Protein S Is a Cofactor for Platelet TFPIα, but Not Cell Surface-Associated TFPI
Authors: Wood Jeremy P. , Ellery Paul E. R. , Maroney Susan A. , Mast Alan E. 
Published: October 2019

Abstract
Abstract 494

Background:
Tissue factor pathway inhibitor (TFPI), the primary physiologic inhibitor of the tissue factor (TF)/factor VIIa (FVIIa) enzymatic complex, is found in plasma, on endothelial cells, and within platelets. There are two alternatively-spliced isoforms of TFPI, TFPIα and TFPIβ. TFPIα is secreted from endothelial cells, present in plasma and stored in circulating platelets, while TFPIβ is the primary cell surface-expressed isoform of TFPI. TFPIα is composed of three Kunitz-type inhibitor domains: K1 and K2 bind to and inhibit the serine proteases FVIIa and factor Xa (FXa), respectively, and K3 binds to protein S (PS). TFPIβ contains the same K1 and K2 domains and a glycosylphosphatidylinositol (GPI)-anchored C-terminus. PS serves as a cofactor for plasma TFPIα in the inhibition of factor Xa (FXa), as measured by amidolytic activity assays. However, TFPIα is a poor inhibitor of thrombin generation by the prothrombinase complex (FXa and its cofactor, factor Va (FVa)) regardless of whether PS is present. Thus, the physiologic function of FXa inhibition by the TFPI/PS anticoagulant system is not entirely clear, particularly on the platelet surface where prothrombinase assembly occurs. In addition, it is not known if PS cofactor activity is mediated by localizing TFPI to the membrane surface, thereby enhancing its interaction with membrane-bound FXa, or by directly enhancing the interaction between TFPI and FXa once both are on the membrane surface.

Methods/Results:
The cofactor activity of PS toward recombinant TFPIα (rTFPIα), platelet TFPIα, and cell surface-associated forms of TFPI was measured. As previously published, low concentrations of PS (5 nM) enhanced the inhibitory activity of rTFPIα in a FXa amidolytic activity assay. PS also substantially delayed thrombin generation in assays where rTFPIα, FXa, and PS were incubated prior to addition of FVa, but not when FVa was first incubated with FXa (before rTFPIα and PS addition). PS was also found to enhance the inhibition of FXa by platelet TFPIα (measured in platelet lysates), in a dose-dependent manner, with enhancement apparent at concentrations as low as 5 nM. Unlike with experiments performed using recombinant or platelet TFPIα, PS (at concentrations up to 80 nM) had no impact on the inhibition of either FXa amidolytic activity or thrombin generation when assays were performed using CHO cells expressing TFPIβ or a GPI-anchored TFPIα (K1K2K3-GPI). Additionally, PS did not act as a cofactor for endogenous TFPI on the surface of EA.hy926 cells or primary HUVECs. However, TFPIα released from EA.hy926 cells by treatment with heparin was enhanced by addition of PS, to a similar extent as observed with rTFPIα. TFPIβ, released by treatment with phosphoinositol-specific phospholipase C, displayed little inhibitory activity either in the presence or absence of PS.

Conclusions:
(1) PS is a cofactor for the inhibition of FXa by soluble TFPIα, including platelet TFPIα, but not for surface-associated forms of TFPI, regardless of the presence of the K3 domain. (2) PS functions by localizing and retaining TFPIα at the membrane surface, rather than through a direct enhancement of the interaction between TFPIα and FXa. (3) PS stabilizes the TFPIα/FXa inhibitory complex. In this manner, PS may act physiologically to sequester FXa away from FVa and slow assembly of prothrombinase on the surface of activated platelets.

Disclosures:
Mast: Novo Nordisk: Research Funding.