The Hydroxyurea Effects on Plasma Levels and Surface Expression of Adhesion Molecules ICAM-1 VCAM-1, E-SELECTIN and on Plasma Levels of Protein C and Protein S in Patients with Sickle Cell Disease and β-Thalassemia Intermedia
Authors: Giuffrida Gaetano , Cingari maria Rocca , Parrinello Nunziatina Laura , Figuera Amalia 
Published: April 2020

Abstract
The pathophysiological mechanism of vaso-occlusion in Sickle Cell disease (SCA) is complex, involving adhesive processes between sickle red blood, leukocyte, and the activated endothelium. Reduced activity of the protein C and protein S may contribute to vaso-occlusion. We investigated the effect of Hydroxyurea (HU) on surface expression of the circulating endothelial cells (CEC) ICAM-1 (CD54), VCAM-1 (CD106), E-SELECTIN (CD62E), on plasma levels of soluble sICAM-1, sVCAM-1, sESELECTIN, and on plasma levels of protein C and protein S in patients with SCA and β-Thalassemia Intermedia (TI)
Methods : We studied 10 patients with SCA disease, 15 patients with TI, and 12 healthy controls (HC). Flow cytometric analysis was performed with a FC-500. For surface expression on CEC of CD54, CD106, CD62E whole blood (EDTA) is incubated with a panel of MoAbs. Activated CEC were defined as CD45 negative, CD54, CD106, CD62E positive. Plasma was separated from blood of patients and controls, and sICAM-1, sVCAM-1, sE-SELECTIN levels were quantified using specific kit Flowcytomix human adhesion 6plex (Bender MedSystem) according to the manufacturer’s instructions.
Results : The percentage (%) of positive CEC expressing markers of endothelial-cell activation were significantly higher in patients with SCA and TI no taking HU, comparated with healthy controls, so as plasma levels (ng/ml) of sICAM-1, sVCAM-1, sE-SELECTIN. SCA patients: CD54+ = 10,9±6,5 % vs 1,4±0,7 % respectively, P= 0,001; CD106+ = 19,7±18,5 % vs 2,1±1,1 % P= 0,03; CD62E+ = 21,2± 20,8 % vs 3,7±1,6 % P= 0,02; sICAM-1=1263,12±445,48 (ng/ml) vs 102,24±14,50 (ng/ml), P=0,03; sVCAM= 1978,67± 985,08 (ng/ml) vs 300,81±70,35 (ng/ml) P=0,01; sESELECTIN= 935,04±313,54 (ng/ml) vs 352,59±50,32 (ng/ml) P= 0,03;
TI patients: CD54+ = 12,1±7,1 % vs 1,4±0,7 % P=0,003; CD106+ = 3,68± 2,0 % vs 2,1±1,1 % P=0,42; CD62E+ = 7,08± 9,1 %vs 3,7±1,6 % P=0,87;
sICAM-1 = 1911,16±509,90 (ng/ml) vs 102,24±14,50 (ng/ml) respectively, P=0,05; sVCAM = 1067,61±755,37 (ng/ml) vs 300,81±70,35 (ng/ml) P=0,03; sE-SELECTIN = 746,92± 236,47 (ng/ml) vs 352,59±50,32 (ng/ml) P=0,05.The expression of adhesion molecules on CEC and plasma levels of soluble sICAM-1, sVCAM-1, sE-SELECTIN were significantly reduced during HU therapy.
SCA patients: CD54+ = 5,01± 4,5 % P=0,01; CD106+ = 9,9± 6,7 % P=0,26; CD62E+ = 11,6± 8,4 % P=0,38;sICAM-1 = 511,81±180,95 (ng/ml) P=0,01; sVCAM = 870,41±419,04 (ng/ml) P=0,02; sE-SELECTIN = 587,49± 364,16 (ng/ml) P=0,03.
TI patients: CD54+ = 2,0±2,5% P=0,003; CD106+ = 2,1±0,9 % P=0,42; CD62E+ = 1,8±1,0 % P=0,87;sICAM-1 = 1294,18±350,69 (ng/ml) P=0,05; sVCAM = 1067,61±755,32(ng/ml) P=0,03; sE-SELECTIN = 746,92± 236,47 (ng/ml) P=0,05. The plasma levels of protein C and protein S were increased in SCA and TI patients on HU therapy comparated to those no taking HU; however, this difference was not significant. (SCA patients: Protein C = 106,76±15,79 % vs 77,59±24,73 P=0,14; Protein S = 79,070±1,38 % vs 65,96±6,36 P=0,49; TI patients: Protein C = 83,46±24,99 % vs 64,65±26,67 % P=0,23; Protein S = 80,50±0,77 % vs 74,33±2,36 % P=0,12).
Conclusion : Our results confirm that the vascular endothelium is activated in patients with SCA and TI disease, as evidenced from the higher expression of adhesion molecules CD54, CD106, CD62E on the surface of CEC and from the higher levels of soluble sICAM-1, sVCAM-1, sE-SELECTIN, reflecting an increased capacity for the adhesion of sickle erythrocytes and leucocytes to the endothelium. HU therapy reduce the expression on the endothelial cell surface, so as plasma levels, of adhesion molecules, reducing the activation state of the endothelium. Furthermore, HU therapy increase plasma levels of anticoagulants protein C and protein S, reducing hypercoagulable state, which means smaller endothelial damages and vaso-occlusive-events.