Release Of Tissue Factor Pathway Inhibitor Research Articles

Targeting cancer-derived extracellular vesicles by combining CD147 inhibition with tissue factor pathway inhibitor for the management of urothelial cancer cells

BackgroundExtracellular vesicles (EVs), including microvesicles, hold promise for the management of bladder urothelial carcinoma (BLCA), particularly because of their utility in identifying therapeutic targets and their diagnostic potential using easily accessible urine samples. Among the transmembrane glycoproteins highly enriched in cancer-derived EVs, tissue factor (TF) and CD147 have been implicated in promoting tumor progression. In this in vitro study, we explored a novel approach to impede cancer cell migration and metastasis by simultaneously targeting these molecules on urothelial cancer-derived EVs.MethodsCell culture supernatants from invasive and non-invasive bladder cancer cell lines and urine samples from patients with BLCA were collected. Large, microvesicle-like EVs were isolated using sequential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and flow cytometry. The impact of urinary or cell supernatant-derived EVs on cellular phenotypes was evaluated using cell-based assays following combined treatment with a specific CD147 inhibitor alone or in combination with a tissue factor pathway inhibitor (TFPI), an endogenous anticoagulant protein that can be released by low-molecular-weight heparins.ResultsWe observed that EVs obtained from the urine samples of patients with muscle-invasive BLCA and from the aggressive bladder cancer cell line J82 exhibited higher TF activity and CD147 expression levels than did their non-invasive counterparts. The shedding of GFP-tagged CD147 into isolated vesicles demonstrated that the vesicles originated from plasma cell membranes. EVs originating from invasive cancer cells were found to trigger migration, secretion of matrix metalloproteinases (MMPs), and invasion. The same induction of MMP activity was replicated using EVs obtained from urine samples of patients with invasive BLCA. EVs derived from cancer cell clones overexpressing TF and CD147 were produced in higher quantities and exhibited a higher invasive potential than those from control cancer cells. TFPI interfered with the effect when used in conjunction with the CD147 inhibitor, further suppressing homotypic EV-induced migration, MMP production, and invasion.ConclusionsOur findings suggest that combining a CD147 inhibitor with low molecular weight heparins to induce TFPI release may be a promising therapeutic approach for urothelial cancer management. This combination can potentially suppress the tumor-promoting actions of cancer-derived microvesicle-like EVs, including collective matrix invasion.Graphical

Quantitative TFPI Antigen Release and Functionality After Intravenous Administration of Heparins Sourced From Various Species in Non‐human Primates

IntroductionTissue factor pathway inhibitor (TFPI) is a multi‐domain Kunitz‐type inhibitor that plays an important role in the regulation of the tissue factor pathway through its release from endothelial sites upon heparin administration. Currently, heparin is primarily sourced from porcine tissue, however, recent advancements have allowed for it to be re‐sources from bovine tissue. In this context, we elected to compare the relative abilities of porcine, bovine, and ovine heparins to release endogenous TFPI following intravenous administrations of gravimetric and USP potency‐adjusted heparin dosages.MaterialsActive pharmaceutical ingredient (API) forms of bovine, ovine, and porcine heparins of mucosal origin were obtained from Kinmaster, Brazil, Ronssi, China, and Medifill, USA, respectively. The active pharmaceutical ingredients were dissolved to obtain 10mg/mL solutions of each type. Individual groups of non‐human primates (n=6) were intravenously injected with 0.5mg/kg and 100U/kg dosages of each heparin type. Blood samples were subsequently drawn at 0, 15, 30, 60, and 120 minutes post‐administration. Citrated blood samples were centrifuged to obtain platelet‐poor plasma and stored at ‐ 80C until analysis. A sandwich ELISA method for TFPI antigen and a chromogenic substrate‐based functional method were used to quantify TFPI antigen release and functionality, respectively. All results were calculated in terms of group means ±SD. Applicable statistical methods were used to compare the heparins for their relative effects on TFPI release.ResultsGravimetric dosages of ovine and porcine heparins produced comparable TFPI release in both the functional and immunologic assays while bovine heparin demonstrated slightly lower peak TFPI antigen release and slightly higher peak function, as shown in Figure 1a and 1b, respectively. When compared following administration of USP‐adjusted 100U/kg dosages, all three heparins consistently demonstrated essentially identical TFPI antigen release and functional profiles, as shown in Figure 1c and 1d, respectively. Overall, any potential differences in the gravimetric dosing regimens were adequately corrected following USP potency adjustment in both TFPI antigen release and functionality, as seen in Figure 1a and 1b and Figure 1c and 1d, respectively. Furthermore, all three heparins demonstrated comparable pharmacokinetic profiles in both TFPI antigen release and function levels, as shown in Table 1.ConclusionThese results demonstrate that heparins from various sources are capable of comparatively releasing TFPI antigen. While the gravimetrically‐dosed studies demonstrated less consistent trajectories, potency‐based adjustments of the heparin dosages allowed for improved pharmacokinetic homogeneity. These studies underscore the potential for the development of alternative heparin sources as viable, pharmacokinetic equivalents.

