Heparin-releasable Tissue Factor Pathway Inhibitor Research Articles

Levels of heparin-releasable TFPI are increased in first-ever lacunar stroke patients

New insights in the pathophysiology of lacunar stroke (LS) suggest that it is caused by increased permeability of the blood-brain barrier due to endothelial activation. Because endothelial cells are the major production and storage site of tissue factor pathway inhibitor (TFPI), this protein can be used as marker of endothelial activation. In this observational study we measured the different pools of TFPI, as a marker of endothelial function, in first-ever lacunar stroke patients. We determined antigen levels of total and free full-length (FL) TFPI using ELISA in 149 patients and 42 controls. Heparin-releasable free FL TFPI was determined in a random subset of 17 patients and 15 controls. By brain MRI, we classified LS patients as having isolated lacunar infarct (ILA) or silent ischemic lesions (SILs). Plasma levels of total TFPI were highest in patients with SILs compared with those with ILA, but this association disappeared after correction for age and levels of low-density lipoprotein cholesterol. However, levels of heparin-releasable free FL TFPI were higher in patients than in controls. Although ambient plasma levels of total TFPI were not different in subtypes of LS, the increased levels of heparin-releasable TFPI in patients suggest a role of endothelial activation in the pathogenesis of LS.

Tissue Specific Expression of Alternatively Spliced Forms of Tissue Factor Pathway Inhibitor (TFPI) in Mice

TFPI is an endothelium associated anticoagulant protein that directly inhibits tissue factor (TF) pro-coagulant activity and prevents disease produced by intravascular thrombosis. TFPI is produced in three alternatively spliced isoforms in mice, α, β and γ, that differ in domain structure and mechanism for association with endothelium. TFPIα has three Kunitz-type serine protease inhibitory domains and a basic C-terminal region. It produces anticoagulant activity by direct inhibition of factor Xa (fXa) via the second Kunitz domain (K2) and, in a fXa dependent manner, inhibition of TF-fVIIa via the first Kunitz domain (K1). The third Kunitz domain (K3) and C-terminal region do not directly inhibit proteolysis. However, alignment of sequences from various vertebrate species demonstrates that K3 has maintained a high degree of sequence conservation for over 430 million years suggesting that K3 maintains a physiological function. TFPIα indirectly associates with endothelium by binding to a glycosylphosphatidyl inositol (GPI) anchored protein and binds non-specifically to glycosaminoglycans. TFPIβ has K1 and K2 but lacks K3 and has a distinct C-terminal region that encodes a GPI-anchor attachment sequence. TFPIγ has K1 and K2 but lacks K3 and has a distinct C-terminal region that results in production of a secreted protein. It is not known if these structurally diverse forms of TFPI with different mechanisms for cell surface association have variable efficacy in their ability to inhibit TF activity in vivo. We characterized TFPI isoform expression in mouse tissues in order to define specific physiological functions for alternative splicing of TFPI and how it may hinder diseases mediated by intravascular TF activity. Message for TFPIα is ~16-fold more abundant than message for TFPIβ or TFPIγ in mouse tissues. In situ hybridization studies of mouse heart and lung found that the isoforms are expressed in the same pattern, primarily in endothelial cells. No cell or vascular bed exclusively producing a single isoform was identified. Western blot analyses revealed the somewhat surprising finding that TFPIβ is the predominant isoform produced by adult mouse tissues. Thus, protein synthesis of TFPI isoforms in mice is regulated at least in part at the level of protein translation. Mouse plasma TFPI levels increase only 10–20% following heparin infusion. These data contrast with the 2- to 4-fold increase in plasma TFPI observed following heparin infusion in humans. The presence of only small amounts of heparin releasable TFPI in mouse plasma is consistent with TFPIβ as the major isoform because TFPIβ is directly GPI-anchored to endothelium and lacks the highly basic C-terminal region that allows TFPIα to associate non-specifically with endothelium glycosaminoglycans. To our knowledge, these are the first data demonstrating TFPIβ production in vivo. In contrast to the adult tissues, western blot analysis of mouse placenta revealed that it produces primarily TFPIα. This finding led us to examine mouse fetal tissue to determine if there is developmental regulation of the production of different TFPI isoforms. These studies demonstrated that TFPIα and TFPIβ are produced in approximately equal amounts in E14.5 embryos. The relative proportion of TFPIα in mouse tissues then decreases during development and very little is produced at 12 weeks of age. The evolutionary conservation of TFPIα and its production in mouse placenta and embryonic tissues suggests that unique features of TFPIα, such as K3, the basic C-terminal region and/or its binding to the endothelial surface through a GPI-anchored co-receptor, perform specific functions in the regulation of intravascular TF activity during vasculogenesis/angiogenesis that are not performed by TFPIβ or TFPIγ.

Disposition of tissue factor pathway inhibitor during cardiopulmonary bypass.

