Tissue factor and IL8 production by P-selectin-dependent platelet–monocyte aggregates in whole blood involves phosphorylation of Lyn and is inhibited by IL10

Summary. Background: P‐selectin and CD40L expressed by activated platelets induce tissue factor (TF) and inflammatory cytokines in monocytes, but little is known of the cellular signaling pathways involved. The anti‐inflammatory cytokine IL10 reduces atherosclerotic plaque formation. Objectives: To evaluate the importance of P‐selectin upon platelet–monocyte aggregate (PMA) formation in thrombin receptor activator peptide (TRAP) stimulated whole blood, the P‐selectin–P‐selectin glycoprotein ligand (PSGL)‐1‐induced cellular signaling pathway, and the effects of IL10 on these functions. Methods: TF, IL8, and monocyte chemotactic protein‐1 (MCP‐1) production, PMAs and phosphorylation of Lyn were analyzed in whole blood, purified monocytes, and vitamin D₃‐differentiated U‐937 cells stimulated with TRAP or P‐selectin with or without IL10. Anti‐P‐selectin or anti‐CD40L antibodies (Abs), Src‐kinases inhibitors, SU6656 or PP2, were added in some experiments. Results: TRAP and P‐selectin increased TF, IL8, and MCP‐1 mRNA in whole blood and purified monocytes. Anti‐P‐selectin Ab reduced TRAP‐induced PMA formation by 80 ± 2% (P = 0.001) and production of TF (P = 0.04) and IL8 (P = 0.01). IL10 and SU6656 had no effect on PMA formation, although both significantly reduced TF (P = 0.002 and P = 0.02) and IL8 (P = 0.009 and P = 0.001) mRNA upon TRAP and P‐selectin stimulation. Induced Lyn phosphorylation in monocytes was diminished by SU6656 (P = 0.02), anti‐P‐selectin Ab (P = 0.02), and IL10 (P = 0.03) upon TRAP or P‐selectin stimulation. These results were confirmed in the vitamin D₃‐differentiated U‐937 cells. Conclusions: The formation of PMAs in whole blood was P‐selectin‐dependent in the long term. P‐selectin–PSGL‐1‐induced TF and IL8 expression through Lyn phosphorylation, and part of the inhibitory effect of IL10 depends on reduced phosphorylation.     

Anti-prothrombin antibodies predict thrombosis in patients with systemic lupus erythematosus: a 15-year longitudinal study

Summary. Objective: To evaluate the role of anti‐prothrombin (anti‐PT) antibodies in predicting thrombosis in patients with systemic lupus erythematosus (SLE). Methods: An inception cohort of 101 SLE patients (12 males, 89 females; mean age 30 ± 8 years), was considered. Clinical and laboratory evaluations were regularly performed during a 15‐year follow‐up (median 108 months) with a special focus on thromboembolic events. Serum samples were collected at time of diagnosis and at least once a year thereafter. IgG and IgM anti‐PT, anti‐cardiolipin (aCL) and anti‐β₂glycoprotein I (β₂GPI) antibodies were measured by enzyme‐linked immunosorbent assay (ELISA); lupus anticoagulant (LAC) was assayed by the dilute Russell’s viper venom time and activated partial thromboplastin time tests. The analytical specificity of anti‐PT ELISA was investigated. The timing of thrombosis occurrence was calculated using the Kaplan–Meier method. Results: In the 15‐year follow‐up, thrombosis occurred in 14 out of the 101 patients: venous thrombosis in nine cases and arterial thrombosis in five. IgG and/or IgM anti‐PT, anti‐β₂GPI and aCL antibodies, and LAC activity were detected in ten, nine, seven, and nine cases, with sensitivity for thrombosis of 71.4%, 64.3%, 50% and 64.3%, respectively. Thrombosis‐free survival was 90% at 5 years and 85.8% at 10 and 15 years, respectively. Thrombosis was predicted by anti‐PT (P = 0.001), anti‐β₂GPI antibodies (P = 0.002) and LAC activity (P = 0.001). Moreover, the risk of thrombosis progressively increased with the number of positive antiphospholipid antibody tests. The presence of four positive antibody tests was associated with a risk of thrombosis thirtyfold higher than in their absence. Conclusions: This longitudinal study shows that IgG anti‐PT antibodies are predictors of thrombosis in SLE patients.     

