Tissue Factor In Blood Research Articles

Comparison of standard and global hemostasis assays in cord and peripheral blood of newborns.

Umbilical cord blood is used for the testing of various parameters in newborns. However, data on its applicability for hemostasis assays is insufficient. To evaluate whether umbilical cord blood can be used for standard tests, thromboelastometry and thrombodynamics for preterm and term newborns. 187 newborns were included in the study. Blood was taken from the umbilical cord and by venipuncture of the newborn. Clotting times, fibrinogen, D-dimer, thromboelastometry and thrombodynamics were measured. Clotting times and fibrinogen indicated a hypocoagulable shift, while thromboelastometry and thrombodynamics showed a hypercoagulable shift in hemostasis in umbilical cord blood compared to newborn blood. D-dimer indicated an enhanced process of thrombus lysis in newborn blood compared to cord blood. Collecting blood into a tube with the addition of a contact pathway inhibitor did not significantly change the global assay parameters in either umbilical cord blood or newborn blood. In the thrombodynamics assay, spontaneous clotting was detected but suppressed by the addition of a tissue factor inhibitor. Hemostasis in cord and newborn blood differs for both global and standard tests. Hypercoagulability in newborns registered with the global assay thrombodynamics is associated with the presence of tissue factor in the blood. 1. We found a hypercoagulation shift in newborns compared with the adult references, possibly due to the presence of tissue factor in blood. 2. Blood coagulation is enhanced in cord blood compared with blood sampled from the vein of a newborn according to thromboelastometry and thrombodynamics assays. 3. Clotting times and fibrinogen concentrations in cord blood differ from these parameters in newborn blood. 4. Studying of the (patho)physiological features of hemostasis in newborns should consider differences in cord blood and vein sampled blood.

Associations of tissue factor and tissue factor pathway inhibitor with organ dysfunctions in septic shock

Coagulopathy, microvascular alterations and concomitant organ dysfunctions are hallmarks of sepsis. Attempts to attenuate coagulation activation with an inhibitor of tissue factor (TF), i.e. tissue factor pathway inhibitor (TFPI), revealed no survival benefit in a heterogenous group of sepsis patients, but a potential survival benefit in patients with an international normalized ratio (INR) < 1.2. Since an increased TF/TFPI ratio determines the procoagulant activity specifically on microvascular endothelial cells in vitro, we investigated whether TF/TFPI ratio in blood is associated with INR alterations, organ dysfunctions, disseminated intravascular coagulation (DIC) and outcome in septic shock. Twenty-nine healthy controls (HC) and 89 patients with septic shock admitted to a tertiary ICU were analyzed. TF and TFPI in blood was analyzed and related to organ dysfunctions, DIC and mortality. Patients with septic shock had 1.6-fold higher levels of TF and 2.9-fold higher levels of TFPI than HC. TF/TFPI ratio was lower in septic shock compared to HC (0.003 (0.002–0.005) vs. 0.006 (0.005–0.008), p < 0.001). Non-survivors had higher TFPI levels compared to survivors (43038 (29354–54023) vs. 28041 (21675–46582) pg/ml, p = 0.011). High TFPI levels were associated with acute kidney injury, liver dysfunction, DIC and disease severity. There was a positive association between TF/TFPI ratio and troponin T (b = 0.531 (0.309–0.754), p < 0.001). A high TF/TFPI ratio is exclusively associated with myocardial injury but not with other organ dysfunctions. Systemic TFPI levels seem to reflect disease severity. These findings point towards a pathophysiologic role of TF/TFPI in sepsis-induced myocardial injury.

Tissue Factor Pathway Inhibitors as Potential Targets for Understanding the Pathophysiology of Preeclampsia.

