Tissue Factor Synthesis Research Articles

Plasmin enhances cell surface tissue factor activity in mesothelial and endothelial cells.

Mesothelial cells that line the thoracic cavity play an important role in maintaining the local balance between procoagulant and fibrinolytic activity, a role akin to the endothelial cells in blood vessels. The mechanism(s) responsible for increased tissue factor (TF) expression in mesothelial cells in response to injury are at present unclear. To investigate whether plasmin or thrombin, two major proteases that may be generated on the pleural surface upon injury, induce TF expression in human pleural mesothelial cells (HMC) and elucidate the underlying mechanism(s). Confluent monolayers of HMC and human umbilical vein endothelial cells (HUVEC) were exposed to plasmin or thrombin for varying time periods and TF expression was analyzed by measuring its activity in a factor Xa generation assay, TF antigen levels by immunoblot analysis and TF mRNA by Northern blot analysis. Both plasmin and thrombin treatments increased cell surface TF activity in HMC by 3- to 4-fold. In contrast to thrombin, plasmin-induced TF activity is not dependent on the de novo synthesis of TF. In HUVEC, plasmin had a minimal effect on unperturbed HUVEC whereas it markedly increased TF activity of activated HUVEC. Plasmin treatment neither affected anionic phospholipid levels at the cell surface nor released protein disulfide isomerase, an oxidoreductase protein that was newly described to play a role in TF activation. Plasmin cleaved cell-associated TFPI. Thrombin up-regulates TF activity in HMC through the transcriptional activation of TF whereas plasmin increases TF activity by inactivating the cell-associated TFPI by a limited proteolysis.

Insulin inhibits tissue factor expression in monocytes.

Platelets from healthy subjects are inhibited by insulin but type 2 diabetes mellitus (T2DM) platelets have become insulin-resistant, which might explain their hyperactivity. In the present study we investigated whether monocytes are responsive to insulin. LPS-induced tissue factor (TF) upregulation was measured in human monocytes and monocytic THP-1 cells in a factor Xa generation assay. Insulin (0.1-100 nmol L(-1)) induced a dose-dependent inhibition in both cell types and in monocytes 100 nmol L(-1) insulin inhibited cytosolic, membrane-bound and microparticle TF by 32 +/- 2, 27 +/- 3 and 52 +/- 4% (n = 3). Insulin induced Tyr phosphorylation of the insulin receptor (INS-R) and formation of an INS-R - G(i)alpha(2) complex, suggesting interference with LPS-induced cAMP control. Indeed, insulin interfered with LPS-induced cAMP decrease and TF upregulation in a manner similar to an inhibitor of G(i) (pertussis toxin) and agents that raise cAMP (iloprost, forskolin, IBMX) reduced TF upregulation. Although LPS failed to raise cytosolic Ca(2+), quenching of Ca(2+) increases (BAPTA-AM) reduced and induction of Ca(2+) entry (ionophore, P2X7 activation) enhanced upregulation of TF mRNA and procoagulant activity. Insulin interfered with MCP-1-induced Ca(2+) mobilization but not with ATP-induced Ca(2+) rises. Insulin inhibits TF expression in monocytes and monocyte-derived microparticles through interference with G(i)alpha(2)-mediated cAMP suppression, which attenuates Ca(2+)-mediated TF synthesis.

Drug-eluting stent thrombosis: A lesson in vascular biology

Early vessel closure due to dissection and elastic recoil were reduced substantially after the introduction of coronary stents. However, later restenosis resulting from cell proliferation and matrix deposition remained an important clinical issue. With the introduction of drug-eluting stents, the problem of restenosis has been substantially reduced. However, concerns about the risk of late drug-eluting stent thrombosis remain. The vascular biology of late drug-eluting stent thrombosis can be considered under the topics of abnormalities of coagulation, and of flow. Delayed stent re-endothelialization, stent polymer-induced inflammation and increased tissue factor synthesis increase the risk of stent thrombosis. Malapposition due to late remodelling of the artery, coronary aneurysm formation and abnormal endothelial-dependent vasomotion may contribute to flow abnormalities that could result in thrombus formation. Dual antiplatelet therapy with acetylsalicylic acid and clopidogrel reduces the risk of both early and late stent thrombosis. Consequently, the need for prolonged use of dual antiplatelet therapy is currently under consideration for patients with drug-eluting stents at high risk of very late stent thrombosis.

