Background: Procoagulant endothelial cell (EC) dysfunction is a hallmark of cardiovascular disease. To promote blood coagulation, phosphatidylserine (PS) must be rapidly externalized to the outer leaflet of the cell membrane. Activated platelets are thought to provide the majority of procoagulant PS, whose externalization is regulated by the phospholipid scramblase TMEM16F. It is unknown whether endothelial-derived PS contributes to hemostasis and thrombosis and what regulates its externalization.

Aims: We sought to identify regulators of PS scrambling in ECs and determine the contribution of vessel wall derived PS to thrombosis in vivo.

Methods: We silenced TMEM16 phospholipid scramblases in primary HUVECs and assessed procoagulant activity via tenase and prothrombinase formation on the EC surface. PS scrambling was measured by annexin V binding via immunofluorescence microscopy and flow cytometry. We used laser-induced injury of mouse cremaster arterioles to assess PS externalization during thrombus formation.

Results: Pharmacologic inhibition of TMEM16 phospholipid scramblases reduced factor Xa generation on the EC surface. We identified two TMEM16 family members, TMEM16F, and its closest paralog TMEM16E, that were both required for tenase and prothrombinase activity on ECs. Cells deficient in TMEM16E or TMEM16F displayed reduced PS scrambling in response to TNFα or calcium ionophore but expressed normal levels of tissue factor (Figure 1). Using an intravital laser-injury model of thrombosis, we observed the majority of PS externalization derived from the vessel wall, not platelets. TMEM16 inhibition with CaCCinh-A01 (5 μg/g body weight) decreased vessel-wall PS externalization and fibrin generation in vivo (Figure 2).
TMEM16E and TMEM16F are required for factor Xa and thrombin generation and PS externalization on endothelial cells.Following laser injury, PS externalization occurs on the vessel wall, independent of platelet aggregation and is inhibited by a TMEM16 antagonist.

Conclusions: Endothelial procoagulant activity is regulated by TMEM16F, known to participate in coagulation, and TMEM16E, which is a novel regulator of this process. In a laser injury model of thrombosis, the majority of PS derives from the vessel wall and is independent of platelet aggregation. TMEM16 inhibition may reduce the prothrombotic potential of the vessel wall.

« Back to ISTH 2021 Congress

ISTH Congress Abstracts - https://abstracts.isth.org/abstract/regulation-of-endothelial-cell-procoagulant-activity-by-tmem16-scramblases/