Release Of Tissue Factor Pathway Inhibitor Research Articles

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.

Differentiating Low-Molecular-Weight Heparins Based on Chemical, Biological, and Pharmacologic Properties: Implications for the Development of Generic Versions of Low-Molecular-Weight Heparins

Low-molecular-weight heparins (LMWHs) are polypharmacologic drugs used to treat thrombotic and cardiovascular disorders. These drugs are manufactured using different chemical and enzymatic methods, resulting in products with distinct chemical and pharmacologic profiles. Generic LMWHs have been introduced in Asia and South America, and several generic suppliers are seeking regulatory approval in the United States and the European Union. For simple small-molecule drugs, generic drugs have the same chemical structure, potency, and bioavailability as the innovator drug. Applying this definition to complex biological products such as the LMWHs has proved difficult. One major issue is defining appropriate criteria to demonstrate bioequivalence; pharmacopoeial specifications alone appear to be inadequate. Whereas available generic versions of LMWHs exhibit similar molecular and pharmacopoeial profiles, marked differences in their biological and pharmacologic behavior have been noted. Preliminary studies have demonstrated differences in terms of anti-Xa activity and tissue factor pathway inhibitor release after subcutaneous administration, as well as antiplatelet and profibrinolytic effects. The current data emphasize the need to consider multiple functional parameters when defining bioequivalence of biologic drugs with complex structures and activities and also underscore the importance of further pharmacologic studies involving animal models and human clinical trials. The U.S. Food and Drug Administration and the European Medicine Evaluation Agency are currently developing guidelines for the acceptance of biosimilar agents including LMWHs. Until such guidelines are complete, generic interchange may not be feasible.

The Role of Tissue Factor Pathway Inhibitor in Tumor Growth and Metastasis

Clotting activation occurs frequently in cancer. Tissue factor (TF), the most potent initiator of coagulation, is expressed aberrantly in many types of malignancy and is involved not only in tumor-associated hypercoagulability but also in promoting tumor angiogenesis and metastasis via coagulation-dependent and coagulation-independent (signaling) mechanisms. Tissue factor pathway inhibitor (TFPI) is the natural inhibitor of TF coagulant and signaling activities. Studies have shown that TFPI exhibits antiangiogenic and antimetastatic effects in vitro and in vivo. In animal models of experimental metastasis, both circulating and tumor cell-associated TFPI are shown to significantly reduce tumor cell-induced coagulation activation and lung metastasis. Heparins and heparin derivatives, which induce the release of TFPI from the vascular endothelium, also exhibit antitumor effects, and TFPI may contribute significantly to those effects. Indeed, a non-anticoagulant low-molecular-weight heparin with intact TFPI-releasing capacity has been shown to have significant antimetastatic effect in a similar experimental mouse model. The evidence supporting the dual inhibitory functions on TF-driven coagulation and signaling strengthen the rationale for considering TFPI as a potential anticancer agent. This article primarily summarizes the evidence for antiangiogenic and antimetastatic effects of TFPI and describes its potential mechanisms of action. The possible application of TFPI and other inhibitors of TF as potential anticancer agents is described, and information regarding potential antitumor properties of TFPI-2 (which has structural similarities to TFPI) is also included.

Exercise modulates platelet-nasopharyngeal carcinoma cell aggregation and subsequent tissue factor and matrix metalloproteinase activities

