11,12-EET suppressed LPS induced TF expression and thrombus formation by accelerating mRNA degradation rate via strengthening PI3K-Akt signaling pathway and inhibiting p38-TTP pathway.

Epoxyeicosatrienoic acids (EETs), which are synthesized from arachidonic acid by cytochrome P450 epoxygenases, function primarily as autocrine and paracrine effectors in the cardiovascular system. So far, most research has focused on the vasodilatory, anti-inflammatory, anti-apoptotic and mitogenic properties of EETs in the systemic circulation. However, whether EETs could suppress tissue factor (TF) expression and prevent thrombus formation remains unknown. Here we utilized in vivo and in vitro models to investigate the effects and underlying mechanisms of exogenously EETs on LPS induced TF expression and inferior vein cava ligation induced thrombosis. We observed that the thrombus formation rate and the size of the thrombus were greatly reduced in 11,12-EET treated mice，accompanied by decreased TF and inflammatory cytokines expression. Further in vitro studies showed that by enhancing p38 MAPK activation and subsequent tristetraprolin (TTP) phosphorylation, LPS strengthened the stability of TF mRNA and induced increased TF expression. However, by strengthening PI3K-dependent Akt phosphorylation, which acted as a negative regulator of p38-TTP signaling pathway，11,12-EET reduced LPS-induced TF expression in monocytes. In addition, 11,12-EET inhibited LPS-induced NF-κB nuclear translocation by activating the PI3K/Akt pathway. Further study indicated that the inhibitory effect of 11,12-EET on TF expression was mediated by antagonizing LPS-induced activation of thromboxane prostanoid receptor. In conclusion, our study demonstrated that 11,12-EET prevented thrombosis by reducing TF expression and targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate thrombosis related diseases.

Tissue factor promotes HCC carcinogenesis by inhibiting BCL2-dependent autophagy.

INTRODUCTION: Tissue factor (TF) is an important predictor for poor prognosis of Hepatocellular carcinoma (HCC). TF can also upregulate the expression of BCL2, which is a key inhibitor of autophagy responses. This study aims to explore the role of BCL2-dependent autophagy in TF-regulated HCC carcinogenesis. METHODS: In this study, we explored the roles of TF in HCC using gene overexpression and silencing assays. Besides, we further identified the significance of BCL2-Beclin1-autophagy signaling on TF-regulated HCC tumorigenesis by combining TF silencing with pharmacological autophagy inhibitor (3-MA). RESULTS: The experimental data showed that the overexpressed TF promoted BCL2 protein expression and inhibited the autophagy activity (shown as LC3 conversion rate, p62 expression and autophagosomes) while maintaining the survival in HCC cells. In contrast, the silent TF showed the completely opposite results. Furthermore, TF knockdown promoted the dissociation of Beclin1 from BCL2-Beclin1 complex. In addition, the enhanced autophagy and inhibited survival by TF knockdown could be reversed by autophagy inhibition with 3-MA or spautin-1 (Beclin1 specific inhibitor) in HCC cells. Xenografts assays also showed that TF-silencing HCC cells had stronger tumorigenicity in vivo, which was recovered by spautin-1 administration. CONCLUSIONS: TF inhibits autophagy-related death by enhancing BCL2 expression, whereby promoting HCC tumorigenesis.

Ruscogenin Alleviates Deep Venous Thrombosis and Pulmonary Embolism Induced by Inferior Vena Cava Stenosis Inhibiting MEK/ERK/Egr-1/TF Signaling Pathway in Mice.

