Tissue factor-bearing microparticles and inflammation: a potential mechanism for the development of venous thromboembolism in cancer.

Cancer is associated with an increased risk of venous thromboembolism (VTE); the exact mechanisms for the induction of VTE remain to be fully elucidated, but it is widely acknowledged that tissue factor (TF)-bearing microparticles (TF-MPs) may play a significant role. However, TF-MPs have yet to be accepted as a genuine biomarker for cancer-associated VTE, as the presence of elevated TF-MP levels is not always accompanied by thrombosis; interestingly, in certain cases, particularly in pancreatic cancer, VTE seems to be more likely in the context of acute inflammation. Although several potential mechanisms for the development of VTE in cancer have been postulated, this review explores the homeostatic disruption of TF-MPs, as the main reservoir of bloodborne TF, in the context of cancer and inflammation, and considers the abrogated responses of the activated endothelium and mononuclear phagocyte system in mediating this disruption.

The effect of lipid peroxidation and lipolysis on the ability of lipoproteins to influence thromboplastin activity.

High, low and very low density lipoproteins and lipoprotein (a) were prepared from porcine serum. The apolipoprotein components of the lipoproteins were then isolated and resuspended in soybean lecithin. Apolipoprotein B was also resuspended in lipids more representative of those found in LDL and VLDL. Lipid peroxidation was induced in samples of all the lipoproteins and reconstituted apolipoproteins by incubation with either Cu2+ ions or hedgehog 15-lipoxygenase. Furthermore, aliquots of the samples were incubated with a mixture of lipases. The effect of native preparations and the treated samples on the procoagulant activity of thromboplastin was examined. Native HDL, apo A-II, native LDL, reconstituted LDL and apo B inhibited thromboplastin activity, whereas native VLDL and reconstituted VLDL enhanced this activity. While the ability of HDL and apolipoprotein A-II to inhibit thromboplastin was unaltered by either Cu2+ oxidation, lipoxygenase oxidation or lipolysis, VLDL and particles resembling VLDL, which acted cooperatively with thromboplastin lost their activating potential. On the other hand, LDL and particles resembling LDL changed from being inhibitory to enhancing the thromboplastin activity following oxidation, but not after lipolysis. Apolipoprotein B fragments obtained by mild digestion of this protein, expressed an inhibitory effect towards thromboplastin, while extensive degradation of the protein reduced its inhibitory potential. It is suggested that modifications of lipoproteins in vivo can lead to a hypercoagulable state by modulation of the cofactor activity of thromboplastin to factor VII.

Alterations in the structure of apolipoprotein B-100 determine the behaviour of LDL towards thromboplastin.

Apolipoprotein B-100 acts as an inhibitor of thromboplastin activity independently of the tissue factor pathway inhibitor (TFPI) associated with plasma lipoproteins. Analysis of the primary structure of Apo B-100 showed a higher than expected occurrence of lysine groups in the receptor-binding region. In order to demonstrate the participation of lysine groups of Apo B-100 in the inhibition of thromboplastin, thromboplastin and Apo B-100 were incubated together in the presence of poly-L-lysine, poly-L-arginine, lysine and arginine monomers. The inhibition of thromboplastin by Apo B-100 was completely suppressed in the presence of poly-L-lysine. Poly-L-arginine was found to be less effective and neither lysine or arginine monomers had any significant effect on the inhibitory effect of Apo B-100. Alterations in the structure of Apo B-100 reconstituted in lipid vesicles resembling LDL, brought about by lipid peroxidation and lipid loading were examined by means of Fourier transform infra-red spectroscopy. It was found that, upon oxidation without the addition of cupric ions, the apolipoprotein attains a more exposed conformation with an increase in alpha-helical structure. This increase occurred at the expense of beta-structure. On lipid loading, an increase in beta-structure at the expense of the alpha-helix, was demonstrated. It is therefore proposed that the variable action of LDL towards thromboplastin derives from alterations in the secondary structure of the Apo B-100, particularly the receptor-binding region.

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 cells. 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 cells, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53.

Molecular mimicry and ankylosing spondylitis: possible role of a novel sequence in pullulanase of Klebsiella pneumoniae.