Interaction between platelet and carcinoma cell contributes to pathogenesis of cancer-related thrombosis and metastasis. This study investigated whether physical exercise affects platelet-nasopharyngeal carcinoma cell (NPC) interaction and platelet-promoted tissue factor (TF) and matrix metalloproteinase (MMP) activities of NPC. Thirty sedentary men performed on three occasions moderate-intensity exercise [MIE, 60% maximal oxygen consumption (V(.)o(2max)) for 40 min] and high-intensity exercise (HIE, up to V(.)o(2max)), with and without warm-up exercise (WUE, 60% V(.)o(2max) for 20 min) on a bicycle ergometer. Before and immediately after exercise, platelet-NPC aggregation, the TF, MMP-2 and MMP-9 expressions and activities, and TF pathway inhibitor (TFPI) and tissue inhibitor of MMP-1 levels of NPC and platelet were measured. The results of this study demonstrated that HIE enhanced platelet-NPC aggregation in the presence of fibrinogen and was accompanied by increased platelet-promoted TF activity, expression of NPC, decreased platelet-promoted MMP-2 and MMP-9 activities, and TFPI release of NPC, whereas these alterations to HIE on platelet-NPC interactions were ameliorated by WUE pretreatment. Conversely, MIE reduced the formation of platelet-NPC aggregates, but did not change the TF, TFPI, MMP-2, MMP-9, tissue inhibitor of MMP activities, and/or levels of NPC mediated by platelet. It is concluded that HIE may enhance aggregation and coagulation and reduce MMP bioactivity related to platelet-NPC interactions, by raising the binding affinity to fibrinogen and TF activity and expression and lowering TFPI release and MMP-2 and -9 activities. These effects on HIE diminish after WUE. However, MIE minimizes the risk of thrombosis induced by platelet-NPC interactions.

Active tissue factor pathway inhibitor is expressed on the surface of coated platelets

AbstractThe incorporation of blood-borne forms of tissue factor (TF) into a growing blood clot is necessary for normal fibrin generation and stabilization of the blood clot. Tissue factor pathway inhibitor (TFPI) is the primary physiologic inhibitor of tissue factor and is present within platelets. Expression of TFPI on the platelet surface may be the optimal location for it to abrogate blood-borne TF activity that incorporates within the blood clot, balancing the need for adequate hemostasis while preventing development of occlusive thrombosis. TFPI is produced by megakaryocytes but is not expressed on the platelet surface. Activation of platelets with thrombin receptor activation peptide does not cause release or surface expression of TFPI, demonstrating that TFPI is not stored within platelet α granules. TFPI is expressed on the platelet surface following dual-agonist activation with convulxin plus thrombin to produce coated platelets. In association with its expression on the surface of coated platelets TFPI is also released in microvesicles or as a soluble protein.

Enoxaparin Use in Pregnancy: State of the Art

Low-molecular-weight heparin is the anticoagulant of choice in pregnancy. Enoxaparin has been increasingly used over the past 20 years in pregnant women at risk of thrombosis and pregnancy complications. The main indications are prophylaxis of venous thromboembolism and prevention of pregnancy loss in thrombophilic women. Other indications include treatment of venous thromboembolism, prophylaxis of arterial thrombosis in pregnant women with mechanical heart valves and prevention of late gestational complication such as pre-eclampsia and intrauterine growth restriction. Enoxaparin does not cross the placenta and is safe for the fetus. Maternal side effects are uncommon and include mild localized allergic reactions in 2% and increased bleeding in 2%, which is dose dependent. Heparin-induced thrombocytopenia is very rare and bone resorption is not clinically relevant. The mechanisms of action of enoxaparin in pregnancy are multiple and include anti-factor Xa (anti-Xa) activity in maternal circulation, tissue factor pathway inhibitor release from endothelial cells and trophoblasts at the placental level as well as anti-inflammatory effects.

Exercise Paradoxically Modulates Bioactivities of Tissue Factor and Matrix Metalloproteinase Induced by Platelets Interaction with Nasopharyngeal Carcinoma Cells

