Protein S Is a Cofactor for Platelet and Endothelial Tissue Factor Pathway Inhibitor-α but Not for Cell Surface–Associated Tissue Factor Pathway Inhibitor

Abstract

Objective Tissue factor pathway inhibitor (TFPI) is produced in 2 isoforms: TFPIα, a soluble protein in plasma, platelets, and endothelial cells, and TFPIβ, a glycosylphosphatidylinositol-anchored protein on endothelium. Protein S (PS) functions as a cofactor for TFPIα, enhancing the inhibition of factor Xa. However, PS does not alter the inhibition of prothrombinase by TFPIα, and PS interactions with TFPIβ are undescribed. Thus, the physiological role and scope of the PS–TFPI system remain unclear. Approach and Results Here, the cofactor activity of PS toward platelet and endothelial TFPIα and endothelial TFPIβ was quantified. PS enhanced the inhibition of factor Xa by TFPIα from platelets and endothelial cells and stabilized the TFPIα/factor Xa inhibitory complex, delaying thrombin generation by prothrombinase. By contrast, PS did not enhance the inhibitory activity of TFPIβ or a membrane-anchored form of TFPI containing the PS-binding third Kunitz domain (K1K2K3) although PS did function as a cofactor for K1K2K3 enzymatically released from the cell surface. Conclusions The PS–TFPI anticoagulant system is limited to plasma TFPIα and TFPIα released from platelets and endothelial cells. PS likely functions to localize solution-phase TFPIα to the cell surface, where factor Xa is bound. PS does not alter the activity of membrane-associated TFPI. Because activated platelets release TFPIα and PS, the PS–TFPIα anticoagulant system may act physiologically to dampen thrombin generation at the platelet surface.

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

“…In contrast, protein S does not enhance the activity of membrane-associated TFPI (TFPIb). 35 Because membrane-bound TFPI does not have a Kunitz domain 3, their study confirmed the importance of this domain for the protein S enhancement. Wood et al also created a TFPIb variant containing a Kunitz domain 3 but, for reasons that are not clear, protein S was unable to enhance this TFPI variant.…”

“…It has previously been hypothesized that protein S, through its high affinity for negatively charged phospholipids, brings TFPI in close proximity to FXa on an activated membrane and thereby increases the TFPI/FXa association rate. [15][16][17]35 We propose that protein S and TFPI can associate in plasma through a direct interaction between the protein S SHBG-like domain and the TFPI Kunitz 3 domain, particularly with Glu226. Upon initiation of coagulation on phospholipid surfaces, protein S brings TFPI Kunitz domain 2 into proximity of the active site of the protease domain of FXa, thereby decreasing the concentration of TFPI needed for efficient inhibition of FXa ( Figure 6).…”

“…Wood et al also created a TFPIb variant containing a Kunitz domain 3 but, for reasons that are not clear, protein S was unable to enhance this TFPI variant. 35 Although the protein S interaction site of TFPI is well characterized, the TFPI interaction site of protein S has not been determined. To explore this, we assessed the importance of solvent-exposed amino acid residues within the Gla-TSR-EGF1-EGF2-EGF3-EGF4 domains and the protein S SHBG-like domain, in the protein S enhancement of TFPI activity in thrombin generation assays.…”

Biology of tissue factor pathway inhibitor

“…6 Thus, it appears that PS enhances FXa inhibition primarily by localizing TFPIa to the membrane surface, a notion supported by experiments demonstrating that PS has cofactor activity toward the soluble TFPI variant protein K1K2K3 8,115 but has no cofactor activity toward the same variant protein when it is attached to the cell surface with a GPI anchor. 115 In addition, PS does not enhance inhibition of FXa by cell surface-associated TFPI on primary endothelial cells or endothelial cell lines. 115 Because optimal inhibition of FXa by TFPIa requires both the K3 domain and the basic C terminus 8,64,107-110 (regions which are not present in TFPIb), one might hypothesize that TFPIb would be a relatively poor inhibitor of FXa.…”

“…115 In addition, PS does not enhance inhibition of FXa by cell surface-associated TFPI on primary endothelial cells or endothelial cell lines. 115 Because optimal inhibition of FXa by TFPIa requires both the K3 domain and the basic C terminus 8,64,107-110 (regions which are not present in TFPIb), one might hypothesize that TFPIb would be a relatively poor inhibitor of FXa. However, this is not the case.…”