BACKGROUND: Ruscogenin (RUS) has anti-inflammatory and antithrombotic effects, while its potential effects on deep venous thrombosis (DVT) and pulmonary embolism (PE) remain unclear. OBJECTIVE: We aimed to elucidate the effects of RUS on DVT and PE induced by the inferior vena cava stenosis (IVCS) model and investigate the underlying mechanism. METHODS: Male C57/BL6 mice were used to explore whether IVCS model could be complicated with deep venous thrombosis and pulmonary embolism. Then, effects of RUS on DVT and PE related inflammatory factors and coagulation were examined using H&E staining, ELISA, and real-time PCR. Western blot analysis was used to examine the effects of RUS on MEK/ERK/Egr-1/TF signaling pathway in PE. RESULTS: IVCS model induced DVT and complied with PE 48 h after surgery. Administration of RUS (0.01, 0.1, 1 mg/kg) inhibited DVT, decreased biomarker D-Dimer, cardiac troponin I, N-Terminal probrain natriuretic peptide in plasma to ameliorate PE induced by IVCS model. Meanwhile, RUS reduced tissue factor and fibrinogen content of lung tissue, inhibited P-selectin and C-reactive protein activity in plasma, and suppressed the expressions of interleukin-6 and interleukin-1ß in mice. Furthermore, RUS suppressed the phosphorylation of ERK1/2 and MEK1/2, decreasing the expressions of Egr-1 and TF in the lung. CONCLUSION: IVCS model contributed to the development of DVT and PE in mice and was associated with increased inflammation. RUS showed therapeutic effects by inhibiting inflammation as well as suppressing the activation of MEK/ERK/Egr-1/TF signaling pathway.

Procoagulant activities of skeletal and cardiac muscle myosin depend on contaminating phospholipid.

Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine-binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by ∼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.

Activation of ß1 integrins and caveolin-1 by TF/FVIIa promotes IGF-1R signaling and cell survival.

The tissue factor/coagulation factor VIIa (TF/FVIIa) complex induces transactivation of the IGF-1 receptor (IGF-1R) in a number of different cell types. The mechanism is largely unknown. The transactivation leads to protection from apoptosis and nuclear translocation of the IGF-1R. The aim of this study was to clarify the signaling pathway between TF and IGF-1R after FVIIa treatment with PC3 and DU145 prostate or MDA-MB-231 breast cancer cells as model systems. Protein interactions, levels, and phosphorylations were assessed by proximity ligation assay or flow cytometry in intact cells and by western blot on cell lysates. The transactivation of the IGF-1R was found dependent on TF/FVIIa-induced activation of ß1-integrins. A series of experiments led to the conclusion that the caveolae protein caveolin-1 prevented IGF-1R activation in resting cells via its scaffolding domain. TF/FVIIa/ß1-integrins terminated this inhibition by activation of Src family kinases and subsequent phosphorylation of caveolin-1 on tyrosine 14. This phosphorylation was not seen after treatment with PAR1 or PAR2 agonists. Consequently, the protective effect of FVIIa against apoptosis induced by the death receptor agonist TRAIL and the de novo synthesis of cyclin D1 induced by nuclear IGF-1R accumulation were both significantly reduced by down-regulation of ß1-integrins or overexpression of the caveolin-1 scaffolding domain. In conclusion, we present a plausible mechanism for the interplay between TF and IGF-1R involving FVIIa, ß1-integrins, Src family proteins, and caveolin-1. Our results increase the knowledge of diseases associated with TF and IGF-1R overexpression in general but specifically of TF-mediated signaling with focus on cell survival.

Construction and characterization of a truncated tissue factor­coagulation­based composite system for selective thrombosis in tumor blood vessels.