Preeclampsia is a hypertensive disorder of pregnancy that causes maternal and perinatal morbidity and mortality worldwide. Preeclampsia is associated with complex abnormalities of the coagulation and fibrinolytic system. Tissue factor (TF) is involved in the hemostatic system during pregnancy, while the Tissue Factor Pathway Inhibitor (TFPI) is a major physiological inhibitor of the TF-initiated coagulation cascade. The imbalance in hemostatic mechanisms may lead to a hypercoagulable state, but prior research has not comprehensively investigated the roles of TFPI1 and TFPI2 in preeclamptic patients. In this review, we summarize our current understanding of the biological functions of TFPI1 and TFPI2 and discuss future directions in preeclampsia research. A literature search was performed from inception to 30 June 2022 in the PubMed and Google Scholar databases. TFPI1 and TFPI2 are homologues with different protease inhibitory activities in the coagulation and fibrinolysis system. TFPI1 is an essential physiological inhibitor of the TF-initiated extrinsic pathway of coagulation. On the other hand, TFPI2 inhibits plasmin-mediated fibrinolysis and exerts antifibrinolytic activity. It also inhibits plasmin-mediated inactivation of clotting factors and maintains a hypercoagulable state. Furthermore, in contrast to TFPI1, TFPI2 suppresses trophoblast cell proliferation and invasion and promotes cell apoptosis. TFPI1 and TFPI2 may play important roles in regulating the coagulation and fibrinolytic system and trophoblast invasion to establish and maintain successful pregnancies. Concentrations of TF, TFPI1, and TFPI2 in maternal blood and placental tissue are significantly altered in preeclamptic women compared to normal pregnancies. TFPI protein family may affect both the anticoagulant (i.e., TFPI1) and antifibrinolytic/procoagulant (i.e., TFPI2) systems. TFPI1 and TFPI2 may function as new predictive biomarkers for preeclampsia and navigate precision therapy.

Involvement of monocyte-derived extracellular vesicle-associated tissue factor activity in convallatoxin-induced hypercoagulability.

Convallatoxin (CNT) is a natural cardiac glycoside extracted from lily of the valley ( Convallaria majalis ). Although it is empirically known to cause blood coagulation disorders, the underlying mechanism remains unclear. CNT exerts cytotoxicity and increases tissue factor (TF) expression in endothelial cells. However, the direct action of CNT on blood coagulation remains unclear. Therefore, herein, we investigated the effects of CNT on whole blood coagulation system and TF expression in monocytes. Blood samples were collected from healthy volunteers to measure plasma thrombin-antithrombin complex (TAT) concentration using ELISA and to perform rotational thromboelastometry (ROTEM) and whole-blood extracellular vesicle (EV)-associated TF (EV-TF) analysis. The effects of CNT were also investigated using the monocytic human cell line THP-1. Quantitative real-time PCR and western blotting were performed, and PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, was used to elucidate the action mechanism of CNT-mediated TF production. CNT treatment increased EV-TF activity, shortened the whole blood clotting time in rotational thromboelastometry analysis, and increased TAT levels, which is an index of thrombin generation. Furthermore, CNT increased TF mRNA expression in THP-1 cells and EV-TF activity in the cell culture supernatant. Therefore, CNT may induce a hypercoagulable state with thrombin generation, in which elevated EV-TF activity derived from monocytes might be involved. These procoagulant effects of CNT were reversed by PD98059, suggesting that CNT-induced TF production in monocytes might be mediated by the MAPK pathway. The findings of the present study have further clarified the procoagulant properties of CNT.