Clinical accuracy for diagnosis of antiphospholipid syndrome in systemic lupus erythematosus: evaluation of 23 possible combinations of antiphospholipid antibody specificities

Summary. Objectives: To evaluate the clinical accuracy of antiphospholipid antibody (aPL) specificities both individually and/or in combination, in a wide cohort of systemic lupus erythematosus (SLE) patients in an attempt to identify a panel of tests that may provide the best accuracy for diagnosing antiphospholipid syndrome (APS). Patients and Methods: This study included 230 patients (218 women, mean age 42.7 ± 11.9 years, mean disease duration 12.2 ± 8.7 years), all fulfilling the 1982 criteria for SLE. All patients were tested for lupus anticoagulant (LA), anti‐cardiolipin (aCL), anti‐β₂glycoprotein I (anti‐β2GPI), solid phase anti‐prothrombin (aPT), anti‐phosphatidylserine/prothrombin (aPS/PT), and anti‐phosphatidylethanolamine (aPE) antibodies. Sensitivity, specificity and predictive values were calculated. The diagnostic accuracy for each combination of tests was assessed by ROC and their area under the curve analysis as well as by the Youden’s index (YI). Results: Testing for six aPL derived 23 possible combinations of results. Among them, LA + anti‐β₂GPI + aPS/PT had the best diagnostic accuracy for APS as a whole and individually for both thrombosis and pregnancy loss (AUC 0.712, OR 3.73 [95% CI 1.82–5.38], P = 0.0001, YI = 0.32 and AUC 0.709, OR 3.75 [95% CI 2.13–6.62], P = 0.0001, YI = 0.37 and AUC 0.677, OR 4.82 [95% CI 2.17–10.72], P = 0.0007, YI = 0.38, respectively) and the best specificity when compared with all the other obtainable combination of tests. Triple positivity for LA + anti‐β₂GPI + aPS/PT was more strongly associated with clinical events (thrombosis and/or PL) when compared with double or single positivity (OR 23.2 [95% CI 2.57–46.2] vs. OR 7.3 [95% CI 2.21–25.97], OR 5.7 [95% CI 2.12–17.01] or OR 3.11 [95% CI 1.56–7.8] for single positivity for LA, aPS/PT and anti‐β₂GPI, respectively). Conclusions: Combining LA, anti‐β₂GPI and aPS/PT improves the diagnostic power and helps in stratifying the risk for each patient, according to their aPL profile.     

Influence of different IgG anticardiolipin antibody cut‐off values on antiphospholipid syndrome classification

Summary. Background: While medium to high titers of anticardiolipin (aCL) antibodies, defined as >40 GPL units or >99th percentile, is a laboratory criteria for the ‘definite’ diagnosis of antiphospholipid syndrome (APS), agreement between the two cut‐offs has not been validated. Objective: To validate the current aCL laboratory criterion by verifying the effect of the two cut‐offs on APS classification. Patients/methods: Ninety aCL positive APS patients were selected on the basis of their GPL values above the 99th percentile (17.4 GPL), which was calculated by testing 100 age‐ and sex‐matched healthy subjects. Results: A significant difference in the IgG positivity (P < 0.0001) was found between the APS laboratory profiles as 20 out of the 24 (83.3%) patients with single positivity (aCL alone), six out of the 23 (26.1%) with double positivity (aCL plus lupus anticoagulant or anti‐β₂glycoprotein I), and none out of the 43 with triple positivity (aCL plus lupus anticoagulant and anti‐β₂glycoprotein I) had titers between the 99th percentile and 40 GPL units. Moreover, the rate of aCL values between the 99th percentile and 40 GPL units was significantly higher (P < 0.0001) in patients with pregnancy morbidity (73.7%) as compared to those with vascular thrombosis (16.9%) and those with both conditions (16.7%). Conclusion: The 99th percentile cut‐off level seems more sensitive than the >40 GPL value for APS classification, as it includes subjects with aCL positivity alone as well as patients with pregnancy morbidity.     