Platelets and wound healing

Platelets help prevent blood loss at sites of vascular injury. To do this, they adhere, aggregate and form a procoagulant surface favoring thrombin generation and fibrin formation. In addition, platelets express and release substances that promote tissue repair and influence processes such as angiogenesis, inflammation and the immune response. They contain large secretable pools of biologically active proteins, while newly synthesized active metabolites are also released. Although anucleate, activated platelets possess a spliceosome and can synthesize tissue factor and interleukin-1beta. The binding of secreted proteins within a developing fibrin mesh or to the extracellular matrix can create chemotactic gradients favoring the recruitment of stem cells, stimulating cell migration and differentiation, and promoting repair. The therapeutic use of platelets in a fibrin clot has a positive influence in clinical situations requiring rapid healing. Dental implant surgery, orthopaedic surgery, muscle and tendon repair, skin ulcers, hole repair in eye surgery and cardiac surgery are situations where the use of autologous platelets accelerates healing. We now review the ways in which platelets participate in these processes.

Plasmin Enhances Cell Surface Tissue Factor Activity without Increasing Tissue Factor Antigen Levels at the Cell Surfaces of Mesothelial and Endothelial Cells.

Mesothelial cells that line the thoracic and peritoneal cavities play an important role in protecting the heart, lungs and internal organs and keeping the surfaces free of friction and non-adhesive. Although human mesothelial cells in culture express low levels of tissue factor (TF), TF expression was not detected in vivo in the mesothelial lining of normal pleura but was detected in the mesothelium overlying injured or inflamed lung tissue. Inflammation in the lung parenchyma predisposes to the development of inflammatory exudative pleural effusions and pleural loculation, but the mechanism(s) responsible for TF expression in mesothelial cells under such conditions remains unclear. In the present study, we investigated whether plasmin and thrombin, two major proteases present in exudative pleural effusions, induce TF expression in cultured primary human pleural mesothelial cells (HMC). Confluent monolayers of HMC were treated with plasmin (50 nM) and thrombin (5 nM) for varying time periods. TF expression was analyzed by measurement of cell surface TF activity in FX activation assay, TF antigen by Western blotting and TF mRNA by Northern blot analysis. Both plasmin and thrombin increased cell surface TF activity by 3–4 fold. Thrombin markedly increased TF antigen levels. Surprisingly, only a minimal increase in TF antigen levels was seen in the plasmin-treated cells and no increase in TF mRNA was observed in these cells. Interestingly, 75% of the increase in TF activity was observed within 30 min of plasmin treatment. In thrombin-stimulated cells, TF activity increased slowly, reaching maximum at 4–6 h. Treatment of HMC with actinomycin-D or cycloheximide prior to the addition of the protease failed to inhibit the plasmin-induced TF activity whereas they completely attenuated the thrombin-induced TF activity. These data suggest that plasmin and thrombin enhance TF activity in HMC by different mechanisms, and that plasmin-induced TF activity is independent of de novo synthesis of TF. We also examined the effect of plasmin on TF activity in human umbilical vein endothelial cells (HUVEC). Although plasmin had a minimal effect on naive HUVEC, 30 min plasmin treatment increased cell surface TF activity of thrombin-stimulated endothelial cells by more than two-fold. In our earlier studies, we showed that proteases could mobilize intracellular TF to the cell surface of fibroblasts and thus enhance TF activity independent of de novo synthesis of TF. However, FACS analysis and 125I-FVIIa binding to cell surface TF revealed no significant differences in TF antigen levels at the surfaces of control and plasmin-stimulated HMC. Since exposure of anionic phospholipids at the outer cell surface membrane can increase cell surface TF activity, we next sought to determine if plasmin increases anionic phospholipids on the outer bilayer of the HMC cell membrane. Prothrombin activation assays showed no increase in anionic phospholipids at the surface in plasmin-treated cells. Although recent studies suggest that disulfide bond formation of cysteines in TF enhances TF procoagulant activity, our recent studies in other cell model systems suggest that this mechanism is unlikely to account for plasmin-mediated induction of cell surface TF activity in HMC. While further studies are required to exclude this possibility, our present data indicate that plasmin enhances cell surface TF activity of HMC and HUVEC by a novel mechanism that is yet to be elucidated.