Molecular mimicry has been shown between two sequences of Klebsiella pneumoniae pulD secretion protein (DRDE) with HLA-B27 (DRED) and pulA (pullulanase) enzyme (Gly-X-Pro) with types I, III and IV collagen respectively. IgG antibody levels in AS patients were elevated against 16mer synthetic peptides of HLA-B27 and pulD by enzyme immunosorbent assay (ELISA) compared to controls (P < 0.001). ELISA assays against K. pneumoniae grown in the absence and presence of pullulan demonstrated significant levels of IgA antibody in AS patients compared to controls (P < 0.001). Increased IgA and IgG antibody levels to pulA and types I and IV collagen were observed in AS patients compared to controls (P < 0.001). These observations could be relevant in the sequence of molecular events in AS.

Modification of tissue factor by peroxynitrite influences its procoagulant activity.

Peroxynitrite, a reactive oxidising species resulting from a reaction between nitric oxide and the superoxide anion, modifies proteins by nitration of certain amino acids such as tyrosine. Tissue factor (TF), a transmembrane protein, is expressed on cells under inflammatory conditions and initiates the coagulation cascade. The extracellular domain of TF is rich in tyrosine. Exposure of recombinant TF and cellular TF to peroxynitrite was associated with a reduction in procoagulant activity. This was accompanied by an elevated level of nitrotyrosine residues. Peroxynitrite may have a protective role by attenuation of the thrombotic properties of TF.

The role of the C-terminal domain in the inhibitory functions of tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) inhibits the activity of coagulation factors VIIa and Xa through Kunitz domains, thereby inhibiting the activity of tissue factor. However, it has been shown that the C-terminal of this inhibitor is essential for the maximal anticoagulant activity of TFPI. We have investigated the endogenous ability of the C-terminal of TFPI to influence coagulation. A synthetic peptide corresponding to residues 254-265 within the C-terminal of TFPI was prepared and shown to be capable of inhibiting tissue factor pathway by preventing the activation of factor VII. Mutational analysis of the peptide revealed the identity of the key lysine residues.

Bovine spongiform encephalopathy: is it an autoimmune disease due to bacteria showing molecular mimicry with brain antigens?

Bovine spongiform encephalopathy (BSE) could be an autoimmune disease produced following exposure of cattle to feedstuffs containing bacteria showing molecular mimicry between bacterial components and bovine tissue. Analysis of molecular sequence databases (Genbank and SwissProt) shows that three bacteria (Acinetobacter calcoaceticus,Ruminococcus albus, and Agrobacter tumefaciens) share sequences with the encephalitogenic peptide of bovine myelin, while three molecules in Escherichia coli show molecular mimicry with host-encoded prion protein. Immune responses against these bacteria at both T and B cell levels may cause neurological tissue injury resembling BSE. The role of these bacteria in BSE, if any, merits further investigation.

The inhibition of thromboplastin by apolipoprotein-B and the effect of various associated lipids.

Human apolipoprotein B was purified by barium sulphate adsorption, subsequent delipidation and gel filtration. The protein was then reconstituted in soya bean lecithin and its inhibitory effect towards thromboplastin was assayed by incubation with rabbit brain thromboplastin. The use of an antibody against human apolipoprotein B diminished this inhibition. The level of inhibition of thromboplastin by the reconstituted apolipoprotein was found to be dependent on the concentration of the phospholipids with which it was reconstituted, reaching a maximum inhibition at a lipid: protein ratio of 1:1 (w/w). However, higher phospholipid concentrations or inclusion of cholesterol esters or triglycerides diminished and at certain concentrations reversed the inhibitory effect of the apolipoprotein. These results point towards apolipoprotein B as an inhibitor whose activity towards thromboplastin could be dependent on the complexes it forms with the surrounding lipids.

Antibodies to prion and Acinetobacter peptide sequences in bovine spongiform encephalopathy.

An amino acid sequence homology has been identified between the bovine prion sequence (RPVDQ) and the Acinetobacter calcoaceticus enzyme, uridine-diphosphate-N-acetyl glucosamine-1-carboxy-vinyl-transferase which also contains (RPVDQ). Class-specific IgA, IgG and IgM antibodies against synthetic peptides containing the structurally related sequences present in bovine prion and A. calcoaceticus were measured in 189 bovine spongiform encephalopathy (BSE) positive cattle, 127 BSE negative cattle and 87 healthy control animals using an ELISA technique. Class-specific IgA, IgG and IgM antibodies against the structurally related synthetic peptides were significantly elevated in BSE positive cattle when compared to BSE negative cattle (P < 0.001) and healthy control animals (P < 0.001). These autoantibodies may have a role in the pathogenesis of BSE.

