Detection of tissue factor in platelets: why is it so troublesome?

Tissue factor (TF) is the most important trigger for the extrinsic coagulation pathway. TF, earlier denoted as thromboplastin, has always been a mystery since its discovery due to its abundant presence in most human tissues but not blood. The latter has been extensively studied in a vast quest for possible sources of blood-borne TF yielding many conflicting findings and confusing conclusions regarding the presence of TF mRNA, protein or functional procoagulant activity in virtually all blood cells. Platelets, in particular, have been heavily scrutinized by investigators eager to demonstrate expression of TF. However, some investigators including our own groups have not found evidence for TF in platelets. This article discusses notable reports and possible reasons for erroneous detection of platelet TF antigen and activity including artificially hyper-stimulated platelets, suboptimal purity of cell preparations, flaws in study design and/or choice of reagents.

Normalization of disrupted clock gene expression in males with tetraplegia: a crossover randomized placebo-controlled trial of melatonin supplementation.

STUDY DESIGN: Crossover double blind, randomized placebo-controlled trial. OBJECTIVES: Circadian oscillators are located both in the brain and in peripheral organs. Melatonin, the main brain-derived hormone governing circadian variations, is highly associated with daylight patterns. However, in subjects with tetraplegia the melatonin levels are blunted. Here we studied peripheral oscillators in peripheral blood mononuclear cells (PBMCs) in males with tetraplegia by examining how exogenous melatonin may influence the expression of clock gene mRNAs. SETTING: Sunnaas Rehabilitation Hospital, Nesoddtangen, Norway. METHODS: Six males with tetraplegia received 2 mg of melatonin or placebo 4 days before the study period. We also included six able-bodied men sleeping or kept awake during the night. Plasma samples were collected four times during a 24-h period. The mRNA expression levels of the clock genes PER1, PER2, BMAL1, and REV-ERBα were quantified in PBMCs using quantitative RT-PCR. RESULTS: The mRNA expression levels of PER-1 and -2 and REV-ERBα were increased at 04:00 h compared with the able-bodied controls (p < 0.05). Melatonin supplementation changed mRNA peak-time toward the time of supplementation. CONCLUSIONS: Several peripheral clock genes displayed distorted expression levels in tetraplegia. Supplementation with melatonin changed the mRNA expression levels of these genes toward those observed among able-bodied. SPONSORSHIP: Financial support was provided from the Throne Holst Foundation, Sunnaas Rehabilitation hospital and the University of Ferrara (FAR2016).

Laboratory characterization of leukemic cell procoagulants.

BACKGROUND: In acute myeloid leukemias, there is an increased chance to develop thrombotic disorders. We hypothesized that in addition to leukemic promyelocytes, monocytic leukemia cells may also have a higher procoagulant activity. METHODS: Fibrin formation was assessed by a one-stage clotting assay using a magnetic coagulometer. The thrombin generation test (TGT) of magnetically isolated normal human monocytes, intact leukemic cells and their isolated microparticles was performed by a fluorimetric assay. Phosphatidylserine (PS) expression of leukemic cells and microparticle number determinations were carried out by flow cytometry. RESULTS: All cell lines displayed a significant procoagulant potential compared to isolated normal human monocytes. In the TGT test, the mean of lagtime and the time to peak parameters were significantly shorter in leukemic cells (3.9-4.7 and 9.9-10.3 min) compared to monocytes (14.9 and 26.5 min). The mean of peak thrombin in various monocytic leukemia cell lines was 112.1-132.9 nM vs. 75.1 nM in monocytes; however, no significant difference was observed in the ETP parameter. Factor VII-deficient plasma abolished all procoagulant activity, whereas factor XII-deficient plasma did not affect the speed of fibrin formation and thrombin generation but modulated the amount of thrombin. Factor XI-deficient plasma affected the time to peak values in one leukemic cell line and also attenuated peak thrombin. Leukemia cell-derived microparticles from all three cell lines exerted a procoagulant effect by significantly shortening the lagtime in TGT; there was a nonsignificant difference in case of ETP parameter. CONCLUSIONS: All investigated monocytic leukemia cell lines exhibited significant thrombin generation. This phenomenon was achieved by the procoagulants on the surface of leukemic cells as well as by their microparticles.

Antioxidant and Anti-Inflammatory Activities in Extracts from Minke Whale (Balaenoptera acutorostrata) Blubber.