The selective induction of tumor vascular thrombosis using truncated tissue factor (tTF) delivered via a target ligand is a promising novel antitumor strategy. In the present study, an anti­neuropilin­1 (NRP­1) monoclonal antibody (mAb)­streptavidin (SA):tTF­biotin (B) composite system was established. In this system, anti­NRP­1­mAb located tTF to the tumor vascular endothelial cell surface and induced vascular embolization. Due to their high binding affinity, SA and B were used to enhance thrombogenic activity. mAb was conjugated with SA using a coupling method with water­soluble 1­ethyl­3­(3­dimethylaminopropyl) carbodiimide and N­hydroxysulfosuccinimide. Biotinylated tTF (tTF­B) was prepared using a B­labeling kit subsequent to the generation and purification of fusion protein tTF. Confocal microscopy and flow cytometry indicated that the anti­NRP­1­mAb­SA conjugate retained mAb targeting activity. The preservation of B­conjugate binding capacity was confirmed using a competitive ELISA, and factor X­activation analysis revealed that tTF­B retained the procoagulant activity exhibited by tTF. Live imaging was performed to assess mAb­SA distribution and tumor­targeting capability, and this yielded promising results. The results of in vivo studies in mice with subcutaneous xenografts demonstrated that this composite system significantly induced tumor vascular thrombosis and inhibited tumor growth, whereas these histological changes were not observed in normal organs.

Construction of novel procoagulant protein targeting neuropilin-1 on tumour vasculature for tumour embolization therapy.

The cellular transmembrane receptor Neuropilin-1(NRP-1) is overexpressed in tumour tissue and endothelial cells of tumour vessels, whereas it has limited expression in normal tissues. This study aimed to design a novel recombinant protein tTF-EG3287, which consisting of the truncated tissue factor (tTF) and the NRP-1 targeting peptide EG3287. The procoagulant protein selectively activates blood coagulation in tumour vessels once bound to the cell surface of the tumour vasculature by a targeting peptide EG3287. In this study, procoagulant activity of the recombinant protein tTF-EG3287 was evaluated by Spectozyme FXa assay. NRP-1 targeting ability was analysed by fluorescence confocal microscopy and flow cytometry. The living imaging system was used to assess the tumour targeting ability of recombinant proteins tTF-EG3287 in vivo. Tumour growth inhibition showed effective antitumor activity in HepG2 tumour-bearing nude mice. Histological study showed obvious thrombosis and thromboembolism in tumour vessels and cell necrosis of tumour tissue, without any clear side effect such as thrombosis in other organs.

Protease-activated receptor 2 promotes actomyosin dependent transforming microvesicles generation from human breast cancer.

Apart from blood coagulation, coagulation proteases are involved inextricably in cancer progression/propagation via intra/inter-cellular signaling, mediated predominantly by protease-activated receptors (PARs). Microvesicles (MVs), a plasma membrane shredded component, has recently been identified as an important contributor to human breast cancer metastasis. However, the role of PAR2 in promoting MVs generation from breast cancer cells remains largely unexplored. The objective of this study is to investigate whether coagulation protease-mediated human breast cancer propagation commences via MVs and also to decipher the underlying signaling mechanism. Here, we elicited that coagulation factor-FVIIa and Trypsin activates PAR2, which governs MVs shedding from MDAMB231 cells by altering actomyosin dynamics. Treatment of cells with PAR2 activators facilitate MVs generation by activating three independent (MAPK, P38, and Rho) signaling cascades. MAPK, signals through activating MLCK followed by MLC phosphorylation to alter myosin organization whereas, P38 reorganizes actin dynamics by the sequential activation of MK2 and HSP27. RhoA-dependent ROCK-II activation again contributes to remodeling myosin II activity. Further, both our in vitro and in vivo analyses showed that these MVs potentiate invasive and migratory property to the recipient cells. Breast cancer patients blood show an elevation of TF-bearing, pro-metastatic MVs than normal. These findings give an insight into the detailed signaling mechanism involved in the production of MVs with transforming ability from PAR2-activated human breast cancer cells. Understanding these mechanistic details will certainly help to identify crucial targets for therapeutic interventions in MVs-associated human breast cancer metastasis.

Engineering of a membrane-triggered activity switch in coagulation factor VIIa.

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

CD13 as target for tissue factor induced tumor vascular infarction in small cell lung cancer.