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

Summary. Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti‐β₂‐glycoprotein I (β₂‐GPI) autoantibodies. β₂‐GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish‐hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. β₂‐GPI has been shown to interact with Streptococcus pyogenes. Objective: To evaluate the potential of S. pyogenes‐derived proteins to induce anti‐β₂‐GPI autoantibodies. Methods and results: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen‐like protein A [SclA], and streptococcal collagen‐like protein B [SclB]) were found to interact with β₂‐GPI. Only binding to protein H induces a conformational change in β₂‐GPI, thereby exposing a cryptic epitope for APS‐related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody‐related epitope in domain I of β₂‐GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti‐protein H antibodies also generated anti‐β₂‐GPI antibodies. Conclusions: Our study has demonstrated that a bacterial protein can induce a conformational change in β₂‐GPI, resulting in the formation of antiβ₂‐GPI autoantibodies. This constitutes a novel mechanism for the formation of anti‐β₂‐GPI autoantibodies.     

Splice variants of tissue factor promote monocyte‐endothelial interactions by triggering the expression of cell adhesion molecules via integrin‐mediated signaling

Summary. Background: TF is highly expressed in cancerous and atherosclerotic lesions. Monocyte recruitment is a hallmark of disease progression in these pathological states. Objective: To examine the role of integrin signaling in TF‐dependent recruitment of monocytes by endothelial cells. Methods: The expression of flTF and asTF in cervical cancer and atherosclerotic lesions was examined. Biologic effects of the exposure of primary microvascular endothelial cells (MVEC) to truncated flTF ectodomain (LZ‐TF) and recombinant asTF were assessed. Results: flTF and asTF exhibited nearly identical expression patterns in cancer lesions and lipid‐rich plaques. Tumor lesions, as well as stromal CD68⁺ monocytes/macrophages, expressed both TF forms. Primary MVEC rapidly adhered to asTF and LZ‐TF, and this was completely blocked by anti‐β1 integrin antibody. asTF‐ and LZ‐TF‐treatment of MVEC promoted adhesion of peripheral blood mononuclear cells (PBMCs) under orbital shear conditions and under laminar flow; asTF‐elicited adhesion was more pronounced than that elicited by LZ‐TF. Expression profiling and western blotting revealed a broad activation of cell adhesion molecules (CAMs) in MVEC following asTF treatment including E‐selectin, ICAM‐1 and VCAM‐1. In transwell assays, asTF potentiated PMBC migration through MVEC monolayers by ～3‐fold under MCP‐1 gradient. Conclusions: TF splice variants ligate β1 integrins on MVEC, which induces the expression of CAMs in MVEC and leads to monocyte adhesion and transendothelial migration. asTF appears more potent than flTF in eliciting these effects. Our findings underscore the pathophysiologic significance of non‐proteolytic, integrin‐mediated signaling by the two naturally occurring TF variants in cancer and atherosclerosis.     

Branched chain amino acids supplementation modulates TGF‐β1/Smad signaling pathway and interleukins in CCl4‐induced liver fibrosis

The alterations and low levels of circulating branched chain amino acids (BCAAs), leucine, isoleucine, and valine, are associated with liver diseases. The study was designed to evaluate hepatoprotective effect of BCAAs on CCl₄‐induced liver fibrosis and to investigate the molecular mechanisms underlying these effects in rats. In all, 30 male rats were divided into three groups. Control group (n = 10) and CCl₄ group (n = 10), where rats were injected with CCl₄ (1 mL/kg of 0.5 : 1 v/v injected i.p. twice weekly for 12 weeks). In CCl₄ + BCAAs group (n = 10), rats were injected with similar doses of CCl₄ and supplemented with a mixture of 600 mg/kg BCAAs (2 : 1 : 1.2 leucine : isoleucine : valine) by oral gavage, three times/week for 12 weeks. Liver fibrosis was assessed by measuring total bilirubin, total protein, alanine aminotransferase, and aspartate aminotransferase, hydroxyproline content, and serum IL‐6 and IL‐10. Histopathologic studies and α‐smooth muscle actin (α‐SMA) were detected immunohistochemically in liver. Serum insulin level, blood glucose, liver malodialdehyde concentration (MDA), glutathione peroxidase, and superoxide dismutase (SOD) activities were quantified. TGF‐β1, Smad3, and Smad7 gene expressions were estimated by qRT‐PCR. BCAAs suppressed liver fibrosis induced by CCl₄ treatment. BCAAs modulated liver indices and downregulated TGF‐β1, Smad3, and Smad7 expressions in hepatocytes. BCAAs enhanced liver antioxidant enzyme activities (P < 0.001), reduced serum levels of TGF‐β1, IL‐6, and IL‐10 compared to CCL₄ group and ameliorated histopathologic changes in rat liver. BCAAs may have a protective role against liver fibrosis via antioxidant and anti‐inflammatory mechanisms.     

