Age‐related plasma reference ranges for two heparin‐binding proteins – vitronectin and platelet factor 4

This study was conducted to establish age‐related reference ranges for two heparin‐binding proteins – vitronectin and platelet factor 4 (PF4) – and to determine if the quantitative values of these proteins may contribute to the reported age‐dependent effect of unfractionated heparin (UFH). Plasma samples were obtained from healthy children aged between 1 month and 16 years and from healthy adult volunteers. Two commercial kits were used to measure plasma vitronectin and PF4 levels. Results were reported as mean and boundaries including 95% of the population. Plasma vitronectin levels for children aged 1–5 years were significantly higher compared with adults. Plasma PF4 levels for infants <1 year of age were significantly lower compared with adults. The differences between reference values for both proteins in all other age‐groups were not statistically significant. This study for the first time has established age‐related reference ranges for vitronectin and PF4. In establishing these ranges, the quantitative values of these proteins do not appear to be the major contributory cause for the age‐dependent variation in UFH effect. Future studies are required to evaluate the possible impact of age‐dependent differences in binding between heparin‐binding proteins and UFH.     

Partial depletion of tissue factor pathway inhibitor during subcutaneous administration of unfractionated heparin, but not with two low molecular weight heparins

Tissue factor pathway inhibitor (TFPI) is released to circulating blood after intravenous (i.v.) and subcutaneous (s.c.) injections of heparins, and may thus contribute to the antithrombotic effect of heparins. We have recently shown that total TFPI activity, plasma free TFPI antigen, and heparin releasable TFPI were partially depleted during repeated and continuous i.v. infusion of unfractionated heparin (UFH), but not during s.c. treatment with a low molecular weight heparin (LMWH). The difference may be attributed to a different mode of action or the different mode of administration. In the present randomized cross-over study, s.c. administration of therapeutic doses of UFH was compared with s.c. administration of two LMWHs. 12 healthy male volunteers were treated for 3 d with UFH, 250 U/kg twice daily, dalteparin, 200 U/kg once daily, and enoxaparin, 1.5 mg/kg once daily. Six participants were also treated with UFH, 300 U/kg once daily. On day 5 a single dose of either drug was given. Peak levels of total TFPI activity and free TFPI antigen were detected 1 h after injection, whereas maximal prolongation of activated partial thromboplastin time (APTT) and peak levels of anti-factor Xa activity and anti-factor IIa activity were detected after 4 h. On UFH administered twice daily, free TFPI antigen decreased by 44% from baseline level before the first injection on day 1 to pre-injection level on day 5. On UFH administered once daily, basal free TFPI antigen decreased by 50%, 56% and 27% on day 2, 3 and 5 respectively, compared with day 1. Minimal depletion of TFPI was detected during treatment with LMWHs. The study demonstrates the different modes of action of LMWHs and UFH and may help to explain the superior antithrombotic efficacy of LMWHs.     

Comparative Effects of Unfractionated Heparin and Low Molecular Weight Heparin on Vascular Endothelial Cell Tissue Factor Pathway Inhibitor Release: A Model for Assessing Intrinsic Thromboresistance