Sulfated non-anticoagulant low molecular weight heparin in the prevention of cancer and non-cancer associated thrombosis without compromising hemostasis

IntroductionCancer-associated thrombosis (CAT) accounts for about 20% of all cases of Venous Thromboembolism (VTE). Tissue factor (TF) is documented to be highly expressed on cancer cells and pathological angiogenic endothelial cells. Here, we used a novel oxidized sulfated ultra-LMWH, S-NACH, which is devoid of anti-factor Xa and IIa activities with limited to no systemic anticoagulant effects. This sulfated form has enhanced binding to vascular endothelial cells (EC) and releases and potentiates the action of tissue factor pathway inhibitor (TFPI). S-NACH binds with high affinity to EC, releases and binds to EC TFPI, and promotes vascular antithrombotic effect with limited to no risk of bleeding complications. Materials and methodsWe investigated the effects of S-NACH on clot kinetics in vitro and in vivo. Also, we investigated the effects of S-NACH on CAT mediated by human acute leukemia cells (K562) and human pancreatic cancer cells (SUIT2). ResultsS-NACH was associated with ~3-fold increase of TFPI 2 levels within 3 h. Also, S-NACH reversed the hypercoagulability state that is associated with cancer cells in vitro. In vivo, S-NACH at 20 mg/kg subcutaneously (SC) had no effect on bleeding time compared to both tinzaparin and enoxaparin at 5 mg/kg SC. S-NACH did not show any anti-IIa or anti-Xa activities in comparison to tinzaparin and enoxaparin (p < 0.001). ConclusionData suggest the importance of S-NACH through its EC binding, EC TFPI release and its interaction with TFPI in enhancing its activity in the prevention of cancer and non-cancer associated thrombosis with limited to no bleeding complications.

Tissue Factor Pathway Inhibitor (TFPI) release by bovine, ovine and porcine unfractionated heparins in primates. Comparative studies at mass and potency adjusted intravenous dosing

BackgroundUnfractionated heparin (UFH) is a highly sulfated glycosaminoglycan (GAG) that consists of repeating disaccharide units, containing iduronic acid (or glucuronic acid) and glucosamine, exhibiting variable degrees of sulfation. UFHs release tissue factor pathway inhibitor (TFPI) which inhibits extrinsic pathway of coagulation by rapidly inactivating of Factor Xa and the Factor VIIa/TF complex. Bovine mucosal heparins (BMH) and ovine mucosal heparin (OMH) are currently being developed for re‐introduction for both medical and surgical indications. In contrast to the porcine mucosal heparins (PMH), the active pharmaceutical ingredient (API) BMH exhibits a somewhat weaker anti‐coagulant activity as cross‐referenced against PMH. In this study, we determined the TFPI antigen release level by various dosages of UFHs using ELISA method. Also, we projected that potency adjusted BMH at equivalent dosage has the same TFPI release profile compared to OMH and PMH.Materials &amp; MethodsBMH, OMH and PMH were obtained from various commercial sources. Potencies of each heparin were determined by an amidolytic anti‐Xa assay in relation to the USP heparin standard. Individual groups of primates (n=4) were administered 0.5 mg/kg or (50 or 100) U/kg dosage of bovine, ovine or porcine UFH via intravenous route. Blood samples were collected at baseline, 15, 30, 60 and 120 minutes post‐administration. Tissue Factor Pathway Inhibitor (TFPI) antigen levels were measured using a commercially available sandwich ELISA methods. All results were compiled as mean ± SD.ResultsAt a gravimetric level (0.5 mg/kg), BMH exhibited a lower TFPI level than OMH &amp; PMH at all time points with a maximum value of (200 ng/ml) at 15 mins post drug administration. OMH and PMH showed comparable TFPI levels at all time points with maximum values of (250 ng/ml) and (265 ng/ml) respectively at 15 mins post drug administration. However, USP cross‐referenced anti‐Xa potency adjusted based dosing showed comparable TFPI antigen levels for all three UFHs at all time points with a maximum value of 250 ng/ml at 15 mins post drug administration. At 60 mins post drug administration, potency adjusted BMH showed slightly higher TFPI level of (200 ng/ml) compared to both OMH &amp; PMH.ConclusionTissue factor pathway inhibitor (TFPI) levels at 15 minutes after IV administration of 0.5 mg/kg heparins to non‐human primates showed that both PMH and OMH release comparable TFPI antigen levels which were significantly higher than BMH. Potency equated dosing resulted in comparable TFPI release profiles for all three heparins. Therefore such dosing may provide uniform levels of anticoagulation for the parenteral indications for UFHs. These observations suggest that potency equated bovine heparin has comparable release of TFPI and therefore is biosimilar to the OMH and PMH.