Lupus anticoagulant is significantly associated with inflammatory reactions in patients with suspected deep vein thrombosis

Objective. Lupus anticoagulant (LA) and antiphospholipid antibodies (aPL) are suggested as risk factors for development of deep vein thrombosis (DVT) among patients without systemic lupus erythematosus (SLE). Other conditions, e.g. inflammation, are reported to induce LA and it is uncertain whether the association between LA and DVT is causal. In this study the associations between aPL, LA and inflammation were investigated in 170 consecutive patients without SLE, but with a tentative diagnosis of DVT. Material and methods. DVT was diagnosed in 64 patients. LA was determined according to the criteria of the International Society of Thrombosis and Haemostasis. The concentration of anticardiolipin (aCL) and β₂‐glycoprotein I (anti‐β₂‐GPI) antibodies as well as C‐reactive protein (CRP) was determined with sensitive and precise methods. Results. LA was demonstrated in 8 patients with DVT and in 10 patients without DVT, relative risk 1.33 (CI: 0.55–3.18). No significant association was observed between aCL or anti‐β₂‐GPI and DVT. Patients suffering from DVT had significantly higher concentrations of CRP than patients without DVT. However, CRP was also significantly higher in patients positive for LA than in patients without LA irrespective of the presence of DVT (p<0.001). Conclusions. The present study supports a strong association between inflammatory reactions and development of LA in patients with suspected DVT, whereas no significant association was demonstrated between LA or aPL and DVT.     

The changing face of sympathetic overactivity in hypertension

Background. The precise pathophysiological processes underlying the prothrombotic or hypercoagulable state in atrial fibrillation (AF) remain uncertain. We hypothesized a relationship between abnormal endothelial damage/dysfunction, coagulation, and angiogenic factors, thereby contributing to increased thrombogenicity.

Methods. Plasma levels of von Willebrand factor (vWF, an index of endothelial damage/dysfunction) and tissue factor (TF, an index of coagulation), as well as the angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin‐1 (Ang‐1) and angiopoietin‐2 (Ang‐2), were measured by enzyme‐linked immunosorbant assay (ELISA) in 59 chronic AF patients. Data were compared to 40 age‐ and sex‐matched healthy controls in sinus rhythm.

Results. Plasma vWF, VEGF and Ang‐2 were significantly higher in AF patients compared to healthy controls (P = 0.005, P = 0.0055 and P<0.0001 respectively) but there were no significant differences in plasma Ang‐1 or TF levels between the two groups (P = 0.925 and P = 0.121 respectively). Significant correlations were found between VEGF and vWF levels (Spearman, r = 0.262, P = 0.011) and between VEGF and Ang‐2 (r = 0.333, P = 0.001).

Conclusions. Raised VEGF in association with Ang‐2 and vWF may reflect a link between abnormal endothelial damage/dysfunction and angiogenic factors. These may act together to alter TF expression and endothelial integrity, thereby contributing to the prothrombotic state in AF.     

Platelet‐targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse

Summary. Background: Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics. Methods and results: An N‐terminal‐azido thrombin‐sensitive fluorescent peptide (ThS‐P) with a thrombin‐releasable quencher was linked to anti‐CD41 using click chemistry to generate a thrombin‐sensitive platelet binding sensor (ThS‐Ab). Rapid thrombin cleavage of ThS‐P (K_m = 40.3 μm , k_cat = 1.5 s^?1) allowed thrombin monitoring by ThS‐P or ThS‐Ab in blood treated with 2–25 pm tissue factor (TF). Individual platelets had > 20‐fold more ThS‐Ab fluorescence after clotting. In a microfluidic assay of whole blood perfusion over collagen ± linked TF (wall shear rate = 100 s^?1), ThS‐Ab fluorescence increased between 90 and 450 s for 0.1–1 molecule‐TF μm^?2 and co‐localized with platelets near fibrin. Without TF, neither thrombin nor fibrin was detected on the platelet deposits by 450 s. Using a microfluidic device to control the pressure drop across a thrombus forming on a porous collagen/TF plug (521 s^?1), thrombin and fibrin were detected at the thrombus–collagen interface at a zero pressure drop, whereas 80% less thrombin was detected at 3200 Pa in concert with fibrin polymerizing within the collagen. With anti‐mouse CD41 ThS‐Ab deployed in a mouse laser injury model, the highest levels of thrombin arose between 40 and 160 s nearest the injury site where fibrin co‐localized and where the thrombus was most mechanically stable. Conclusion: ThS‐Ab reveals thrombin locality, which depends on surface TF, flow and intrathrombus pressure gradients.     