Objectives: The purpose of our study was to characterize tissue factor pathway inhibitor (TFPI) release from human vascular endothelial cells following daily exposure to varying concentrations of unfractionated heparin (UFH) and low molecular weight heparin (LMWH). Background: A rebound increase in ischemic/thrombotic events, including myocardial infarction and cardiovascular death, has been observed after the abrupt cessation of UFH. In a single center pilot study of patients with acute coronary syndromes (ACS) we reported that thrombin generation was evident within one (1) hour of UFH cessation, increased progressively over the subsequent 24 hours, correlated directly with factor VII activity and inversely with TFPI (concentration and activity). Methods: Human umbilical vein endothelial cells were grown to confluence and incubated with varying concentrations of UFH or dalteparin, a low molecular weight haparin, for up to 144 hours. Daily samples of the cells supernatant were obtained and assayed for TFPI. Cellular reserve and responsiveness to recombinant endothelial cell growth factor (rEGF) stimulation were determined at 168 hours. Results: In low concentrations (0.5 U/mL) UFH caused a progressive rise in TFPI concentration with a peak level of 6.36 ± 0.5 ng/10⁵ cells at 24 hours. By 72 hours of daily exposure, the levels declined to below control values and TFPI release following rEGF stimulation was reduced by approximately 60% compared to control (1.93 ± 0.42 vs 4.3 ± 0.78 ng/10⁵ cells; p = 0.001). Initial endothelial cell release and rate of decline were more robust with high concentrations of UFH (5.0 U/ml). TFPI levels were above control values at each sampling time point up to 120 hours and cellular responsiveness to stimulation was preserved with dalteparin (compared to UFH) (p < 0.001). Conclusions: Thrombin generation and clinical events that occur during treatment with UFH and following its abrupt cessation may represent an acquired state of transiently impaired thromboresistance to the tissue factor-VIIa complex. The differing effects of UFH and LMWH on vascular endothelial cell TFPI synthesis, release and reserve with prolonged administration require further investigation.     

Differential effect of unfractionated heparin and low molecular weight heparin on intravascular tissue factor pathway inhibitor: evidence for a difference in antithrombotic action

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor (TF)-induced blood coagulation, which is increased several-fold in post-heparin plasma and thought to contribute significantly to the antithrombotic action of heparin. In the present study we investigated whether subcutaneous (s.c.) administration of a low molecular weight heparin (LMWH), enoxaparin, had a different effect on intravascular pools of TFPI compared with continuous i.v. infusion of unfractionated heparin (UFH). 18 healthy male volunteers were randomly assigned to continuous i.v. infusion with UFH (initially 450 U/kg/24 h, n  = 6) or to s.c. treatment with LMWH once daily (enoxaparin, 1.5 mg/kg, n  = 12) for 72 h. A bolus injection of 5000 IU UFH i.v. caused an 8–13-fold increase in plasma-free TFPI antigen (TFPI Ag), followed by a progressive decrease (81 ± 4%, P  < 0.001) during the 72 h infusion with UFH. 4 h after discontinuation of the infusion, basal free TFPI Ag and heparin-releasable TFPI were significantly decreased compared with the concentrations before the infusion (30 ± 9% and 27 ± 7%, respectively). In contrast, LMWH treatment did not reduce either basal or heparin-releasable TFPI Ag. The changes in plasma TFPI Ag by UFH and LMWH were statistically different between groups both in pre- ( P  < 0.001) and post-heparin ( P  < 0.0001) plasma. The differential effect of UFH and LMWH on intravascular pools of TFPI may contribute to the understanding of the apparent superior efficacy of LMWHs in the treatment of both arterial and venous thrombosis.     

Blocking inhibition of prothrombinase by tissue factor pathway inhibitor alpha: a procoagulant property of heparins

Unfractionated heparin (UFH) has procoagulant activity in antithrombin/heparin cofactor II (HCII)‐depleted plasma. UFH prevents tissue factor pathway inhibitor alpha (TFPIα) from inhibiting the procoagulant enzyme complex, prothrombinase, providing a possible mechanism for its procoagulant activity. The procoagulant potential of UFH and various low molecular weight heparins (LMWHs) were characterized for TFPIα dependence, using thrombin generation assays performed with antithrombin/HCII‐depleted plasma. UFH, the LMWHs enoxaparin and dalteparin, and the low anticoagulant LMWH 2‐O, 3‐O desulphated heparin (ODSH) all promoted thrombin generation, but fondaparinux did not, and this activity was blocked by a TFPIα antibody. UFH, enoxaparin, and dalteparin were anticoagulant in reactions containing 1–2% normal plasma. In prothrombinase activity assays, UFH, enoxaparin, dalteparin and ODSH blocked prothrombinase inhibition by TFPIα, while again fondaparinux did not. In both the plasma and purified assays, LMWHs displayed greater procoagulant potential than UFH, even when normalized to saccharide concentration. These biochemical data reveal that UFH and LMWHs, but not fondaparinux, block prothrombinase inhibition by TFPIα, thereby producing their paradoxical procoagulant activity observed in the absence of antithrombin/HCII. The findings may help to understand the complex pathophysiology and treatment of patients that are simultaneously bleeding and clotting, such as those with disseminated intravascular coagulation.     

