VLA-4 Blockade by Low Molecular Weight Heparin: A Novel Therapeutic Approach in Sickle Cell Disease.

Vaso-occlusion in sickle cell disease (SCD) that leads to painful events and complications is caused in part by adhesion of sickled erythrocytes (SSRBCs) to components of the vascular wall and to circulating leukocytes (WBC). Very late activation antigen-4 (VLA-4) or α4β1 integrin expressed on RBCs and WBCs mediates SSRBC-WBC adhesion as well as SSRBCs adhesion to the endothelium via vascular cell adhesion molecule (VCAM-1). VLA-4 blockade has been used to treat pathologic inflammation in multiple sclerosis and Crohn's Disease by inhibiting the interaction between VLA-4 on the leukocyte and endothelial VCAM-1. Enoxaparin, a low molecular weight heparin (LMWH), has been recently shown to exhibit anti-adhesive properties via VLA-4 inhibition. The potential role of enoxaparin as an anti-adhesive therapy for SCD has not been explored. The objective of this study was to determine if enoxaparin could decrease VLA-mediated SS RBC adhesive interactions.

Peripheral blood samples were obtained from pediatric patients (n=6) with homozygous SCD (5–18 years) at steady state. Normal control samples (n=3) were obtained from healthy volunteers.

We evaluated the interaction between VLA-4 on isolated SSRBCs and immobilized VCAM-1 in a flow adhesion assay under physiologic flow conditions. RBCS were isolated from whole blood after ficoll density gradient separation. We observed adhesion of SSRBCs to immobilized VCAM-1 under three conditions: 1) unstimulated, 2) stimulated with Manganese (Mn²⁺) – an experimentally accepted method of integrin stimulation that puts VLA-4 in its active confirmation and 3) Mn²⁺ stimulated RBCs pretreated with enoxaparin.

SSRBC-WBC aggregates were identified by classical flow cytometry. Whole blood samples were processed under three conditions: 1) unstimulated, 2) stimulated with Mn²⁺ and 3) stimulated with Mn²⁺ and pretreated with enoxaparin. Samples were separated on a ficoll density gradient to obtain an enriched WBC layer. Within this WBC layer, components of multicellular aggregates were identified using antibodies staining for WBCs (anti-CD45), or RBCs (anti-glycophorin A), and CD71 for reticulocytes.

Mn²⁺ treatment enhanced VLA-4 adhesion of isolated SSRBCs to immobilized VCAM-1 in 5/6 patient samples. When Mn²⁺ stimulated samples were treated with enoxaparin, 4/6 patient samples showed a significant decrease in adhesion (p < 0.02).

Mn²⁺ stimulation induced SS RBC-WBC aggregate formation in 3/4 patient samples tested. Both mature red blood cells and reticulocytes were involved in these aggregates. Aggregates were variable in size. All 4 patient samples treated with enoxaparin showed decreased aggregates. 3/4 patient samples showed aggregates decreased to levels at or below that observed in unstimulated samples.

Our data show that enoxaparin inhibits adhesion of pediatric SSRBCs to immobilized VCAM-1, presumably via blockade of activated VLA-4 on the SS RBC surface. Further, our data demonstrate that stimulation of VLA-4 can increase adhesive interactions of circulating SSRBCs and WBCs, and that these interactions can be disrupted by enoxaparin. The complications and sequelae in SCD begin in childhood warranting the need to find more treatment strategies for pediatric SCD. The fact that numerous adhesive interactions responsible for vaso-cclusive events in SCD are VLA-4-mediated makes enoxaparin an attractive therapeutic possibility to prevent and treat vaso-occlusive complications in SCD.

No relevant conflicts of interest to declare.