Studies on Tissue Factor Pathway Inhibitor Antigen Release by Bovine, Ovine and Porcine Heparins Following Intravenous Administration to Non-Human Primates.

Unfractionated heparin (UFH) is a sulfated glycosaminoglycan that consists of repeating disaccharides, containing iduronic acid (or glucuronic acid) and glucosamine, exhibiting variable degrees of sulfation. UFHs release tissue factor pathway inhibitor (TFPI) which inhibits the extrinsic pathway of coagulation by inactivating factor Xa and the factor VIIa/TF complex. Most heparins used clinically are derived from porcine intestinal mucosa however, heparins can also be derived from tissues of bovine and ovine origin. Currently there are some concerns about the shortage of the porcine heparins as they are widely used in the manufacturing of the low molecular weight heparins (LMWHs). Moreover, due to cultural and religious reasons in some countries, alternative sources of heparins are needed. Bovine mucosal heparins (BMH) are currently being developed for re-introduction to the US market for both medical and surgical indications. Compared to porcine mucosal heparin (PMH), BMH exhibits a somewhat weaker anti-coagulant activity. In this study, we determined the TFPI antigen level following administration of various dosages of UFHs from different origins. These studies demonstrated that IV administration of equigravemetric dosages of PMH and ovine mucosal heparin (OMH) to non-human primates resulted in comparable TFPI antigen release from endothelial cells. In addition, the levels of TFPI were significantly higher than TFPI antigen levels observed after BMH administration. Potency adjusted dosing resulted in comparable TFPI release profiles for all 3 heparins. Therefore, such dosing may provide uniform levels of anticoagulation for the parenteral indications for UFHs. These observations warrant further clinical validation in specific indications.

The clinical use of Fondaparinux: A synthetic heparin pentasaccharide.

Fondaparinux is a synthetic heparin pentasaccharide with a sequence identical to that found in anticoagulant heparin. It is a pure compound with a molecular weight of 1728Da. Fondaparinux catalyzes the conformational change of a serpin or serine protease inhibitor antithrombin III to accelerate the suicidal inactivation of factor Xa over 340-fold, which in turn inhibits thrombin generation in the coagulating signal transduction pathway. Fondaparinux does not inhibit thrombin activity, release tissue factor pathway inhibitor, or possess other properties of heparin such as anti-inflammatory, anti-viral, anti-angiogenesis, anti-neoplastic, and anti-metastatic effects though high affinity interactions with a variety of proteases, protease inhibitors, chemokines, cytokines, growth factors, and their respective receptors. Low antithrombin III levels in blood circulation also affects the efficacy of Fondaparinux. Thus, Fondaparinux represents a refined use of the anti-factor Xa property of heparin. As an anti-factor Xa drug, Fondaparinux has complete bioavailability subcutaneously, instant onset of action, a half-life of 15-20h, and a direct renal excretion without any metabolism. Fondaparinux has been shown to be superior to low molecular weight heparin in preventing deep vein thrombosis. Clinically, Fondaparinux is used for the prevention of deep vein thrombosis in patients who have had orthopedic surgery as well as for the treatment of deep vein thrombosis and pulmonary embolism with limitations of use in elderly, low weight, renal impaired patients and in those receiving spinal anesthesia. Clinical studies showed that Fondaparinux acts in prevention and treatment of venous thromboembolism and in ischemic heart disease without significant risk of bleeding.