Increased Thrombin-Activatable Fibrinolysis Inhibitor and Decreased Tissue Factor Pathway Inhibitor and Thrombomodulin Levels in Children with Hypothyroidism

Objective: We determined the profile of coagulation/fibrinolytic and vascular endothelial cell function parameters including plasminogen activator inhibitor (PAI) and thrombin-activatable fibrinolysis inhibitor (TAFI), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM), and tissue plasminogen activator (tPA) levels in children with hypothyroidism. Methods: Forty children with hypothyroidism aged 0-16 months who presented for the first time to our hospital and 29 age-and sex-matched healthy controls were enrolled in the study. All coagulation tests were performed with ELISA method. One year after Na-L-thyroxine treatment, the study parameters were re-evaluated in 25 euthyroid children out of the 40 patients diagnosed with hypothyroidism.Results: Although no significant effect was detected regarding PAI antigen (Ag) and tPA Ag, the levels of TAFI, TM, and TFPI were consistent with subclinical hypercoagulability and hypofibrinolysis. There was a significant increase in TAFI Ag levels and a significant decrease in TFPI Ag and TM Ag levels in hypothyroid patients compared to healthy controls. As a result of correlation tests, the largest impact of hypothyroidism on coagulation system was on TFPI. In accordance with these findings, TAFI Ag levels decreased and TFPI Ag and TM Ag levels increased with hormonal replacement therapy. Conclusions: Increased TAFI and decreased TFPI and TM in patients with hypothyroidism may indicate a potential hypercoagulable and hypofibrinolytic state as well as possible endothelial dysfunction, which may increase the risk of atherosclerotic and atherothrombotic complications. Thyroid hormone levels should also be checked in patients with a predisposition to coagulation, and thyroid replacement therapy should be initiated. Conflict of interest:None declared.     

Effects of unfractionated and low molecular weight heparins on plasma levels of hemostatic factors in patients with acute coronary syndromes.

BACKGROUND AND OBJECTIVES: Unfractionated heparin (UFH) and enoxaparin (low molecular weight heparin) constitute fundamental therapies in the treatment of patients with acute coronary syndrome (ACS). Since enoxaparin appears to offer clinical advantages over UFH in managing ACS, markers of thrombin generation, endothelial function and acute phase response could manifest different responses to UFH or enoxaparin. The purpose of the present study was to investigate the effect that treatment with either UFH or enoxaparin has on plasma hemostatic markers in 24 patients with ACS. DESIGN AND METHODS: The patients were randomized to receive 5,000 IU intravenous bolus and continuous infusion of 18 IU/Kg/h UFH (n=11) or 1 mg/kg/12h subcutaneous enoxaparin (n=13). The plasma levels of fibrinogen (Fg), prothrombin fragment 1+2 (F1+2), thrombin antithrombin complex (TAT), von Willebrand factor (vWF), tissue factor (TF) and tissue factor pathway inhibitor (TFPI) were assayed at admission and 6, 12, 24 and 48 hours after heparin treatment. RESULTS: Upon admission, UFH and enoxaparin patients showed a significant increase in all the hemostatic parameters measured with respect to the levels in the control subjects. In comparison with the baseline levels of the UFH- and enoxaparin-treated patients, Fg showed a significant increase at 48 h and TFPI at 6, 12 and 24 hours. However, at 48 hours TFPI levels were not significantly higher than the basal values. There were no significant changes in F1+2, TAT, vWF or TF. INTERPRETATION AND CONCLUSIONS: Markers of thrombin generation, endothelial function and acute-phase reactants manifest a similar response to UFH and enoxaparin. An increase in thrombin generation may be a result of persistently activated inflammatory and endothelial processes, despite UFH and enoxaparin treatment.     