Suppression of HUVEC tissue factor synthesis by antisense oligodeoxynucleotide

Tissue factor (TF) is an important regulator and effector molecule of coagulation. It is primary known as a cofactor for factor VIIa-mediated triggering of blood coagulation, which proceeds in a cascade of extracellular reactions, ultimately resulting in thrombin formation. In sepsis, expression of TF by activated monocytes, macrophages and endothelial cells may lead to disseminated intravascular coagulation. Further studies have suggested that TF also plays non-haemostatic roles in blood vessel development, tumor angiogenesis, metastasis and inflammation. In the present study we examined the feasibility of inhibiting lipopolysaccharide (LPS)-induced TF expression in cultured human umbilical vein endothelial cells (HUVECs) using a modified phosphorothioate antisense oligodeoxynucleotide targeted to the TF mRNA. CD31 receptor-mediated endocytosis was used as a means of delivering TF antisense oligomer to HUVECs. This DNA carrier system consists of anti-CD31 antibody conjugated to the antisense. Co-exposure of HUVECs with TF antisense and LPS resulted in 54.6 ± 3.2% suppression of TF activity when compared with control LPS stimulated cells. The antisense also reduced the LPS-induced TF mRNA level. Control experiments with TF sense and mismatched antisense oligomers were performed to exclude non-specific inhibitory effects. The cytotoxicity of the antisense oligomer conjugate was also evaluated. Results demonstrate that this TF antisense oligomer specifically suppressed the synthesis of biologically active endothelial TF and that antisense oligomers might represent a useful tool in the investigation of endothelial TF function/biology.

Human platelets synthesize and express functional tissue factor

AbstractThe source and significance of bloodborne tissue factor (TF) are controversial. TF mRNA, protein, and TF-dependent procoagulant activity (PCA) have been detected in human platelets, but direct evidence of TF synthesis is missing. Nonstimulated monocyte-free platelets from most patients expressed TF mRNA, which was enhanced or induced in all of them after platelet activation. Immunoprecipitation assays revealed TF protein (mainly of a molecular weight [Mr] of approximately 47 kDa, with other bands of approximately 35 and approximately 60 kDa) in nonstimulated platelet membranes, which also increased after activation. This enhancement was concomitant with TF translocation to the plasma membrane, as demonstrated by immunofluorescence–confocal microscopy and biotinylation of membrane proteins. Platelet PCA, assessed by factor Xa (FXa) generation, was induced after activation and was inhibited by 48% and 76% with anti-TF and anti-FVIIa, respectively, but not by intrinsic pathway inhibitors. Platelets incorporated [35S]-methionine into TF proteins with Mr of approximately 47 kDa, approximately 35 kDa, and approximately 60 kDa, more intensely after activation. Puromycin but not actinomycin D or DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) inhibited TF neosynthesis. Thus, human platelets not only assemble the clotting reactions on their membrane, but also supply their own TF for thrombin generation in a timely and spatially circumscribed process. These observations simplify, unify, and provide a more coherent formulation of the current cell-based model of hemostasis.