Intake of long-chain omega-3 polyunsaturated fatty acids (LC-n3-PUFA) is commonly recognized to reduce cardiovascular disease (CVD). In previous studies, cold-pressed whale oil (CWO) and cod liver oil (CLO) were given as a dietary supplement to healthy volunteers. Even though CWO contains less than half the amount of LC-n3-PUFA of CLO, CWO supplement resulted in beneficial effects on anti-inflammatory and CVD risk markers compared to CLO. In the present study, we prepared virtually lipid-free extracts from CWO and CLO and evaluated the antioxidative capacity (AOC) and anti-inflammatory effects. Oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays were used to test the AOC, and the results indicated high levels of antioxidants present in all extracts. The anti-inflammatory effects of the extracts were tested with lipopolysaccharide- (LPS-) treated THP-1 cells, measuring its ability to reduce cytokine and chemokine secretion. Several CWO extracts displayed anti-inflammatory activity, and a butyl alcohol extract of CWO most effectively reduced TNF-α (50%, p < 0.05) and MCP-1 (85%, p < 0.001) secretion. This extract maintained a stable effect of reducing MCP-1 secretion (60%, p < 0.05) even after long-term storage. In conclusion, CWO has antioxidant and anti-inflammatory activities that may act in addition to its well-known LC-n3-PUFA effects.

The immunomodulatory effects of barettin and involvement of the kinases CAMK1α and RIPK2.

BACKGROUND: Barettin is a marine natural compound with reported anti-inflammatory and antioxidant properties. The combination of these effects led us to explore barettin further as an inhibitor of atherosclerosis development. METHODS: The effect of barettin on MCP-1 and IL-10 secretion from activated immune cells was detected by ELISA. Determination of cell viability of oxidized low-density lipoprotein (oxLDL) and barettin exposed HUVEC cells were investigated by using CellTiter 96® AQ(ueous) One Solution. The kinase inhibition assays were performed using a radioactive ((33)P-ATP) filter binding assay at the University of Dundee, UK. RESULTS: Barettin reduces the secretion of monocyte chemotactic protein-1 (MCP-1) from LPS-stimulated monocytes, but was not able to prevent oxLDL-induced cell death in HUVEC. Barettin has inhibitory activity against two protein kinases related to inflammation, namely the receptor-interacting serine/threonine kinase 2 (RIPK2) and calcium/calmodulin-dependent protein kinase 1α (CAMK1α). We also demonstrate that barettin reduce the production of the anti-inflammatory cytokine interleukin-10 (IL-10) in a dose and time-dependent manner, possibly by inhibiting CAMK1α. CONCLUSIONS: The anti-inflammatory activity of barettin is exerted through the regulation of inflammatory mediators such as MCP-1 and IL-10, possibly via inhibition of kinases.

Extracellular vesicles from activated platelets possess a phospholipid-rich biomolecular profile and enhance prothrombinase activity.

BACKGROUND: Extracellular vesicles (EVs), in particular those derived from activated platelets, are associated with a risk of future venous thromboembolism. OBJECTIVES: To study the biomolecular profile and function characteristics of EVs from control (unstimulated) and activated platelets. METHODS: Biomolecular profiling of single or very few (1-4) platelet-EVs (control/stimulated) was performed by Raman tweezers microspectroscopy. The effects of such EVs on the coagulation system were comprehensively studied. RESULTS: Raman tweezers microspectroscopy of platelet-EVs followed by biomolecular component analysis revealed for the first time 3 subsets of EVs: (i) protein rich, (ii) protein/lipid rich, and (iii) lipid rich. EVs from control platelets presented a heterogeneous biomolecular profile, with protein-rich EVs being the main subset (58.7% ± 3.5%). Notably, the protein-rich subset may contain a minor contribution from other extracellular particles, including protein aggregates. In contrast, EVs from activated platelets were more homogeneous, dominated by the protein/lipid-rich subset (>85%), and enriched in phospholipids. Functionally, EVs from activated platelets increased thrombin generation by 52.4% and shortened plasma coagulation time by 34.6% ± 10.0% compared with 18.6% ± 13.9% mediated by EVs from control platelets (P = .015). The increased procoagulant activity was predominantly mediated by phosphatidylserine. Detailed investigation showed that EVs from activated platelets increased the activity of the prothrombinase complex (factor Va:FXa:FII) by more than 6-fold. CONCLUSION: Our study reports a novel quantitative biomolecular characterization of platelet-EVs possessing a homogenous and phospholipid-enriched profile in response to platelet activation. Such characteristics are accompanied with an increased phosphatidylserine-dependent procoagulant activity. Further investigation of a possible role of platelet-EVs in the pathogenesis of venous thromboembolism is warranted.

