Recent studies of the anticoagulant activities of the tissue factor (TF) pathway inhibitor (TFPI) isoforms, TFPIα and TFPIβ, have provided new insight into the biochemical and physiological mechanisms that underlie bleeding and clotting disorders. TFPIα and TFPIβ have tissue-specific expression patterns and anticoagulant activities. An alternative splicing event in the 5′ untranslated region allows for translational regulation of TFPIβ expression. TFPIα has 3 Kunitz-type inhibitor domains (K1, K2, K3) and a basic C terminus, whereas TFPIβ has the K1 and K2 domains attached to a glycosylphosphatidyl inositol–anchored C terminus. TFPIα is the only isoform present in platelets, whereas endothelial cells produce both isoforms, secreting TFPIα and expressing TFPIβ on the cell surface. TFPIα and TFPIβ inhibit both TF–factor VIIa–dependent factor Xa (FXa) generation and free FXa. Protein S enhances FXa inhibition by TFPIα. TFPIα produces isoform-specific inhibition of prothrombinase during the initiation of coagulation, an anticoagulant activity that requires an exosite interaction between its basic C terminus and an acidic region in the factor Va B domain. Platelet TFPIα may be optimally localized to dampen initial thrombin generation. Similarly, endothelial TFPIβ may be optimally localized to inhibit processes that occur when endothelial TF is present, such as during the inflammatory response.

Summary Background Tissue factor pathway inhibitor (TFPI) is an alternatively spliced protein with two isoforms, TFPIα and TFPIβ, which differ in their carboxy-terminal structure and cellular localization. Detailed characterization of their inhibitory activity is needed to define potentially unique inhibitory roles in tissue factor (TF) mediated thrombotic and inflammatory disease and to understand how pharmaceuticals targeted to different structural regions of the TFPI isoforms alter hemostasis in hemophilia patients. Methods The TF inhibitory activity of TFPIβ localized to the surface of CHO cells was compared to soluble TFPIα using in vitro and in vivo assays. Results In TF-FVIIa-mediated FXa generation assays, TFPIβ was a slightly better inhibitor than TFPIα, which was ~3-fold better than TFPI- 160, a soluble, altered form of TFPI similar to TFPIβ. In direct FXa inhibitory assays, TFPIβ had an IC50 2.5-fold lower than TFPIα and 56-fold lower than TFPI-160. TFPIβ inhibited TF-mediated CHO cell migration though Matrigel, while TFPIα or TFPI-160 were poor inhibitors, demonstrating that TFPIβ effectively blocks TF-initiated signaling events during cellular migration through matrices not permeable to soluble forms of TFPI. Further, TFPIβ inhibited TF-dependent CHO cell infiltration into lung tissue following tail vein injection into SCID mice and blocked development of consumptive coagulopathy. Conclusions When compared to TFPIα, TFPIβ is a slightly better inhibitor of TF procoagulant activity. As a surface associated protein, TFPIβ is a much better inhibitor of TF-mediated cellular migration than soluble TFPIα and may distinctly act in the inhibition of TF-mediated signaling events on inflamed endothelium and/or monocytes.

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.

SummaryBackgroundPlasma Tissue Factor Pathway Inhibitor (TFPI) circulates bound to factor V (fV) and Protein S (PS). Estrogen therapy decreases plasma TFPI and PS. TFPI, fV, and PS circulate within platelets, and are released upon activation to modulate thrombus formation.ObjectiveIdentify factors affecting the concentrations of plasma and platelet TFPI, fV, and PS.MethodsBlood samples were obtained from 435 healthy individuals. Plasma total TFPI, TFPIɑ, fV, and PS, and platelet TFPI, fV, and PS were quantified. Correlations between these protein concentrations and age, gender, race, and estrogen use were established.ResultsIn males, only plasma fV increased with age, while in females, all plasma analytes increased with age. Males had higher plasma total TFPI, TFPIα, and PS than females. The platelet proteins in either sex remained relatively stable with increasing age. Platelet TFPI and PS were comparable in both sexes, while platelet fV was higher in females. Estrogen use was associated with decreased plasma total TFPI and TFPIα, and platelet PS, but not with platelet TFPI concentration. Racial differences in plasma and platelet proteins were observed, some of which were larger than inter-individual differences observed within racial groups. TFPI, fV and PS concentrations correlated in plasma, while only fV and PS correlated in platelets.ConclusionsPlasma and platelet TFPI, fV and PS differ in their: (i) in vivo association; (ii) demographic correlates; and (iii) alteration by estrogen therapies. Therefore, the plasma and platelet pools of these proteins may modulate hemostasis and thrombosis via different biochemical pathways.