Differential effects of somatostatin on circulating tissue factor procoagulant activity and protein

The tissue factor (TF) pathway is the primary mechanism for initiation of blood coagulation. Circulating blood contains TF, which originates mainly from monocytes and is thrombogenic. The presence of somatostatin (SMS) receptors on monocytes suggests the possibility that SMS may regulate TF synthesis and/or release. Circulating TF procoagulant activity (TF-PCA), factor VIIa activity (FVIIa; clotting assays), TF antigen (TF-Ag; ELISA), prothrombin fragment 1.2 (F1.2), thrombin-antithrombin complexes (ELISAs), CD40 ligand expression on platelets, and monocyte-platelet aggregates (flow cytometry) were determined in blood from normal volunteers undergoing 24 h of basal glucose/basal insulin (BG/BI) clamps and high-glucose/high-insulin (HG/HI) clamps with and without SMS. Infusions of SMS under basal conditions (BG/BI) raised TF-PCA 1.8-fold (P < 0.03), TF-Ag 2.3-fold (P < 0.001), and TF expression on monocytes by 36% (P < 0.001) and decreased plasma levels of FVIIa by 30% (P < 0.001). Infusion of SMS reduced the 8.6-fold HG/HI-induced increase in TF-Ag by 26% and the 8.6-fold increase in TF-PCA by 100%. SMS also prevented the 60% increase in TF expression on monocytes, the 2.2-fold increase in F1.2, the 40% increase in CD40L expression on platelets, and the 17% increase in monocyte-platelet aggregates seen during HG/HI. We conclude that SMS completely prevented HG/HI-induced TF activation in normal volunteers and may be of use to reduce the procoagulant state and acute vascular events in hyperinsulinemic insulin-resistant patients with type 2 diabetes.

Tissue factor expression by a human kidney proximal tubular cell line in vitro: a model relevant to urinary tissue factor secretion in disease?

Aim: To study baseline and stimulated tissue factor (TF) production from a normal, albeit immortalised, human kidney proximal tubular cell line (HKC-5), in order to establish a model for investigating...

Long‐term incubation with IL‐4 and IL‐10 oppositely modifies procoagulant activity of monocytes and modulates the surface expression of tissue factor and tissue factor pathway inhibitor

Monocytes can be induced to express both tissue factor (TF) and its inhibitor, TF pathway inhibitor-1 (TFPI-1). A short incubation (<6 h) with interleukin (IL)-4 and IL-10, two potent deactivators of monocyte functions, has been shown to modulate the synthesis and expression of TF by monocytes activated by lipopolysaccharide, but the consequences of longer incubations (up to 96 h) on both TF and TFPI-1 are unknown. The results of this study showed that adherent monocytes in culture spontaneously expressed TF and TFPI and that prolonged incubation with IL-10 induced a time- and dose-dependent decrease of monocyte TF synthesis, and an accumulation of TF/TFPI-1 complexes at the moncyte surface, suggesting a decreased clearance of these complexes. In contrast, IL-4 induced a time- and dose-dependent increase in TF synthesis, which remained intracytoplasmic, as shown by confocal microscopy. Surprisingly, TF:antigen (Ag) was decreased at the monocyte surface, but the procoagulant activity (PCA) of IL-4-treated monocytes was increased, as a result of more pronounced decrease of TFPI-1:Ag expression than that of TF. In conclusion, prolonged incubation with IL-4 and IL-10 oppositely modified PCA of cultured monocytes, and altered TF and TFPI trafficking and clearance. These data explain the respective deleterious or benefit effects of IL-4 or IL-10 in atherothrombosis.

Intravascular Tissue Factor (TF) Is Predominantly Platelet-Associated during the Aplastic Phase of Hematopoietic Stem Cell Transplantation (HSCT).

