteins of the expected molecular weights in the two donor populations (Figure 3). Likewise, no differences in G protein levels were observed (Figure 3). Previous studies have indicated that the levels of residual platelet mRNA may correlate with protein expression. Therefore, as a complementary approach to examine differences in LPA signaling systems in the 2 populations, quantitative PCR (qPCR) was used to measure gene expression of specific LPA receptors in 4 responders and 4 nonresponders. Expression of mRNA for Lpa1 and Lpa2 did not vary between the populations (Table 1), which is in agreement with immunoblot results indicating no difference in protein levels. Lpa3 mRNA was low or undetectable in 1 sample from a responsive donor and in 2 of 4 samples from "nonresponsive" donors. Interestingly, we found that expression of Lpa4 and PPAR was elevated in samples from nonresponders by 6.6±3.3-fold and 2.9±1.3-fold, respectively, over levels in responders (Table 1). The increase in PPAR protein in "nonresponsive" platelets was confirmed by immunoblot analysis (Figure 3). An antibody for LPA4 is presently not available to us.
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LPA has been proposed to serve as a potential mediator of atherothrombotic vascular disease. Therefore, we examined LPA responses in 70 patients with stable coronary artery disease. The average age was 68±12 years; 36% were females, 41% were diabetic, 60% were current or former smokers, 56% had suffered a previous MI, and 17% a previous stroke or transient ischemic attack. Platelets isolated from 69 CAD patients aggregated in response to LPA, whereas platelets from only 1 patient (95% CI 0.004 to 0.077 for nonresponse rate) failed to aggregate to LPA. These results differ from the nonresponse rate of 0.2 observed in our population of healthy donors (95% CI 0.096 to 0.346 for nonresponse rate) and suggest that patients with CAD may be less likely to be nonresponders. However, a number of confounders, such as age, medication use, or medical conditions, could account for the difference observed in these 2 populations.
To investigate the association of platelet LPA responses and CAD further, we analyzed platelet responses in 33 individuals presenting for diagnostic cardiac catheterization. The average age of these individuals was 61±11 years; 24% were female, 76% had high blood pressure, 18% diabetes, 42% were current or former smokers, 73% had hyperlipidemia or were taking medication to lower cholesterol. Platelets from 28 of the patients (85%) aggregated in response to LPA, whereas 5 patients met criteria for being LPA nonresponders. Among the responders, 25 of 28 (89%) had angiographic evidence for CAD, whereas CAD was diagnosed in only one of 5 (20%) LPA nonresponders.
As shown in Table 2, only CAD itself was identified as having a bivariate association with responder/nonresponder status (probability value=0.0039). Among the 26 patients with coronary artery disease, only 1 was a nonresponder (3.9%). On the other hand, 4 of 7 patients without coronary artery disease were nonresponders (57.1%). Given the small sample size, however, there is a possibility that some genuine bivariate associations may not have been detected. Coronary artery disease was also the only variable chosen for inclusion in a multivariate logistic regression model. Again, in view of the modest sample size, some genuine associations may not have been detected. The estimated odds ratio (odds of cath laboratory patient with CAD being a nonresponder/odds of cath laboratory patient without CAD being a nonresponder) was 0.030 (95% CI 0.002 to 0.364, probability value=0.0059 by a Wald tests.
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Discussion |
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Atherosclerotic plaque rupture exposes prothrombotic substrates in the lipid-rich core that stimulate platelet activation and can result in platelet-dependent thrombus formation. Previous studies by others have demonstrated that the lipid rich core of atherosclerotic plaque contains LPA and have supported a key role for LPA in platelet activation on contact with plaque lipid core in an in vitro setting.17,18 However, the role that LPA may play in platelet activation in a pathophysiologically relevant context is not known. We report heterogeneity in the ability of platelets from different donors to aggregate in response to LPA and have used this variability to gain insight into signaling systems involved in platelet LPA responses and to delineate potential physiological roles for platelet LPA signaling.
Platelets isolated from approximately 20% of normal, healthy donors failed to aggregate when exposed to LPA and were termed LPA "nonresponsive". Aggregation responses to a variety of other platelet agonists were normal. Nonresponders tested multiple times over 2 to 3 years consistently failed to aggregate in response to LPA, whereas repeated testing of selected responders always yielded aggregation to LPA. Further investigation revealed that LPA promoted inhibitory pathways including production of cAMP and VASP phosphorylation, and that pharmacological inhibition of adenylyl cyclase partially restored LPA-induced aggregation in platelets from "nonresponsive" donors. The additive effects of LPA and PGI2 and the inability of adenylyl cyclase inhibitors to completely restore LPA-induced aggregation suggest that the inhibitory effects of LPA may occur through cAMP-dependent and cAMP-independent pathways.
Taken together, our results suggest that LPA may be able to promote pathways that both inhibit and stimulate platelet function and that the relative balance between these pathways may determine the ultimate platelet response to LPA. The ability of LPA to attenuate its own platelet signaling is most likely attributable to the fact that it is a relatively weak platelet agonist.
