Polymorphism in the tissue factor region is associated with basal but not endotoxin-induced tissue factor-mRNA levels in leukocytes

OBJECTIVE: Tissue factor (TF) plays a central role during disseminated intravascular coagulation (DIC) in sepsis. We hypothesized that a frequent D/I polymorphism, at nucleotide position -1208 in the promoter region, could influence TF-mRNA and downstream coagulation. METHODS: Basal- and lipopolysaccharide (LPS)-induced TF-mRNA expression, microparticle-associated TF-procoagulant activity and coagulation were determined in healthy men (n = 74) before and after endotoxin (LPS) infusion (2 ng kg(-1)). Basal values of TF-mRNA ranged between 34 and > 37.5 cycles. RESULTS: Baseline TF-mRNA levels significantly differed between genotypes: I/I carriers had almost 2-fold higher TF-mRNA levels compared to D/D carriers at baseline (P < 0.01). In accordance, higher levels of microparticle-associated TF-procoagulant activity could be seen in I/I carriers. However, the genotype did not affect basal or LPS-induced levels of prothrombin fragment F1+2, D-dimer or cytokines including tumor necrosis factor and interleukin-6. CONCLUSION: The TF-1208 polymorphism is functional in that it regulates basal TF-mRNA in circulating monocytes and circulating microparticle-associated TF-procoagulant activity in vivo, but does not influence the relative increase in TF-mRNA or coagulation activation during low-grade endotoxemia.     

Racial differences in endotoxin-induced tissue factor-triggered coagulation

Summary.  Background: Racial differences in coagulation are poorly understood. While some studies suggest a 'prothrombotic' coagulation profile in blacks compared with whites, others report an increased bleeding risk for blacks in various clinical settings. Moreover, preclinical data suggest a link between the Duffy antigen (= DARC, Duffy antigen receptor of chemokines) and coagulation. Objectives: Based on our previous research in Duffy antigen negative Africans, we hypothesized that Africans have an attenuated procoagulant response compared with Caucasians in a model of lipopolysaccharide (LPS)-induced, tissue factor (TF)-triggered coagulation activation. Patients/methods: Healthy male volunteers (16 Duffy-negative Africans, 16 Duffy-positive Caucasians) received 2 ng kg⁻¹ LPS, and outcome parameters were measured using enzyme immunoassays and real-time polymerase chain reaction (RT-PCR, Taqman). Results: LPS increased microparticle (MP)-associated TF procoagulant activity (PCA) less in Africans than Caucasians. Africans had reduced in vivo thrombin formation compared with Caucasians: they generated less thrombin–antithrombin (TAT) complexes (10.4 pg mL⁻¹ vs. 23.0 pg mL⁻¹, P  < 0.0001) and less prothrombin fragments (F₁₊₂) (337 pmol mL⁻¹ vs. 819 pmol mL⁻¹, P  < 0.0001). Consistently, Africans also had decreased fibrin formation ( d -dimer: 0.3 pg mL⁻¹ vs. 0.5 pg mL⁻¹, P  = 0.02). Conclusion: Duffy-negative subjects of African descent have a markedly reduced procoagulant response in a model of LPS-induced, TF-triggered coagulation activation compared with Duffy-positive healthy Caucasians.     

Insulin inhibits tissue factor expression in monocytes

Summary.  Objectives:  Platelets from healthy subjects are inhibited by insulin but type 2 diabetes mellitus (T2DM) platelets have become insulin-resistant, which might explain their hyperactivity. In the present study we investigated whether monocytes are responsive to insulin. Methods and results:  LPS-induced tissue factor (TF) upregulation was measured in human monocytes and monocytic THP-1 cells in a factor Xa generation assay. Insulin (0.1–100 nmol L⁻¹) induced a dose-dependent inhibition in both cell types and in monocytes 100 nmol L⁻¹ insulin inhibited cytosolic, membrane-bound and microparticle TF by 32 ± 2, 27 ± 3 and 52 ± 4% ( n  = 3). Insulin induced Tyr phosphorylation of the insulin receptor (INS-R) and formation of an INS-R – G_iα₂ complex, suggesting interference with LPS-induced cAMP control. Indeed, insulin interfered with LPS-induced cAMP decrease and TF upregulation in a manner similar to an inhibitor of G_i (pertussis toxin) and agents that raise cAMP (iloprost, forskolin, IBMX) reduced TF upregulation. Although LPS failed to raise cytosolic Ca²⁺, quenching of Ca²⁺ increases (BAPTA-AM) reduced and induction of Ca²⁺ entry (ionophore, P2X7 activation) enhanced upregulation of TF mRNA and procoagulant activity. Insulin interfered with MCP-1-induced Ca²⁺ mobilization but not with ATP-induced Ca²⁺ rises. Conclusions:  Insulin inhibits TF expression in monocytes and monocyte-derived microparticles through interference with G_iα₂-mediated cAMP suppression, which attenuates Ca²⁺-mediated TF synthesis.     