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. 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. 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. 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.

Characterization of the association of tissue factor pathway inhibitor with human placenta.

Tissue factor pathway inhibitor (TFPI) is an endothelial-associated inhibitor of blood coagulation. Because the mechanism for attachment of TFPI to endothelium is not clear, we investigated its association with human placenta. Western blots demonstrate that treatment with phosphatidylinositol-specific phospholipase C (PIPLC) removes more placental TFPI than either PBS or heparin, a finding confirmed by immunohistochemistry. The amounts of heparin-releasable and PIPLC-releasable TFPI activity on placental endothelium were measured in placentas from 5 individuals. PIPLC removes >10-fold more TFPI activity from the placental fragments than 10 U/mL heparin and >100-fold more than 1 U/mL heparin. Pretreatment of the placental fragments with PIPLC increases the amount of heparin-releasable TFPI by approximately 3-fold. An antibody specific for the C-terminal region of TFPI recognizes PIPLC-releasable TFPI in Western blots. GPI-anchored TFPI is the predominant form on placental endothelium. Heparin-releasable TFPI likely represents only a small portion of the total TFPI on endothelium that remains attached to cell-surface glycosaminoglycans after cleavage of the GPI anchor by endogenous enzymes. The predominance of GPI-anchored TFPI suggests that heparin infusion does not significantly redistribute TFPI within the vasculature. The intact C-terminus in GPI-anchored TFPI indicates it is not directly attached to a GPI anchor. Rather, it most likely associates with endothelium by binding to a GPI-anchored protein.

Analysis of the tissue factor pathway inhibitor gene and antigen levels in relation to venous thrombosis.

Tissue factor pathway inhibitor (TFPI) inhibits tissue factor-induced coagulation. The major part of TFPI is releasable by heparin. We recently found eight patients with thrombosis and low levels of heparin-releasable TFPI in whom we investigated the TFPI gene for mutations. A transition of G to A coding for Valine264Methionine in the heparin-binding domain was found. The Val264Met polymorphism had an allele frequency of 3% in 96 healthy individuals. A silent polymorphism was identified in TFPI exon IV (T-->C), which does not alter Tyrosine 56. Apart from Val264Met, which was detected in one out of the eight patients, no other mutations in the TFPI gene were found in patients with low heparin-releasable TFPI. Analysis of Val264Met in 317 patients with deep vein thrombosis (DVT) and 292 controls showed no association between Val264Met and DVT. However, a study of total TFPI antigen levels in 122 DVT patients and 126 controls demonstrated an association between TFPI levels and venous thrombosis (P = 0.0001). These results provide evidence for a relationship between venous thrombosis and total TFPI level as a possible risk factor, whereas they do not support a link between DVT and mutations in the nine exons of the TFPI gene.

Low Levels of Heparin-releasable Tissue Factor Pathway Inhibitor in Young Patients with Thrombosis

An association between deficiency of tissue factor pathway inhibitor (TFPI) and thrombosis has not been clearly demonstrated in humans, but previous studies have focused on the measurement of plasma TFPI, which is only a small part of the total body TFPI. The major fraction of this natural anticoagulant can be measured in plasma after release by heparin injection. To investigate if deficiency of heparin-releasable TFPI is associated with thrombosis. we measured TFPI activity in plasma before and 10 min after intravenous injection of 7500 IU unfractionated heparin in 64 young patients with venous thrombosis, 49 young patients with arterial thrombosis and 38 healthy individuals. Post-heparin TFPI activity levels were significantly lower in the group of patients with venous thrombosis than in controls (mean+/-SD: 230%+/-39 vs 260%+/-34, p = 0.0002), whereas there was no difference for patients with arterial thrombosis. Defining the normal range as the mean+/-2 SD of TFPI activity in controls, twelve patients had low post-heparin TFPI activity levels, seven with venous and five with arterial thrombosis. Low levels of TFPI activity were confirmed by immunoassay in six of the seven patients with venous thrombosis and two of the five patients with arterial thrombosis, and were present also in at least one first degree relative of six patients, suggesting that the defect might be inheritable. However, the causative role of low heparin-releasable TFPI remains uncertain, because co-segregation of the defect with thrombotic symptoms could not be demonstrated in the small number of families studied.

