Introduction

Optimal platelet reactivity (PR) inhibition is critical to prevent thrombotic events (cardiovascular deaths, myocardial infarction, stent thrombosis, or stroke) in stented patients, especially in the aftermath of acute coronary syndromes (ACS). Therefore, several antiplatelet agents have been developed in order to preclude such complications.

Aspirin was the first antiplatelet agent used in patients presenting with coronary artery disease. Although effective in low-risk patients, it offered only a very limited protection in high-risk patients (ACS patients), leading to the development of new antiplatelet agents targeting another platelet activation pathway. Ticlopidine and then clopidogrel are thienopyridines targeting the platelets P2Y12 receptor to adenosine diphosphate (ADP). They were shown to reduce (in combination with aspirin) major adverse cardiac events (MACE) in ACS and patients undergoing percutaneous coronary interventions (PCI) [1, 2].

Therefore, dual antiplatelet therapy with aspirin in addition to a P2Y12 inhibitor (e.g., clopidogrel, prasugrel, or ticagrelor) was recommended in stented patients by international guidelines [3, 4, 5].

However, dual antiplatelet therapy failed to eradicate MACE following PCI. In the early days of clopidogrel, stent thrombosis at 1 month occurred in about 2% of stented patients raising concern of clinical resistance to clopidogrel [6].

Järemo et al. were the first to demonstrate the interindividual variability in response to clopidogrel, thanks to a flow cytometer assessing the rate of platelets bound to fibrinogen reflecting PR [7]. Nevertheless, this method lacks specificity and does not allow to analyze specifically the response to P2Y12 inhibitors such as clopidogrel. More specific tests are required.

The VASP index assay

Vasodilator-stimulated phosphoprotein (VASP) is an intraplatelet actin-binding protein [8, 9] is involved in the transduction of signal between the P2Y12 ADP receptor antagonists, which participate in the amplification process of platelet aggregation. When activated, the P2Y12 ADP receptor induces a dephosphorylation of the VASP index through the inactivation of a cyclic adenosine monophosphate-dependent protein kinase or guanosine monophosphate-dependent protein kinase [10]. This dephosphorylated VASP induces the activation of glycoprotein GPIIb/IIIa, which is involved in platelet aggregation [11]. Prostaglandin E1 (PGE1)-activated adenylyl cyclase and P2Y12 inhibitors cause VASP phosphorylation [12].

The VASP index assay is a flow cytometer assessment of the ratio of phosphorylated and dephosphorylated VASP. This test is performed on a citrated blood sample. The blood is incubated with either PGE1 or with PGE1 associated with ADP for about 10 min. After being fixed with formaldehyde, platelets are permeabilized by a detergent. Afterward, platelets are marked with a fluorescent monoclonal antibody against phosphorylated VASP [8]. The median fluorescence intensity (MFI) is assessed by a flow cytometer. Thus, the VASP index is expressed in platelet reactivity index (PRI) and is determined with this calculation:

The main limitations of this test are the need for a trained operator, the use of a flow cytometer (which can be an economic limitation), and the need for a compatible rate of red cells.

A VASP index ELISA assay has been recently validated and may overcome these limitations [13, 14].

VASP index and clinical events

Barragan et al. were the first to demonstrate the relation between thrombotic events (stent thrombosis) and high on-treatment platelet reactivity (HTPR) [15]. They compared the VASP index of 16 prospectively included patients with 30 stented patients free from stent thrombosis. Stent thrombosis patients had a higher VASP index than control (63.28 ± 9.56% vs. 39.80 ± 10.9%, p < 0.0001). Several studies followed and clearly confirmed the relation between MACE in stented patients and HTPR after the clopidogrel loading dose [16, 17, 18, 19, 20, 21].

Further, using a ROC curve analysis on the data from several studies, a consensus determined the cutoff value of VASP PRI greater than 50% to define patients presenting with high on-clopidogrel PR. A VASP index above this threshold is a risk marker of thrombotic events (i.e., stent thrombosis, myocardial infarction, or cardiovascular deaths) in stented patients. This threshold yields to this test an excellent negative predictive value although the positive predictive value is low [19, 22].

Using different tests to assess PR, recent studies suggested a link between low on-treatment PR (« hyperresponders ») and bleeding events in patients treated with dual antiplatelet therapy. These observations lead to the concept of therapeutic window for PR inhibition in patients receiving a P2Y12 ADP receptor antagonist and treated with PCI or CABG [23, 24, 25].