In-Cell Western assay: a new approach to visualize tissue factor in human monocytes

BACKGROUND: Tissue factor (TF) is an integral membrane protein essential for the initiation of the extrinsic pathway of hemostasis. A precise understanding of the TF regulation is still limited and dependent on the availability of methodological tools. Here, we describe a new approach for assessing TF amounts in human mononuclear cells (MNCs) by using the whole blood experimental conditions. AIM: In order to study TF antigen levels in human MNCs, we applied a quantitative immunostaining technique-- in-cell Western (ICW) assay using an Odyssey Infrared Imaging System. METHODS AND RESULTS: The ICW assay of TF in resting or lipopolysaccharide (LPS)-stimulated human MNCs was performed. Several sample preparation conditions were tested, namely the plating of MNCs prior to immunostaining, paraformaldehyde fixation, and an adequate cell number was used in the assay. By the use of recombinant human TF standards, it was possible, for the first time, to measure TF amounts in LPS-stimulated MNCs as 0.09 +/- 0.02 ng and 0.43 +/- 0.15 ng 10(-6) cells of surface and total TF, respectively. The concentrations of TF in resting MNCs, however, were below the detection limit. CONCLUSIONS: A novel TF ICW assay is a reproducible, time- and cost-saving method, which could become useful for studies in the fields of physiology and pathophysiology of human hemostasis.     

Elevated tissue factor expression contributes to exacerbated diabetic nephropathy in mice lacking eNOS fed a high fat diet

Summary. Background: Human eNOS (NOS3) polymorphisms that lower its expression are associated with advanced diabetic nephropathy (DN), and the lack of eNOS accelerates DN in diabetic mice. Diabetes is associated with fibrin deposition. Lack of nitric oxide and fatty acids stimulates the NF‐kB pathway, which increases tissue factor (TF). Objectives: To test the hypothesis that TF contributes to the severity of DN in the diabetic eNOS^?/? mice fed a high‐fat diet (HF). Methods: We made eNOS^?/? and wild‐type mice diabetic with streptozotocin. Half of them were placed on HF. Results: Blood glucose levels were not affected by either the diet or eNOS genotype. Lack of eNOS in the diabetic mice increased urinary albumin excretion, glomerulosclerosis, interstitial fibrosis, and glomerular basement membrane thickness. HF by itself did not affect DN in the wild‐type mice, but significantly enhanced DN in eNOS^?/? mice. More than half of diabetic eNOS^?/? mice on HF died prematurely with signs of thrombotic complications. Diabetic kidneys contained fibrin and TF, and their levels were increased by the lack of eNOS and by HF in an additive fashion. The HF diet increased the kidney expression of inflammatory genes. The increase in TF preceded DN, and administration of an anti‐mouse TF antibody to diabetic mice reduced the expression of inflammatory genes. Conclusion: Together, these data indicate a causal link between TF and the exacerbation of DN in eNOS^?/? mice. The condition is significantly worsened by enhanced inflammatory responses to an HF diet via TF.     

Fluvastatin inhibits up-regulation of tissue factor expression by antiphospholipid antibodies on endothelial cells

BACKGROUND: Mechanisms of thrombosis induced by antiphospholipid (aPL) antibodies include up-regulation of tissue factor (TF) expression on endothelial cells (ECs). Statins have been shown to reduce levels of TF induced by tumor necrosis factor (TNF-alpha) and lipopolysaccharide (LPS) on ECs. In a recent study, fluvastatin inhibited thrombogenic and proinflammatory properties of aPL antibodies in in vivo models. The aim of this study was to determine whether fluvastatin has an effect on aPL-induced expression of TF on ECs. METHODS: IgGs were purified from four patients with APS (IgG-APS) and from control sera (IgG-NHS). Cultured human umbilical vein endothelial cells (HUVEC) were treated with IgG-APS or IgG-NHS or with medium alone or with phorbol myristate acetate (PMA), as a positive control. In some experiments, cells were pretreated with fluvastatin (2.5, 5 or 10 micro m) with and without mevalonate (100 micro m). TF expression on HUVECs was measured by ELISA. RESULTS: PMA and the four IgG-APS preparations increased the expression of TF on EC significantly (4.9-, 2.4-, 4.2-, 3.5- and 3.1-fold, respectively), in a dose-dependent fashion. Fluvastatin (10 micro m) inhibited the effects of PMA and the four IgG-APS on TF expression by 70, 47, 65, 22 and 68%, respectively, and this effect was dose-dependent. Mevalonate (100 micro m) completely abrogated the inhibitory effects of fluvastatin on TF expression induced by aPL. CONCLUSION: Because of the suggested pathogenic role of aPL on induction of TF on ECs, our data provide a rationale for using statins as a therapeutic tool in treatment of thrombosis in APS.     