PB1844: THROMBOTIC EVENTS DURING REMISSION INDUCTION OF ACUTE MYELOID LEUKEMIA – FREQUENCY AND RELATED FACTORS

Background: Thrombotic events in newly diagnosed patients with acute myeloid leukemia (AML) have been considered rare and have not received a great deal of attention. These events may occur at the diagnosis, during the remission induction, or later. The incidence of thromboembolism in AML varied markedly among studies, ranging from 2 to 13%, and are usually divided between arterial and venous events. Limited evidence points that using markers of disseminated intravascular coagulation (DIC) might predict thrombosis in AML, and the Khorana score is not useful for this purpose. Aims: In this work, we evaluated a cohort of intensively treated adults with non-promyelocytic AML in order to assess the thrombosis rate and baseline factors that might be associated with this event. Methods: This is a retrospective cohort encompassing newly diagnosed AML adult patients (pts) treated at Instituto do Cancer do Estado de Sao Paulo, Brazil, between June 2011 and June 2020. All patients received the “7 + 3” regimen as the frontline regimen, and all thrombotic events until day 60 from diagnosis were captured. Logistic regression was used to find risk factors for thrombosis. Cutoffs for numeric variables were defined by Akaike’s information criteria. Results: Overall, 204 pts were included, with a median age of 54 years (range,17-74). The main baseline features of this cohort are summarized in Table 1. Overall, 24 thrombotic events were registered – the thrombotic rate of 11.9% (95% CI 7.9-17.4%). Most events were catheter-related (9/24), followed by upper (10) and lower (3) limb events. Two patients presented spontaneous jugular vein thrombosis. DIC score (Taylor et al., 2001) was calculated for 107 patients with laboratory data fully available and resulted in overt DIC (≥ 5) in 45.2%. By univariate analysis, morphologic subtype, AML genetic classification, fibrinogen, d-dimer, and DIC score were not associated with thrombosis during induction in AML. Excluding those catheter-related cases, we noticed a higher monocyte count in patients who developed thrombosis (15.8 vs. 4.7x109/L, p=0.009). Incidence of thrombosis in patients with monocyte count >8.5x109/L was 16% vs. 6.2%. The overall 60-day mortality was 25.8%, with no death related to thrombosis. DIC score was not associated with early mortality during AML induction (p=0.19). Remaining coagulation parameters when individually assessed were also not linked with thrombosis or mortality during induction. Image:Summary/Conclusion: In this study with intensively treated newly diagnosed AML, we noticed a similar incidence of thrombosis to that previously reported. While most events reported in this cohort were related to catheter devices, spontaneous thrombosis was associated with a higher baseline monocyte count. While monocytic AML may present with DIC at the diagnosis, monocytic cells are a major source of blood tissue factor, pointing it as an independent marker for thrombosis in AML. Our data, although limited, highlights that the understanding of the thrombosis landscape in AML is complex, and the sole use of already recognized markers of coagulation activation might not be sufficient for prediction.

Coagulation markers and functional outcome in acute ischemic stroke: Impact of intensive versus standard hyperglycemia control

ObjectiveAlterations in coagulation could mediate functional outcome in patients with hyperglycemia after acute ischemic stroke (AIS). We prospectively studied the effects of intensive versus standard glucose control on coagulation markers and their relationships to functional outcomes in patients with AIS. ApproachThe Insights on Selected Procoagulation Markers and Outcomes in Stroke Trial measured the coagulation biomarkers whole blood tissue factor procoagulant activity (TFPCA); plasma factors VII (FVII), VIIa (FVIIa), and VIII (FVIII); thrombin‐antithrombin (TAT) complex; D‐dimer; tissue factor pathway inhibitor, and plasminogen activator inhibitor‐1 (PAI‐1) antigen in patients enrolled in the Stroke Hyperglycemia Insulin Network Effort trial of intensive versus standard glucose control on functional outcome at 3 months after AIS. Changes in biomarkers over time (from baseline ≈12 hours after stroke onset) to 48 hours, and changes in biomarkers between treatment groups, functional outcomes, and their interaction were analyzed by two‐way analysis of variance. ResultsA total of 125 patients were included (57 in the intensive treatment group and 68 in the standard treatment group). The overall mean age was 66 years; 42% were women. Changes from baseline to 48 hours in coagulation markers were significantly different between treatment groups for TFPCA (P= 0.02) and PAI‐1 (P= .04) and FVIIa (P= .04). Increases in FVIIa and decreases in FVIII were associated with favorable functional outcomes (P= .04 and .04, respectively). In the intensive treatment group, reductions in TFPCA and FVIII and increases in FVIIa were greater in patients with favorable than unfavorable outcomes (P= .02, 0.002, 0.03, respectively). In the standard treatment group, changes in FVII were different by functional outcome (P= .006). ConclusionsIntensive glucose control induced greater alterations in coagulation biomarkers than standard treatment, and these were associated with a favorable functional outcome at 3 months after AIS.

Functional characterization of tissue factor in von Willebrand factor-dependent thrombus formation under whole blood flow conditions.