Comparison of assays measuring extracellular vesicle-tissue factor in plasma samples: Communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays. MATERIALS AND METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

Antioxidant and anti-inflammatory activities of barettin.

In this paper, we present novel bioactivity for barettin isolated from the marine sponge Geodia barretti. We found that barettin showed strong antioxidant activity in biochemical assays as well as in a lipid peroxidation cell assay. A de-brominated synthetic analogue of barettin did not show the same activity in the antioxidant cell assay, indicating that bromine is important for cellular activity. Barettin was also able to inhibit the secretion of the inflammatory cytokines IL-1ß and TNFα from LPS-stimulated THP-1 cells. This combination of anti-inflammatory and antioxidant activities could indicate that barettin has an atheroprotective effect and may therefore be an interesting product to prevent development of atherosclerosis.

A wax ester and astaxanthin-rich extract from the marine copepod Calanus finmarchicus attenuates atherogenesis in female apolipoprotein E-deficient mice.

The aim of this study was to investigate the effect of dietary supplementation with an oil extracted from the zooplankton copepod Calanus finmarchicus [calanus oil (CO)] on atherosclerosis in apoE-deficient (apoE(-/-)) mice. Thirty 6-wk-old female apoE(-/-) mice (n = 10/group) were fed: 1) a Western-type, high-fat diet (HFD); 2) HFD supplemented with 1% (wt:wt) CO; or 3) HFD supplemented with 0.88% (wt:wt) corn oil + 0.12% (wt:wt) EPA+DHA ethyl esters (EPA+DHA) for 13 wk. Dietary CO supplementation lowered total aorta atherogenesis by 36.5% compared to the HFD (P < 0.01), whereas the reduction in the lesion prone aortic arch was 34.8% (P < 0.01). The degree of aortic atherogenesis was intermediate in mice fed EPA+DHA compared to those fed HFD and CO. The effect on atherogenesis was paralleled by reduced expression of hepatic genes for the proinflammatory cytokines, Ccl2, Icam1, Il1b, and Nfkb1, in mice fed CO compared to those fed HFD. For mice fed EPA+DHA, gene expression did not differ compared to those fed CO or HFD. Plasma concentrations of total cholesterol, TG, and cytokines did not differ between the groups at the end of the study. However, mice fed CO gained more weight compared to those fed HFD but not compared to those fed EPA+DHA. In conclusion, dietary CO supplementation attenuated atherosclerotic lesion formation in female apoE(-/-) mice and may be an effective and safe dietary intervention to reduce the development of atherosclerosis. However, further studies are warranted to elucidate the underlying physiological and molecular mechanisms.

Heparin exerts a dual effect on murine lupus nephritis by enhancing enzymatic chromatin degradation and preventing chromatin binding in glomerular membranes.

OBJECTIVE: Association of nucleosome-IgG immune complexes with glomerular basement membranes (GBMs) is an important event in the development of lupus nephritis. Preventing this binding and/or increasing nuclease sensitivity of nucleosomes may be viable strategies for the prevention of the disease. Theoretically, heparin may alter nucleosomal structure and increase sensitivity to proteinases and nucleases, and may also inhibit binding of nucleosomes and nucleosome-IgG complexes to basement membrane structures. The aim of this study was to investigate whether and eventually how heparin prevents murine lupus nephritis. METHODS: Surface plasmon resonance was used to analyze if heparin inhibits binding of nucleosomes to laminin and collagen. The effect of heparin on nuclease- and proteinase-mediated degradation of nucleosomes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrophoresis. In vitro results were compared with analyses in vivo in heparin-treated (NZB × NZW)F(1) mice. Anti-double-stranded DNA antibody production, deposition of nucleosome-IgG complexes in GBMs, and development of proteinuria were monitored, and circulating chromatin fragments were quantified using quantitative polymerase chain reaction. RESULTS: In vitro studies demonstrated that heparin increased enzymatic degradation of nucleosomes and almost completely inhibited binding of nucleosomes to laminin and collagen. (NZB × NZW)F(1) mice treated with heparin demonstrated delayed or no antibody production and higher variation of circulating chromatin levels compared with untreated control mice. This effect was accompanied by highly reduced nucleosome-IgG complexes in GBMs and delayed development of nephritis. CONCLUSION: Increasing the degradation of nucleosomes, reducing their immunogenicity, and preventing binding of nucleosome-IgG complexes in glomeruli together provide an alternative basis for the treatment of lupus nephritis.

Enhanced Blood Clotting After Rewarming From Experimental Hypothermia in an Intact Porcine Model.