Marked increase in vascular endothelial growth factor concentrations during Escherichia coli endotoxin-induced acute inflammation in humans

BACKGROUND: Bacterial endotoxins can induce the synthesis and release of vascular endothelial growth factor (VEGF), which may alter vascular permeability and cause vascular leakage. MATERIALS AND METHODS: The effect of acute systemic inflammation on VEGF concentration was measured in healthy males after an intravenous bolus infusion of Escherichia coli endotoxin (lipopolysaccharide, LPS, 20 IU kg-1) in a double-blind, placebo-controlled parallel group study. LPS administration was followed by an infusion of lepirudin (bolus 0.1 mg kg-1, continuous infusion of 0.1 mg kg-1 h-1, n = 12) or saline (n = 12). RESULTS: Plasma VEGF increased from a mean of 15.1 pg mL-1 to 74.6 pg mL-1 5 h after LPS (P < 0.003). Body temperature, pulse rate, leukcytes, prothrombin fragment 1 + 2 (F1 + 2) and lactoferrin increased and platelets decreased after LPS (P < 0.05). The LPS-induced increase in VEGF was paralleled by the neutrophil cell degranulation marker lactoferrin but not by F1 + 2, and was not affected by lepirudin, which blunted F1 + 2 formation (P < 0.05). CONCLUSIONS: Inflammation-induced activation of leukcytes rather than platelets plays a role in the marked increase in VEGF, which cannot be abrogated by antithrombotic therapy.     

The tissue factor–factor VIIa complex: procoagulant activity, regulation, and multitasking

Greater understanding of the cellular interactions associated with tissue factor (TF), activated factor (F) VII and TF-FVIIa complexes is likely to provide considerable clinical benefit. This article reviews current knowledge on the function and regulation of TF and its role in a range of biological processes, including hemostasis, thrombosis and inflammation.     

Enhanced monocyte expression of tissue factor by oxidative stress in patients with antiphospholipid antibodies: effect of antioxidant treatment

Summary. In a first study, we performed a cross‐sectional analysis of urinary excretion of isoprostanes, IPF_2α‐III and _VI, and monocyte tissue factor (TF) antigen and activity between 11 antiphospholipid (APL) antibody‐positive patients and 13 APL negative subjects. In a second study, 11 APL positive patients were randomly supplemented either with (n = 6) or without (n = 5) antioxidants (vitamin E at 900 IU day^?1, vitamin C at 2000 mg day^?1) for 6 weeks. In a third study, TF and superoxide anion were measured in human monocytes incubated with anti‐β₂ glycoprotein 1 (β₂GP₁) or control IgG, either with or without vitamin E. APL‐positive patients had higher values of isoprostanes (P < 0.05) and monocyte TF antigen (P = 0.001) and activity (P = 0.0001) than APL‐negative subjects. Only in APL positive patients did monocyte TF antigen correlate significantly with IPF_2α‐III (rho 0.79; P < 0.003) and IPF_2α‐VI (rho = 0.87; P < 0.0001). In patients who received antioxidant supplementation, we found a significant decrease of isoprostanes (P < 0.05) and monocyte TF antigen (P < 0.01) and activity (P < 0.007). In vitro experiments demonstrated that anti‐β₂GP₁ antibodies dose‐dependently enhanced the monocyte production of the superoxide anion and TF, which were significantly inhibited by vitamin E. This study demonstrates that in APL‐positive patients, oxidative stress contributes to activate the clotting system via over‐expression of monocyte TF. We suggest that anti‐β₂GP₁ antibodies could play a pivotal role by enhancing the monocyte production of oxygen free radicals.     