Characterisation of red and white muscle myosin heavy chain gene coding sequences from antarctic and tropical fish.

To understand molecular adaptation for locomotion at different environmental temperatures, we have studied the myosin heavy chain genes as these encode the molecular motors involved. For this purpose, cDNA libraries from white (fast) and red (slow) myotomal muscle of an Antarctic and a tropical fish were constructed and from these different myosin heavy chain cDNAs were isolated. Northern and in situ hybridisation confirmed in which type of muscle these isoform genes are expressed. The cDNAs were sequenced and the structure of the ATPase sites compared. There was a marked similarity between the tropical fast myosin and the Antarctic slow myosin in the loop 1 region, which has similar amino acid side chains, charge distribution and conformation. These findings help to explain why the myofibrils isolated from white muscle of tropical fish show a lower specific ATPase activity than the white muscle of Antarctic fish but a similar activity to the Antarctic red (slow) muscle. It also provides insight into the way molecular motors in Antarctic fish have evolved to produce more power and thus ensure effective swimming at near zero temperatures by the substitution or addition of a few residues in strategic regions, which include the ATPase site.

Microparticle-associated tissue factor is recycled by endothelial cells resulting in enhanced surface tissue factor activity.

In this study the uptake of tissue factor (TF)-positive microparticles by endothelial cells and the recycling of the TF component were examined. Human dermal blood endothelial cells (HDBEC) were incubated with microparticles derived from cancer cell lines for up to 6 hours. Measurement of HDBEC cell surface TF antigen revealed two distinct peaks at 30 and 180-240 minutes post-incubation with TF-positive, but not TF-deficient microparticles. However, only the second peak was concurrent with high TF activity as determined by a chromogenic thrombin-generation assay. Annexin V-labelling of HDBEC showed phosphatidylserine exposure following 90 minutes incubation with microparticles, which explains the high TF activity associated with the second antigen peak. Analysis of TF mRNA levels revealed no de novo expression of TF mRNA in response to microparticles, and pre-incubation of cells with cycloheximide did not prevent the appearance of TF. However, blocking endocytosis with a dynamin inhibitor prolonged the disappearance and prevented the reappearance of TF antigen on the cell surface. Incubation of HDBEC with microparticles containing TF-GFP revealed the early co-localisation of TF with Rab4 and Rab5, followed by co-localisation with the late endosomal/trans-Golgi network marker Rab9, and the recycling endosome marker Rab11. siRNA-mediated suppression of Rab11 reduced the reappearance of TF on the cell surface. These data suggest a mechanism by which TF-containing microparticles are internalised by endothelial cells and the TF moiety recycled to the cell surface. Together with the exposure of phosphatidylserine, this is capable of inducing a substantial increase in the procoagulant potential of the surface of endothelial cells.

Gemcitabine versus gemcitabine plus dalteparin thromboprophylaxis in pancreatic cancer.

BACKGROUND: Annualised figures show an up to 7-fold higher incidence of vascular thromboembolism (VTE) in patients with advanced pancreatic cancer (APC) compared to other common malignancies. Concurrent VTE has been shown to confer a worse overall prognosis in APC. METHODS: One hundred and twenty three APC patients were randomised to receive either gemcitabine 1000 mg/m(2) or the same with weight-adjusted dalteparin (WAD) for 12 weeks. Primary end-point was the reduction of all-type VTE during the study period. NCT00462852, ISRCTN: 76464767. FINDINGS: The incidence of all-type VTE during the WAD treatment period (<100 days from randomisation) was reduced from 23% to 3.4% (p = 0.002), with a risk ratio (RR)of 0.145, 95% confidence interval (CI) (0.035-0.612) and an 85% risk reduction. All-type VTE throughout the whole follow-up period was reduced from 28% to 12% (p = 0.039), RR = 0.419, 95% CI (0.187-0.935) and a 58% risk reduction. Lethal VTE <100 days was seen only in the control arm, 8.3% compared to 0% (p = 0.057), RR = 0.092, 95% CI (0.005-1.635). INTERPRETATION: Weight adjusted dalteparin used as primary prophylaxis for 12 weeks is safe and produces a highly significant reduction of all-type VTE during the prophylaxis period. The benefit is maintained after dalteparin withdrawal although decreases with time.