Heparin-Releasable Endothelial Cell-Associated Tissue Factor Pathway Inhibitor (Tfpi) is Increased in the Coronary Circulation After Coronary Spasm in Patients with Coronary Spastic Angina

Tissue factor pathway inhibitor (TFPI) is a physiological regulator of the extrinsic coagulation cascade. Coronary spasm can alter endothelial cell properties in the coronary artery with resultant thrombosis. To determine whether coronary spasm affects plasma TFPI level, we measured the heparin-releasable endothelial cell-associated TFPI (heparin-releasable TFPI) (ng/ml) in the coronary sinus and the aortic root before and after coronary spasm induced by an injection of acetylcholine in 18 patients with coronary spastic angina, and before and after myocardial ischemia induced by rapid atrial pacing in 18 patients with stable exertional angina, and in 17 control subjects with normal coronary arteries and no coronary spasm. Heparin-releasable TFPI level in the coronary spastic angina group significantly increased in the coronary sinus (122±46 to 147±63, p<0.001) after the ischemic event but not in the aortic root (113±44 to 121±58). The level in the coronary sinus and the aortic root remained unchanged after the ischemic event in the stable exertional angina group and after the injection of acetylcholine in the control group. The coronary sinus-arterial difference in the amount of the heparin-releasable TFPI significantly increased after the ischemic event only in the coronary spastic angina group (10±18 to 26 ±18, p<0.002). Our result suggested that heparin-releasable TFPI is increased in the coronary circulation after coronary spasm.

BINDING OF TISSUE FACTOR PATHWAY INHIBITOR TO CULTURED ENDOTHELIAL CELLS-INFLUENCE OF GLYCOSAMINOGLYCANS

Tissue factor pathway inhibitor (TFPI) is mainly bound to the vessel wall and is released to circulating blood after injections of heparin. It has been suggested that the highly positively charged carboxy terminal end of heparin releasable TFPI is bound to negatively charged binding molecule(s), presumably glycosaminoglycans (GAGs), on the luminal surface of endothelial cells. The aim of the present study was to characterize this binding. Confluent monolayers of human umbilical vein endothelial cells (HUVECs) and Ea·hy926 cells were incubated with 125I-labelled recombinant TFPI (rTFPI). Two different rTFPI preparations were used in the experiments; one preparation was full-length rTFPI and one preparation was truncated at the C-terminal end (rTFPI 1–161). Binding of 125I-rTFPI reached equilibrium conditions after 2 hours incubation at room temperature. Scatchard plots indicated a single class of binding sites with a mean K d value of 164±16 nmol/L for HUVECs and a K d value of 296±10 nmol/L for Ea·hy926 cells. The number of rTFPI binding sites per cell were approximately 1.10 7. Binding of 125I-rTFPI 1–161 was non-specific. GAGs reduced binding of 125I-rTFPI in a dose-dependent manner by 50–75%. The potency of different GAGs to displace bound rTFPI was in the following order: Unfractionated heparin (UF) > low-molecular weight (LMW) heparin > hexadecasaccharides/octasaccharides/dodecasaccharides > heparan sulfate > dermatan sulfate. Treatment of the cells with heparinase III, with chondroitinase ABC lyase, or with sodium chlorate (to prevent sulfation) did not influence the binding of TFPI. We conclude that the C-terminal end is necessary for binding of TFPI to endothelial cells, but the binding is weak and does not involve GAGs. Copyright © 1996 Elsevier Science Ltd

Depletion of Intravascular Pools of Tissue Factor Pathway Inhibitor (TFPI) during Repeated or Continuous Intravenous Infusion of Heparin in Man

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of the extrinsic coagulation system. TFPI is increased several-fold in postheparin plasma and thereby thought to contribute significantly to the antithrombotic action of heparin. The present study was conducted to investigate how repeated (n = 8) and continuous (n = 6) administration of heparin affect plasma TFPI and the inhibition of tissue factor (TF)-induced coagulation ex vivo in humans. Free TFPI antigen (TFPI Ag) increased from 19.2 +/- 4.0 ng/ml to 204.7 +/- 31.7 ng/ml after intravenous injection of 5000 IU of unfractionated heparin. Five repeated injections of 5000 IU of heparin at 4 h intervals caused a progressive decrease (-45 +/- 8%, p < 0.0001 for time effect) in heparin-releasable TFPI and a progressive shortening of the clotting time as determined in a dilute prothrombin time assay (dPT) (-8.7 +/- 6.1 s, p < 0.0001). The basal concentration of TFPI Ag in plasma collected immediately before each heparin injection was decreased by 29 +/- 15% (p < 0.0001), whereas the dPT was decreased by 6.9 +/- 3.5 s (p < 0.0001). During a 24 h continuous infusion of heparin TFPI Ag decreased from 161.5 +/- 26.0 ng/ml to 35.6 +/- 4.7 ng/ml (-77.3 +/- 5.1%) (p < 0.0001). The contribution of TFPI to the inhibition of TF-induced coagulation during heparin infusion was estimated to decrease from 60 +/- 15% to 20 +/- 10% (p < 0.0001). The present data indicate partial depletion of intravascular pools of TFPI by repeated and continuous heparin administration and thereby attenuation of its contribution to the antithrombotic action of heparin.

Loss of Heparin-Releasable Tissue Factor Pathway Inhibitor in Patients Undergoing PTCA

Loss of Heparin-Releasable Tissue Factor Pathway Inhibitor in Patients Undergoing PTCA