OBJECTIVES: Zinc-binding protease aminopeptidase N (CD13) is expressed on tumor vascular cells and tumor cells. It represents a potential candidate for molecular targeted therapy, e.g. employing truncated tissue factor (tTF)-NGR, which can bind CD13 and thereby induce tumor vascular infarction. We performed a comprehensive analysis of CD13 expression in a clinically well characterized cohort of patients with small cell lung cancer (SCLC) to evaluate its potential use for targeted therapies in this disease. MATERIAL AND METHODS: CD13 expression was analyzed immunohistochemically in 27 SCLC patients and correlated with clinical course and outcome. In CD-1 nude mice bearing human HTB119 SCLC xenotransplants, the systemic effects of the CD13-targeting fusion protein tTF-NGR on tumor growth were tested. RESULTS AND CONCLUSION: In 52% of the investigated SCLC tissue samples, CD13 was expressed in tumor stroma cells, while the tumor cells were negative for CD13. No prognostic effect was found in the investigated SCLC study collective with regard to overall survival (p>0.05). In CD-1 nude mice, xenografts of CD13 negative HTB119 SCLC cells showed CD13 expression in the intratumoral vascular and perivascular cells, and the systemic application of CD13-targeted tissue factor tTF-NGR led to a significant reduction of tumor growth. We here present first data on the expression of CD13 in SCLC tumor samples. Our results strongly recommend the further investigation of tTF-NGR and other molecules targeted by NGR-peptides in SCLC patients. Considering the differential expression of CD13 in SCLC samples pre-therapeutic CD13 analysis is proposed for testing as investigational predictive biomarker for patient selection.

Combinatorial effects of doxorubicin and retargeted tissue factor by intratumoral entrapment of doxorubicin and proapoptotic increase of tumor vascular infarction.

Truncated tissue factor (tTF), retargeted to tumor vasculature by GNGRAHA peptide (tTF-NGR), and doxorubicin have therapeutic activity against a variety of tumors. We report on combination experiments of both drugs using different schedules. We have tested fluorescence- and HPLC-based intratumoral pharmacokinetics of doxorubicin, flow cytometry for cellular phosphatidylserine (PS) expression, and tumor xenograft studies for showing in vivo apoptosis, proliferation decrease, and tumor shrinkage upon combination therapy with doxorubicin and induced tumor vascular infarction. tTF-NGR given before doxorubicin inhibits the uptake of the drug into human fibrosarcoma xenografts in vivo. Reverse sequence does not influence the uptake of doxorubicin into tumor, but significantly inhibits the late wash-out phase, thus entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with doxorubicin in vitro increases PS concentrations in the outer layer of the cell membrane as a sign of early apoptosis. Cells expressing increased PS concentrations show comparatively higher procoagulatory efficacy on the basis of equimolar tTF-NGR present in the Factor X assay. Experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in athymic nude mice indeed show a combinatorial tumor growth inhibition applying doxorubicin and tTF-NGR in sequence over single drug treatment. Combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by new mechanisms, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in tumor endothelial and tumor cells.

Transport physics and biorheology in the setting of hemostasis and thrombosis.

The biophysics of blood flow can dictate the function of molecules and cells in the vasculature with consequent effects on hemostasis, thrombosis, embolism, and fibrinolysis. Flow and transport dynamics are distinct for (i) hemostasis vs. thrombosis and (ii) venous vs. arterial episodes. Intraclot transport changes dramatically the moment hemostasis is achieved or the moment a thrombus becomes fully occlusive. With platelet concentrations that are 50- to 200-fold greater than platelet-rich plasma, clots formed under flow have a different composition and structure compared with blood clotted statically in a tube. The platelet-rich, core/shell architecture is a prominent feature of self-limiting hemostatic clots formed under flow. Importantly, a critical threshold concentration of surface tissue factor is required for fibrin generation under flow. Once initiated by wall-derived tissue factor, thrombin generation and its spatial propagation within a clot can be modulated by γ'-fibrinogen incorporated into fibrin, engageability of activated factor (FIXa)/activated FVIIIa tenase within the clot, platelet-derived polyphosphate, transclot permeation, and reduction of porosity via platelet retraction. Fibrin imparts tremendous strength to a thrombus to resist embolism up to wall shear stresses of 2400 dyne cm(-2) . Extreme flows, as found in severe vessel stenosis or in mechanical assist devices, can cause von Willebrand factor self-association into massive fibers along with shear-induced platelet activation. Pathological von Willebrand factor fibers are A Disintegrin And Metalloprotease with ThromboSpondin-1 domain 13 resistant but are a substrate for fibrin generation due to FXIIa capture. Recently, microfluidic technologies have enhanced the ability to interrogate blood in the context of stenotic flows, acquired von Willebrand disease, hemophilia, traumatic bleeding, and drug action.