The activated clotting time can be used to monitor the low molecular weight heparin dalteparin after intravenous administration

OBJECTIVES: This study was designed to compare the dose response of dalteparin versus unfractionated heparin (UFH) on the activated clotting time (ACT), and to determine whether the ACT can be used to monitor intravenous (IV) dalteparin during percutaneous coronary intervention (PCI). BACKGROUND: The use of low molecular weight heparin (LMWH) during PCI has been limited by the presumed inability to monitor its anticoagulant effect using bedside assays. METHODS: This study was performed in three phases. In vitro, ACTs were measured on volunteer (n = 10) blood samples spiked with increasing concentrations of dalteparin or UFH. To extend these observations in vivo, ACTs were then measured in patients (n = 15) who were sequentially treated with IV dalteparin and then UFH. Finally, a larger monitoring study was undertaken involving patients (n = 110) who received dalteparin 60 or 80 international U (IU)/kg alone or followed by abciximab. We measured ACT (Hemochron), activated partial thromboplastin time (aPTT), plasma anti-Xa and anti-IIa levels, tissue factor pathway inhibitor (TFPI) concentration, and plasma dalteparin concentration. RESULTS: Dalteparin induced a significant rise in the ACT with a smaller degree of variance as compared to UFH. Five min after administration of IV dalteparin 80 IU/kg the ACT increased from 125 s (122 s, 129 s) to 184 s (176 s, 191 s) (p < 0.001). The aPTT, anti-Xa and anti-IIa activities, and TFPI concentration also demonstrated significant increases following IV dalteparin. CONCLUSIONS: The ACT and aPTT are sensitive to IV dalteparin at clinically relevant doses. These data suggest that the ACT may be useful in monitoring the anticoagulant effect of intravenously administered dalteparin during PCI.     

Pharmacodynamic considerations in the selection of dosage of tinzaparin for various indications: experimental studies in primates

Like unfractionated heparin (UFH), low-molecular-weight heparins (LMWHs) are polypharmacologic agents that can modulate the hemostatic system at multiple points. Thus, to select an optimal dose of LMWH for a given indication, it is necessary to consider multiple actions of the drug. In this study, nonhuman primates were treated with intravenous or subcutaneous boluses of the LMWH tinzaparin or UFH. Doses were selected on the basis of the expected prophylactic (75 U/kg) and therapeutic (175 U/kg) dosing of tinzaparin. Blood samples were drawn periodically up to 24 hours after administration. Circulating anti-Xa and anti-thrombin (anti-IIa) activities determined using amidolytic assays were used to estimate plasma tinzaparin (heparin) concentrations. In addition, total tissue factor pathway inhibitor (TFPI) levels were measured in these primates. Subcutaneous administration of 75 U/kg tinzaparin resulted in plasma levels of approximately 0.2 to 0.3 U/mL, concentrations sufficient for DVT prophylaxis. Such drug levels were not associated with a significant release of TFPI. Intravenous administration of the same dose resulted in a peak drug level of approximately 1.5 anti-Xa U/mL. The elimination half-life was approximately 1 hour. Thus, intravenously administered tinzaparin may be useful for providing anticoagulation during coronary interventions. Subcutaneous administration of 175 U/kg resulted in tinzaparin levels of approximately 0.7 anti-Xa U/mL and a significant increase in TFPI levels. Interestingly, the increase in TFPI levels occurred over a different time frame than anticoagulant activity. Intravenous administration of 175 U/kg resulted in peak drug concentrations of almost 5 anti-Xa U/mL. The pharmacokinetic behavior of intravenously administered tinzaparin was comparable to that of UFH. The data show that the pharmacokinetic and pharmacodynamic effects measured using different assays widely differ. For a proper pharmacodynamic analysis, multiple assays should be considered, given that both UFH and LMWHs are polycomponent in nature.     