This investigation explores how different exercise regimens affect heterotypic aggregation, as well as expressions/activities of tissue factor (TF)/tissue factor pathway inhibitor (TFPI) and matrix metalloproteinase (MMP)-2 and -9/tissue inhibitor of metalloproteinase-1 (TIMP-1) induced by platelet-nasopharyngeal carcinoma cell (NPC) interactions. Thirty sedentary men performed on three occasions moderate-intensity exercise (MIE, 60% VO2max for 40 min) and high-intensity exercise (HIE, up to VO2max) with and without warm-up exercise (WUE, 60% VO2max for 20 min) on a bicycle ergometer. The results of this study demonstrated that HIE enhanced platelet-NPC aggregation and platelet-promoted TF activity/expression of NPC in the presence of fibrinogen, and was accompanied by an increase in TIMP-1 level and decreases in MMP-2 and -9 activities and TFPI content under conditions of platelet-NPC co-incubation, whereas these alterations to HIE on platelet-NPC interactions were ameliorated by WUE pretreatment. Conversely, MIE lowered the formation of platelet-NPC aggregates induced by fibrinogen, but did not change TF, TFPI, MMP-2 and -9, and TIMP-1 activities or/and expressions induced by platelet-NPC interactions. We conclude that HIE enhances aggregation and coagulation, and reduce matrix degradation related with platelet-NPC interactions, by raising the binding affinity to fibrinogen, TF expression/activity and TMIP release, and lowering TFPI release and MMP-2/-9 activities. These effects on HIE diminish after WUE. However, MIE minimizes the risk of thrombosis induced by platelet-NPC interactions.

Molecular weight dependent tissue factor pathway inhibitor release by heparin and heparin oligosaccharides

Heparin and low molecular weight heparins exert their vascular effects by mobilizing tissue factor pathway inhibitor (TFPI) from the vascular endothelium into the blood circulation. We compared the influence of molecular weight on the TFPI release by heparin and its fractions in a non-human primate model. Primates were treated with unfractionated heparin, a low molecular weight heparin (gammaparin), or a heparin-derived oligosaccharide mixture (C3). Endothelial TFPI release was determined using both immunologic and functional assays. After intravenous administration, all agents significantly increased TFPI levels ( p < 0.05) in a dose dependent manner. The increase produced by unfractionated heparin and gammaparin was greater than that by C3 at an equal dosage ( p < 0.05). With subcutaneous injection, all agents produced less TFPI release. Repeated administration of heparin-derived oligosaccharides gradually increased TFPI release. A 1.89 fold increase in TFPI levels was observed 4 days after C3 treatment (2.5 mg/kg). Our findings indicated that TFPI release is dependent on the molecular weight of heparin and its derivatives. Heparin oligosaccharides exert their vascular effects through increased TFPI release after long-term repeated administration.

Modulatory Effects of Escherichia coli Capsular–Derived Sulfaminoheparosans and Heparins on Tissue Factor–Mediated Activation of Platelets: Flow Cytometric Analysis

Sulfaminoheparosans (alternatively known as bioheparins) represent sulfated derivatives obtained from the K5 capsular polysaccharide of Escherichia coli. Previous studies have shown that these agents are structurally comparable to heparins and capable of exerting anticoagulant and antiprotease effects like heparins. Furthermore they are also able to release tissue factor pathway inhibitor (TFPI). Tissue factor (TF) plays a vital role in the pathogenesis of thrombotic and cardiovascular disorders. Anticoagulants such as heparins and bioheparins inhibit this thrombogenic mediator and thereby downregulate the activation of prothrombin and factor X. This study was carried out to determine the effects of several bioheparin fractions and heparins on TF-mediated platelet activation and their direct effect on platelets using human whole blood flow cytometry. Four different sulfaminoheparosan fractions with mean molecular weights of 20, 9, 7, and 6 kDa were tested for their inhibitory effects on platelet activation at two different concentrations (100 and 10 microg/mL). Unfractionated heparin and a low-molecular-weight heparin, tinzaparin, were also tested under the same experimental conditions for comparative modulatory responses. Fresh whole blood from healthy female and male volunteers (n = 5) was mixed with each of these agents and incubated with TF (diluted thromboplastin C) to activate platelets. Platelets were labeled with the antibodies CD61 FITC (GP IIIa) and CD62 PE (P-selectin). The data were analyzed in terms of percent platelet aggregation and platelet P-selectin expression. At 100 microg/mL, all of these agents strongly and significantly inhibited (approximately 40%) the platelet activation induced by TF in comparison to saline control. The inhibitory effects of each of these agents were slightly weaker (approximately 24% inhibition) at 10 microg/mL. The inhibitory effects of these agents on P-selectin expression correspond to their effects on platelet aggregation. At 100 microg/mL all the agents produced greater than 80% inhibition of P-selectin expression whereas at 10 microg/mL, the inhibition is greater than 70% except for bio-20 kDa, which produced less than 50% of inhibition. No molecular weight dependence was observed with bioheparin fractions in terms of inhibitory effects on platelet aggregation or P-selectin expression. None of the bioheparins and heparins exhibited any direct effects on platelets. These observations suggest that both the bioheparins and heparins are capable of inhibiting TF-mediated activation of platelets. Thus the therapeutic effects of bioheparins in the TF-mediated pathogenesis of platelet activation may be similar to those of heparins.

