Antiphospholipid Antibodies Increase Von Willebrand Factor-Platelet String Formation From Human Endothelial Cells Under Physiologic Flow Conditions

The antiphospholipid syndrome (APS) is characterized by a predisposition to arterial and venous thromboembolism. The underlying mechanism of thrombosis in APS is uncertain, but is thought to involve a multifactorial activation of various components of the blood and vasculature. The role of von Willebrand factor (VWF), a plasma protein expressed by endothelial cells in response to endothelial cell damage or activation, is not clearly defined in APS. Previous studies have demonstrated that antiphospholipid antibodies increase the release of soluble VWF from human umbilical vein endothelial cells. However, these experiments were performed in static conditions and not in a flow based model. Recent investigations of expressed ultra large VWF (ULVWF) under flow have led to a new model of VWF-platelet strings that remain anchored to endothelial cells and provide a nidus for the formation of thrombi. Consistent with this, increases in VWF and variations in ADAMTS13, the enzyme that modulates ULVWF, have been associated with venous and arterial thrombosis.

We hypothesized that antiphospholipid antibodies would induce VWF-platelet strings on endothelial cells and that this mechanism contributes to APS-associated thrombosis.

Human umbilical vein endothelial cells were seeded in flow chambers prepared with a collagen substrate. All experiments were performed with cells of passage 5 or under. Confluent cells were incubated in serum-free medium with APS-derived IgG or anti-β2-GPI antibodies vs. control IgG. After incubation, the slides were perfused with lyophilized platelets and washed with Tyrode’s buffer prior to image acquisition. Shear stresses were varied from 2-10 dyn/cm² to assess for shear stress dependent variations in string formation. Pre-determined sites of imaging were set via a standardized method with relief contrast brightfield microscopy. Between 50-100 images were collected per condition. A VWF-platelet string-unit was defined as a 25 µm length of an individual VWF-platelet string. Images were analyzed for the number of VWF-platelet string units per image; quantification of string units was performed blinded to sample vs. control IgG treatment status. Data are shown as means +/- SEM, and significance was determined as p<0.05 by paired t-test.

Endothelial cells treated with human antiphospholipid antibodies demonstrated increased VWF-platelet strings at two different shear stresses, one mimicking venous flow and one mimicking arterial flow. At 2 dyn/cm², cells incubated for 1 hour with 100 µM of APS patient–derived IgG demonstrated 23.04 +/- 2.139 (SEM) VWF-platelet string units/image vs. 17.64 +/- 1.203 in cells treated with 100 µM control human IgG (p=0.0247). At 10 dyn/cm², cells incubated for 6 hours with 600 nM of human anti-β2-GPI antibodies demonstrated 1.848 +/- 0.2866 (SEM) VWF-platelet string units/image vs 1.048 +/- 0.2350 in cells treated with 600 nM control human IgG (p=0.0395).

Antiphospholipid antibodies, in aggregate (IgG from an APS patient) or specific (anti-β2-GPI), induce the formation of VWF-platelet strings from human endothelial cells. This finding suggests the potential role of VWF-platelet strings in thrombosis associated with APS. The clinical predisposition to arterial and venous thrombi in APS and the observation of increased VWF-platelet strings at arterial and venous shear stresses suggest that modulation of VWF-platelet strings may provide a future therapeutic target.

Manco-Johnson:Bayer HealthCare: Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Baxter BioScience: Membership on an entity’s Board of Directors or advisory committees; Biogen Idec: Membership on an entity’s Board of Directors or advisory committees; Novo Nordisk: Membership on an entity’s Board of Directors or advisory committees; Eisai: Research Funding. Di Paola:Pfizer: DSMB, DSMB Other; CSL Behring: Consultancy.