Efficacy of unfractionated heparin, low molecular weight heparin and both combined for releasing total and free tissue factor pathway inhibitor.

Unfractionated heparin (UFH) exerts its anticoagulant properties by increasing the inactivation of thrombin and activated factor X by antithrombin III. Apart from this main action release of tissue factor pathway inhibitor (TFPI) from endothelial cells could also be important for the antithrombotic activity of heparins. Four different heparin preparations were injected subcutaneously into 5 healthy volunteers 1 week apart: (1) UFH 2,500 IU fix dose (FixUFH), (2) 1 mg/kg body weight of low molecular weight heparin (LMWH), (3) the combined LMWH-adjusted dose plus UFH 2,500 IU fix dose (ComHep) and (4) UFH 2,500 IU/10 kg body weight (UFHvar). Plasma samples were drawn before and 1, 2, 4, 6, 12 and 24 h afterwards. FixUFH did not affect the concentration of total and free TFPI. Total TFPI increased in the 1st hour after LMWH injection from 74 to 124 ng/ml (p < 0.01), after ComHep from 82 to 144 ng/ml (p < 0.01), and after UFHvar from 91 to 113 ng/ml (p < 0.05). All observed elevations were significant at the peak value (+/- 2 h, p < 0.01 compared with baselines). The increase of free TFPI produced by UFHvar (74.5 and 70.5 ng/ml) was significantly higher than with LMWH (42.8 and 38.0 ng/ml) at 2 and 4 h (p < 0.001 and p < 0.01, respectively). UFHvar and ComHep but not LMWH produced a statistically significant increase of free TFPI compared with FixUFH at 2, 4 and 6 h (p < 0. 01). We concluded that at comparable therapeutic doses, subcutaneous UFHvar released more free TFPI than LMWH and ComHep. A synergism between LMWH and low dose of UFH was found in 4-, 6- and 12-hour blood samples.     

Tissue factor pathway inhibitor (TFPI) release after heparin stimulation is increased in Type 1 diabetic patients with albuminuria.

AIMS: To study heparin-stimulated TFPI release in relation to complications in Type 1 diabetic patients. SUBJECTS AND METHODS: Nineteen uncomplicated Type 1 diabetic patients (group I) were compared with 18 patients with retinopathy (group II), and nine patients with retinopathy and albuminuria (group III). Blood samples were taken before (basal) and till 30 min after 5000 IU of heparin i.v. (post-heparin). TFPI activity was measured chromogenically. Von Willebrand factor, tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and thrombomodulin were also measured. RESULTS: Basal TFPI activity was higher in group III (121 +/- 10%) compared with group II (111 +/- 8%) or group I (110 +/- 13%) (P < 0.05), and strongly correlated with albuminuria (r = 0.66, P < 0.05). At all time points after heparin administration, TFPI activity in group III was significantly higher than in group I. TFPI activity was also higher in group III than in group II 5-30 min post-heparin. The increase in post-heparin TFPI activity, measured as the incremental area under the curve, was higher in group III compared with group I (65 +/- 7 vs. 59 +/- 4; P < 0.05). Of the other parameters, only thrombomodulin was higher in group III (44 +/- 24 vs. 26 +/- 7 (group II) and 28 +/- 9 ng/ml (group I); P < 0.01). CONCLUSIONS: We conclude that basal and post-heparin TFPI activity is increased in albuminuric patients. The increase in heparin-stimulated TFPI release in patients with albuminuria is higher than in patients with retinopathy or without complications. This could be the result of altered endothelial glycosaminoglycan characteristics.     