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.

Impact of native VLDL on tissue factor pathway inhibitor in endothelial cells and interactions between TFPI and lipoprotein lipase

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor (TF)-induced blood coagulation. A positive association between very low density lipoproteins (VLDLs) and TFPI has been reported in vivo. In contrast, one in vitro study indicates that TFPI may enhance lipoprotein lipase (LPL) activity, thereby increasing triglyceride hydrolysis. The current study was conducted to investigate how native VLDL influenced the synthesis and release of TFPI in endothelial cells, and how TFPI affected the LPL-induced hydrolysis of VLDL in vitro and at the endothelial surface. A spontaneously transformed immortal endothelial cell line (ECV304) and primary coronary artery cells (CoEc) were used, and VLDL was isolated from healthy volunteers by density gradient ultracentrifugation. Sequential free fatty acid (FFA) measurements were used to evaluate the kinetics of the LPL-induced hydrolysis. The levels of TFPI mRNAs in the stimulated cells were determined by quantitative real-time reverse transcription-ploymerase chain reaction (qPCR) using the ABI PRISM 7700 Sequence Detection System. Stimulation of ECV304 cells for 24 hours with native VLDL (0-100 microg/mL) caused a dose-dependent increase of TFPI in the medium (6.7-23.8 ng/10(6) cells, P < 0.001), without affecting the cellular content of TFPI. The expression of TFPI mRNA was significantly upregulated after 10 minutes of stimulation with n-VLDL. Both recombinant TFPI (r-TFPI) and LPL showed a dose-dependent binding to ECV 304 cells without saturation, and no competitive binding interactions between LPL and TFPI were observed at the endothelial surface. The addition of increasing concentrations of r-TFPI to ECV 304 cells, preincubated with LPL, did not affect the hydrolysis of VLDL triglycerides. The maximal reaction velocity (V(max)) of LPL-induced hydrolysis of n-VLDL was not affected by the addition of increasing concentrations of r-TFPI to the reaction mixture in vitro. The current experimental study indicates an upregulation of TFPI synthesis and release by VLDL. LPL-induced hydrolysis of VLDL in vitro was not influenced by TFPI neither in suspension nor at the endothelial surface.

BETAINE INDUCED RELEASE OF TISSUE FACTOR PATHWAY INHIBITOR AND NITRIC OXIDE: IMPLICATIONS IN THE MANAGEMENT OF CARDIOVASCULAR DISEASE

Betaine (trimethyglycine), isolated from sugar beet (Beta vulgaris) is a widely distributed natural compound and acts as a substrate in the betaine-homocysteine methyltransferase process converting homocysteine to methionine. Homocysteine is a recognized risk factor for occlusive vascular disease. Betaine is currently registered as an orphan drug to control homocystinuria (Cystadane®, Orphan Medical Inc.). Although Betaine is also known to produce certain effects on the vascular system, the mechanism of these effects are not known. When repeatedly administered orally to normal human volunteers (n=12) at 6 g/OD for one week, Betaine exhibited hypocoagulant responses as measured by thrombelastographic methods. Blood samples drawn included a baseline, one week post oral administration of Betaine administration. Analysis of the plasma samples did not show any significant anticoagulant effects on the global clotting assays such as the PT, APTT. However, tissue factor pathway inhibitor (TFPI) antigen and functional levels were increased as shown below. Nitric oxide (NO) levels were also measured in all of the samples by using a chromogenic method. Betaine administration resulted in an increase (20-90%). However, unlike the TFPI after the cessation of Betaine administration NO levels dropped to baseline level. These observations suggest that Betaine may have multiple mechanisms by which it facilitates vascular function. The release of TFPI and generation of NO during Betaine administration may be helpful in restoring vascular function.