To date, 5 G protein–coupled receptors have been proposed to mediate LPA signaling.20 LPA may also be an endogenous agonist of PPAR,34 which is of interest given a recent report that PPAR agonists exhibit inhibitory effects on certain platelet functions. In our qPCR analysis of gene expression levels of the known LPA receptors, Lpa5 was the most abundantly expressed in platelets (Z. Pamuklar, H.Y. Cheng, S.S. Smyth, unpublished data, 2007). Using a combination of immunoblot analysis and qPCR, we observed no difference in any of the major G proteins in platelets or in levels of expression of LPA receptors 1 and 2 in samples from responders and nonresponders. However, our results indicate that expression of LPA4 and PPAR mRNA may be elevated in nonresponders. This is interesting because, in some35,36 but not all systems,37 LPA4 couples through G
s to stimulate adenylyl cyclase and elevate cAMP levels. In addition, PPAR agonists reduce expression of CD40L and thromboxane release,37,38 2 important positive-feedback events that occur during platelet activation. Thus, both LPA4 or PPAR are attractive candidates to mediate inhibitor signaling by LPA, and their expression may be influenced by genetic or other factors. To our knowledge a direct link between PPAR signaling and platelet cAMP levels has not been observed, which could make this an attractive candidate mediator of cAMP-independent effects. As has been previously reported,18,27 we observed that alkyl-LPA was more potent in stimulating platelets than was LPA. Platelets from 4 of the LPA "nonresponsive" donors also failed to respond to alkyl-LPA, whereas platelets from 1 LPA-responsive donor did not aggregate in response to alkyl-LPA (Z. Pamuklar and S.S. Smyth, unpublished data, 2007). These findings suggest that different receptor systems may potentially mediate agonist activation of platelets by LPA and alkyl-LPA.
Our results in patients with CAD suggest that the incidence of platelet LPA responsiveness is higher in individuals with CAD and that LPA nonresponders may be less likely to have CAD. Our sample size of 33 provided 95% power to detect an association between responder/nonresponder status and CAD. Based on the observed proportions of responders and nonresponders presenting for diagnostic coronary angiography having the risk factors/conditions in Table 2, the following points can be made. An estimated sample size of 83 patients (70 responders, 13 nonresponders) would provide 80% power to detect an association between responder/nonresponder status and age. For detecting an association between responder/nonresponder status and hyperlipidemia, an estimated sample size of 86 (73 responders, 13 nonresponders) would provide 80% power. For detecting an association between responder/nonresponder status and hypertension, or between responder/nonresponder status and gender, an estimated sample size of 349 (296 responders, 53 nonresponders) would provide 80% power. As for diabetes, smoking, medication for hyperlipidemia, and family history of coronary artery disease, the sample size estimates for 80% power are all above 600 and the power estimates for a sample size of 33 are all below 10%. Based on these computations, the nonsignificant probability values in Table 2 should not necessarily be viewed as implying negligible associations.
In addition, we note that the relationship we perceived between CAD and LPA nonresponsiveness may have been mildly attenuated by survivor bias, but even the attenuated relationship was strong enough to be perceived with modest sample sizes. The inclusion criteria for the diagnostic catheterization study were very broad, as we wished to minimize bias; however, some exclusion criteria were necessary because LPA nonresponsiveness cannot be assessed reliably in patients taking certain medications.
It is possible that the presence of CAD alters platelet reactivity to attenuate the inhibitory pathway and render platelets more susceptible to the stimulatory effects of LPA. A number of other studies have suggested that CAD enhances platelet reactivity39,40 directly or indirectly as a result of endothelial dysfunction and reduction in local or systemic platelet inhibitors,41 such as prostaglandins or ectonucleotidase activity. Longitudinal studies will be necessary to determine whether LPA nonresponders display vascular protection from development of atherosclerosis.
Although considerable progress has been made in the identification of G protein–coupled receptors that mediate LPA responsiveness in a variety of cells,20 the identity of the LPA receptor responsible for platelet activation remains to be established. Work from several groups suggests that this receptor exhibits a pharmacological selectivity that is distinct from that of presently characterized LPA receptors. For example, Tokumura et al previously reported that platelets preferentially respond to LPA having a mono-unsaturated fatty acyl group and ether linked fatty acid (alkyl-LPA) rather than the more commonly found acyl-linked fatty acid.27 As noted above, we also observed that alkyl-LPA was more potent in inducing platelet aggregation, but did not find perfect correlation between LPA and alkyl-LPA responses. The most parsimonious explanation for our findings is that platelets express at least 2 distinct LPA receptors that couple selectively to pathways that promote and inhibit platelet activation. The existence of antagonistic receptor signaling systems has been documented for many other G protein–coupled receptor agonists including agents such as adrenergic agonists and angiotensin that play important roles in cardiovascular regulation. There is also precedent for a platelet agonist to have both stimulatory and inhibitory effects: the prostanoid PGE2 at low doses acts through the EP3 receptor to stimulate platelet function and at high doses through the IP receptor to increase cAMP and inhibit platelet function. Further work will be required to identify the platelet LPA receptors and definitively determine their roles in modulating platelet activation by LPA.
In conclusion, our findings support the contention that LPA may elicit both stimulatory and inhibitory effects on platelet function, with the net outcome determined by a relative balance of signaling systems. An individual’s platelet response to LPA may be influenced by both factors that affect the degree of systemic platelet activation, such as the presence of vascular disease, and genetic factors that contribute to the expression of LPA receptors. Additional studies will be required to establish the nature of the receptor systems mediating these responses and whether LPA nonresponsiveness protects from the development of vascular disease.
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Acknowledgments |
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Sources of Funding
This work was support by NIH grants HL078663, HL070304, HL07419 (S.S.S.), and GM050388 (A.J.M.). S.S.S. was the recipient of an Atorvastatin Physician-Scientist Award from Pfizer.
Disclosures
S.R.S. has received honoraria for serving as an advisor or consultant to the Medicine’s Company, Sanofi Aventis, AstraZeneca, Lilly, and Daiichi Sankyo. S.S.S. has received research grant support from Pfizer, Takeda, and The Medicine’s Company.
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Footnotes |
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Original received August 7, 2007; final version accepted December 23, 2007.
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