Reference values for thrombotic markers in children

Thromboembolic events are an increasingly common problem encountered in children. The laboratory diagnosis of thrombotic disorders in children differs from that in adults. To establish the normal reference of natural anticoagulant parameters in children of different age groups, plasma from healthy children between the ages of 2 months and 16 years (n = 127) and adults (n = 30) were assayed for a disintegrin-like and metalloprotease with thrombospondin type 1 domain 13 (ADAMTS-13), von Willebrand factor collagen-binding activity (vWF:CB), tissue factor pathway inhibitor (TFPI), homocyteine and natural anticoagulants. Children were divided into four age groups: less than 1 year, 1-5 years, 6-10 years, and 11-16 years. The reference values for ADAMTS 13, homocysteine, and protein C activity were significantly lower in children of all age groups compared with those in the adults. Similarly, those for protein C antigen, total protein S, free protein S and antithrombin III (AT III) for children less than 1 year were significantly lower than in the adults. On the contrary, TFPI levels were significantly higher in the children for all age groups when compared with the adults. vWF:CB levels were comparable across all groups. There are age-related physiologic differences in ADAMTS-13, TFPI, homocysteine and natural anticoagulants between children and adults. Our data will provide physicians with a useful reference guide in interpreting test results of inhibitors of hemostatic parameters in children suspected of thrombotic disorders.     

Decreased tissue factor pathway inhibitor (TFPI)‐dependent anticoagulant capacity in patients with cirrhosis who have decreased protein S but normal TFPI plasma levels

Protein S acts as a cofactor for tissue factor pathway inhibitor (TFPI) in the down regulation of thrombin generation, and acquired and congenital protein S deficiencies are associated with a concomitant TFPI deficiency. In contrast, in patients with liver diseases, decreased protein S, but normal or increased levels of TFPI have been reported. We compared TFPI and protein S plasma levels between 26 patients with cirrhosis and 20 healthy controls and found that TFPI levels were comparable between patients (111 ± 38%) and controls (108 ± 27%), despite reduced protein S levels (74 ± 23% in patients vs. 98 ± 10% in controls). Subsequently, we quantified the activity of the TFPI‐protein S system by measuring thrombin generation in the absence and presence of neutralizing antibodies to protein S or TFPI. Ratios of peak thrombin generation in the absence and presence of these antibodies were calculated. Both the protein S and the TFPI ratios were increased in patients with cirrhosis compared to controls. Protein S ratios were (0·62 [0·08–0·93] in patients vs. 0·32 [0·20–0·54] in controls; TFPI ratios were 0·50 [0·05–0·90] in patients vs. 0·18 [0·11–0·49] in controls). Thus, although the acquired protein S deficiency in patients with cirrhosis is not associated with decreased TFPI levels, the TFPI/protein S anticoagulant system is functionally impaired.     

Low‐Molecular‐Weight Heparins in Thrombosis and Cancer: Emerging Links

Heparin as well as low‐molecular‐weight heparins (LMWHs) have polypharmacological actions at various levels. Earlier studies focused on the plasma anti‐Xa and anti‐IIa pharmacodynamics (PD) for the different LMWHs. Other important PD parameters for heparin and LMWHs might explain the diverse clinical impacts of this class of agents in thrombosis and beyond: the release of the vascular tissue factor pathway inhibitor (TFPI), inhibition of key matrix‐degrading enzymes, and other mechanisms. There is much evidence for the key role of LMWHs in hypercoagulation in thrombosis and cancer, angiogenesis, and inflammatory disorders. Many cancer patients reportedly have a hypercoaguable state, with recurrent thrombosis due to the impact of cancer cells and chemotherapy or radiotherapy on the coagulation cascade. Studies have demonstrated that unfractionated heparin (UFH) or its low molecular weight fractions interfere with various processes involved in tumor growth and metastasis. Clinical trials have suggested a clinically relevant and improved efficacy of LMWHs, as compared to UFH, on the survival of cancer patients with deep vein thrombosis. Our laboratory has demonstrated a significant role for LMWHs and for LMWH‐releasable TFPI on the regulation of angiogenesis, tumor growth, and tumor metastasis; we have also seen potent inhibition of matrix‐degrading enzymes by LMWHs but not by TFPI. The antiangiogenesis effect of LMWHs or non‐anticoagulant LMWH derivatives was shown to be reversed by anti‐TFPI. Thus, modulation of tissue factor/Vila noncoagulant activities by LMWH‐releasable TFPI and the inhibitory effects on matrix‐degrading enzymes beside the anticoagulant efficacy have provided an expanded clinical utility for LMWHs in angiogenesis‐associated disorders, including human tumor growth and metastasis.     