TF is a 47 kDa cell-bound glycoprotein that initiates coagulation upon contact with factor VII(a). Both monocytes and cells derived from the vessel wall (such as endothelial cells) are capable of TF synthesis, and both intact cells and microparticles (MPs) derived from them are considered to be the principal source(s) of intravascular TF. Data from in vitro systems and animal models suggest that MP-borne TF may be transferred to activated platelets via a P-selectin dependent pathway. We previously demonstrated that during the aplastic phase of HSCT, about 55% of baseline (pre-transplant) levels of circulating whole blood TF procoagulant activity (PCA) remained detectable (Thromb & Haemost 2001; 85:250). This study was therefore undertaken to identify whether this residual TF activity is accounted for by intact cells or MPs, and to determine whether cells derived from the vessel wall or the hematopoietic compartment are responsible. MP-associated TF PCA was measured using a recently described MP capture assay (Blood 2004; 103:4545). MP-associated TF PCA declined by 89% at day 0 and by 77% at day +7 compared to baseline levels measured at day −7, with a subsequent normalization of activity paralleling engraftment. TF PCA was assayed on fractionated platelets and mononuclear cells obtained on days −7,0,7,14 and 21 from 11 patients undergoing HSCT. Mononuclear cell and platelet fractions were obtained by differential centrifugation and exposed to calcium ionophore to maximally ‘de-encrypt' TF PCA. Following pre-incubation with FVIIa, the clotting time of added plasma was recorded, and the amount of TF calculated by reference to a standard curve generated using Innovin™. In parallel with the drop in circulating monocyte counts, a significant decline in TF PCA in both the mononuclear cell (1855±690 at day −7 vs. 74±37 pg/ml at day +7, p=0.003) and platelet (260±45 at day −7 vs. 51±16 pg/ml at day +7, p=0.05) fractions was noted. TF PCA in these fractions subsequently returned towards baseline levels with engraftment. Notably, while platelet-associated TF PCA accounted for only 12% of the total cell-associated TF PCA at baseline (day −7), it accounted for fully 59% at day 0 and 41% at day +7. This led us to postulate that transfused platelets might contribute to circulating TF during the aplastic phase. Within 1 hour of receiving leuco-depleted platelet transfusion, total cell-associated (platelet + mononuclear cell) TF PCA had increased significantly (142 ± 43 pg/ml to 317 ± 55 pg/ml, p=0.015), confirming that transfused platelets are indeed a source of TF. While this augmentation of cell-associated TF PCA was primarily seen in the platelet fraction, an increase in mononuclear cell-associated TF PCA was also noted. Conclusion: The majority of cell-associated TF PCA is associated with the mononuclear cell fraction in whole blood. However, with the onset of severe leucopenia during HSCT, the platelet pool becomes the dominant location for TF. Our data are in agreement with recent transgenic mouse model data demonstrating that the bone marrow - not the vessel wall - is the dominant source of both cell- and MP-associated intravascular TF.

Thrombin and Tumor Necrosis Factor α Synergistically Stimulate Tissue Factor Expression in Human Endothelial Cells: REGULATION THROUGH c-Fos AND c-Jun

Tissue factor is critically important for initiating the activation of coagulation zymogens leading to the generation of thrombin. Quiescent endothelial cells do not express tissue factor on their surface, but many stimuli including cytokines and coagulation proteases can elicit tissue factor synthesis. We challenged human endothelial cells simultaneously with tumor necrosis factor alpha (TNFalpha) and thrombin because many pathophysiological conditions, such as sepsis, diabetes, and coronary artery disease, result in the concurrent presence of circulating inflammatory mediators and activated thrombin. We observed a remarkable synergy in the expression of tissue factor by thrombin plus TNFalpha. This was due to altered regulation of the transcription factors c-Jun and c-Fos. The activation of c-Jun was greater and more sustained than that obtained with either thrombin or TNFalpha alone. Thrombin-stimulated expression of c-Fos was both enhanced and prolonged by the concurrent presence of TNFalpha. These changes support the increased availability of c-Jun/c-Fos AP-1 complexes for mediating transcription at the tissue factor promoter. Transcription factors downstream of the extracellular signal-regulated kinases as well as changes in NFkappaB regulation were not involved in the synergistic increase in tissue factor expression by thrombin and TNFalpha. Thus, concurrent exposure of vascular endothelial cells to cytokines and procoagulant proteases such as thrombin can result in greatly enhanced tissue factor expression on the endothelium, thereby perpetuating the prothrombotic phenotype of the endothelium.

The long pentraxin PTX3 up-regulates tissue factor in activated monocytes: another link between inflammation and clotting activation.