The IgG autoimmune response in postpartum acquired hemophilia A targets mainly the A1a1 domain of FVIII

Summary. Background: Acquired hemophilia A (AHA) is a severe life‐threatening autoimmune disease due to the development of autoantibodies that neutralize the procoagulant activity of factor VIII (FVIII). In rare cases, AHA occurs in the postpartum period as a serious complication of an otherwise normal pregnancy and delivery. Due to its rarity, little is known about the features of the antibody response to FVIII in AHA. Objectives: Our study wanted to (i) determine the epitope specificity and the immunoglobulin (Ig) subclasses of anti‐FVIII autoantibodies in plasma samples from a large cohort of AHA patients, and (ii) compare the epitope specificity of anti‐FVIII autoantibodies in plasma samples from postpartum AHA and other AHA patients. Patients/Methods: Seventy‐three plasma samples from patients with postpartum AHA (n = 10) or associated with malignancies (n = 16) or autoimmune diseases (n = 11) or without underlying disease (n = 36) were analyzed with three multiplexed assays. Results and Conclusions: Our results showed a stronger response against the A1a1‐A2a2‐B fragments of FVIII and more specifically against the A1a1 domain in patients with postpartum AHA than in the other AHA groups (P < 0.01). Moreover, although IgG₄ was the predominant IgG subclass in all groups, anti‐A1a1‐A2a2‐B and anti‐A1a1 domain autoantibodies of the IgG₁ and IgG₃ subclasses were more frequently detected in postpartum AHA than in the other AHA groups. These findings support the involvement of the Th1‐driven response in the generation of autoantibodies in women with postpartum AHA compared with the other groups of AHA patients in whom production of Th2‐driven IgG₄ was predominant.     

A two‐step coagulation test to identify antiβ2‐glycoprotein I lupus anticoagulants

Summary. Lupus anticoagulants (LA) are immunoglobulins which inhibit phospholipid (PL)‐dependent coagulation tests. LA are not specific, as they may reflect the presence of antibodies to human prothrombin, human β₂‐Glycoprotein I (β₂GPI), an association of previous antibodies or other antibodies. Antibodies to human β₂GPI act as in vitro anticoagulants by enhancing the binding of β₂GPI to PL, and this binding may be influenced by calcium ion concentration. A reduction in final calcium concentration, from 10 mm to 5 mm , increased coagulation times in both dilute Russell Viper Venom Time (dRVVT) and dilute Prothrombin Time (dPT) when plasmas of patients with antiβ₂GPI antibodies were used. Ten LA patients showed increased dRVVT and dPT ratios from means of 1.5 to 1.7 (P < 0.001) and 2.4 to 4.3 (P = 0.002), respectively. Instead, all LA‐positive antiβ₂GPI antibody‐negative patients showed decreased coagulation times from mean ratios of 1.5 to 1.3 (P = 0.004) in dRVVT and from 2.0 to 1.5 (P = 0.01) in dPT. These results are confirmed by running dRVVT of normal plasma spiked with affinity purified IgG antiβ₂GPI antibodies. Therefore, when a PL–dependent coagulation test is run twice, at different final calcium concentrations, antiβ₂GPI LA can be identified.     

The CD14++CD16+ monocyte subset and monocyte‐platelet interactions in patients with ST‐elevation myocardial infarction