Anti-tissue factor pathway inhibitor activity in patients with primary antiphospholipid syndrome

The association between antiphospholipid antibodies and an increased risk of thrombosis in antiphospholipid syndrome (aPS) patients is probably caused by numerous mechanisms, including the effects of antibodies to phospholipid-binding proteins such as beta(2)-glycoprotein I and prothrombin. In this study, we investigated the inhibition of tissue factor pathway inhibitor (TFPI) in 33 patients with primary antiphospholipid syndrome (PAPS). TFPI was measured in PAPS patients using an amidolytic assay, dependent on the generation of activated factor X (Fxa), and this was compared with 55 healthy subjects. Functional levels of TFPI (mean +/- SD) were significantly lower in PAPS patients (0.89 +/- 0.37 U/ml) than the control group (1.05 +/- 0.15 U/ml) (P = 0.02). The difference was caused by a subset of five patients who had TFPI levels below the lower 99% confidence interval of the normal reference range, representing increased FXa generation in the assay system. IgG fractions were isolated from these five patients and five control subjects, then incorporated into normal plasma to measure FXa generation in the TFPI assay system. FXa generation was increased when polyclonal rabbit anti-human TFPI IgG (P < 0.0001) or PAPS IgG (P = 0.0001) were added to normal plasma, demonstrating inhibition of TFPI. The apparent anti-TFPI activity demonstrated in the five subjects with PAPS in this study may represent a significant new mechanism for thrombosis in patients with aPS, as it implies that increased tissue factor FVIIa-mediated thrombin generation might occur.     

Molecular weight-dependent influence of heparin on the form of tissue factor pathway inhibitor circulating in plasma.

The increase of circulating tissue factor pathway inhibitor (TFPI) in plasma by heparins is thought to contribute to their overall antithrombotic activity. In a clinical study in healthy volunteers, we recently found that the specific potency of a heparin to mobilize TFPI from the vessel wall increases with its molecular weight (MW). The released TFPI originally is not associated with lipoproteins, but it is not known whether it remains free circulating in plasma. A further question is whether the MW of heparin influences not only the release of TFPI but also its potential association with lipoproteins. In the present study, the release of free TFPI was compared with the release of total TFPI after application of four heparins with different MWs. Only the TFPI released by unfractionated heparin (UFH) circulated completely as free TFPI. With decreasing heparin MW, the percentage of released free TFPI on released total TFPI decreased to 57%. As a consequence, the longer the heparin chains are, the better they are at preventing the binding of the released, originally free, TFPI to plasma lipoproteins. Because only free TFPI is known to exhibit anticoagulant activity, the activity of released TFPI is better the higher the MW of the applied heparin is. In conclusion, in addition to the potency of heparin to mobilize TFPI, there is its influence on the circulating form, and thus the anticoagulant activity of the released TFPI depends on its MW.     