Pro- and non-coagulant forms of non-cell-bound tissue factor in vivo

Summary.  Background : Concentrations of non-cell-bound (NCB; soluble) tissue factor (TF) are elevated in blood collecting in the pericardial cavity of patients during cardiopulmonary bypass (CPB). Previously, we reported microparticles supporting thrombin generation in such blood samples. In this study we investigated the extent of microparticle association of the NCB form of TF in pericardial and systemic blood, and whether this microparticle-associated form is active in thrombin generation compared with non-microparticle-bound, (fluid-phase) TF. Methods : Systemic and pericardial blood samples were collected before and during CPB from six patients undergoing cardiac surgery. Microparticles were isolated by differential centrifugation and their thrombin-generating capacity measured in a chromogenic assay. Microparticle-associated and fluid-phase forms of NCB TF were measured by ELISA. Microparticle-associated TF was visualized by flow cytometry. Results : In pericardial samples, 45–77% of NCB TF was microparticle-associated, and triggered factor VII (FVII)-mediated thrombin generation in vitro . Microparticles from systemic samples triggered thrombin generation independently of FVII, except at the end of bypass ( P  = 0.003). The fluid-phase form of TF did not initiate thrombin generation. Both forms of NCB TF were, at least in part, antigenically cryptic. Conclusions : We demonstrate the occurrence of two forms of NCB TF. One form, which is microparticle-associated, supports thrombin generation via FVII. The other form, which is fluid-phase, does not stimulate thrombin formation. We hypothesize that the microparticle-associated form of NCB TF may be actively involved in postoperative thromboembolic processes when pericardial blood is returned into the patients.     

Plasminogen activator inhibitor‐1 inhibits factor VIIa bound to tissue factor

Summary. Background and objective: A growing body of experimental evidence supports broad inhibitory and regulatory activity of plasminogen activator inhibitor 1 (PAI‐1). The present study was designed to investigate whether PAI‐1 inhibits factor (F) VIIa complexed with tissue factor (TF), a well‐known procoagulant risk factor. Methods and results: The ability of PAI‐1 to inhibit FVIIa‐TF activity was evaluated in both clotting and factor X (FX) activation assays. PAI‐1 and its complex with vitronectin inhibit: (i) clotting activity of FVIIa‐TF (PAI‐1_IC50, 817 and 125 nm , respectively); (ii) FVIIa‐TF‐mediated FX activation (PAI‐1_IC50, 260 and 50 nm , respectively); and (iii) FVIIa bound to TF expressed on the surface of stimulated endothelial cells (PAI‐1_IC50, 260 and 120 nm , respectively). The association rate constant (k_a) for PAI‐1 inhibition of FVIIa‐TF was determined using a chromogenic assay. K_a for PAI‐1 inhibition of FVIIa bound to relipidated TF is 3.3‐fold higher than that for FVIIa bound to soluble TF (k_a = 0.09 ± 0.01 and 0.027 ± 0.03 μm ^?1 min^?1, respectively). Vitronectin increases k_a for both soluble and relipidated TF by 3.5‐ and 30‐fold, respectively (to 0.094 ± 0.020 and 2.7 ± 0.2 μm ^?1 min^?1). However, only a 3.5‐ to 5.0‐fold increase in the acylated FVIIa was observed on SDS PAGE in the presence of vitronectin for both relipidated and soluble TF, indicating fast formation of PAI‐1/vitronectin/FVIIa/relipidated TF non‐covalent complex. Conclusions: Our results demonstrate potential anticoagulant activity of PAI‐1 in the presence of vitronectin, which could contribute to regulation of hemostasis under pathological conditions such as severe sepsis, acute lung injury and pleural injury, where PAI‐1 and TF are overexpressed.     

Levels of microparticle tissue factor activity correlate with coagulation activation in endotoxemic mice

Summary.  Background: Tissue factor (TF) is present in blood in various forms, including small membrane vesicles called microparticles (MPs). Elevated levels of these MPs appear to play a role in the pathogenesis of thrombosis in a variety of diseases, including sepsis. Objective: Measure levels of MP TF activity and activation of coagulation in control and endotoxemic mice. Materials and methods: MPs were prepared from plasma by centrifugation. The procoagulant activity (PCA) of MPs was measured using a two-stage chromogenic assay. We also measured levels of thrombin-antithrombin and the number of MPs. Results: Lipopolysaccharide (LPS) increased MP PCA in wild-type mice; this PCA was significantly reduced by an anti-mouse TF antibody (1H1) but not with an anti-human TF antibody (HTF-1). Conversely, in mice expressing only human TF, MP PCA was inhibited by HTF-1 but not 1H1. MPs from wild-type mice had 6-fold higher levels of PCA using mouse factor (F)VIIa compared with human FVIIa, which is consistent with reported species-specific differences in FVIIa. Mice expressing low levels of human TF had significantly lower levels of MP TF activity and TAT than mice expressing high levels of human TF; however, there were similar levels of phosphatidylserine (PS)-positive MPs. Importantly, levels of MP TF activity in wild-type mice correlated with levels of TAT but not with PS-positive MPs in endotoxemic mice. Conclusion: These results suggest that the levels of TF-positive MPs can be used as a biomarker for evaluating the risk of disseminated intravascular coagulation in endotoxemia.     

