A comparative study of functional assays for tissue factor pathway inhibitor using normal plasma and clinical samples

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type inhibitor that regulates the initiation of tissue factor-mediated coagulation. Recent reports in the literature have described variable results using different methodologies for TFPI measurement. In this study, we used one clotting and two amidolytic methodologies to assess TFPI functional levels. The study groups included normal healthy donors as well as patients with acute hepatitis, diabetes, coronary artery bypass graft operations, deep vein thrombosis, and prior to and during heparin therapy. The aims were to compare the results obtained in normal plasma using different assay systems, to compare TFPI levels in a range of clinical samples, including those previously not determined using a clotting methodology, and to report TFPI levels in patient groups previously not investigated. The results clearly demonstrate poor correlation between functional TFPI values using the different methodologies, highlighting the requirement for standardization

Further insight into the heparin-releasable and glycosylphosphatidylinositol-lipid—anchored forms of tissue factor pathway inhibitor

The release of tissue factor pathway inhibitor (TFPI) from human umbilical vein endothelial cells (HUVECs) was investigated using heparin and phospholipase C. The experiment included incubating HUVECs with 0, 1, or 10 U/mL heparin diluted in Dulbecco Modified Eagle's Medium plus 5% fetal calf serum for 1 or 24 hours. A statistically significant increase in TFPI activity levels was seen at 1 hour, but not at 24 hours. A 20-fold increase in the release of TFPI after phospholipase C treatment of HUVECs was demonstrated, confirming that it is glycosylphosphatidylinositol-lipid (GPI) anchored. Sequential treatment of HUVECs with phospholipase C and heparin was performed, and a trend was observed where GPI-anchored TFPI levels were increased after 1 hour of pretreatment with heparin but were decreased after 24 hours. Serum is a requirement for the heparin-dependent release of TFPI from HUVECs. Heparin pretreatment of HUVECs may affect levels of GPI anchored TFPI in a time and dose-dependent manner

Tissue factor pathway inhibitor: Regulator of the tissue factor pathway of coagulation and a future antithrombotic agent?

Tissue factor pathway inhibitor (TFPI) is a recently 'rediscovered' inhibitor of tissue factor mediated coagulation that has a central role in the modern hypothesis of coagulation. The regulatory role of this inhibitor has redefined the classical extrinsic and intrinsic pathways of coagulation and helps to explain why haemophiliacs bleed. Comprehensive investigations have been performed during the last 15 years to elucidate the structure, distribution, function and physiological characteristics of TFPI. More recent work has focused on the therapeutic potential of TFPI as an antithrombotic agent. Augmentation of TFPI levels may provide treatment for thrombotic disorders such as disseminated intravascular coagulation and deep vein thrombosis. Full length and truncated forms of TFPI molecules are also being considered as antithrombotic agents. These have been used in a number of animal models with initial results demonstrating the alleviation of thrombotic tendencies. Trials are currently ongoing

Further investigations of lupus anticoagulant interference in a functional assay for tissue factor pathway inhibitor

Tissue factor pathway inhibitor (TFPI) is a three domain Kunitz type inhibitor that regulates the initiation of coagulation by inhibiting tissue factor - activated factor VII (TF - FVIIa) in the presence of activated factor X (FXa) (1). Although the precise role of TFPI has yet to be determined, its ability to inhibit the TF - FVIIa complex in the presence of FXa suggests it does have major physiological significance. Animal studies support this theory and have shown that depletion of TFPI sensitises rabbits to disseminated intravascular coagulation induced by TF (2). TFPI levels have been reported to be normal in a variety of clinical conditions, including patients with lupus anticoagulants (3). Lupus anticoagulants (LA) are acquired inhibitors, generally considered to be immunoglobulins, that interfere with in vitro phospholipid dependent coagulation tests (4). The literature has reported LA to be present in many disorders, but with a particularly high frequency in recurrent pregnancy loss and venous and arterial thrombosis (5–7). In this study two previously reported functional assays for TFPI (8–9) were modified and used to assess TFPI levels in plasma. We assessed TFPI levels in normal and LA positive plasmas using both assays and report an interference by LA in one of these assay systems, which is not evident in the other

Anti-tissue factor pathway inhibitor activity in subjects with antiphospholipid syndrome is associated with increased thrombin generation

BACKGROUND AND OBJECTIVES: Immunoglobulin G (IgG) fractions from subjects with antiphospholipid syndrome (aPS) have previously been demonstrated to have inhibitory activity against tissue factor pathway inhibitor (TFPI). This may contribute to the development of a prothrombotic state by impaired regulation of the tissue factor (TF) pathway. This study investigated the effect that IgG fractions from aPS subjects containing anti-TFPI activity have on in vitro TF-induced thrombin generation. DESIGN AND METHODS: TFPI and anti-TFPI activities were determined in normal controls (n=29) and aPS subjects (n=57). TFPI activity was determined using an amidolytic assay based on the generation of factor Xa. Anti-TFPI activity was determined after incubating IgG isolated from a control or subject plasma with pooled normal plasma, using the TFPI activity assay. The influence of IgG fractions from controls (n=10) and subjects (n=23) on TF-induced in vitro thrombin generation was determined using a chromogenic assay of thrombin activity. RESULTS: TFPI activity in controls (1.13+/-0.25 U/mL) was significantly lower than in subjects (1.30+/-0.42 U/mL) (p 0.05), with increased levels of each demonstrated in 5 subjects. INTERPRETATION AND CONCLUSIONS: Anti-TFPI activity was confirmed in 65% of aPS subjects. IgG fractions demonstrated a variable ability to interfere with TFPI function and TF-induced thrombin generation. Cross-reacting antiphospholipid antibodies and/or other entities may interfere with TFPI function, resulting in a net increase in thrombin generation and an increased thrombotic risk

Hypercoagulability in chronic kidney disease is associated with coagulation activation but not endothelial function

Introduction Patients with chronic kidney disease exhibit features of a hypercoagulable state and have endothelial dysfunction, which may contribute to their increased cardiovascular risk. We examined the relationship between coagulation activation and vascular function in patients with chronic kidney disease. Materials and Methods We measured parameters of the tissue factor pathway of blood coagulation (tissue factor, factor VIIc and factor X); natural inhibitors (tissue factor pathway inhibitor, protein C, free and total protein S, antithrombin III) and markers of coagulation activation (thrombin-antithrombin complexes, prothrombin fragment 1 + 2) in 66 stage 4&5 chronic kidney disease patients and 36 healthy controls. Their relationship with markers of vascular function (flow mediated dilatation, soluble E-selectin and thrombomodulin) and a mediator of inflammation (interleukin-6) was determined. Results Up-regulation of the tissue factor pathway (increased tissue factor and factor VIIc), increased prothrombin fragment 1 + 2 and significant reductions in antithrombin III and the ratio of free protein S: total protein S were found in patients compared to healthy controls. Increased tissue factor antigen was significantly and independently correlated with creatinine and interleukin-6 (P < 0.001). Factor X and antithrombin III were both reduced in chronic kidney disease and correlated (r = 0.58; P < 0.001). Changes in coagulation and anti-coagulation were independent of all measures of endothelial function. Conclusions Significant activation of the TF pathway of coagulation and depletion or reduction of some natural anticoagulants in chronic kidney disease was correlated with the degree of renal dysfunction, but not correlated with the abnormalities of vascular function. These data are consistent with a hypercoagulable state in chronic kidney disease that may be independent of endothelial based regulation but associated with an inflammatory state