Extracellular vesicles from malignant effusions induce tumor cell migration: inhibitory effect of LMWH tinzaparin.

Elevated levels of extracellular vesicles (EVs) have been correlated with inflammatory diseases as well as progressive and metastatic cancer. By presenting tissue factor (TF) on their membrane surface, cellular microparticles (MPs) activate both the coagulation system and cell-signaling pathways such as the PAR/ERK pathway. We have shown before that malignant effusions are a rich source of tumor cell-derived EVs. Here, we used EVs from malignant effusions from three different patients after serial low-speed centrifugation steps as recommended by the ISTH (lsEV). Significant migration of human pancreatic carcinoma cells could be induced by lsEVs and was effectively inhibited by pre-incubation with tinzaparin, a low-molecular-weight heparin. Tinzaparin induced tissue factor pathway inhibitor (TFPI) release from tumor cells, and recombinant TFPI inhibited EV-induced tumor cell migration. EVs also induced ERK phosphorylation, whereas inhibitors of PAR2 and ERK suppressed EV-induced tumor cell migration. LsEVs have been characterized by high-resolution flow cytometry and, after elimination of smaller vesicles including exosomes, by further high-speed centrifugation (hsEV). The remaining population consisting primarily of MPs is indeed the main migration-inducing population with tenase activity. Compared to other LMWHs, tinzaparin is suggested to have high potency to induce TFPI release from epithelial cells. The migration-inhibitory effect of TFPI and the interruption of tumor cell migration by inhibitors of PAR2 and ERK suggest that lsEVs induce tumor cell migration by activating the PAR2 signaling pathway. Tinzaparin might inhibit this process at least partly by inducing the release of TFPI from tumor cells, which blocks PAR-activating TF complexes. The clinical relevance of the results is discussed.

The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin.

Objective:Sepsis syndrome is usually accompanied by activation of blood coagulation mechanisms. Earlier studies found deficiencies of the 3 main natural anticoagulants, antithrombin, protein C, and protein S. However, none of these inhibitors block tissue factor, the prime trigger of coagulation during sepsis that is controlled specifically by the tissue factor pathway inhibitor (TFPI). The aim of this study was to characterize the fluctuations in the levels of natural anticoagulants, particularly TFPI, in the course of sepsis and to find out their association with the anticoagulant action of the low-molecular-weight heparin enoxaparin.Materials and Methods:We studied 51 consecutive patients with sepsis. Blood samples were collected from patients at baseline (0 h) and at 4, 12, and 24 h after enoxaparin administration. The following assays were undertaken using commercial kits: activated partial thromboplastin time, prothrombin time, thrombin time, total and free TFPI, protein C and protein S, antithrombin, fibrinogen, and anti-factor Xa.Results:Before enoxaparin administration, there was significant prolongation of the prothrombin time and activated partial thromboplastin time, and this remained the case in the 3 subsequent samples. There was marked reduction in the levels of antithrombin, protein C, and total and free protein S to below control values throughout the study. In contrast, plasma levels of both total and free TFPI were markedly elevated and increased after enoxaparin therapy. Anti-factor Xa levels were within the therapeutic range throughout. There was no difference in TFPI levels between those patients who died and those who survived.Conclusion:Sepsis triggered marked release of TFPI from endothelial cells. This persisted and was increased further following the administration of enoxaparin. In contrast, there was marked consumption of the natural coagulation inhibitors antithrombin, protein C, and protein S. These results go some way towards explaining why the therapeutic use of recombinant TFPI fails to correct sepsis-associated coagulopathy.

Tissue factor and tissue factor pathway inhibitor in nasal mucosa and nasal secretions of chronic rhinosinusitis with nasal polyp.