Anti-metastatic effect of a non-anticoagulant low-molecular-weight heparin versus the standard low-molecular-weight heparin, enoxaparin

Low-molecular-weight heparins (LMWH) exhibit potent anticoagulant efficacy via their plasmatic effects on thrombin and factor Xa. These agents are also effective in releasing endothelial tissue factor pathway inhibitor (TFPI), the natural inhibitor of tissue factor, and exhibit significant anti-metastatic effects in experimental animal models. However, the potential for bleeding complications has slowed down the more widespread adoption of LMWH therapy in cancer patients. In this study, the effect of a non-anticoagulant form of LMWH (NA-LMWH) on experimental lung metastasis and tumor cell-induced platelet aggregation in vivo was compared to the LMWH enoxaparin. Using the B16 melanoma mouse model of metastasis, subcutaneous (s.c.) injection of NA-LMWH or enoxaparin (10 mg/kg), three hours before intravenous (i.v.) injection of metastatic melanoma cells, followed by daily doses for 14 days, reduced lung tumor formation by 70% (P < 0.001). I.v. injection of tumor cells resulted in a significant (50-62%, P < 0.01) fall in platelet counts. Pre-injection (i.v.) of enoxaparin completely abolished the tumor cell-induced thrombocytopenia, whereas NA-LMWH had no effect. Four hours after a single s.c. dose, enoxaparin but not NA-LMWH prolonged the clotting time three-fold and delayed the time to clot initiation more than 10-fold as measured by a Sonoclot analyzer and by thromboelastography, respectively. Enoxaparin but not NA-LMWH demonstrated a significant anticoagulant effect in mice. Both NA-LMWH and enoxaparin caused similar TFPI release from endothelial cells in vitro. These data provide evidence to support the potential of NA-LMWH as an anti-metastatic agent without any significant impact on coagulation.

Co-Interaction and Increased Release of Tissue Factor Pathway Inhibitor by Heparanase.

Background and objectives. Tissue factor pathway inhibitor (TFPI) is a potent direct inhibitor of factor Xa and factor VIIa-tissue factor complex. In addition, TFPI was shown to be an inhibitor of angiogenesis and metastasis. Heparanase is an endo-beta-D-glucuronidase of 65 kDa that cleaves heparan sulfate chains on cell surfaces and in the extra-cellular matrix an activity that closely correlates with cell invasion, angiogenesis and tumor growth. The study hypothesis was that heparanase may reduce the level of TFPI or release it from the cell surface in an attempt to increase heparanase prometastatic potential.Material and methods. The effect of exogenous heparanase on TFPI expression and release to the medium was studied in HUVEC by immunoblotting, real time RT-PCR, and flow-cytometry. Human cell lines (MDA-MB-435 breast carcinoma; U87 glioma; HEK-293 embryonic kidney) were transfected to over express heparanase and the effect on TFPI was studied. TFPI expression was explored in heparanase transgenic mice by immunoblotting and immunostaining. Transfections with various modified forms of heparanase were used to further explore the effect of heparanase. Interaction between TFPI and heparanase was studied by co-immunoprecipitation analysis.Results. Heparanase was found to increase the release of TFPI to the medium, reduce the level of TFPI at the cell surface, and to up-regulate its expression in the cells. These results were verified in HUVEC, tumor cell lines, and in the animal model. The effect was independent of heparanase activity or interaction with heparan sulfate, and dependent on heparanase secretion. A protein co-interaction between TFPI and heparanase was found.Conclusions. Overall, a cell surface interaction is suggested in which heparanase impose increased release of TFPI from the cell surface to the medium, providing a local procoagulant and a systemic anticoagulant environment.

