Fabry disease, an X-linked metabolic disorder, is caused by insufficient activity of the lysosomal enzyme alpha galactosidase A (α-gal A). This results in impaired catabolism of glycosphingolipids and their subsequent accumulation (Gb3) leading to endothelial dysfunction. Currently, enzyme replacement therapy (ERT) is the only available specific treatment for patients with Fabry disease. The identification of markers that could serve as indicators of disease severity, prognosis and possible effects of specific therapies is highly desirable. Elevated endothelial cell membrane microparticles in blood have been documented for various diseases with a vascular injury component. Thus, we investigated the presence of endothelial cell membrane derived microparticles (EMPs) in peripheral venous blood samples of 10 pediatric Fabry patients (9 males and 1 female) before they were started on ERT and at the end of 6 and 12 months into the treatment regimen of infusions every other week with agalsidase alfa (Shire Human Genetic Therapies, Cambridge, MA) at 0.2 mg/kg. Both the 6 and 12 month samples were collected 14 days after the last enzyme infusion, thus right before the next infusion. Median age of patients was 12 (range: 10 – 18); renal function, urinary protein excretion, and cardiac function and structure were normal. Carbamazepine or gabapentin were common medications. Patients were stable except for one who suffered recurrent symptomatic and lacunar ischemic strokes. Plasma from healthy age-matched volunteers (Ctrl, n=19) was used for comparison. For EMP analysis, which was done at the same time and blindly, we used a three-color flow cytometric assay (

Simak et al, J Thromb Haemost 4, 1296–1302, 2006
). The analyzed MPs of 0.3 – 1.0 μm in size (mean 0.45 μm) were sedimented from plasma at 20,000 g. EMPs were identified by antibodies to both endothelial antigen CD105 (endoglin) and the more specific CD144 (VE-cadherin). Annexin V was used to identify MPs exposing phosphatidylserine (PS). Our results demonstrated that CD144+CD105+ EMPs were elevated in Fabry patients prior to ERT (522±177/μL) as compared to the control group (393±190/μL, p=0.031). Similarly, CD144+PS+CD41− EMPs were also significantly elevated in Fabry patients (122±55/μL vs. 83±40/μL, p=0.037). It should be noted that the patient experiencing ischemic strokes was not an outlier. In contrast, there were no significant differences in red blood cell (CD235a+), leukocyte (CD45+) or platelet (CD41a+) MP-counts between Fabry patients and the control group. Interestingly, we found a highly significant decrease (p= 0.004) in EMP-counts after 6 months (406±147/μL) and 12 months (282±112/μL) of ERT (see Fig), while platelet, leukocyte and red blood cell MPs did not change significantly. Our results suggest that ERT may influence the counts of circulating EMPs in pediatric Fabry patients. These finding warrant further studies to elucidate how circulating EMPs may correlate with severity and characteristics of endothelial injury in Fabry disease and whether EMP analysis could be used for monitoring the effects of ERT on endothelial functions. The views of the authors represent a scientific opinion and should not be construed as FDA policy.

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Topics:
cell-derived microparticles, endothelium, enzyme replacement therapy, pediatrics, fabry disease, massively-parallel genome sequencing, mucopolysaccharidoses, enzymes, infusion procedures, ischemic stroke

Disclosure: No relevant conflicts of interest to declare.

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2006, The American Society of Hematology
2006
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