Studies on platelet function and microvesicles in acute coronary syndrome

INTRODUCTION: The cornerstone of treatment of acute coronary syndrome (ACS) is percutaneous coronary intervention (PCI) and dual antiplatelet therapy (DAT) with aspirin and the ADP-receptor inhibitor clopidogrel. However, some ACS patients will suffer from recurrent cardiovascular events, and it has been shown that this may partly be due to an insufficient antiplatelet response to DAT. In this thesis, we investigated platelet function in ACS. The overarching aim was to further investigate clopidogrel non-responsiveness with established and new methods to assess platelet function.

METHODS AND RESULTS: In study I, we investigated if arterial and venous sampling give comparable results with respect to detection of poor antiplatelet responsiveness to clopidogrel and aspirin with the established method multiple electrode whole blood impedance aggregometry (MEA). Twenty-eight patients with coronary artery disease were investigated in connection to PCI. Identical number of patients with poor responsiveness to clopidogrel (7/28; i.e. 25 %) and aspirin (3/28; i.e. 11 %) were detected in arterial and venous blood samples with MEA. Thus, arterial and venous sampling can be used interchangeably in the detection of poor responsiveness to clopidogrel and aspirin when MEA is used to study platelet function. In study II, we investigated ADP-induced platelet aggregation by MEA in 183 patients with ACS on DAT at discharge (3–5 days after admission). We also measured circulating microvesicles (MVs) and platelet derived MV (PMVs) in 154 of the ACS patients. Of note, MVs are small membrane buds released from various cells in response to activation or apoptosis. We found that around 20 % (36/183) of ACS patients were nonresponders (“high on-treatment platelet reactivity”; HPR) to clopidogrel. Flow cytometry measurements showed that circulating PMV levels were significantly higher in HPR compared to those with “sufficient” clopidogrel responsiveness (“normal on-treatment platelet reactivity”; NPR). Furthermore, in patients with strong inhibition to clopidogrel (low on-treatment platelet reactivity; LPR) PMV levels were significantly lower than in NPR patients. Thus, levels of circulating PMVs reflect platelet responsiveness to clopidogrel. Study III was performed to elucidate if MVs from ACS patients on DAT influence platelet aggregation. Thus, we added MVs from samples of patients with ACS with HPR and from patients with Non-HPR (i.e, LPR or NPR) to clopidogrel, to platelet rich plasma obtained from healthy volunteers. Results showed that MVs from HPR patients significantly enhanced spontaneous platelet aggregation as compared to MVs from patients with Non-HPR. In addition, we could also show that MVs from ACS patients with diabetes and DAT, enhanced platelet aggregation compared to MVs from ACS patients on DAT without diabetes. MVs have the potential to enhance platelet aggregation, supporting the idea that MVs may not only be “markers” but also “makers” of physiological or pathophysiological processes. In study IV, we investigated if circulating MVs expose a molecule of the humoral immune system pentraxin-3 (PTX3). Thus, we measured the presence of PTX3+--MVs in plasma in patients with acute myocardial infarction (AMI). We found that PTX3+--MVs were elevated at admittance in patients with acute ST-elevation myocardial infarction (STEMI; n=23) and that levels decreased after PCI. Further, the circulating PTX3+--MVs levels were even lower in AMI patients at discharge (3–5 days after admission; n=153), but not as low as in healthy subjects (n=15). Thus, PTX3 is exposed on circulating MVs in the acute setting of AMI, and the levels fall significantly over the days after the acute event. The PTX3+--MVs should be further phenotyped with our flow cytometry method to elucidate the origin of PTX3 and its possible role in acute coronary artery disease.

CONCLUSION: Clopidogrel non-responsiveness is present in every fourth to every fifth ACS patient on clopidogrel. This can be detected either in arterial or venous samples by MEA. The number of circulating MVs reflect platelet responsiveness to clopidogrel, and circulating MVs in ACS patients have the capacity to enhance platelet aggregation, indicating a possible functional role of circulating MVs in the setting of ACS. In acute AMI, MVs exposing PTX3 circulate in an elevated concentration. The role of these PTX3+--MVs deserves to be further investigated.

List of scientific papers

I. Sam Kafian, Fariborz Mobarrez, Majid Kalani, Håkan Wallén & Bassem A. Samad. Comparison of venous and arterial blood sampling for the assessment of platelet aggregation with whole blood impedance aggregometry. Scand J Clin Lab Invest. 2011;71(8):637–40.
https://doi.org/10.3109/00365513.2011.604731

II. Sam Kafian, Fariborz Mobarrez, Håkan Wallén & Bassem Samad. Association between platelet reactivity and circulating platelet-derived microvesicles in patients with acute coronary syndrome. Platelets. 2015;26(5):467–73.
https://doi.org/10.3109/09537104.2014.940304

III. Sam Kafian, Håkan Wallén, Bassem A. Samad, Fariborz Mobarrez. Microvesicles from patients with acute coronary syndrome enhance platelet aggregation. [Manuscript]

IV. Sam Kafian, Håkan Wallén, Gundars Rasmanis, Charlotte Thålin, Bassem A. Samad, Fariborz Mobarrez. Exposure of pentraxin-3 on microvesicles in patients with acute myocardial infarction. [Manuscript]