A novel anticoagulant activity assay of tissue factor pathway inhibitor I (TFPI).

Tissue factor (TF) pathway inhibitor I (TFPI) is the physiological inhibitor of TF-induced blood coagulation. Circulating blood contains full-length TFPI and TFPI truncated at the C-terminal end. Previous studies have shown that full-length TFPI exerts a stronger anticoagulant effect on diluted prothrombin time (DPT) than truncated TFPI, and it has been suggested that full-length TFPI is biologically more important in vivo. The objective of this study was to develop and validate an assay of TFPI anticoagulant activity. TFPI anticoagulant activity was assayed using a modified DPT assay. Plasmas were incubated in the absence and the presence of TFPI-blocking antibodies. Results were expressed as a ratio with the clotting time in the presence of anti-TFPI as the denominator. The ratio was normalized against a ratio obtained with a reference plasma. The assay was compared with assays of TFPI free antigen, total antigen, and bound TFPI, and TFPI chromogenic substrate activity. We performed all tests in 436 healthy individuals. The normalized TFPI anticoagulant ratio was strongly associated with TFPI free antigen (r = 0.73) but was weakly associated with TFPI chromogenic substrate activity (r = 0.46), TFPI total antigen (r = 0.48), and bound TFPI (r = 0.30). TFPI chromogenic substrate activity was strongly associated with TFPI total antigen (r = 0.73). We have developed a novel assay of TFPI anticoagulant activity in plasma, which may be considered a functional assay of full-length TFPI. Further studies are needed to establish the role of TFPI anticoagulant activity for thrombotic disorders.     

Evolutionary conservation of the allosteric activation of factor VIIa by tissue factor in lamprey

Essentials

• Tissue factor (TF) enhances factor VIIa (FVIIa) activity through structural and dynamic changes.
• We analyzed conservation of TF‐activated FVIIa allosteric networks in extant vertebrate lamprey.
• Lamprey Tf/FVIIa molecular dynamics show conserved Tf‐induced structural/dynamic FVIIa changes.
• Lamprey Tf activation of FVIIa allosteric networks follows molecular pathways similar to human.

Summary

Background

Previous studies have provided insight into the molecular basis of human tissue factor (TF) activation of activated factor VII (FVIIa). TF‐induced allosteric networks of FVIIa activation have been rationalized through analysis of the dynamic changes and residue connectivities in the human soluble TF (sTF)/FVIIa complex structure during molecular dynamics (MD) simulation. Evolutionary conservation of the molecular mechanisms for TF‐induced allosteric FVIIa activation between humans and extant vertebrate jawless fish (lampreys), where blood coagulation emerged more than 500 million years ago, is unknown and of considerable interest.

Objective

To model the sTf/FVIIa complex from cloned Petromyzon marinus lamprey sequences, and with comparisons to human sTF/FVlla investigate conservation of allosteric mechanisms of FVIIa activity enhancement by soluble TF using MD simulations.

Methods

Full‐length cDNAs of lamprey tf and f7 were cloned and characterized. Comparative models of lamprey sTf/FVIIa complex and free FVIIa were determined based on constructed human sTF/FVIIa complex and free FVIIa models, used in full‐atomic MD simulations, and characterized using dynamic network analysis approaches.

Results

Allosteric paths of correlated motion from Tf contact points in lamprey sTf/FVIIa to the FVIIa active site were determined and quantified, and were found to encompass residue–residue interactions along significantly similar paths compared with human.

Conclusions

Despite low conservation of residues between lamprey and human proteins, 30% TF and 39% FVII, the structural and protein dynamic effects of TF activation of FVIIa appear conserved and, moreover, present in extant vertebrate proteins from 500 million years ago when TF/FVIIa‐initiated extrinsic pathway blood coagulation emerged.