Pentraxin-3 (PTX3), an acute-phase protein that belongs to the family of the PTXs, is found elevated in septic shock and increased in patients with acute myocardial infarction. As tissue factor (TF) plays a key role in thrombosis and inflammation associated with atherosclerosis and as we have recently reported that PTX3 increases TF synthesis in endothelial cells, we tested whether PTX3 could modulate TF expression in monocytes. Monocytes from peripheral blood of healthy donors were incubated with highly purified PTX3 with or without lipopolysaccharide (LPS). Cells were then disrupted, and procoagulant activity was assessed by a one-stage clotting time. PTX3 enhanced TF activity and antigen from LPS-stimulated monocytes in a dose-dependent way. The effect was specific, as other PTXs, such as C-reactive protein and serum amyloid P component, were ineffective. Moreover, the increase in activity was specific for LPS, as in the presence of other TF-inducing agents such as interleukin-1beta and tumor necrosis factor alpha, PTX3 was not effective. The increase in TF activity requires mRNA synthesis, as assessed by polymerase chain reaction. The mechanism by which PTX3 modulates TF synthesis resides in an enhanced IkappaB, alpha phosphorylation and degradation and increased migration of the transacting factor c-Rel/p65 into the nucleus, as determined by Western blot and electro-mobility shift assay. These results show that PTX3 is an enhancer of the expression of TF by mononuclear cells. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. PTX3 increases TF expression, thus potentially playing a role in thrombogenesis and wound healing.

Identifying patients at risk of coronary vascular disease: the potential role of inflammatory markers

Inflammation is intimately involved in the pathogenesis of cardiovascular disease (CVD). The measurement of inflammatory markers may be a potent method for identifying individuals with increased inflammation at risk of future cardiovascular events. High-sensitivity C-reactive protein (hs-CRP), one marker of inflammation, is a strong independent predictor of CVD and can be used to identify individuals at increased risk who would otherwise be missed if standard lipid measurements only were used. Furthermore, CRP may actively participate in the pathogenic progression of atherosclerotic disease by activating complement, inducing synthesis of tissue factor, influencing nitric oxide levels, and enhancing foam cell uptake of low-density lipoprotein cholesterol. Simple, inexpensive methods for measuring hs-CRP are commercially available, making the measurement of CRP levels feasible in clinical settings. Together, these data suggest that the ability to accurately predict CVD risk may be significantly increased by including CRP measurements in risk assessment models. In addition, the identification of increased inflammation suggests that rational therapy should be applied and favours treatments with anti-inflammatory action.

C-reactive protein does not directly induce tissue factor in human monocytes.

It is generally assumed that C-reactive protein (CRP) induces synthesis of tissue factor (TF) in monocytic cells, thereby potentially initiating intravascular blood coagulation. We aimed to elucidate the mechanism of CRP-induced TF expression in monocytes and monocyte-derived macrophages (MDMs) in vitro. Monocytes were isolated from the blood of healthy donors and cultured with or without CRP or lipopolysaccharide (LPS) to study the time course of TF antigen and TF mRNA expression. Addition of 100 microg/mL CRP did not result in a significant increase in TF antigen (range: 9 to 163 pg/10(6) cells, n=11) and TF mRNA (relative number of TF transcripts; N(TF)=0.01 to 0.33), when compared with nonstimulated cells (TF antigen 7 to 46 pg/10(6) cells, N(TF)=0.01 to 0.13). Variation of CRP concentration and exposure time did not affect the TF response. Similar results were obtained in monocytes cultured in suspension and in MDMs. In contrast, TF was strongly induced by 10 microg/mL LPS (TF antigen 1125 to 6120 pg/10(6) cells, N(TF)=5.94 to 23.43). Cultured monocytes did express FcRgammaII, a putative CRP receptor, and addition of CRP induced a 7-fold increase in the production of monocyte chemoattractant protein-1 (MCP-1). Interestingly, CRP addition to peripheral blood mononuclear cells (PBMCs) did result in TF expression on monocytic cells. The absence of TF induction after incubation of purified monocytes with CRP indicates that CRP is unable to induce TF expression in monocytes and MDMs directly. The presence of CRP-induced TF expression in PBMCs suggests that CRP can induce TF indirectly, probably through cross-talk between cells.

Tissue factor in neutrophils: no

Tissue factor in neutrophils: no

M type 1 and 3 group A streptococci stimulate tissue factor-mediated procoagulant activity in human monocytes and endothelial cells.

