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Plasma levels of the growth arrest‐specific gene 6 product (Gas6) and antiplatelet drug responsiveness in healthy subjects

2006; Elsevier BV; Volume: 4; Issue: 10 Linguagem: Inglês

10.1111/j.1538-7836.2006.02155.x

1538-7933

Laurent Burnier, Delphine Borgel, Anne Angelillo‐Scherrer, Pierre Fontana,

Diabetes Treatment and Management

Aspirin targets the thromboxane (Tx) A2 pathway and clopidogrel the adenosine diphosphate (ADP) pathway of platelet activation process. The biological effects of aspirin and clopidogrel vary from one subject to another and this variability has been implicated in the risk of recurrent ischemic events [1Cattaneo M. Aspirin and clopidogrel: efficacy, safety, and the issue of drug resistance.Arterioscler Thromb Vasc Biol. 2004; 24: 1980-7Crossref PubMed Scopus (406) Google Scholar]. We recently examined aspirin and clopidogrel responsiveness in 96 healthy subjects [2Fontana P. Nolli S. Reber G. De Moerloose P. Biological effects of aspirin and clopidogrel in a randomized cross‐over study in 96 healthy volunteers.J Thromb Haemost. 2006; 4: 813-9Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar] and found aspirin pseudo‐resistance (APR) in 30% of cases. As in the other subjects, the production of TxB2 (the stable breakdown product of TxA2) was abolished by aspirin in these pseudo‐resistant subjects, but their platelet closure times (CT) determined with the PFA‐100® device (collagen/epinephrine cartridge) were normal, despite aspirin intake (100 mg day−1) for 7 days. This suggested that platelets continued to aggregate despite adequate inhibition of TxA2 production by aspirin, and that additional platelet amplification pathways therefore compensated for the lack of TxA2. Several studies have linked low PFA‐100® (Dade Behring, Düdingen, Switzerland) CT values to vascular events in aspirin‐treated patients [3Grundmann K. Jaschonek K. Kleine B. Dichgans J. Topka H. Aspirin non‐responder status in patients with recurrent cerebral ischemic attacks.J Neurol. 2003; 250: 63-6Crossref PubMed Scopus (259) Google Scholar, 4Frossard M. Fuchs I. Leitner J.M. Hsieh K. Vlcek M. Losert H. Domanovits H. Schreiber W. Laggner A.N. Jilma B. Platelet function predicts myocardial damage in patients with acute myocardial infarction.Circulation. 2004; 110: 1392-7Crossref PubMed Scopus (226) Google Scholar, 5Pamukcu B. Oflaz H. Nisanci Y. The role of platelet glycoprotein IIIa polymorphism in the high prevalence of in vitro aspirin resistance in patients with intracoronary stent restenosis.Am Heart J. 2005; 149: 675-80Crossref PubMed Scopus (72) Google Scholar], suggesting that APR may be a risk factor for recurrent cardiovascular events. The importance of the growth arrest‐specific gene 6 product (Gas6) in platelet activation and thrombus stabilization has been demonstrated in mice [6Angelillo‐Scherrer A. De Frutos P. Aparicio C. Melis E. Savi P. Lupu F. Arnout J. Dewerchin M. Hoylaerts M. Herbert J. Collen D. Dahlback B. Carmeliet P. Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.Nat Med. 2001; 7: 215-21Crossref PubMed Scopus (365) Google Scholar, 7Gould W.R. Baxi S.M. Schroeder R. Peng Y.W. Leadley R.J. Peterson J.T. Perrin L.A. Gas6 receptors Axl, Sky and Mer enhance platelet activation and regulate thrombotic responses.J Thromb Haemost. 2005; 3: 733-41Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 8Angelillo‐Scherrer A. Burnier L. Flores N. Savi P. DeMol M. Schaeffer P. Herbert J.M. Lemke G. Goff S.P. Matsushima G.K. Earp H.S. Vesin C. Hoylaerts M.F. Plaisance S. Collen D. Conway E.M. Wehrle‐Haller B. Carmeliet P. Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy.J Clin Invest. 2005; 115: 237-46Crossref PubMed Scopus (209) Google Scholar], but the expression of Gas6 and its receptors in human platelets is controversial [6Angelillo‐Scherrer A. De Frutos P. Aparicio C. Melis E. Savi P. Lupu F. Arnout J. Dewerchin M. Hoylaerts M. Herbert J. Collen D. Dahlback B. Carmeliet P. Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.Nat Med. 2001; 7: 215-21Crossref PubMed Scopus (365) Google Scholar, 9Balogh I. Hafizi S. Stenhoff J. Hansson K. Dahlback B. Analysis of Gas6 in human platelets and plasma.Arterioscler Thromb Vasc Biol. 2005; 25: 1280-6Crossref PubMed Scopus (105) Google Scholar]. We and others have detected Gas6 protein with an ELISA method in human and mouse plasma [9Balogh I. Hafizi S. Stenhoff J. Hansson K. Dahlback B. Analysis of Gas6 in human platelets and plasma.Arterioscler Thromb Vasc Biol. 2005; 25: 1280-6Crossref PubMed Scopus (105) Google Scholar, 10Borgel D. Clauser S. Bornstain C. Bieche I. Bissery A. Remones V. Fagon J.Y. Aiach M. Diehl J.L. Elevated growth‐arrest‐specific protein 6 plasma levels in patients with severe sepsis.Crit Care Med. 2006; 34: 219-22Crossref PubMed Scopus (81) Google Scholar, 11Clauser S. Bachelot‐Lozat C. Fontana P. Gaussem P. Remones V. Aiach M. Borgel D. Physiological plasma Gas6 levels do not influence platelet aggregation.Arterioscler Thromb Vasc Biol. 2006; 26: e22Crossref PubMed Scopus (19) Google Scholar]. We recently showed that plasma Gas6 levels in healthy subjects do not influence platelet aggregation ex vivo in response to selected platelet agonists [11Clauser S. Bachelot‐Lozat C. Fontana P. Gaussem P. Remones V. Aiach M. Borgel D. Physiological plasma Gas6 levels do not influence platelet aggregation.Arterioscler Thromb Vasc Biol. 2006; 26: e22Crossref PubMed Scopus (19) Google Scholar], but the role of Gas6 in the variability of biological responses to antiplatelet drugs has not been investigated. Plasma Gas6 levels were determined in the 96 healthy volunteers enrolled in the above‐mentioned study [2Fontana P. Nolli S. Reber G. De Moerloose P. Biological effects of aspirin and clopidogrel in a randomized cross‐over study in 96 healthy volunteers.J Thromb Haemost. 2006; 4: 813-9Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar] with an ELISA method [10Borgel D. Clauser S. Bornstain C. Bieche I. Bissery A. Remones V. Fagon J.Y. Aiach M. Diehl J.L. Elevated growth‐arrest‐specific protein 6 plasma levels in patients with severe sepsis.Crit Care Med. 2006; 34: 219-22Crossref PubMed Scopus (81) Google Scholar] in baseline samples, and were expressed as a percentage of the Gas6 level in a normal plasma pool [11Clauser S. Bachelot‐Lozat C. Fontana P. Gaussem P. Remones V. Aiach M. Borgel D. Physiological plasma Gas6 levels do not influence platelet aggregation.Arterioscler Thromb Vasc Biol. 2006; 26: e22Crossref PubMed Scopus (19) Google Scholar]. Statistical analysis used the Kruskall–Wallis test and a logistic regression model to test the relationship between antiplatelet drug responsiveness and quartiles of Gas6 levels, after adjustment for potentially confounding variables. Plasma Gas6 levels ranged from 47.7% to 167.1% of the value obtained with the normal plasma pool [median 85%, interquartile range (IR): 68–103%]. These values are compatible with those found in another population of healthy male volunteers [11Clauser S. Bachelot‐Lozat C. Fontana P. Gaussem P. Remones V. Aiach M. Borgel D. Physiological plasma Gas6 levels do not influence platelet aggregation.Arterioscler Thromb Vasc Biol. 2006; 26: e22Crossref PubMed Scopus (19) Google Scholar]. Subjects in the first and second quartiles of plasma Gas6 levels had median values of 64.3% and 79.2%, respectively, while subjects in the third and fourth quartiles had values of 88.9% and 115.7% (P < 0.001). Subjects with APR had a median Gas6 level of 88% (IR: 78–133%), while aspirin‐sensitive subjects had a median Gas6 level of 79% (IR: 57–132%, P = 0.033). The proportion of APR subjects increased from 8.0% in quartile 1 of plasma Gas6 levels to 21.7%, 45.8% and 43.4% in quartiles 2–4, respectively (P = 0.009). Table 1 shows the results of univariate and multivariate logistic regression analysis testing the association of APR and quartiles of Gas6 levels with and without adjustment variables. Subjects in quartiles 2 through 4 had Odds ratios of 7.0 to 10.2 compared to subjects in quartile 1, after adjustment for von Willebrand factor, basal