Introduction: Due to functional alterations of blood platelets and coagulation enzymes at low temperatures, excessive bleeding is a well-recognized complication in victims of accidental hypothermia and may present a great clinical challenge. Still, it remains largely unknown if hemostatic function normalizes upon rewarming. The aim of this study was to investigate effects of hypothermia and rewarming on blood coagulation in an intact porcine model. Methods: The animals were randomized to cooling and rewarming (n = 10), or to serve as normothermic, time-matched controls (n = 3). Animals in the hypothermic group were immersion cooled in ice water to 25°C, maintained at 25°C for 1 h, and rewarmed to 38°C (normal temperature in pigs) using warm water. Clotting time was assessed indirectly at different temperatures during cooling and rewarming using a whole blood coagulometer, which measures clotting time at 38°C. Results: Cooling to 25°C led to a significant increase in hemoglobin, hematocrit and red blood cell count, which persisted throughout rewarming. Cooling also caused a transiently decreased white blood cell count that returned to baseline levels upon rewarming. After rewarming from hypothermia, clotting time was significantly shortened compared to pre-hypothermic baseline values. In addition, platelet count was significantly increased. Discussion/Conclusion: We found that clotting time was significantly reduced after rewarming from hypothermia. This may indicate that rewarming from severe hypothermia induces a hypercoagulable state, in which thrombus formation is more likely to occur.

A rapid, sensitive, and specific assay to measure TF activity based on chromogenic determination of thrombin generation.

BACKGROUND: Most tissue factor (TF) activity assays are based on measurement of factor X (FX) activation by TF in the presence of factor VII (FVII)/FVIIa. This requires long incubation, which may result in TF-independent activity of FX and inaccurate measurement of TF activity. AIM: To develop a sensitive and specific TF activity assay, which does not register a non-specific TF activity, using commercial coagulation factors. METHODS: Tissue factor activity was measured based on the ability of TF to accelerate the activation of FX by FVIIa in the presence of factor V (FV)/Va, prothrombin, and phospholipids. Following 4 min incubation at 37°C, TF activity was quantified in test samples of different nature by thrombin generation using a chromogenic substrate. RESULTS: The TF activity assay proved high sensitivity (low fM range) and specificity, assessed by neutralization of TF activity by anti-TF antibody and the use of FVIIai. TF activity was detected in extracellular vesicles (EVs) derived from HAP1-TF+cells, while no activity was measured in EVs from HAP1-TF/KO cells. The assay was applicable for measurement of TF activity on the surface of live endothelial cells and monocytes activated in vitro, and cell lysates. Infusion of low dose lipopolysaccharide (2 ng/kg bodyweight endotoxin) caused a transient 8-fold increase (peaked at 4 h) in TF activity in EVs isolated from plasma of healthy volunteers. CONCLUSION: Our assay provides a fast, sensitive, and specific measurement of TF activity. It reliably quantifies TF activity on cell surface, cell lysate, and isolated EVs. The assay can be used for laboratory and clinical research.

Anti-ß2-glycoprotein I autoantibodies influence thrombin generation parameters via various mechanisms.

INTRODUCTION: Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterised by recurrent thrombotic events, pregnancy loss and thrombocytopenia and the presence of antiphospholipid antibodies (APL). The exact pathomechanism of APS is still unknown, thus we investigated the effect of anti-ß2-glycoprotein I (anti-ß2GPI) on thrombin generation in different plasma samples. METHODS: For the separation of anti-ß2GPI IgG, overall 12 APS patients were selected. The criteria were the existence of lupus anticoagulant, and the presence of anti-CL and anti-ß2GPI, the latter exceeding at least 25 times the upper reference limit. We purified anti-ß2GPI IgG antibodies from APS patients by affinity chromatography and added the antibodies to normal pooled, and heterozygous forms of inherited thrombophilia plasma samples (prothrombin G20210A, factor V Leiden). To further specify the mechanism of the effect, we also used factor deficient plasmas in the thrombin generation assay. RESULTS: In normal pooled plasma, the anti-ß2GPI significantly prolonged Lag Time according to the lupus anticoagulant effect, in contrast, it also elevated Peak Thrombin significantly, which suggests a procoagulant effect. The antibody was also able to exert this multi-faceted effect both in FVLeiden heterozygous plasma and prothrombin G20210A heterozygous polymorphism, however, the prolonging effect was more remarkable in the latter. By using factor deficient plasmas, it was found that FVII is required for the prolongation, while intrinsic factors are needed for the elevation of the Peak Thrombin. CONCLUSION: The anti-ß2GPI autoantibodies exert their effect in both normal and thrombophilic plasmas via various mechanisms.