Streptococcal toxic shock syndrome (StrepTSS) is an invasive infection characterized by marked coagulopathy, multiple organ failure, and rapid tissue destruction and is strongly associated with M type 1 and 3 group A streptococci (GAS). Initiation of the coagulation cascade with formation of microvascular thrombi contributes to multiple organ failure in human cases of gram-negative bacteremia; however, little is known regarding the mechanism of coagulopathy in StrepTSS. Thus, we investigated the abilities of several strains of M type 1 and 3 GAS isolated from human cases of StrepTSS to stimulate production of tissue factor (TF), the principal initiator of coagulation in vivo. Washed, killed M type 1 and 3 GAS, but not M type 6 GAS, elicited high-level TF-mediated procoagulant activity from both isolated human monocytes and cultured human umbilical vein endothelial cells. M type 1 GAS consistently elicited higher levels of TF from monocytes than did M type 3 GAS. GAS-induced TF synthesis in monocytes did not correlate with production of tumor necrosis factor alpha or interleukin-8. Conversely, M type 3 GAS were consistently more potent than M type 1 GAS in stimulating endothelial cell TF synthesis. These results demonstrate that (i) M type 1 and 3 strains of GAS are potent inducers of TF synthesis, (ii) GAS-induced TF synthesis is not simply an epiphenomenon of cytokine generation, and (iii) induction of TF in endothelial cells and monocytes may be M type specific. In total, these findings suggest that a novel interaction between GAS and host cells contributes to the observed coagulopathy in StrepTSS.

Antiphospholipid antibodies and thrombosis: pathogenesis of antiphospholipid syndrome

Antiphospholipid antibodies are present in a wide range of infectious and autoimmune diseases. Antiphospholipid antibodies, in particular anticardiolipin antibodies (aCL), lupus anticoagulants and antiprothrombin antibodies, are of considerable clinical importance because of the close association with predominant clinical features of venous and arterial thrombosis and pregnancy morbidity. The term antiphospholipid syndrome (APS) has been used to define this set of pathologic features. Recognition of this syndrome is now better understood worldwide as related clinical implications are now more well defined. aCL found in APS patients are directed against phospholipid-binding plasma or serum proteins, in particular, β2-glycoprotein I (β2-GPI). Such aCL (anti-β2-GPI autoantibodies) recognized epitopes appearing on the β2-GPI molecule when β2-GPI interacts with a lipid membrane composed of negatively charged phospholipids or when β2-GPI is adsorbed on a polyoxygenated polystyrene plate treated with γ-irradiation or electrons. Anti-β2-GPI antibodies have been found to activate endothelial cells by inducing a proinflammatory and procoagulant phenotype sustained by the upregulation of adhesion molecule (E-selectin, intracellular adhesion molecule-1 and vascular cell adhesion molecule-1) expression, synthesis and secretion of cytokines, chemokines, endothelin-1 and tissue factor. Anti-β2-GPI antibody binding has been shown to induce NF-κB translation, leading to proinflammatory cell phenotypes similar to that elicited by interaction with lipopolysaccharide and proinflammatory cytokines (IL-1β, tumor necrosis factor alpha). Very recently, it was reported that anti-β2-GPI antibodies activate cells through the MyD88-dependent pathway, therefore implicating members of the Toll-like receptors family. In this lecture, we will discuss the relation between the anti-β2-GPI antibodies, the Toll-like receptors/IL-1 receptor family on the cell surface and the pathogenesis of APS.

Effects of HMG-CoA reductase inhibitors on coagulation and fibrinolysis processes.

