Background/Purpose: Adhesion of leukocytes to the endothelium is an initiating event in the thrombosis inherent to antiphospholipid syndrome (APS). Over the years, a number of groups have demonstrated that antiphospholipid antibodies (aPL) upregulate both selectins and integrin receptors on endothelial cells, thereby increasing the adhesive potential of the endothelium. Here, we posit that factors intrinsic to leukocytes may also play a pivotal role in adhesive interactions. For example, transcriptome analysis of APS patient neutrophils has revealed an activated, adhesive signature. Furthermore, leukocyte adhesion in our mouse model of aPL-mediated large vein thrombosis is regulated by leukocyte (more so than endothelial) factors. In this study, we functionally characterize leukocyte adhesion in APS, with the goal of ultimately testing the therapeutic potential of putative targets.

Methods: Patients had primary APS (classified by Sydney criteria), while healthy controls were matched for age and gender. Freshly-isolated human umbilical vein endothelial cells (HUVECs) were used. Anticoagulated whole blood (or purified cellular components) was introduced into a flow channel via a programmable syringe pump, and perfused across an unstimulated HUVEC monolayer in a pulsatile flow profile with wall shear rate set to 1000 s-1. After 15 minutes of perfusion, the chamber was flushed to remove nonadherent cells, and images were captured with an inverted microscope.

Results: Perfusion of APS blood across unstimulated HUVECs resulted in significantly more leukocyte adhesion as compared with control blood (mean 5.0-fold increase; p<0.0001 with n=18 patients). To assess the role of leukocytes themselves (versus plasma factors that might activate HUVECs), leukocytes were washed free of plasma, resuspended in buffer, and then perfused across HUVECs. In this plasma-free context, there was still significantly more adhesion of APS leukocytes (mean 2.2-fold increase; p<0.01 with n=10 patients), suggesting an intrinsic increase and/or activation of leukocyte adhesion factors. Furthermore, treating control leukocytes with APS patient plasma (which was then washed away before perfusion) resulted in increased adhesion of the control leukocytes (mean 2.5-fold increase; p<0.0001 with n=12 plasma samples). Experiments are underway to delineate the factors on the leukocyte surface that dictate the increased adhesive potential (as well as the factors in APS plasma that mediate their upregulation). In particular, we are characterizing both selectin ligands and beta-2 integrins on leukocytes, as even unstimulated HUVECs demonstrate baseline expression of potential binding partners (selectins and ICAM-1, respectively). We are also determining the relative adhesive potential of different leukocyte subpopulations (i.e., monocytes versus neutrophils).

Conclusion: While there is a known role for aPL in increasing the adhesive potential of endothelial cells, we now report that leukocytes themselves have an intrinsic increase in their adhesive potential. The ultimate goal of this work is to identify the most relevant pharmacologic targets on the leukocyte surface.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/antiphospholipid-syndrome-leukocytes-demonstrate-increased-adhesive-potential-a-search-for-novel-therapeutic-targets/