Summary. Aim: Monocytes contribute to both myocardial damage and repair by virtue of subset heterogeneity. The dynamics and functional characteristics of the three human monocyte subsets, including the unique CD14++CD16+ subset, and their contributions to monocyte platelet aggregates (MPAs) following ST‐elevation myocardial infarction (STEMI) are unknown. We aimed to examine dynamic changes and relation to left ventricular ejection fraction (LVEF) of the three human monocyte subsets and their aggregates with platelets following STEMI. Methods: Three monocyte subsets, CD14++CD16?CCR2+ (‘classical’, Mon1), CD14++CD16+CCR2+ (‘intermediate’, Mon2) and CD14+CD16++CCR2? (‘non‐classical’, Mon3), and their contribution to MPAs were analyzed by flow cytometry in 50 patients with STEMI, 40 patients with stable coronary artery disease (CAD) and 40 healthy volunteers. Study parameters were measured within 24 h of primary percutaneous coronary intervention (PCI) (day1) and on days 3, 7 and 30. Monocyte activation was assessed by measuring the nuclear factor κB (NFκB) pathway. LVEF was assessed 6 weeks after STEMI. Correlations between monocyte subsets/MPAs and plasma cytokines and troponin were assessed. Results: We observed marked differences in subset dynamics, with a prominent increase in Mon2 (P < 0.0001) but no changes in Mon3. Significant increases in Mon2 CD14 (P = 0.002) and CCR2 (P < 0.0001) expression, and reduction in CD16 expression (P = 0.001) were seen. NFκB pathway activity increased most prominently in Mon2 (P = 0.007). Mon2 count correlated with peak troponin (r = 0.31, P = 0.04) and plasma interleukin (IL)‐6 (r = 0.65, P < 0.0001) and IL‐10 (r = 0.34, P = 0.017). Mon1 correlated with IL‐6 (r = 0.55, P < 0.0001). Reduced Mon2 expression of CD16 on day 1 was independently predictive of higher LVEF (β = ?0.37, P = 0.013). The increase in MPA count following STEMI persisted at 1 month. Conclusion: The Mon2 ‘intermediate’ subset has unique dynamic and functional characteristics following STEMI and significant correlations with troponin, plasma cytokines and convalescent left ventricular function. The persistent increase in MPA count 30 days after STEMI may affect monocyte subset functional activity.     

Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner

Summary. Background: Circulating microparticles of various cell types are present in healthy individuals and, in varying numbers and antigenic composition, in various disease states. To what extent these microparticles contribute to coagulation in vivo is unknown. Objectives: To examine the in vivo thrombogenicity of human microparticles. Methods: Microparticles were isolated from pericardial blood of cardiac surgery patients and venous blood of healthy individuals. Their numbers, cellular source, and tissue factor (TF) exposure were determined using flow cytometry. Their in vitro procoagulant properties were studied in a fibrin generation test, and their in vivo thrombogenicity in a rat model. Results: The total number of microparticles did not differ between pericardial samples and samples from healthy individuals (P = 0.786). In both groups, microparticles from platelets, erythrocytes, and granulocytes exposed TF. Microparticle‐exposed TF antigen levels were higher in pericardial compared with healthy individual samples (P = 0.036). Pericardial microparticles were strongly procoagulant in vitro and highly thrombogenic in a venous stasis thrombosis model in rats, whereas microparticles from healthy individuals were not [thrombus weights 24.8 (12.2–41.3) mg vs. 0 (0–24.3) mg median and range; P < 0.001]. Preincubation of pericardial microparticles with an inhibitory antibody against human TF abolished their thrombogenicity [0 (0–4.4) mg; P < 0.01], while a control antibody had no effect [19.6 (12.6–53.7) mg; P > 0.05]. The thrombogenicity of the microparticles correlated strongly with their TF exposure (r = 0.9524, P = 0.001). Conclusions: Human cell‐derived microparticles promote thrombus formation in vivo in a TF‐dependent manner. They might be the direct cause of an increased thromboembolic tendency in various patient groups.     

Microparticle‐associated tissue factor activity,venous thromboembolism and mortality in pancreatic,gastric, colorectal and brain cancer patients

