The Effect of Vitamin D Supplementation on HIV-associated Platelet Hyperreactivity

1. Study design A prospective intervention study. HIV-infected patients using ART and who come for follow-up at the Teratai HIV clinic will be asked to participate in the study. All study participants will receive vitamin D supplementation (soft gel capsule containing cholecalciferol 400 IU) to be consumed 2 soft gel capsules daily for three consecutive months. The effect on the platelet parameters specified above will be determined before start cholecalciferol supplementation and after three months.
2. Duration of study Usually, 150 HIV-infected patients are treated with ART every week at the Teratai HIV clinic of the Hasan Sadikin Hospital Bandung Indonesia. Between half January 2014 and end of February 2014, 50 patients will be enrolled in this study. We hypothesize that this period is long enough to include 50 patients for the present study. Supplementation will take place between March and May and analysis will be done in June and July. A summary of duration of study is depicted in the study timeline.
3. Study population The study population consists of adult HIV-infected patients treated with ART enrolled at HIV polyclinic at Hasan Sadikin General Hospital, Bandung, Indonesia.
4. Sample size calculation Our sample size calculation is based on the primary outcome, which is the area under the curve (AUC) of the platelet reactivity curve with P-selectin (CD62P) as platelet activation marker and ADP as platelet agonist. In a cohort of Dutch HIV-infected patients we found a mean AUC of 2500 with an SD of 660. Assuming that a decrease in AUC of 2500 to 2100 upon supplementation of vitamin D is clinically relevant, a sample size of 23 would be sufficient using a paired design with α 0.05 and a power of 80%.
5. Methods Study parameters/endpoints

There are two study parameters applied in this study:

1. Platelet reactivity, expressed as the membrane expression of the platelet activation markers CD62P (P-selectin) and fibrinogen binding to integrin αIIbβ3, to stimulation with increasing concentration of platelet agonists ADP.
2. Platelet-monocyte complexes.
3. Study procedures

a. Enrolment of patients During a scheduled routine polyclinic visit, the treating internist will identify patients fulfilling inclusion criteria. He/she will explain the background and objectives of the study to the patient. In case patients opt to participate, the patient will then be asked to read and undersign the informed consent form.

Venous blood (three tubes of 3 mL each) will then drawn for study purpose at enrolment and after three months supplementation for:

1. 25-hydroxyvitamin D level (3 ml serum)
2. Platelet assays as specified above (3 ml CTAD)
3. Full blood count and CD4 cells (3 ml EDTA) In many instances, this will not require an extra venepuncture as blood is usually collected for planned blood analysis for routine clinical care during regular polyclinic visits.

Measurements and analysis consist of 25-hydroxyvitamin D level, platelet reactivity, and platelet-monocyte complexes will be done when the patients participate in the study. The study participants will receive 180 soft gel cholecalciferol capsules, to be taken 2 capsules once a day for 90 days as stated above.