The Usefulness of Platelet-derived Microparticle as Biomarker of Antiplatelet Therapy in Kawasaki Disease

Kawasaki disease (KD) is an acute febrile, systemic vasculitic syndrome of unknown etiology that occurs primarily in children younger than 5 years of age. The standard treatment of acute KD is a combination of high-dose (2 g/kg) intravenous immunoglobulin (IVIG) and oral aspirin (50 mg/kg/day). The acute phase of KD involves platelet activation, and antiplatelet therapy is included in the treatment protocol for this phase (1). The finding of low levels of antithrombin III and depletion of the fibrinolytic system supports the concept that active intravascular clot formation and degradation occur in these patients and are most notable early in the acute phase of the disease (2). Thrombosis mainly occurs in the artery and is associated with platelet activation in KD (3). Following the cessation of fever for 2 to 3 days and improvement of all acute symptoms, the dosage of acetylsalicylic acid (ASA) can be reduced to the minimum level necessary to inhibit platelet aggregation.

Low-dose ASA has an antithrombotic effect through inhibiting thromboxane A₂ (TxA₂). Aspirin is continued for its antithrombotic effect until 6–8 weeks after KD onset, when the erythrocyte sedimentation rate (ESR) and thrombocytosis have normalized in patients that have not developed coronary artery lesion (CAL), as detected by echocardiography. However, the decision in the total duration of antiplatelet therapy in KD patients is dependent on a routine blood testing, including the level of platelets.

Microparticles, also referred to as microvesicles or more rarely ectosomes, are submicron fragments that are shed from the plasma membrane of stimulated or apoptotic cells (4). Platelet-derived microparticles (PDMPs) have recently been reported as a platelet activation marker. Therefore, the PDMP levels can be used as an index of platelet activation (5, 6). PDMPs are a heterogeneous population of vesicles generated from the plasma membrane upon platelet activation by various stimuli. An increase in the total numbers of PDMPs is associated with disease states such as atherosclerosis, diabetes, cancer, sepsis, and pulmonary hypertension (7). However, little is known about these microparticles being linked to platelet dynamics in patients with KD. The purpose of this study was to prove that a state of platelet activation exists in patients with KD from the acute stage to the convalescent phase and to demonstrate the usefulness of the PDMP levels in evaluating the effect of antiplatelet therapy in KD.

This study was conducted to determine the role of PDMPs as a biomarker of antiplatelet agents in KD and to investigate the serial change of plasma PDMP levels per stage of KD. Our study showed that the PDMP levels were significantly elevated in patients with KD immediately after IVIG treatment, but the levels of PDMP decreased from the subacute stage to the convalescent phase during antiplatelet therapy.

Platelets are well-established components of the hemostatic system and are recognized as having a role in innate immunity (11). Recently, several additional functions of platelets have been demonstrated in the pathogenesis of various diseases in which inflammation is an important clinical sign. Activation of blood platelets is a typical finding in patients with systemic inflammation and sepsis. Under inflammatory conditions, platelets also show robust interactions with leukocytes (12). Activated platelets adhere to a damaged vessel wall and promote local recruitment of leukocytes.

Thrombocytosis is a universal finding in patients with subacute stage of KD, but it is not specific for KD. In previous epidemiological studies, platelet counts were found to be elevated in patients with chronic inflammatory diseases, malignancies, and myeloproliferative disorders (13). The degree of platelet activation was closely associated with the presence of coronary artery complications in the acute stage of KD (2). In the subacute stage of KD, the platelet count increases rapidly and severe stenosis or obstruction is reported to occur due to thrombi in the main coronary arteries with aneurysms. In the present study, the degree of thrombocytosis was not significantly different between groups of KD with and without CALs.

PDMPs are endoplasmic reticulum-derived vesicles ranging from 0.02 to 0.10 μm in size that are discharged from platelets upon activation by some sort of stimulus. PDMPs are the most abundant microparticles in the blood stream constituting approximately 70% to 90% of circulating microparticles (14). Detectable in the peripheral blood of normal individuals, microparticles are elevated in clinical situations where the thrombotic risk is increased. No significant difference in PDMP levels were observed between the KD patients with and without CALs in the present study. Because recent research has focused on microparticles in pathological conditions, the vesicles can easily be mistaken as purely detrimental. However, these microparticles can be beneficial and are present in healthy individuals. The present study showed that PDMP levels in control groups were not significantly different from those in the pre-IVIG KD group. It is probably because the control group in our study does not have healthy controls but disease controls.

Under most pathophysiologic conditions, PDMPs appear to be derived mostly from procoagulant circulating species with leukocytic or endothelial origins being less representative. Inflammation and coagulation are linked processes in many diseases such as septicemia and PDMPs are known to amplify the response by activating the endothelium (15). PDMPs themselves can initiate inflammation. Barry and coworkers demonstrated that PDMPs deliver arachidonic acid to endothelial cells, which results in the upregulation of CD54 (intercellular adhesion molecule-1 [ICAM-1]) and subsequent adhesion of monocytes (15). Therefore, these microparticles are not only a consequence of the disease but also have a casual role in the pathogenesis of inflammatory diseases.