Activation of the coagulation system with an increase in thrombin generation is involved in the pathogenesis of tissue remodeling in chronic rhinosinusitis (CRS). Tissue factor (TF) is an important protein for initiation of the extrinsic coagulation pathway, and TF pathway inhibitor (TFPI) is a regulator of TF-induced coagulation. This study was conducted to elucidate the roles of TF and TFPI in the pathogenesis of CRS. Tissue localization of TF, TFPI, and fibrin was determined by immunostaining of nasal polyps and inferior turbinates obtained during endonasal surgery in patients with CRS with nasal polyp (CRSwNP). Nasal secretions were collected from patients with CRSwNP, allergic rhinitis, and from control patients. The concentrations of TF and TFPI were measured in nasal secretions from each group. The concentrations of TF and TFPI released into culture medium by normal human nasal epithelial cells treated with thrombin, protease-activated receptor 1 agonist peptide, or tumor necrosis factor α were also measured. TF expression was localized in nasal epithelial cells and in infiltrating eosinophils of nasal mucosa. TFPI expression was localized in nasal epithelial cells, and fibrin deposition was observed in nasal secretions and the lamina propria of nasal polyps. Nasal secretions contained significant concentrations of TF and TFPI. The concentration of TFPI in nasal secretions correlated positively with thrombin activity and the concentration of thrombin-antithrombin III complex. Treatment with thrombin, protease-activated receptor 1 agonist peptide, or tumor necrosis factor α stimulated significant release of TF and TFPI from cultured nasal epithelial cells. By upregulating the coagulation system, TF and TFPI play an important role in the pathogenesis of CRSwNP.

Comparative Studies on Branded Enoxaparin and a US Generic Version of Enoxaparin

Enoxaparin, a complex, biologically derived low-molecular-weight heparin, is approved for a range of clinical indications. This study was carried out to compare the potency profile and pharmacodynamic responses of branded enoxaparin (Lovenox; Sanofi, US) with a generic enoxaparin (enoxaparin sodium injection, USP). Five batches of each product were tested. Although the average molecular weight, anti-factor Xa, and anti-factor IIa potencies were similar for the two products, differences were observed in the in vitro thrombin generation and kinetics of clot formation (P = .01) and in the ex vivo pharmacodynamics regarding thrombin generation inhibition (P = .029), tissue factor pathway inhibitor release (P = .006), and inhibition of the active form of thrombin-activated fibrinolysis inhibitor (P = .023). These findings suggest that simple analytical characterization can establish good quality control in manufacturing, but they may not assure similarity in biological performance between the branded and the generic enoxaparin.

The effect of tissue factor pathway inhibitor release and interactions with growth factors on the antitumor effects of ultra low molecular weight heparin semuloparin.

e13117 Background: Heparins are known to produce anti-tumor effects beside other pharmacologic actions. More recently an ultra LMWH has been developed for the prevention of thrombosis in cancer patients. Initial studies demonstrated that this agent also exhibits anti-tumor effects in animal models. To understand the mechanism of anti-tumor effects of this agent studies were designed to fractionate this ULMWH in to various AT affinity fractions. Methods: An ULMWH (semuloparin, Sanofi-aventis) and its high (HAF) and low (LAF) affinity fractions were prepare by AT affinity chromatography. These agents were administered at 1 mg/kg to primates via intravenous route. Blood samples were collected for a period of up to 6 hours. TFPI release and anti-Xa activities were measured. The interactions of ULMWH and its fractions with various growth factors such as FGF, VEGF, EGF and beta TGF were measured using a mass spectral method employing SELDI technique. The anti-tumor effects of each of these agents were measured using the mouse model of Lewis lung carcinoma. Results: In comparison to ULMWH the LAF showed minimal anti Xa activity whereas HAF exhibited a higher activity. All agents exhibited a similar release of the TFPI; however, HAF exhibited slightly higher release. All agents also showed varying degrees of binding to growth factors as measured by peak intensities. All agents produce anti-tumor effects in the mouse model resulting in a significant reduction in tumor size. Conclusions: Semuloparin has been clinically proven to prevent venous thromboembolism in patients with cancer (SAVE ONCO). These effects are attributable to the inhibition of factor Xa and the generation of thrombin. The fact that LAF mediates its anti-tumor effects without producing anti Xa effects suggest that these may be related to the release of TFPI. Furthermore the ULMWH, HAF and LAF all interact with growth factors suggest that these interactions may also contribute to anti-tumor effects of semuloparin, therefore, beside antithrombotic effects this agent also exhibits oncostatic effects.