NG2 proteoglycan as a pericyte target for anticancer therapy by tumor vessel infarction with retargeted tissue factor.

tTF-TAA and tTF-LTL are fusion proteins consisting of the extracellular domain of tissue factor (TF) and the peptides TAASGVRSMH and LTLRWVGLMS, respectively. These peptides represent ligands of NG2, a surface proteoglycan expressed on angiogenic pericytes and some tumor cells. Here we have expressed the model compound tTF-NGR, tTF-TAA, and tTF-LTL with different lengths in the TF domain in E. coli and used these fusion proteins for functional studies in anticancer therapy. We aimed to retarget TF to tumor vessels leading to tumor vessel infarction with two barriers of selectivity, a) the leaky endothelial lining in tumor vessels with the target NG2 being expressed on pericytes on the abluminal side of the endothelial cell barrier and b) the preferential expression of NG2 on angiogenic vessels such as in tumors. Chromatography-purified tTF-TAA showed identical Factor X (FX)-activating procoagulatory activity as the model compound tTF-NGR with Km values of approx. 0.15 nM in Michaelis-Menten kinetics. The procoagulatory activity of tTF-LTL varied with the chosen length of the TF part of the fusion protein. Flow cytometry revealed specific binding of tTF-TAA to NG2-expressing pericytes and tumor cells with low affinity and dissociation KD in the high nM range. In vivo and ex vivo fluorescence imaging of tumor xenograft-carrying animals and of the explanted tumors showed reduction of tumor blood flow upon tTF-TAA application. Therapeutic experiments showed a reproducible antitumor activity of tTF-TAA against NG2-expressing A549-tumor xenografts, however, with a rather small therapeutic window (active/toxic dose in mg/kg body weight).

TETIS study: evaluation of new topical hemostatic agent TT-173 in tooth extraction.

OBJECTIVES: TT-173 is a new hemostatic agent consisting of yeast-derived microvesicles containing a modified version of recombinant human tissue factor. In the present work, the procoagulant activity of TT-173 has been evaluated for the first time in humans. METHODS: This is a phase I, randomized, placebo-controlled study to evaluate the efficacy, safety, systemic absorption, and immunogenicity of TT-173 in healthy volunteers undergoing tooth extraction. Subjects received TT-173 or placebo into the alveolar cavity, just after tooth extraction. Time to clot formation, bleeding time, and adverse events were recorded. RESULTS: Treatment with TT-173 reduced the bleeding time and the time to clot formation. No adverse events related with product administration were reported. In the same way, neither systemic absorption nor immunogenic reaction against the product was detected. Our findings pave the way to evaluate the usefulness of this new topical hemostatic agent in more complex oral surgeries and in those patients affected with coagulation disorders that may compromise the realization of dental procedures. CONCLUSION: The new hemostatic agent TT-173 has proven efficacious and safe in healthy subjects undergoing tooth extraction supporting its further evaluation in more complex surgeries. CLINICAL RELEVANCE: The development of this new topical hemostatic agent could contribute to bleeding control in oral and maxillofacial surgery.

pHLIP-mediated targeting of truncated tissue factor to tumor vessels causes vascular occlusion and impairs tumor growth.