Although PDMP has recently received a great deal of attention as a marker of platelet activation in adults (5, 6), there is little data on PDMP levels in pediatric patients. Taki et al. (16) reported that enhanced platelet aggregation was noted at a high frequency before treatment of KD by using the particle counting method. They suggested that platelet activation continues during the second stage (i.e., from the start of IVIG therapy to 20 days), but platelet activation is inhibited by antiplatelet agents. A recent study demonstrated that IVIG treatment significantly decreased PDMP levels in 18 patients with acute KD (17). They showed that the PDMP levels both immediately and 10–14 days after IVIG treatment were significantly lower than the pre-IVIG level. In contrast, we could find significant increase in PDMP level 2–5 days after IVIG treatment compared with the pre-IVIG level. We speculate that this difference was probably due to differences across ELISA kits used in this study and the timing of changes in ASA dosage. In the above-mentioned reports, they immediately changed the dose of ASA from an anti-inflammatory dose (30–50 mg/kg/day) to an anti-thrombotic dose (5 mg/kg/day) when the patients became afebrile. However, we maintained a high dose of ASA (50 mg/kg/day) until the patient had been afebrile for 2 to 3 days and all acute symptoms were improving. The ELISA test depends on the quality of reagents, usually antibodies. In addition, the possible interference of soluble antigens may result in underestimation of microparticle levels by antigenic capture.

In the present study, we used the Zymuphen MP-activity (Medicago) ELISA to measure prothrombinase activity with an ELISA reader (Bio-Rad, Hercules, CA, USA), measuring samples at a wavelength of 405 nm (18, 19). The diluted assayed plasma sample, supplemented with calcium, factor Xa, and thrombin inhibitors were introduced into 1 of the microplate wells coated with streptavidin and biotinylated annexin V, and then incubated. The phospholipid concentration is the limiting factor in this ELISA test. Even if flow cytometry is considered the “gold standard” of microparticle detection, there are still technical limitations concerning detection of small microparticles (100 nm to 500 nm). Therefore, microparticle activity can be analyzed by prothrombinase assay (ELISA), which is easily performed and shows a significant correlation of results (20).

In the present study, the PDMP levels of 17 patients rebounded after aspirin was discontinued even though they had no CALs. These results suggest that vascular inflammation may persist after the convalescent phase even if the morphology of the coronary arteries appears normal onechocardiography. The causes and clinical implications of the persistent platelet activation after the acute stage in patients with KD were probably due to the persistence of subclinical low-grade inflammation in KD (21). A recent study reported that children with a previous episode of KD showed increased platelet activation when compared with healthy participants despite no apparent vascular abnormality at follow-up (22).

ASA was the first antiplatelet agent used for both the primary and secondary prevention of cardiovascular events. By irreversibly binding to cyclooxygenase-1, ASA inhibits TxA₂, thus blocking TxA₂-induced platelet aggregation and inducing vasoconstriction (23). In KD patients, low-dose ASA is continued for its antithrombotic effect until 6–8 weeks after KD onset. Moreover, recent evidence supports the fact that antiplatelet therapy affects host immunity. Several studies have shown that ASA and clopidogrel not only diminish the risk of atherothrombotic events, but also reduce markers of systemic inflammation, including CRP, and proinflammatory cytokines (24). Antiplatelet medications such as clopidogrel and ticagrelor were also associated with reduced mortality in patients with sepsis, without causing excessive bleeding in these patients (25, 26).

In cardiovascular diseases, the inhibition of the shedding of microparticles account for the efficacy of various drugs (27). Abciximab, a platelet glycoprotein IIb–IIIa antagonist acts as a potent antiplatelet agent for KD patients with giant coronary artery aneurysm or thrombosis. A recent study showed that abciximab treatments have an important role in the management of severe complications of KD, although prospective randomized controlled studies are needed to fully evaluate their efficacy in preventing thrombotic complications and promoting vascular remodeling (28).

The current study has some limitations. First, we enrolled a relatively small number of patients. Therefore, we could not determine whether our results are also applicable to different geographic populations. Second, there were no coronary aneurysms, such as a sequela in the KD group. Lastly, we could not perform the platelet function tests such as adenosine diphosphate (ADP)-induced platelet aggregation test in patients with KD and could not check the follow-up PDMP levels in control patients because this would have required a large amount of additional blood to be drawn from these small infants and young children.

In conclusion, enhanced platelet activation is noted before treatment of KD until the convalescent phase of KD as long as 4 or 5 months after the onset of KD. These results indicate that the timing of ASA discontinuation should be evaluated in each individual patient with KD. Monitoring the biomarker of platelet activation is important because they can be used as sentinels for patients with a high risk for thrombosis such as those with intracardiac stents, complex congenital heart disease, and KD. Further studies with a larger sample size and longer follow-up period are required to clarify the clinical significance of the PDMPs in the pathogenesis and treatment of KD.