Plasma tissue factor is a predictor for restenosis after femoropopliteal angioplasty.

BACKGROUND: In vitro studies suggest an association between raised levels of tissue factor and restenosis after coronary percutaneous transluminal angioplasty (PTA). This prospective, controlled study examined the association between plasma tissue factor concentrations and restenosis after femoropopliteal PTA. METHODS: Plasma samples from ten healthy controls and 36 patients with unilateral claudication undergoing femoropopliteal PTA were collected at baseline and, in the patients with claudication, at 24 h and 1, 3 and 6 months after PTA. Clinical assessment and arterial duplex imaging were performed before and at the same time points after PTA to identify restenosis. Plasma tissue factor was measured using a specific enzyme-linked immunosorbent assay. RESULTS: Baseline plasma tissue factor concentrations were significantly higher in patients with claudication (median 3.4 (interquartile range (i.q.r.) 1.3-7.4) ng/ml) than in controls (median 1.2 (i.q.r. 0.5-1.8) ng/ml) (P < 0.050). Baseline tissue factor concentrations were significantly higher in the ten patients with claudication who developed restenosis after PTA (median 7.0 (i.q.r. 3.4-183.5) ng/ml) than in those who did not (median 1.7 (i.q.r. 1.3-7.2) ng/ml) (P < 0.050). In addition, plasma tissue factor levels increased significantly over time in the patients who developed restenosis after PTA. CONCLUSION: High baseline and progressive increases in the plasma tissue factor concentration were useful predictors of restenosis after femoropopliteal angioplasty.

Heat shock proteins in vascular disease--a review.

INTRODUCTION: There is growing evidence that heat shock proteins (HSPs), a family of stress-inducible proteins may be involved in the pathogenesis of atherosclerotic vascular diseases. Here, we systematically review the evidence behind this notion. METHODS: A detailed literature search and extensive bibliographic review of literature relating to HSPs and atherosclerotic vascular disease. RESULTS: Atherosclerotic vascular disease is classified into four main areas of presentation: carotid, coronary, aortic and peripheral vascular disease, for consideration in this review. In each of these vascular diseases, the evidence linking HSPs and atherosclerosis is outlined in a systematic manner. Current evidence suggests that components of the immune system may be involved in the pathogenesis of atherosclerosis, with HSPs acting as auto-antigens in the immune response. HSPs are detected in atherosclerotic lesions and antibodies to HSPs are increased in patients with vascular disease; the rise often correlating with the severity of atherosclerosis. The levels of anti-HSP antibodies have been shown to be independent predictors of risk and have prognostic value. CONCLUSION: There is a strong link between heat shock protein expression and the principal manifestations of atherosclerotic vascular diseases. A better understanding of this involvement could lead to the development of new and improved treatment strategies.

Inhibition of tissue factor activity reduces the density of cellular network formation in an in vitro model of angiogenesis.

Tissue factor (TF) is a transmembrane glycoprotein that was originally recognized for its ability to initiate the extrinsic pathway of coagulation. More recently, additional functions of TF in cellular signalling have emerged, notably the role of TF in vasculogenesis and angiogenesis. We have described previously the ability of a peptide derived from the apolipoprotein B100 (apoB100) moiety of low-density lipoproteins (KRAD14) to inhibit the procoagulant function of TF. In this study, we demonstrate the ability of the KRAD14 peptide to attenuate the density of cellular network structures of T24 cells grown on specialized matrix (Matrigel). In addition, an alternative inhibitor of TF activity, the TF8 5G9 antibody, also reduces the density of cellular network formation. Targeted use of a stable structural equivalent of the KRAD14 peptide may thus prove useful in the prophylactic treatment of diseases whose pathologies feature the formation of neovascular tissue, e.g. tumour growth and metastasis, rupture of atherosclerotic plaques and retinopathy secondary to diabetes.

Comparison of the inhibitory effects of ApoB100 and tissue factor pathway inhibitor on tissue factor and the influence of lipoprotein oxidation.