Melatonin stimulates release of tissue factor pathway inhibitor from the vascular endothelium

We previously found an association between the circadian variation of free tissue factor pathway inhibitor (TFPI) and melatonin in able-bodied males and in men with complete cervical spinal cord injuries. We therefore examined whether melatonin modifies production and/or secretion of TFPI in endothelial cells. We sampled supernatants from cultures of primary human umbilical vein endothelial cells (HUVECs) and of human coronary artery endothelial cells (HCAECs), that had been exposed to varying doses (0-300 pg/ml) of melatonin for 0.5-24 h. We then measured the protein concentrations of free TFPI, tissue factor and plasminogen activator inhibitor type 1 (PAI-1). We also measured endothelial TFPI, tissue factor and PAI-1 transcripts using quantitative real-time PCR. Melatonin dose dependently increased free TFPI levels about 25-30-fold in supernatants of both HUVEC and HCAEC, and independent of incubation duration. In contrast, TF and PAI-1 remained unaltered upon increasing doses of melatonin. Neither TFPI mRNAs nor tissue factor mRNAs nor PAI-1-mRNAs were changed in cell cultures added melatonin. The ratio of free TFPI in cell supernatants to free TFPI in cell lysates about doubled upon addition of melatonin, indicating that melatonin increased release from intracellular storages of free TFPI or from membrane-bound free TFPI. Our data indicate that melatonin stimulates vascular endothelial cells to secrete TFPI without altering transcription of the TFPI gene. If melatonin increases TFPI release in a similar fashion in vivo as in vitro, this could have potential clinical implications in both prophylaxis and treatment of thromboembolic events.

Involvement of heparanase in vaginal and cesarean section deliveries

Background Heparanase is an endo-β-D-glucuronidase dominantly involved in cell invasion, tumor angiogenesis and metastasis. Recently, we demonstrated increased levels of heparanase, tissue factor pathway inhibitor (TFPI)-2 and vascular endothelial growth factor (VEGF)-A in early miscarriages (Nadir et al., Thromb Res, 2010). Herein, we investigated the role of heparanase in third trimester placentas, in correlation to tissue factor (TF), TFPI, TFPI-2, and VEGF-A. Methods Twenty five third-trimester placenta samples (weeks 36-41) were studied applying real time RTPCR and immunostaining. Ten cases were placentas of elective cesarean sections, 8 cases were of normal vaginal deliveries and 7 samples were placentas of intra-uterine growth restriction (IUGR) fetuses. Skin and lung tissues of heparanase over-expressing mice and heparanase knock-out mice were subjected to immunostaining. Results Sections obtained from vaginal and IUGR placentas revealed 2 folds increased levels of heparanase, TFPI-2 and VEGF-A compared to placentas from elective cesarean sections in maternal decidua as well as in fetal placenta elements. Interestingly, abundance of TFPI staining in the intra-villous blood was observed in placentas of vaginal and IUGR deliveries. Heparanase effect on TFPI release to the blood was supported by immunostaining of heparanase over-expressing and heparanase knock-out mice tissues. Conclusions In regard to our previous data on early pregnancy losses, the present data strengthen the understanding that in placental vascular complications levels of heparanase, TFPI-2 and VEGF-A increase. In the process of delivery, heparanase may have a regulatory role on TFPI release to fetal circulation.

Novel interactions between UFH and TFPI in children

The impact of age upon therapeutic response to unfractionated heparin (UFH) in children is proposed to reflect quantitative and potentially qualitative differences in coagulation proteins across childhood. This study explores the UFH-dependent tissue factor pathway inhibitor (TFPI) release in children compared to previously published data in adults. Children <16 years of age undergoing cardiac angiography formed the population for this prospective cohort study. TFPI release was measured prior to (baseline) and at 15, 30, 45 and 120 min post-UFH dose. This study demonstrated that, whilst the immediate release of TFPI post-UFH was similar in children compared to adults, TFPI release in children remained increased and consistent for a significantly longer period post-UFH administration compared to adults. Plasma TFPI levels in children did not demonstrate an UFH concentration -dependent reduction, as has been previously reported in adults. The prolonged TFPI-mediated anticoagulant levels observed in children administered UFH may contribute to the increased rate of major bleeding reported in children compared to adults. Furthermore, we postulate that this sustained UFH-dependent increase in TFPI levels in children may influence the binding of UFH to competitive plasma proteins, such as those involved in the immunological response to UFH associated with heparin-induced thrombocytopenia.