Non-anti-coagulant heparins: A promising approach for prevention of tumor metastasis (Review)

The metastatic spread of solid tumors is responsible for most cancer-related deaths, and unfortunately no specific anti-metastatic therapy exists. Heparin is widely used in the clinic today as an anti-coagulant therapy for cancer patients at risk for venous thrombo-embolism. Recent clinical trials with low molecular weight heparin and meta-analyses of earlier clinical trials with unfractionated heparin indicate that heparin has an anti-metastatic activity. Animal studies using non-anti-coagulant heparin (NAC heparin) suggest that it is possible to separate the anti-metastatic and anti-coagulant activities of heparin. The use of heparin as an anti-metastatic agent is limited due to its potent anti-coagulant activity. NAC heparins have clinical potential because they could be administered at a higher dose, thereby fully exploiting the anti-metastatic component of heparin activity, and because they could be used with cancer patients with bleeding complications, where the use of heparin is currently precluded. The mechanism by which NAC heparin inhibits metastasis is not yet fully understood. Possible mechanisms include the inhibition of selectin-mediated cell-cell interactions, heparanase and angiogenesis, and stimulation of tissue factor pathway inhibitor release. The promising results from clinical trials and animal studies clearly warrant further investigation of NAC heparin as a therapeutic for the prevention of tumor metastasis.

Comparative Tissue Factor Pathway Inhibitor Release Potential of Heparins

Tissue factor pathway inhibitor (TFPI) is released following the administration of unfractionated heparin, low-molecular-weight heparins, defibrotide and PI-88. In this study, the comparative effects of heparin, a low-molecular-weight heparin-gammaparin and a heparin-derived oligosaccharide mixture-subeparin (C3) were studied on functional and immunologic tissue factor pathway inhibitor activity levels in a non-human primate (Macaca mulatta) model. The dose-dependent effect was studied following intravenous and subcutaneous administration. Following the administration of 1 mg/kg of heparin, gammaparin, and C3, the functional levels of TFPI at 5 minutes were 2.40, 2.56, and 1.08 U/mL and the corresponding TFPI immunologic levels were 4.3-, 4.0-, and 2.1-fold, increased, respectively, over the baseline value. From these results, it can be concluded that heparin and gammaparin produced similar levels of TFPI release. Hence, gammaparin and heparin have similar TFPI release potential despite their differences in molecular weight. The influence of molecular weight, charge density, and interactions with heparin cofactor II on TFPI release are also discussed.

Low-Molecular-Weight Heparin in Thrombosis and Cancer

Venous thromboembolism (VTE) is a recognized complication of malignant disease and multiple risk factors contribute to the hypercoagulability that commonly accompanies malignancy. Thromboprophylaxis with antithrombotic drugs such as the low-molecular-weight heparins (LMWHs) can be used to control the hypercoagulable state and to reduce the incidence of VTE in patients with cancer. Clinical and biochemical data suggest that LMWHs may also inhibit tumor growth, leading to a survival benefit in these patients. 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 in cancer patients with thromboembolic disorders have suggested a clinically relevant effect of LMWHs (as compared with UFH) on the survival of cancer patients with deep vein thrombosis. Similarly, the impact of warfarin on the survival of cancer patients with thromboembolic disorders was demonstrated in certain tumor types. Studies from our laboratory demonstrated a significant role for LMWH, warfarin, anti-VIIa, and LMWH releasable tissue factor pathway inhibitor on the regulation of angiogenesis, tumor growth, and tumor metastasis. However, a direct anticancer effect for heparin in cancer patients without thrombosis still remains to be clinically documented.

Inhibition of endothelial cell tube formation by the low molecular weight heparin, tinzaparin, is mediated by tissue factor pathway inhibitor.

Heparin and low molecular weight heparins (LMWHs) have both antithrombotic and anti-angiogenic activities. The anti-angiogenic activity of LMWH may be associated with the release of endothelial tissue factor pathway inhibitor (TFPI), an important endogenous inhibitor of tissue factor/Factor VIIa (TF/fVIIa). To evaluate the effects of LMWH, tinzaparin, and TFPI in a model of angiogenesis-mediated processes, we compared the effects of tinzaparin, and recombinant TFPI in inhibiting either basic fibroblast growth factor-2 (FGF2)- or TF/fVIIa-induced endothelial cell tube formation in human umbilical vein endothelial cells (HUVEC). Our results show that tinzaparin and recombinant TFPI both blocked endothelial tube formation induced by either FGF2 or TF/fVIIa, in a concentration-dependent manner. Endothelial tube formation was only marginally inhibited by a potent and specific anti-Factor Xa, recombinant tick anticoagulant protein (rTAP). A monoclonal anti-TFPI anti-body reversed the inhibitory effects of either tinzaparin or recombinant-TFPI on HUVEC tube formation. Tinzaparin fractions in the range of 8,000 to 12,600 Da were most effective in stimulating the release of TFPI from HUVEC. These results suggest that the inhibitory effect of the LMWH tinzaparin on endothelial tube formation is associated with stimulation of the release of TFPI, but not to anti-Factor Xa activity.