The procoagulant activity of tissue factor is regulated by circulating inhibitors such as tissue factor pathway inhibitor (TFPI) and LDL. These 2 inhibitors also readily associate making the distinction between their activities difficult. We have examined the relative contributions of intact and C-terminal truncated TFPI and ApoB100. By following the inhibitory potential of the preparations, over a period of 120 minutes, it was demonstrated that TFPI and LDL-resembling particles inhibited tissue factor at different rates. TFPI was found to be a short, fast-acting inhibitor, whereas the action of LDL-resembling particles was more prolonged but slower. The oxidation of LDL has been closely associated with the development of cardiovascular disease, including atherosclerosis and thrombosis. Positively charged amino acids, particularly lysine residues, are prone to alterations via the formation of adducts by lipid peroxidation products. These residues are important in the inhibition of tissue factor activity by ApoB100. They also play an important role in the inhibitory Kunitz domains of TFPI. We have shown that the decline in the ability of LDL to inhibit tissue factor was as a result of modifications in LDL arising from oxidation. By examining the effects of oxidation on full-length and C-terminal truncated TFPI bound to LDL-resembling particles, we found that TFPI is only affected when in close association with ApoB100. C-terminal truncated TFPI was not affected significantly by oxidation. Finally, chemical modification of lysine and arginine residues reduced the overall inhibition of tissue factor by TFPI. We propose that TFPI and LDL act separately to inhibit tissue factor in vivo. However, the oxidation of LDL can alter both the endogenous activity of ApoB100 and reduce that of closely associated TFPI, compromising normal hemostasis.

The mechanism of inhibition of factor III (thromboplastin) activity by apolipoprotein B-100. Protein-protein interactions.

Factor III (thromboplastin) activity is inhibited by apoB-100, but the mechanism of inhibition is unknown. By examining the effect of purified apoB-100 on factor III activity, we showed that apoB-100 can inhibit factor III via a different mechanism from that caused by the issue-factor pathway-inhibitor, which is mainly carried on the surface of lipoproteins. Although the presence of calcium ions and factors X and VII may enhance the rate of inhibition, they are not a prerequisite for the inhibition of factor III by apoB-100. In addition, by investigating the changes in the UV spectra of apoB-100 on interaction with factor III and factors X and VII and by assigning the shifts in absorption spectra to particular amino acids, we showed that these interactions involve negative and positive residues within these proteins. By following the rates of interactions between apoB-100 and either factors III, X, VII, a two-step mechanism for the inhibition process involving factors X and VII was postulated. In this mechanism, the primary interaction of apoB-100 with factor III is followed by a rate-limiting step that can be accelerated by the presence of either factor X or VII and leads to the inhibition of factor III. Furthermore, a computer-based analysis of the sequences of factor III revealed a possible binding site for apoB-100.

Identification of a domain in apolipoprotein B-100 that inhibits the procoagulant activity of tissue factor.

The ability of low-density lipoprotein (LDL) to inhibit the procoagulant activity of tissue factor is mediated by a direct protein-protein interaction involving apolipoprotein (apo) B-100. A lysine-rich sequence within apo B-100 (residues 3121-3217), which we have termed lysine-rich apo B-100-derived (KRAD)-98 peptide, may be responsible for its activity. Within this region, residues 3147-3160 (KRAD-14) contain an exceptionally high proportion of positive amino acids. Both recombinant KRAD-98 and KRAD-14 peptides inhibited the procoagulant activity of tissue factor by preventing the activation of factor VII. KRAD-14 also inhibited the prothrombinase components, factors Xa and V. In comparison with the parent protein (apo B-100), KRAD-14 peptide displayed a 20-fold enhancement in the rate of inhibition, whereas KRAD-98 peptide exhibited a rate closer to that of apo B-100. Mutational analysis of KRAD-14 peptide revealed three adjacent amino acids, alteration of which greatly reduced the inhibitory potential of this peptide. A peptide derived from tissue factor (residues 58-66) was found to act co-operatively with tissue factor itself, but also augmented the inhibition of tissue-factor activity by apo B-100. In conclusion, LDL may be a physiological regulator of haemostatic mechanisms through the interactions of lysine-rich domains of apo B-100 with tissue factor.