Mechanism for Multiple Ligand Recognition by the Human Transferrin Receptor

Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf

Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets

Although tissue factor (TF), the principial initiator of physiological coagulation and pathological thrombosis, has recently been proposed to be present in human blood, the functional significance and location of the intravascular TF is unknown. In the plasma portion of blood, we found TF to be mainly associated with circulating microvesicles. By cell sorting with the specific marker CD42b, platelet-derived microvesicles were identified as a major location of the plasma TF. This was confirmed by the presence of full-length TF in microvesicles acutely shedded from the activated platelets. TF was observed to be stored in the α-granules and the open canalicular system of resting platelets and to be exposed on the cell surface after platelet activation. Functional competence of the blood-based TF was enabled when the microvesicles and platelets adhered to neutrophils, as mediated by P-selectin and neutrophil counterreceptor (PSGL-1, CD18 integrins) interactions. Moreover, neutrophil-secreted oxygen radical species supported the intravascular TF activity. The pools of platelet and microvesicle TF contributed additively and to a comparable extent to the overall blood TF activity, indicating a substantial participation of the microvesicle TF. Our results introduce a new concept of TF-mediated coagulation crucially dependent on TF associated with microvesicles and activated platelets, which principally enables the entire coagulation system to proceed on a restricted cell surface

Analysis of the potential of cancer cell lines to release tissue factor-containing microvesicles: correlation with tissue factor and PAR2 expression

BackgroundDespite the association of cancer-derived circulating tissue factor (TF)-containing microvesicles and hypercoagulable state, correlations with the incidence of thrombosis remain unclear.MethodsIn this study the upregulation of TF release upon activation of various cancer cell lines, and the correlation with TF and PAR2 expression and/or activity was examined. Microvesicle release was induced by PAR2 activation in seventeen cell lines and released microvesicle density, microvesicle-associated TF activity, and phoshpatidylserine-mediated activity were measured. The time-course for TF release was monitored over 90 min in each cell line. In addition, TF mRNA expression, cellular TF protein and cell-surface TF activities were quantified. Moreover, the relative expression of PAR2 mRNA and cellular protein were analysed. Any correlations between the above parameters were examined by determining the Pearson’s correlation coefficients.ResultsTF release as microvesicles peaked between 30–60 min post-activation in the majority of cell lines tested. The magnitude of the maximal TF release positively correlated with TF mRNA (c = 0.717; p

Induction of endothelial cell proliferation by recombinant and microparticle-tissue factor involves β1-integrin and extracellular signal regulated kinase activation

Objective: Increased levels of circulating tissue factor (TF) in the form of microparticles increase the risk of thrombosis. However, any direct influence of microparticle-associated TF on vascular endothelial cell proliferation is not known. In this study, the influence of recombinant and microparticle- associated TF on endothelial cell proliferation and mitogen-activated protein kinase signaling mechanisms was examined. Methods and Results: Incubation of human coronary artery endothelial cells with lipidated recombinant full-length TF, or TF-containing microparticles (50 to 200 pmol/L TF), increased the rate of cell proliferation and induced phosphorylation of extracellular signal regulated kinase 1 in a TF-dependent manner. Inhibition of extracellular signal regulated kinase 1/2 using PD98059 or extracellular signal regulated kinase 1/2 antisense oligonucleotides or inhibition of c-Jun N-terminal kinase reduced recombinant TF-mediated cell proliferation. PD98059 also reduced cell proliferation in response to TF-containing microparticles. Inclusion of FVIIa (5 nmol/L) and FXa (10 nmol/L) or preincubation of cells with an inhibitory anti-FVIIa antibody had no additional influence on TF-mediated cell proliferation. However, preincubation of exogenous TF with a β1-integrin peptide (amino acids 579 to 799) reduced TF-mediated proliferation. Conclusion: High concentrations of recombinant or microparticle-associated TF stimulate endothelial cell proliferation through activation of the extracellular signal regulated kinase 1/2 pathway, mediated through a novel mechanism requiring the interaction of exogenous TF with cell surface β1-integrin and independent of FVIIa. © 2010 American Heart Association, Inc

Accumulation of tissue factor in endothelial cells induces cell apoptosis, mediated through p38 and p53 activation

We previously reported that high levels of tissue factor (TF) can induce cellular apoptosis in endothelial. In this study, TF-mediated mechanisms of induction of apoptosis were explored. Endothelial cells were transfected to express wild-type TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued TF to enhance or prevent TF release respectively. Alternatively, cells were pre-incubated with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein were only increased in cells pre-incubated with TF-rich microvesicle and in cells expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53 using pifithrin-α suppressed the expression of Bax. Finally, siRNA–mediated suppression of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels, p53 nuclear localisation and transcriptional activity, suppressed Bax expression and prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial cell, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53

Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles

We previously reported that the incorporation of tissue factor (TF) into cell-derived microvesicles (MVs) is regulated by the phosphorylation of the cytoplasmic domain of TF. Since the cytoskeletal protein filamin-A is known to bind to the cytoplasmic domain of TF in a phosphorylation-dependent manner, the involvement of filamin-A in the incorporation of TF into MVs was examined. Endothelial cells were transfected to express TF, whereas MDA-MB-231 cells were used to examine endogenously expressed TF. MV release was induced by activating protease-activated receptor-2 (PAR2). Partial suppression of filamin-A expression using two different filamin-A siRNA sequences resulted in significant reductions in the incorporation of TF antigen into MVs as determined by TF-ELISA and western blot analysis, and was reflected in reduced thrombin-generation and FXa-generation capacities of these MVs. Deletion of the cytoplasmic domain of TF also resulted in reduced incorporation of TF into MVs, whereas the suppression of filamin-A expression had no additional effect on the incorporation of truncated TF into MVs. Partial suppression of filamin-A expression had no effect on the number and size distribution of the released MVs. However, >90 % suppression of filamin-A expression resulted in increased MV release, possibly as a result of increased instability of the plasma membrane and underlying cytoskeleton. In conclusion, the presence of filamin-A appears to be essential for the incorporation of TF into MVs following PAR2 activation, but is not required for the process of MV formation and release following PAR2 activation

Circulating tissue factor-positive procoagulant microparticles in patients with type 1 diabetes

Aim: To investigate the count of circulating tissue factor-positive (TF+) procoagulant microparticles (MPs) in patients with type 1 diabetes mellitus (T1DM). Methods: This case-control study included patients with T1DM and age and sex-matched healthy volunteers. The counts of phosphatidylserine-positive (PS+) MPs and TF(+)PS(+)MPs and the subgroups derived from different cell types were measured in the peripheral blood sample of the two groups using multicolor flow cytometric assay. We compared the counts of each MP between groups as well as the ratio of the TF(+)PS(+)MPs and PS(+)MPs (TF(+)PS(+)MPs/PS(+)MPs). Results: We recruited 36 patients with T1DM and 36 matched healthy controls. Compared with healthy volunteers, PS(+)MPs, TF(+)PS(+)MPs and TF(+)PS(+)MPs/PS(+)MPs were elevated in patients with T1DM (PS(+)MPs: 1078.5 +/- 158.08 vs 686.84 +/- 122.04/mu L, P &lt;0.001; TF(+)PS(+)MPs: 202.10 +/- 47.47 vs 108.33 +/- 29.42/mu L, P &lt;0.001; and TF(+)PS(+)MPs/PS(+)MPs: 0.16 +/- 0.04 vs 0.19 +/- 0.05, P = 0.004), mostly derived from platelet, lymphocytes and endothelial cells. In the subgroup analysis, the counts of total and platelet TF(+)PS(+)MPs were increased in patients with diabetic retinopathy (DR) and with higher HbA1c, respectively. Conclusion: Circulating TF(+)PS(+)MPs and those derived from platelet, lymphocytes and endothelial cells were elevated in patients with T1DM.De tre första författarna delar förstaförfattarskapet.</p

Multiuser Detection Assisted Time- and Frequency-Domain Spread Multicarrier Code-Division Multiple-Access

In this contribution, we study a reduced-complexity multiuser detection aided multicarrier direct-sequence code-division multiple-access (MC DS-CDMA) scheme, which employs both time (T)-domain and frequency (F)-domain spreading. We investigate the achievable detection performance in the context of synchronous TF-domain spread MC DS-CDMA when communicating over an additive white Gaussian noise (AWGN) channel. Five detection schemes are investigated, which include the single-user correlation based detector, the joint TF-domain decorrelating multiuser detector (MUD), the joint TF-domain MMSEMUD, the separate TF-domain decorrelating/MMSE MUD, and the separate TF-domain MMSE/decorrelating MUD. Our simulation results show that the separate TF-domain MUD schemes are capable of achieving a similar bit error rate (BER) performance to that of the significantly more complex joint TF-domain MUD schemes. Index Terms—Code-division multiple-access (CDMA), decorrelating, frequency-domain spreading, joint detection, minimum mean square error (MMSE), multicarrier (MC), multiuser detection, separate detection, time-domain spreading