Comparative Studies on the Topical Administration of Mucopolysaccharide and Heparin Ointments in Nonhuman Primates

Mucopolysaccharide polysulfate (MPS) represents a mammalian-derived sulfated polysaccharide. Because the origin and structure of heparins is similar to MPS, this study was conducted to compare 2 ointment formulations containing MPS or heparin with a placebo ointment on tissue factor pathway inhibitor (TFPI) released in nonhuman primates (Macaca mulatta). A primate colony composed of 18 animals, housed at Loyola University Medical Center, was used in compliance with an Institutional Animal Care and Use Committee (IACUC)-approved protocol. Mucopolysaccharide polysulfate (4.5%), heparin (4.5%), and a placebo ointment were topically applied to individual groups of primates in a crossover study for periods of up to 2 weeks. Blood samples were drawn on days 1, 2, 5, 7, and 10. The anticoagulant effects (activated partial thromboplastin time [APTT], Heptest, thrombin time [TT]), TFPI antigen and functional levels, thrombin activatable fibrinolytic inhibitor (TAFI), and antiheparin platelet factor 4 antibodies (AHPF4 abs) were measured in citrated plasma. All data were compiled as mean +/- 1 standard deviation and compared in groups. Topical administration of both the MPS and heparin ointments resulted in no measurable anticoagulant effects in the primate model; however, MPS produced a concentration-dependent release of TFPI antigen and a functional activity that was stronger than the effects observed with heparin. A decrease in TAFI activation was also observed in the MPS-treated primates. In addition, in the heparin-treated group, a slight increase in AHPF4 abs was observed. In conclusion, MPS showed a stronger release of TFPI than heparin that was not associated with a strong anticoagulant effect. Moreover, MPS downregulated TAFI, resulting in an enhanced fibrinolytic effect.

Comparative pharmacodynamic time-course of bemiparin and enoxaparin in healthy volunteers

Low-molecular-weight heparins (LMWHs) are antithrombotic drugs that differ on biochemical and pharmacological properties. This study was conducted to compare the pharmacodynamic time-course of two LMWHs, bemiparin and enoxaparin, at high prophylactic doses. This was an open, randomized, single-blind, cross-over study to compare the pharmacodynamic time-course, safety and tolerability of two LMWHs, bemiparin 3500 IU and enoxaparin 4000 IU at subcutaneous single doses in 12 healthy male volunteers. Anti-Xa activity (main biomarker of heparin activity), anti-IIa activity, total and free tissue factor pathway inhibitor (TFPI), activated partial thromboplastin time (APTT), thrombin time (TT) and thromboplastin-thrombomodulin mediated time (Tp-TmT) were investigated. Bemiparin 3500 IU achieved more anti-Xa activity than enoxaparin 4000 IU, measured by the area under the curve (geometric mean AUC0t) (bemiparin 3.69 vs. enoxaparin 3.33 IU h/ml; p < 0.001). Maximum anti-Xa activity was reached at 3 hours and there were anti-Xa measurable levels up to 16 h after subcutaneous administration. Anti-Xa activity half-life was 5.44 hours for bemiparin and 4.71 hours for enoxaparin. Anti-IIa activity was above the limit of quantification (0.05 IU/ml) in only 2 volunteers after bemiparin and in 8 after enoxaparin. The "in-vivo" anti-Xa:IIa ratios were: bemiparin 37.9 (95% CI: 28.0 - 55.3, n = 2) and enoxaparin 16.3 (95% CI: 12.2 - 23.4, n = 8). Enoxaparin induced a higher release of total TFPI, but not on free TFPI, and a longer prolongation of APTT and TT (Emax) than bemiparin, with no differences between groups on Tp-TmT. Adverse events (one in each group) were mild and transient. Bemiparin 3500 IU showed more anti-Xa activity and higher anti-Xa: anti-IIa relationship than enoxaparin 4000 IU in healthy volunteers. Both treatments were well tolerated.