     

A novel nitric oxide-releasing statin derivative exerts an antiplatelet/antithrombotic activity and inhibits tissue factor expression.

BACKGROUND: NO-releasing statins are new chemical entities, combining HMG-CoA reductase inhibition and slow NO release, that possess stronger anti-inflammatory and antiproliferative activities than the native statins. OBJECTIVE: We evaluated the antithrombotic effects of nitropravastatin (NCX-6550) by assessing its activity on platelet activation and tissue factor (TF) expression by mononuclear cells in vitro and in vivo. METHODS AND RESULTS: In vitro, NCX-6550 inhibited (1) U46619- and collagen-induced platelet aggregation in buffer and plasma; (2) collagen-induced P-selectin expression in whole blood and (3) platelet adhesion to collagen-coated coverslips under high shear stress. These effects were displayed at concentrations of NCX-6550 ranging from 25 to 100 mum, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 microm). Equimolar concentrations of pravastatin had no influence on these parameters of platelet function. LPS- and PMA-induced TF expression by blood mononuclear cells was also inhibited by NCX-6550 (IC50 13 microm), but not by pravastatin, as assessed by functional and immunological assays and by real-time PCR. In a mouse model of platelet pulmonary thromboembolism, induced by the i.v. injection of collagen plus epinephrine, pretreatment with NCX-6550 (24-48 mg kg(-1)) significantly reduced platelet consumption, lung vessel occlusion and mortality. Moreover, nitropravastatin markedly inhibited the generation of procoagulant activity by spleen mononuclear cells and peritoneal macrophages in mice treated with LPS. In these in vivo models too, pravastatin failed to affect platelet activation and monocyte/macrophage procoagulant activity. CONCLUSIONS: Our results show that nitropravastatin exerts strong antithrombotic effects in vitro and in vivo, and may represent an interesting antiatherothrombotic agent for testing in acute coronary syndromes.     

Disposition of tissue factor pathway inhibitor during cardiopulmonary bypass

BACKGROUND: The tissue factor (TF) factor (F) VIIa complex activates coagulation FIX and FX to initiate coagulation, and also cleaves protease activated receptors (PARs) to initiate inflammatory processes in vascular cells. Tissue factor pathway inhibitor (TFPI) is the only specific inhibitor of the TF-FVIIa complex, regulating both its procoagulant and pro-inflammatory properties. Upon heparin infusion during cardiopulmonary bypass (CPB), a heparin releasable pool of endothelial associated TFPI circulates in plasma. Following protamine neutralization of heparin, the plasma TFPI level decreases, but does not return completely to baseline, suggesting that during CPB a fraction of the plasma TFPI becomes heparin-independent. We have investigated the structural and functional properties of plasma TFPI during CPB to further characterize how TFPI is altered during this procedure. METHODS: We enrolled 17 patients undergoing first-time cardiac surgery involving CPB. Plasma samples were obtained at baseline, 5 min and 1 h after start of CPB (receiving heparin), 10 min after protamine administration (off CPB) and 24 h following surgery. Samples were analyzed for full-length and free (non-lipoprotein bound) TFPI antigen by enzyme-linked immunosorbent assay (ELISA) and for TFPI anticoagulant activity using an amidolytic assay. Western blot analysis was used to identify TFPI species of varying molecular weights in three additional patients. Dunnett's test for post hoc comparisons was used for statistical analysis. RESULTS: The ELISA and Western blot data indicated that an increase in full-length TFPI accounted for most of the heparin releasable TFPI. Following heparin neutralization with protamine, the full-length TFPI antigen returned to baseline levels while the free TFPI antigen and the total plasma TFPI activity remained elevated. This was associated with the appearance of a new 38 kDa form of plasma TFPI identified by Western blot analysis. The 38 kDa form of TFPI did not react with an antibody directed against the C-terminal region of TFPI indicating it has undergone proteolysis within this region. All TFPI measurements returned to baseline 24 h following CPB. CONCLUSIONS: During CPB the full-length form of TFPI is the predominant form in plasma because of its prompt release from the endothelial surface following heparin administration. Upon heparin neutralization with protamine, full-length TFPI redistributes back to the endothelial surface. However, a new 38 kDa TFPI fragment is generated during CPB and remains circulating in plasma, indicating that TFPI undergoes proteolytic degradation during CPB. This degradation may result in a decrease in endothelium-associated TFPI immediately post-CPB, and may contribute to the procoagulant and proinflammatory state that often complicates CPB.