Physiological function of tissue factor pathway inhibitor and interaction with heparins

Tissue factor pathway inhibitor (TFPI) is now recognized as a major physiological anticoagulant. Its main role is to modulate factor VIIa/tissue factor catalytic activity. Another important role is to potentiate the effect of heparins. TFPI is released from the vascular endothelium after injection of either unfractionated heparin (UFH) or low-molecular-weight heparins (LMWHs), which may then provide high concentrations of TFPI at sites of tissue damage and ongoing thrombosis. In dilute prothrombin-time-based assays, released TFPI contributes approximately one-third to the anticoagulant effect of heparin, the remaining being accounted for by antithrombin. Released TFPI, but not plasma TFPI, contains the basic carboxy-terminal tail which is important for the anticoagulant effect. UFH and LMWH exert differential effects on intravascular TFPI. UFH, but not LMWH, given in therapeutic doses, is associated with a progressive depletion of TFPI, which is associated with a strong rebound activation of coagulation after cessation of treatment. Such depletion may explain the apparent superior efficacy of LMWH observed in clinical trials.     

Probable regulation of factor VIIa–tissue factor and prothrombinase by factor Xa–TFPI and TFPI in vivo

Given that factor VIIa–tissue factor (TF) probably initiates coagulation in vivo , this study investigated the relationship between plasma concentrations of factor VIIa and prothrombin fragment 1+2 in plasma (the latter as an index of prothrombinase activity in vivo ). The relationships between these two parameters and the concentrations of tissue factor pathway inhibitor (TFPI) and factor Xa–TFPI in plasma were also investigated. TFPI inactivates factor Xa in a reaction accelerated by heparin, whereas factor Xa–TFPI inactivates factor VIIa–TF and prothrombinase. Established enzyme-linked immunosorbent assays (ELISAs) were used to quantify TFPI and prothrombin fragment 1+2, whereas we developed an ELISA to quantify factor Xa–TFPI using affinity purified rabbit (anti-human TFPI)-IgG and chicken anti-(human factor Xa–TFPI)-IgY as the capture and detector antibodies, respectively. Plasma factor VIIa was quantified using truncated tissue factor. The concentrations of factor VIIa and prothrombin fragment 1+2 increased in parallel in the plasmas of up to 145 healthy adults assayed ( P  = 0.007), as did the concentrations of factor VIIa and TFPI ( P  = 0.0039), and prothrombin fragment 1+2 and TFPI ( P  = 0.013). In contrast, there was an inverse relationship between the concentrations of free factor Xa–TFPI and factor VIIa ( P  <0.0001) and free factor Xa–TFPI and prothrombin fragment 1+2 ( P =0.0095). These results are consistent with factor Xa–TFPI regulating factor VIIa–tissue factor and prothrombinase in vivo .     

Vascular Endothelial Tissue Factor Pathway Inhibitor Kinetics in Culture Following Exposure to DX-9065a—A Selective and Direct Factor Xa Inhibitor

Background: Tissue factor (TF), a membrane-bound glycoprotein that initiates blood coagulation by allosteric activation of factor (f) VII, is regulated predominantly by tissue factor pathway inhibitor (TFPI). Because vascular endothelial cells synthesize and constitutively secrete TFPI and fXa may directly influence its cellular clearance, we sought to determine the effects of DX-9065a, a direct and selective fXa inhibitor, on TFPI kinetics in culture. Methods/Results: Human umbilical vein endothelial cells were grown to confluence and incubated with unfractionated heparin (1.0 U/mL), enoxaparin (1.5 U/mL), or DX-9065a at low (10 ng/ml), moderate (30 ng/ml), or high (90 ng/ml) concentrations. Compared to control, increases in TFPI were seen with both unfractionated heparin (182% higher, p < 0.001) and enoxaparin (194% higher, p < 0.001). Low concentration DX-9065a did not increase TFPI levels above control (0.8% higher, p = 0.91). In contrast, moderate and high concentrations produced 124% higher (p < 0.001) and 198% higher (p < 0.001) TFPI concentrations than control, respectively. Conclusions: DX-9065a increases vascular endothelial cell TFPI concentrations in human tissue culture. Although the mechanism has yet to be established, decreased fXa availability may limit fXa-TFPI complex formation and its subsequent cellular uptake. Whether increased surface TFPI contributes to the overall anticoagulant profile of DX-9065a will require further investigation.This revised version was published online in May 2005 with a corrected cover date.