Differential Anticoagulant Effects of Protein S on Vascular Cells and Platelets.
Authors: Brinkman Herm Jan , Sellink Erica , de Laat Bas , Mertens Koen 
Published: April 2020

Background: Protein S is a vitamin K-dependent plasma protein and involved in down-regulation of the coagulation process. Protein S serves as a cofactor of activated protein C (APC) in the proteolytic inactivation of activated factor V and VIII. Protein S is also able to exert its anticoagulant activity independent of APC, e.g. by supporting the anticoagulant activity of tissue factor pathway inhibitor (TFPI). The anticoagulant properties of protein S have been thoroughly characterized by in vitro methods. However, fewer studies focus on protein S function on vascular cells. These studies are limited to model systems employing purified coagulation factors. The aim of this study was to investigate the role of protein S in plasma that is in contact with natural cell membranes, including endothelial cells, smooth muscle cells and platelets.

Method: We employed thrombography to evaluate protein S function in 50 % v/v recalcified citrated plasma in the presence of washed platelets, cultured umbilical vein endothelial cells or cultured umbilical artery smooth muscle cells. Since we aimed at a comparison between different cellular membranes, micro-particle free plasma was used. As a reference, we also examined synthetic phospholipid membranes composed of phosphatidyl serine, phosphatidyl choline and phosphatidyl ethanolamine in a 2/6/2 molar ratio. Thrombin activity was measured employing the fluorogenic substrate z-Gly-Gly- Arg-AMC. Protein S activity was probed with CLB-PS13, an antibody directed against the protein S Gla-domain. The APC-independent activity of protein S was assayed in the presence of an inhibitory antibody against protein C. In studies employing phospholipids, thrombin generation was triggered with relipidated tissue factor (TF). Expression of TF on endothelial cells was induced during a 6-hour preincubation with PMA.

Results: In the presence of CLB-PS13, the APC-independent activity of protein S became apparent as an increase in peak height in the thrombogram. Lag time, time to peak and area under the curve remained essentially unaffected. Peak height was increased two-fold when examining phospholipids at standard conditions (4 μM lipids and 1 pM TF). This increase in peak height by CLB-PS13 was concentration dependent and complete at 10 μg/ml IgG. Increasing the TF concentration from 1 to 5 pM resulted in loss of the APC-independent activity of protein S on these membranes. APC cofactor activity was assessed in the presence of APC. Addition of APC resulted in inhibition of the thrombin formation on phospholipids with an IC50 of 0.4 nM. CLB-PS13 completely abolished this decrease in thrombin generation up to 50 nM APC, irrespective whether 1 or 5 pM TF was present. Our results are compatible with the view that at high procoagulant stimuli the TFPI-cofactor activity of protein S is abolished. Furthermore, these results show that in plasma APC is completely dependent on protein S. Protein S activity on platelets was studied in the presence of 1 pM TF. As for synthetic lipid membranes and in the absence of APC, CLB-PS13 increased the peak height in the thrombogram. APC inhibited platelet mediated thrombin generation (IC50 = 19 nM), and this inhibition was completely abolished by CLB-PS13. These observations suggest that platelets support both APC-dependent and APC-independent anticoagulant activities of protein S. Thrombography on TF-expressing endothelial cells and smooth muscle cells revealed massive thrombin generation that could not be enhanced with CLB-PS13, indicating that protein S does not contribute to regulation of thrombin formation under these conditions. The APC-dependent activity of protein S became apparent as an abolished inhibition by APC in the presence of CLB-PS13. However, APC proved relatively inefficient in inhibiting thrombin generation on TF-expressing vascular cells (IC50 &gt; 50 nM). Inhibition of TF restored the APC-independent protein S activity.

Conclusion: Our results indicate that, in plasma, vascular cells and platelets are able to support the APC-dependent as well as the APC-independent anticoagulant activities of protein S. The APC-independent activity on vascular cells is abolished upon increasing TF expression, while the APC-dependent activity of protein S is limited by the relatively low anticoagulant activity of APC on these cell surfaces. We conclude that protein S activity on cells require relatively high levels of APC or low expression of TF.