PFA‐100® CT, platelet, hematocrit, fibrinogen, and basal collagen lagtime values.Table 1Logistic regression analysis of the association between aspirin pseudo‐resistance and quartiles of plasma gas6 levels. Quartile 1 is the reference group. Adjustment variables are the plasma fibrinogen, hematocrit, platelet count, von Willebrand factor, basal PFA‐100® closure time and basal collagen lagtime valuesGas6 levelsUnadjustedAdjustedORCIPORCIPQuartile 23.20.5–18.40.1947.00.9–51.00.056Quartile 39.71.9–50.80.0079.41.5–57.90.015Quartile 48.81.7–46.70.01010.21.6–63.00.012OR, Odds ratio; CI, 95% confidence interval. Open table in a new tab OR, Odds ratio; CI, 95% confidence interval. Quartiles of clopidogrel responsiveness did not correlate with quartiles of Gas6 levels (P = 0.4, data not shown). Likewise, median Gas6 levels did not differ across quartiles of clopidogrel responsiveness (87.5%, 85.8%, 81.7% and 83.9% in quartiles 1 through 4, respectively, P = 0.6). Platelet activation triggers numerous amplification pathways [12Brass L.F. Zhu L. Stalker T.J. Minding the gaps to promote thrombus growth and stability.J Clin Invest. 2005; 115: 3385-92Crossref PubMed Scopus (114) Google Scholar], and antiplatelet therapy targeting a single pathway (TxA2 pathway with aspirin, ADP pathway with clopidogrel) may not therefore adequately impair platelet aggregation in some subjects. Amplification pathways not targeted by current antiplatelet drugs include Gas6, CD40 ligand, Eph kinase and ephrins, junctional adhesion molecules, endothelial cell‐specific adhesion molecules, CD226, platelet endothelial cell molecule‐1, signaling lymphocytic activation molecule, CD84 and semaphorin 4D [12Brass L.F. Zhu L. Stalker T.J. Minding the gaps to promote thrombus growth and stability.J Clin Invest. 2005; 115: 3385-92Crossref PubMed Scopus (114) Google Scholar]. The biological effectiveness of clopidogrel is tightly related to the inhibition of cyclic adenosine monophosphate production by blockade of the ADP receptor P2Y12 [13Aleil B. Ravanat C. Cazenave J.P. Rochoux G. Heitz A. Gachet C. Flow cytometric analysis of intraplatelet VASP phosphorylation for the detection of clopidogrel resistance in patients with ischemic cardiovascular diseases.J Thromb Haemost. 2005; 3: 85-92Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar], and this diminishes the potential influence of other amplification pathways on clopidogrel responsiveness. In contrast, aspirin is equally potent on TxB2 production in aspirin pseudo‐resistant and aspirin‐sensitive subjects [2Fontana P. Nolli S. Reber G. De Moerloose P. Biological effects of aspirin and clopidogrel in a randomized cross‐over study in 96 healthy volunteers.J Thromb Haemost. 2006; 4: 813-9Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar]. As a result, inhibition of the TxA2 pathway by aspirin might unveil the role of platelet amplification pathways such as Gas6 [11Clauser S. Bachelot‐Lozat C. Fontana P. Gaussem P. Remones V. Aiach M. Borgel D. Physiological plasma Gas6 levels do not influence platelet aggregation.Arterioscler Thromb Vasc Biol. 2006; 26: e22Crossref PubMed Scopus (19) Google Scholar]. The role of the Gas6 pathway in platelet activation might therefore become crucial when a major amplification pathway such as TxA2 is abolished by aspirin. We show here that healthy subjects with Gas6 levels in the third and fourth quartiles have a 9‐ to 10‐fold higher risk of being aspirin pseudo‐resistant compared to subjects in the first quartile. The implications of these findings for the outcome of antiplatelet therapy in clinical practice are under investigation. The authors state that they have no conflict of interest. We thank Dr Thomas Perneger for help with the statistical analysis and Rocco Sugamele for excellent technical assistance. This work was supported by grants from the Internal Medicine Department of Geneva hospitals (PRD‐03‐II‐18), the Swiss Society of Angiology, the Leenaards Foundation, the Ingeborg Naegeli Foundation (PF) and the Swiss National Foundation for Scientific Research PP00B‐106690/1 (AAS).