Unfractionated and Low-Molecular-Weight Heparins, Basic Mechanism of Action and Pharmacology

Unfractionated heparin has enjoyed sole anticoagulant status for nearly 50 years. Despite a dramatic growth in the development and the introduction of many newer anticoagulant and antithrombotic drugs and polytherapeutic approaches during the past decade, unfractionated and low-molecular-weight heparins remain the drugs of choice for many indications, including surgical anticoagulation, interventional cardiology, and in several additional considerations. Unfractionated heparin has a major role in the areas of vascular medicine and surgery, and it is the only parenteral anticoagulant drug that can be empirically neutralized by such agents as protamine sulfate. The development of low and ultra low-molecularweight heparins, which are a class of depolymerized heparin derivatives with distinct pharmacologic profiles that are largely determined by their composition, represents a refinement for the use of heparin. These drugs produce their major effects by combining with antithrombin Ill and exerting antithrombin and anti-Xa inhibition. The low-molecular-weight heparins also increase non-antithrombin III-dependent effects, such as tissue factor pathway inhibitor release, modulation of adhesion molecules, and the release of profibrinolytic and antithrombotic mediators from the blood vessels. Each of the low-molecular-weight heparins has different cumulative effects, and each product exhibits a distinct profile. Initially developed for the prophylaxis of postsurgical deep vein thrombosis, these drugs are now also used for the treatment of both venous and arterial thrombotic disorders. To a large extent, the low-molecular-weight heparins have replaced unfractionated heparin in most of the subcutaneous indications. This has resulted in a dramatic evolution in anticoagulant management that allows patients with thrombotic disorders to be treated in an outpatient setting. Thus, the introduction of low-molecular-weight heparins represents a major advance in improving the use of heparin. Generic versions of these drugs are likely to be developed as their patents expire. Currently, there are no clear guidelines for the acceptance of the generic versions of branded products. To avoid safety and efficacy-related problems, a generic drug must meet both the chemical and biologic equivalence criterion. Synthetically and biosynthetically derived agents such, as pentasaccharide, will also be introduced for clinical use; however, these drugs will have a narrower therapeutic spectrum due to their monotherapeutic nature. Heparin and its derivatives will continue to have a crucial role in the management of thrombotic and cardiovascular disorders in years to come.

Pharmacodynamic Properties of the Low Molecular Weight Heparin, Tinzaparin: Effect of Molecular Weight Distribution on Plasma Tissue Factor Pathway Inhibitor in Healthy Human Subjects

Low molecular weight heparins (LMWHs) stimulate the release of endothelial tissue factor pathway inhibitor (TFPI). An inhibitor of tissue factor-induced coagulation, TFPI may contribute to the efficacy of LMWHs. The release of TFPI may be affected by characteristics of LMWHs, such as molecular weight (MW) distribution. The authors measured plasma levels of TFPI after single, subcutaneous injections of tinzaparin (mean MW = 6500 Daltons [Da]; 3.5% of fractions < 2000 Da) or a tinzaparin-like LMWH (mean MW = 5650 Da; 18.3% of fractions < 2000 Da) in 30 healthy subjects. Maximum mean plasma levels of TFPI approached 200 to 230 ng/mL 0.8 hours after administration of either drug. Plasma levels of TFPI remained elevated compared with baseline values for 12 to 16 hours after drug administration. These results suggest that LMWH fractions < 2000 Da do not affect the release of TFPI.