Occluding tumor blood supply by delivering the extracellular domain of coagulation-inducing protein tissue factor (truncated tissue factor, tTF) to tumor vasculature has enormous potential to eliminate solid tumors. Yet few of the delivery technologies are moved into clinical practice due to their non-specific tissue biodistribution and rapid clearance by the reticuloendothelial system. Here we introduced a novel tTF delivery method by generating a fusion protein (tTF-pHLIP) consisting of tTF fused with a peptide with a low pH-induced transmembrane structure (pHLIP). This protein targets the acidic tumor vascular endothelium and effectively induces local blood coagulation. tTF-pHLIP, wherein pHLIP is cleverly designed to mimic the natural tissue factor transmembrane domain, triggered thrombogenic activity of the tTF by locating it to the endothelial cell surface, as demonstrated by coagulation assays and confocal microscopy. Systemic administration of tTF-pHLIP into tumor-bearing mice selectively induced thrombotic occlusion of tumor vessels, reducing tumor perfusion and impairing tumor growth without overt side effects. Our work introduces a promising strategy for using tTF as an anti-cancer drug, which has great potential value for clinical applications.

Low-Energy Ultrasound Treatment Improves Regional Tumor Vessel Infarction by Retargeted Tissue Factor.

OBJECTIVES: To enhance the regional antitumor activity of the vascular-targeting agent truncated tissue factor (tTF)-NGR by combining the therapy with low-energy ultrasound (US) treatment. METHODS: For the in vitro US exposure of human umbilical vein endothelial cells (HUVECs), cells were put in the focus of a US transducer. For analysis of the US-induced phosphatidylserine (PS) surface concentration on HUVECs, flow cytometry was used. To demonstrate the differences in the procoagulatory efficacy of TF-derivative tTF-NGR on binding to HUVECs with a low versus high surface concentration of PS, we performed factor X activation assays. For low-energy US pretreatment, HT1080 fibrosarcoma xenotransplant-bearing nude mice were treated by tumor-regional US-mediated stimulation (ie, destruction) of microbubbles. The therapy cohorts received the tumor vessel-infarcting tTF-NGR protein with or without US pretreatment (5 minutes after US stimulation via intraperitoneal injection on 3 consecutive days). RESULTS: Combination therapy experiments with xenotransplant-bearing nude mice significantly increased the antitumor activity of tTF-NGR by regional low-energy US destruction of vascular microbubbles in tumor vessels shortly before application of tTF-NGR (P < .05). Mechanistic studies proved the upregulation of anionic PS on the outer leaflet of the lipid bilayer of endothelial cell membranes by low-energy US and a consecutive higher potential of these preapoptotic endothelial cells to activate coagulation via tTF-NGR and coagulation factor X as being a basis for this synergistic activity. CONCLUSIONS: Combining retargeted tTF to tumor vessels with proapoptotic stimuli for the tumor vascular endothelium increases the antitumor effects of tumor vascular infarction. Ultrasound treatment may thus be useful in this respect for regional tumor therapy.

In vivo production of mesenchymal stromal cells after injection of autologous platelet-rich plasma activated by recombinant human soluble tissue factor in the bone marrow of healthy volunteers.

Autologous mesenchymal stromal cell (MSC)-based therapies offer one of the most promising and safe methods for regeneration or reconstruction of tissues and organs. Routine procedures to obtain adequate amount of autologous stem cells need their expansion through culture, with risks of contamination and cell differentiation, leading to the loss of cell ability for therapies. We suggest the use of human bone marrow (BM) as a physiological bioreactor to produce autologous MSC by injection of autologous platelet-rich plasma activated by recombinant human soluble tissue factor (rhsTF) in iliac crest. A trial on 13 healthy volunteers showed the feasibility and harmlessness of the procedure. The phenotype and cellularity of BM cells were not modified, on day 3 after injection. Endothelial progenitor cells (EPC) were mobilized to the bloodstream, without stimulation of hematopoietic stem cells (HSC). MSC level in BM increased with a specific commitment to preosteoblastic cell population both in vivo and in vitro. This self-stimulation system of BM seems thus to be a promising feasible process 3 days before clinical cell therapy applications.