Structural and Functional Characterization of Low-molecular-weight Heparins: Impact on the Development of Guidelines for Generic Products

Low-molecular-weight heparins (LMWHs) are poly-pharmacologic drugs used to treat thrombotic and cardiovascular disorders. Recently, several generic versions of branded LMWHs have been introduced. Although generic versions of LMWHs exhibit similar profiles, marked differences in their biological and pharmacologic properties have been demonstrated. Several studies have demonstrated differences in terms of anti-Xa activity and tissue factor pathway inhibitor release. The current data emphasize the need to consider multiple functional parameters when defining bioequivalence of biologic drugs and also underscore the importance of further pharmacologic studies involving animal and human clinical trials. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) are currently developing guidelines for the acceptance of complex biological drugs including LMWHs. The US FDA considers these drugs as follow-on agents whereas the EMEA classifies these drugs as biosimilar agents. Until clear guidelines are developed, generic interchange of LMWHs may not be feasible.

Tissue factor pathway inhibitor release and depletion by sulodexide in humans

Anticoagulant tissue factor pathway inhibitor (TFPI) is released from its endothelial stores by heparin, which may lead to its untoward depletion. We investigated the effects of sulodexide--a commercially available mixture of heparan and dermatan sulfate, on plasma TFPI release and depletion. An open-label pilot trial of intravenous and/or oral sulodexide effects on plasma immunoreactive total TFPI antigen level was performed in 11 healthy men. The drug was initially administered i.v. at a single dose of 120 mg, thenorally for 12 days (50 mg b.i.d), and again by i.v route after 2 weeks. Sulodexide injections induced marked increases in plasma TFPI; they were more pronounced on day 14 than on study initiation (3-fold vs. 2-fold after 10 min) and still evident after 120 min. TFPI levels did not change when measured at 120 min after oral sulodexide administration. The percentage increment in plasma TFPI after 10 min from initial sulodexide injection inversely correlated with baseline TFPI levels (r = - 0.664, P = 0.026). On day 14, the association became strong (r = - 0.970, P < 0.0001) and evident also after 120 min (r = - 0.810, P < 0.002). Baseline TFPI levels decreased over the trial; on day 14 they were lower by 34% than on study initiation (P = 0.001). TFPI release by i.v. sulodexide and its depletion during oral administration of this heparinoid compound constitute novel and likely important hemostatic effects of the drug.

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.

Biomarkers and Coagulation Tests for Assessing the Biosimilarity of a Generic Low‐Molecular‐Weight Heparin: Results of a Study in Healthy Subjects With Enoxaparin

Low-molecular-weight heparins (LMWHs) differ considerably in their influence on clotting tests and release of tissue factor pathway inhibitor (TFPI). Biosimilarity therefore becomes an issue when generic forms of LMWHs are developed. So far, no bioequivalence study with a generic LMWH has been reported. A generic enoxaparin (test) was compared with the originator (reference) in 20 volunteers after single-dose subcutaneous administration (40 mg enoxaparin sodium, 4000 IU/mL anti-factor Xa (anti-FXa; activity). Target variables were anti-FXa and anti-FIIa activity, activated partial thromboplastin time (aPTT), prothrombinase-induced clotting time (PiCT), and TFPI over 24 hours. The statistical evaluation of the anti-FXa activity profile demonstrated bioequivalence of test and reference with confidence intervals of area under the plasma concentration-time curve (AUC0-tlast) (93%-99%) and Amax (88%-95%). Confidence intervals of AUC(0-tlast) (89%-102%) and Amax (90%-103%) of anti-FIIa activity also fulfill bioequivalence criteria. The 90% confidence interval for the maximum concentration of TFPI ranged from 90% to 113%. The claim of similarity was also supported by aPTT and PiCT profiles. Bioequivalence with the originator enoxaparin could be demonstrated by ex vivo inhibition of FXa and FIIa activity, by coagulation tests (aPTT and PiCT), and by in vivo release of TFPI. Whether such data also prove biosimilarity of the generic enoxaparin needs to be determined.