Summary. Background: Tissue factor (TF) expression by tumors contributes to tumor growth. Release of TF‐positive microparticles (MPs) may contribute to venous thromboembolism (VTE). Objectives: To conduct a prospective cohort study to determine whether elevated MP‐associated TF (MP‐TF) activity is predictive of VTE and mortality in four cancer types. Patients/Methods: We determined MP‐TF activity in pancreatic, gastric, colorectal and brain cancer patients. We used a chromogenic endpoint assay for all patients and also a chromogenic kinetic assay for patients with pancreatic and brain cancer. Results: During follow‐up, 12/60 (20%) pancreatic, 6/43 (14%) gastric, 12/126 (10%) colorectal and 19/119 (16%) brain cancer patients developed VTE; 46/60 (77%), 30/43 (70%), 47/126 (37%) and 67/119 (56%), respectively, died. MP‐TF activity levels were highest in pancreatic cancer. We did not find a statistically significant association of MP‐TF activity with the risk of VTE in any of the four cancer types by using two statistical methods. An association of MP‐TF activity with the risk of mortality was detected in pancreatic cancer with the endpoint assay (hazard ratio [HR] 1.8 and 95% confidence interval [CI] 1.4–2.3 per doubling of activity, P < 0.001) and the kinetic assay (HR 1.2, 95% CI 1.1–1.4, P < 0.001); adjustment for type of treatment was not performed. In pancreatic cancer, MP‐TF activity correlated with D‐dimer level (endpoint assay, r = 0.51; chromogenic assay, r = 0.48), and a correlation between assays (r = 0.61) was found. Conclusion: MP‐TF activity was not associated with future VTE in pancreatic, gastric, colorectal and brain cancer. However, we found a strong association of MP‐TF activity with mortality in pancreatic cancer. MP‐TF activity might be reflective of an aggressive pancreatic cancer phenotype.     

Staphylococcus aureus‐induced complement activation promotes tissue factor‐mediated coagulation

Essentials

• Complement, Toll‐like receptors and coagulation cross‐talk in the process of thromboinflammation.
• This is explored in a unique human whole‐blood model of S. aureus bacteremia.
• Coagulation is here shown as a downstream event of C5a‐induced tissue factor (TF) production.
• Combined inhibition of C5 and CD14 efficiently attenuated TF and coagulation.

Summary

Background

There is extensive cross‐talk between the complement system, the Toll‐like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression.

Objectives

To study the relative roles of complement, TLRs and TF in Staphylococcus aureus‐induced coagulation.

Methods

Lepirudin‐anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF_{1 + 2}) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively.

Results

All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF_{1 + 2}, and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF_{1 + 2} levels to baseline levels.

Conclusions

S. aureus‐induced coagulation in human whole blood was mainly attributable to C5a‐induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus‐induced coagulation.     

Polypoidal choroidal vasculopathy treatment options: A meta‐analysis

Background

Combined treatment with intravitreal anti‐vascular endothelial growth factor (anti‐VEGF) and verteporfin photodynamic therapy (PDT) is widely used for patients with polypoidal choroidal vasculopathy (PCV), although clinical evidence regarding the therapeutic efficacy and safety of such treatment remains lacking.

Design/Methods

We performed a meta‐analysis of previously reported studies comparing combination treatment, PDT monotherapy, and anti‐VEGF monotherapy. Primary outcome measures included changes in best‐corrected visual acuity (BCVA) and central retinal thickness (CRT). The proportion of patients with polyp regression was regarded as the secondary outcome measure.

Results

Twenty studies (three RCTs and 19 retrospective studies) involving 1,178 patients with PCV were selected. Significant differences in the proportion of patients with polyps were observed between the PDT and anti‐VEGF monotherapy groups at 3 and ≥6 months (P < .00001; and P = .0001, respectively). Significantly greater reductions in CRT were observed in the anti‐VEGF than in the PDT group at the 3‐month follow‐up (P = .04). Significantly greater improvements in BCVA were observed in the combined therapy group than in the PDT monotherapy group at 3, 6, 12, and 24 months (P = .03; P = .005; P = .02; and P < .00001, respectively). Combined treatment also resulted in significantly greater improvements in BCVA than monotherapy with anti‐VEGF at 6 and 24 months (P = .001; P < .00001, respectively), and significantly greater polyp regression than that observed following anti‐VEGF treatment at 3 and ≥6 months (P < .00001; P < .0001, respectively).

Conclusions

Combined therapy involving anti‐VEGF agents and PDT may be more effective in improving long‐term outcomes for patients with PCV than monotherapy.     