Soluble guanylate cyclase agonists inhibit expression and procoagulant activity of tissue factor.

OBJECTIVE: Tissue factor (TF), a major initiator of blood coagulation, contributes to inflammation, atherosclerosis, angiogenesis, and vascular remodeling. Pharmacological agonists of soluble guanylate cyclase (sGC) attenuate systemic and pulmonary hypertension, vascular remodeling, and platelet aggregation. However, the influence of these novel pharmacophores on TF is unknown. METHODS AND RESULTS: We evaluated effects of BAY 41-2272 and BAY 58-2667 on expression and activity of TF in human monocytes and umbilical vein endothelial cells (HUVECs). Both compounds reduced expression of active TF protein in monocytes stimulated with lipopolysaccharide, as demonstrated by immunoblotting and a TF procoagulant activity assay. In-cell Western assay revealed that this effect was associated with a marked reduction of total and surface TF presentation. Furthermore, BAY 41-2272 and BAY 58-2667 decreased TF protein expression and the TF-dependent procoagulant activity in HUVECs stimulated with TNF-alpha. The sGC agonists also suppressed transcriptional activity of NF-kappaB. A siRNA-mediated knockdown of the alpha1-subunit of sGC in monocytes and HUVECs confirmed that the inhibitory effect of BAY 41-2272 and BAY 58-2667 on TF expression is mediated through the sGC-dependent mechanisms. CONCLUSIONS: Inhibition of TF expression and activity by sGC agonists might provide therapeutic benefits in cardiovascular diseases associated with enhanced procoagulant and inflammatory response.

Improving scientific practice in sports-associated drug testing.

Antidoping work is heavily based on scientific analyses of biological material, such as urine and blood. Because of the high stakes both for sports and for the athletes involved it is important that analyses are performed and interpreted in agreement with established scientific standards and professional norms. This is not always the case, as we document here. It is our experience that the antidoping movement does not appear willing to consider that errors can occur and should be corrected. The consequences of the lack of transparency and responsibility are carried by unlucky athletes. Scientific, ethical and legal considerations urge the antidoping movement to reform some of their rules and regulations and to include the possibility that the World Anti-Doping Agency position could, in some cases, be incorrect.

Increased thrombin generation in persons with echogenic carotid plaques.

Echolucent carotid plaques are associated with higher risk for future ischemic cerebrovascular events (CVE) than echogenic plaques independent of the degree of stenosis. Elevated markers of thrombin generation are associated with atherosclerotic plaques and are increased in the acute and chronic phases of CVE. The present study was conducted to investigate the influence of plaque morphology on thrombin generation in persons with carotid stenosis. One hundred twenty-eight persons with carotid stenosis (>or=35% lumen diameter reduction) and 136 matched controls without stenosis were recruited from the health survey of the Tromsø Study. Blood samples were collected and plaque morphology determined by ultrasonography. Thrombin generation was assessed by thrombin-antithrombin complexes (TAT) and by prothrombin fragment 1+2 (F1+2). Persons with echogenic plaques (n = 63) had significantly higher levels of TAT (5.24 microg/l, 4.33-6.14) (mean, 95%CI) than persons with echolucent plaques (n = 65) (3.44 microg/l, 2.91-3.96, p < 0.001) and controls (n = 136) (3.33 microg/l, 3.06-3.60, p < 0.001). They also had significantly higher levels of F1+2 (2.14 nM, 1.83-2.45) than persons with echolucent plaques (1.54 nM, 1.38-1.71, p < 0.001) and controls (1.49 nM, 1.40-1.58, p < 0.001). TAT and F1+2 increased linearly with plaque echogenicity (p = 0.002 and p = 0.001, respectively) independent of the degree of stenosis. Increased thrombin generation was associated with a significant increase in plasma factor V levels among persons with echogenic plaques compared to echolucent plaques (p = 0.049) and controls (p = 0.025). The present findings indicate that increasing plaque echogenicity, rather than plaque echolucency and the degree of stenosis, is associated with thrombin generation in persons with carotid stenosis.

Regulation of tissue factor procoagulant activity by post-translational modifications.

Post-translational modification of amino acid residues is a common way to regulate localization, stability and ultimately the function of the protein. Tissue factor (TF), the major initiator of blood coagulation cascade, receives several post-translational modifications, like glycosylation, phosphorylation, palmitoylation and nitrosylation. Recent studies have demonstrated that these processes play important roles in modulating biological functions of TF. The present review highlights the mechanisms of several common protein post-translational modifications of TF with the special reference on the recent knowledge about their roles in regulation of trafficking, stability as well as procoagulant and signaling functions of TF.