Clearance of Platelet Microvesicles by Endothelium. the Role of Developmental Endothelial Locus–1 (Del-1)

Abstract 4303

Activation of platelets with certain agonists induces release of small submicron size membrane-enclosed compartments of platelet content called microvesicles. During microvesiculation, phosphatidylserine, an anionic phospholipid, moves from the inner to the outer leaflet of membrane bilayer accounting for the procoagulant effect of microvesicles. Microvesicles circulate in the blood with a short half-life of less than 5–10 minutes and the exposure of phosphatidylserine may mediate its rapid clearance. Developmental endothelial locus–1 (del-1) is a 52kDa glycoprotein secreted by endothelial cells. It contains three epidermal growth factor (EGF)-like repeats in amino terminus and two discoidin I-like domains in carboxy terminus, similar to the macrophage opsonin lactadherin. The second EGF like repeat contains the canonical Arg-Gly-Asp (RGD) motif that enables Del-1 to bind integrin αVβ3 and αVβ5. Here, we tested the role of Del-1 in endothelial uptake of platelet-derived microvesicles.

In vitro, resting human umbilical vein endothelial cells engulf BODIPY-labeled phosphatidylserine expressing platelets microvesicles avidly. Evidence of uptake was obtained by confocal microscopy and by flow cytometry. Following uptake, the microvesicles were distributed throughout the cytoplasm of the endothelial cells. We quantified the internalization of fluorescent microvesicles by measuring the intracytoplasmic fluorescence associated with endothelial cells in a flow cytometer after quenching cell surface fluorescence with Trypan Blue. A monoclonal antibody to Del-1 inhibits (p=0.03) the uptake while exogenous Del-1 promotes the uptake by endothelial cells. Abciximab inhibited -dependent phagocytosis to about ∼40 % while under similar conditions an irrelevant control antibody had no effect (p=0.016). In addition, annexin A5 also inhibited the uptake of platelet-derived microvesicles by endothelial cells (p=0.002). These results show Del-1 mediates phosphatidylserine and integrin-dependent uptake of microvesicles by endothelial cells. To assess the in vivo significance, we infused BODIPY labeled platelet-derived microvesicles into the inferior vena cava of the mouse and harvested endothelial cells from the pulmonary circulation. Microvesicles were detected within the endothelial cells. In Del-1 deficient mice has impaired uptake of microvesicles compared wild type littermate controls (P=0.02). We also measured the levels of microvesicles six after administration lipopolysaccharide. Del-1 deficient mice have increased microvesicles compared to their littermate controls (p=0.02).

These studies show a physiological role for Del-1 in the clearance of microvesicles by endothelium.