Prevalence of autoantibodies in patients of psoriasis

Psoriasis is a common chronic inflammatory disease of the skin and joints. Autoantibodies have been reported in psoriasis patients. Objective of the study was to see the prevalence of various autoantibodies in patients of psoriasis and its correlation with gender, age, and type. Anti‐nuclear antibody and antibody to double‐stranded deoxyribonucleic acid were studied by indirect enzyme linked immunosorbent assay, rheumatoid factor was done by latex agglutination, whereas anti‐thyroid microsomal antibody (anti‐TMA) was by gelatin agglutination method. About 28.8% of psoriasis cases were positive for atleast one autoantibody. Age of onset (P=0.033) and types of psoriasis (P=0.037) had significant association with gender. Anti‐double‐stranded deoxyribonucleic acid (P=0.029) and anti‐thyroid microsomal antibody (P=0.002) had significant association with types of psoriasis. Gender wise distribution of psoriasis in age group had significant (P=0.03) association with anti‐TMA. This study concludes that the autoantibodies are found to be present in psoriasis patients or latent autoimmune diseases develop in psoriasis patients without any clinical symptoms. J. Clin. Lab. Anal. 24:44–48, 2010. © 2010 Wiley‐Liss, Inc.     

Phosphatidylserine exposure and procoagulant activity in acute promyelocytic leukemia

Summary. Background: Acute promyelocytic leukemia (APL) frequently causes disseminated intravascular coagulation that can worsen with cytotoxic chemotherapy but improve with the therapeutic differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As₂O₃). APL cells display tissue factor but the relationship of tissue factor and other procoagulant activity to phosphatidylserine (PS) exposure is largely unknown. Methods: Lactadherin, a milk protein with stereospecific binding to phosphatidyl‐L‐serine, was used as a probe for PS exposure on an immortalized APL cell line (NB4) and on the cells of eight patients with APL. PS exposure was evaluated with flow cytometry, confocal microscopy, coagulation assays, and purified prothrombinase and factor (F) Xase assays. Results: Plasma procoagulant activity of NB4 and APL cells increased approximately 15‐fold after exposure to etoposide or daunorubicin and decreased 80% after treatment with ATRA or As₂O₃. Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure decreased after treatment with ATRA or As₂O₃ and increased after treatment with daunorubicin or etoposide. Excess lactadherin inhibited 80–85% of intrinsic FXase, FVIIa‐tissue factor and prothrombinase activities on both NB4 cells and APL cells. Confocal microscopy identified membrane patches that stained with lactadherin, but not annexin V, demonstrating focal, low‐level PS exposure. Conclusions: PS is exposed on viable APL cells and is necessary for approximately 80% of procoagulant activity.     

Thromboxane and prostacyclin biosynthesis in heart failure of ischemic origin: effects of disease severity and aspirin treatment

Summary. Background: Thromboembolism is a relatively common complication of chronic heart failure (HF) and the place of antiplatelet therapy is uncertain. Objectives: We characterized the rate of thromboxane and prostacyclin biosynthesis in chronic HF of ischemic origin, with the aim of separating the influence of HF on platelet activation from that of the underlying ischemic heart disease (IHD). Patients and Methods: We compared urinary 11‐dehydro‐thromboxane (TX)B₂, 2,3 dinor 6‐keto‐PGF_1α, 8‐iso‐prostaglandin (PG)F_2α, and plasma N‐terminal pro‐brain natriuretic peptide (NT‐pro‐BNP), asymmetric dimethylarginine (ADMA), and soluble CD40 ligand (sCD40L), in 84 patients with HF secondary to IHD, 61 patients with IHD without HF and 42 healthy subjects. Results: HF patients not on aspirin had significantly higher urinary 11‐dehydro‐TXB₂ as compared with healthy subjects (P < 0.0001) and IHD patients not on aspirin (P = 0.028). They also showed significantly higher 8‐iso‐PGF_2α (P = 0.018), NT‐pro‐BNP (P = 0.021) and ADMA (P < 0.0001) than IHD patients not on aspirin. HF patients on low‐dose aspirin had significantly lower 11‐dehydro‐TXB₂ (P < 0.0001), sCD40L (P = 0.007) and 2,3‐dinor‐6‐keto‐PGF_1α (P = 0.005) than HF patients not treated with aspirin. HF patients in NYHA classes III and IV had significantly higher urinary 11‐dehydro‐TXB₂ than patients in classes I and II, independently of aspirin treatment (P < 0.05). On multiple linear regression analysis, higher NT‐pro‐BNP levels, lack of aspirin therapy and sCD40L, predicted 11‐dehydro‐TXB₂ excretion rate in HF patients (R² = 0.771). Conclusions: Persistent platelet activation characterizes HF patients. This phenomenon is related to disease severity and is largely suppressable by low‐dose aspirin. The homeostatic increase in prostacyclin biosynthesis is impaired, possibly contributing to enhanced thrombotic risk in this setting.