Recent large clinical trials have demonstrated that HMG-CoA reductase inhibitors, or statins, markedly reduce morbidity and mortality when used in the primary and secondary prevention of cardiovascular disease. It has been established that the benefits of statin therapy in cardiovascular disease can be explained not only by the lipid-lowering potential of statins but also by nonlipid-related mechanisms (so-called "pleiotropic effects") that contribute to the positive effect of statins on the incidence of cardiovascular events. The coagulation and fibrinolytic systems are two separate but reciprocally linked enzyme cascades that regulate the formation and breakdown of fibrin. Numerous studies have demonstrated that disturbances of coagulation and fibrinolysis contribute to the development and progression of atherosclerosis, and that they affect the incidence of atherosclerosis-related clinical events. High plasma levels or activities of fibrinogen, factor VII, factor VIII, von Willebrand factor (vWF), soluble thrombomodulin, tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) are thought to be associated with increased morbidity and mortality related to cardiovascular disease. Experimental studies and many clinical studies have recently shown that statins produce favourable effects on haemostatic parameters, including those that are risk factors for cardiovascular disease. Statins diminish procoagulant activity, which is observed at different stages of the coagulation cascade, including tissue factor (TF) activity, conversion of prothrombin to thrombin and thrombin activity. In some studies, statins also reduced fibrinogen levels. By altering the levels and activities of tPA and PAI-1, statins seem to stimulate fibrinolysis. The data on the effects of combined treatment with statins and other drugs on haemostasis are rather limited. They suggest that statins combined with fibric acid derivatives, omega-3 fatty acids and 17beta-estradiol are superior to statins alone. The only two clinical studies performed in patients with acute coronary syndromes showed a relatively weak effect of statins on haemostasis in those patients. Although various statins may produce different effects on individual variables, there are no convincing data showing that differences in their physicochemical and pharmacokinetic properties significantly alter their net effect on excessive procoagulant activity. Apart from the lipid-lowering effect, statins suppress the synthesis of several important nonsterol isoprenoids derived from the mevalonate pathway, especially farnesyl and geranylgeranyl pyrophosphates, which via enhanced protein prenylation, are involved in the regulation of many cellular processes. It is presumed that the inhibitory effect of statins on the mevalonate pathway is involved in the regulation of some key steps of coagulation and fibrinolysis processes. In this way they probably regulate the synthesis of TF, tPA and PAI-1, and perhaps they also control the generation and activity of thrombin. The beneficial effects of statins on coagulation and fibrinolysis may be responsible for their ability to decrease the number of cardiovascular events. The lipid-independent effects of statins on haemostasis may contribute to the marked decrease in the incidence rates of mortality, hospitalisation and revascularisation in patients treated with these drugs.

Demonstration of a tissue factor pathway inhibitor 2 messenger RNA synthesis by pure villous cytotrophoblast cells isolated from term human placentas.

Tissue factor pathway inhibitor 2 (TFPI-2), a Kunitz-type proteinase inhibitor, might play an important role during placenta growth by regulating trophoblast invasion and differentiation. Many TFPI-2 transcripts have been detected in syncytiotrophoblast cells, but conflicting results have been reported concerning TFPI-2 synthesis by the cytotrophoblast. To address this issue, we developed a method to isolate pure preparations of human villous cytotrophoblast cells from normal term placentas, and the synthesis of tissue factor, TFPI-1, and TFPI-2 mRNAs was then evaluated. Cells were isolated by trypsin-DNase-EDTA digestion, followed by Percoll gradient separation and immunodepletion of human leukocyte antigen-positive cells. The quality of villous cytotrophoblast cells was verified by electron microscopy. Purity of cell preparations was assessed by labeling cells with GB25, a monoclonal antibody specific to villous trophoblast cells, and by checking the absence of contaminating cells using anti-CD9 antibody. The lack of hCG, CD32 mRNA, and tissue factor mRNA also indicated the absence of contaminating cells. Using competitive reverse transcription polymerase chain reaction, we showed that freshly isolated villous cytotrophoblast cells synthesized significant levels of TFPI-1 mRNA and larger amounts of TFPI-2 mRNA. TFPI-1 and TFPI-2 mRNA synthesis remained unchanged when cytotrophoblast cells were cultured in complete medium and evolved as a multinucleated syncytiotrophoblast. These results indicate that the villous cytotrophoblast and syncytiotrophoblast are both important sites of TFPI-2 synthesis in the human placenta. This study also indicates that tissue factor detection should be used systematically to check the purity of cytotrophoblast cell preparations because it allows detection of contamination by monocytes/macrophages and by syncytial fragments.