Non-invasive monitoring of tumor-vessel infarction by retargeted truncated tissue factor tTF-NGR using multi-modal imaging.

The fusion protein tTF-NGR consists of the extracellular domain of the thrombogenic human tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA (NGR), a ligand of the surface protein CD13 (aminopeptidase N), upregulated on endothelial cells of tumor vessels. tTF-NGR preferentially activates blood coagulation within tumor vasculature, resulting in tumor vessel infarction and subsequent tumor growth retardation/regression. The anti-vascular mechanism of the tTF-NGR therapy approach was verified by quantifying the reduced tumor blood-perfusion with contrast-enhanced ultrasound, the reduced relative tumor blood volume by ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging, and by in vivo-evaluation of hemorrhagic bleeding with fluorescent biomarkers (AngioSense(680)) in fluorescence reflectance imaging. The accumulation of tTF-NGR within the tumor was proven by visualizing the distribution of the iodine-123-labelled protein by single-photon emission computed tomography. Use of these multi-modal vascular and molecular imaging tools helped to assess the therapeutic effect even at real time and to detect non-responding tumors directly after the first tTF-NGR treatment. This emphasizes the importance of imaging within clinical studies with tTF-NGR. The imaging techniques as used here have applicability within a wider scope of therapeutic regimes interfering with tumor vasculature. Some even are useful to obtain predictive biosignals in personalized cancer treatment.

Tissue factor-mediated activation of the prothrombin complex concentrate (PCC) is differently inhibited by dabigatran, rivaroxaban, and apixaban: potential clinical implication.

Currently, several newer oral anticoagulants namely dabigatran (anti-IIa), rivaroxaban (anti-Xa), and apixaban are available for various clinical implications. Another oral anti-Xa edoxaban is under development. A parenteral anti-Xa drug namely otamixaban is also under development for cardiovascular interventions. Bleeding complications have been reported in the new oral anticoagulants and have been managed by conventional approaches with limited success. Prothrombin complex concentrates (PCCs) are reported to neutralize the anticoagulant activity of these agents. The PCCs are also able to generate endogenous factor Xa and IIa along with other proteases that are capable of neutralizing the circulating anti-Xa or anti-IIa activities of the newer anticoagulants. The generation of Xa and IIa is also dependent on the type of tissue factor available for their activation. These reported studies suggest that different tissue factors differentially activate a PCC namely Profilnine SD. Furthermore, dabigatran differs from rivaroxaban and other factor Xa inhibitors in its inhibitory profile.

Protease-activated receptor-2 signalling by tissue factor on dendritic cells suppresses antigen-specific CD4+ T-cell priming.

The precise function of tissue factor (TF) expressed by dendritic cells (DC) is uncertain. As well as initiating thrombin generation it can signal through protease-activated receptor 2 (PAR-2) when complexed with factor VIIa. We investigated the expression and function of TF on mouse bone marrow (BM) -derived DC; 20% of BM-derived DC expressed TF, which did not vary after incubation with lipopolysaccharide (LPS) or dexamethasone (DEX). However, the pro-coagulant activity of DEX-treated DC in recalcified plasma was 30-fold less than LPS-treated DC. In antigen-specific and allogeneic T-cell culture experiments, the TF on DEX-treated DC provided a signal through PAR-2, which contributed to the reduced ability of these cells to stimulate CD4(+) T-cell proliferation and cytokine production. In vivo, an inhibitory anti-TF antibody and a PAR-2 antagonist enhanced antigen-specific priming in two models where antigen was given without adjuvant, with an effect approximately 50% that seen with LPS, suggesting that a similar mechanism was operational physiologically. These data suggest a novel TF and PAR-2-dependent mechanism on DEX-DC in vitro and unprimed DC in vivo that contributes to the low immunogenicity of these cells. Targeting this pathway has the potential to influence antigen-specific CD4(+) T-cell activation.