Von Willebrand factor (VWF) plays an important role in mediating platelet adhesion and aggregation under high shear rate conditions. Such platelet aggregates are strengthened by fibrin-network formation triggered by tissue factor (TF). However, little is known about the role of TF in VWF-dependent thrombus formation under blood flow conditions. We evaluated TF in thrombus formation on immobilized VWF under whole blood flow conditions in an in vitro perfusion chamber system. Surface-immobilized TF amplified intra-thrombus fibrin generation significantly under both low and high shear flow conditions, while TF in sample blood showed no appreciable effects. Furthermore, immobilized TF enhanced VWF-dependent platelet adhesion and aggregation significantly under high shear rates. Neutrophil cathepsin G and elastase increased significantly intra-thrombus fibrin deposition on immobilized VWF-TF complex, suggesting the involvement of leukocyte inflammatory responses in VWF/TF-dependent mural thrombogenesis under these flow conditions. These results reveal a functional link between VWF and TF under whole blood flow conditions, in which surface-immobilized TF and VWF mutually contribute to mural thrombus formation, which is essential for normal hemostasis. By contrast, TF circulating in blood may be involved in systemic hypercoagulability, as seen in sepsis caused by severe microbial infection, in which neutrophil inflammatory responses may be active.

Validation of a modified thromboelastometry approach to detect changes in fibrinolytic activity.

BackgroundThus far, validated whole blood assays used in in vitro fibrinolysis experiments using thromboelastometry (ROTEM) are lacking or have yet to be tested in humans.The objective was first, to establish a standardized modified ROTEM approach to detect both hypo- and hyperfibrinolysis. And second, to perform a technical and clinical validation of the assay.MethodsBlood was used of healthy volunteers, patients with sepsis, patients after cardiothoracic surgery, pregnant women, and cirrhotic liver disease patients. A whole blood tissue factor (TF) activated ROTEM assay with and without the addition of recombinant tissue plasminogen activator (rTPA) was developed. Plasma fibrinolysis determinants were measured in all volunteers and patients.ResultsThirty five pM TF and additions of 125 and 175 ng/ml rTPA resulted in full lysis within 60 min in healthy volunteers. Coefficients of variation were below 10 % without and below 20 % with rTPA addition. In sepsis the hypofibrinolytic ROTEM profiles with 175 ng/ml rTPA were in line with the plasma determinants (high PAI-1, high fibrinogen, low tPA activity, and high d-dimers). After cardiothoracic surgery, reduced fibrinogen and platelet levels accounted for the reduced maximum clot firmness. The hypofibrinolytic profile is attributed to tranexamic acid use and elevated PAI-1 levels. The lowest rTPA concentration in cirrhosis resulted in hyperfibrinolysis in only few of the patients. In pregnancy normal profiles were found.DiscussionOur high rTPA concentration demonstrates hypofibrinolytic profiles adequately in sepsis and after cardiothoracic surgery. Our low rTPA concentration of 125 ng/ml seems too high for demonstrating hyperfibrinolysis in cirrhotic liver disease.ConclusionsWe were able to present a validated whole blood ROTEM approach to fibrinolysis testing using added rTPA, which can be of added value next to classical plasma based fibrinolysis assays.

Type-2 Phosphatidylserine (PS)-Positive Erythrocytes and Their Association with Markers of Hemolysis and Hemostatic Activation in Children with Sickle Cell Disease (SCD)

Introduction: Type-2 phosphatidylserine (PS)-positive red cells are a subpopulation of erythrocytes that are highly positive for PS, contain low levels of fetal hemoglobin, are specific for sickle cell disease (SCD) and have been identified in the dense red cell fraction. Studies have implicated PS-positive red cells in enhancing anemia due to phagocytosis and hemolysis. Shielding of red cell PS by diannexin, a synthetic homodimer of human annexin-V, has been demonstrated to provide protection against hemolysis and prevent activation of prothrombinase.Methods: Using flow cytometry, we measured the levels of type-1 (red cells with low PS positivity) and type-2 PS-positive red cells in 50 children with SCD (31 with HbSS and 19 with HbSC), and assessed their association with various markers of hemolysis and hemostatic activation. Markers of hemolysis evaluated included plasma lactate dehydrogenase (LDH), reticulocyte count, and hemoglobin. Whole blood tissue factor (WBTF), pro-thrombin fragment F1+2, and D-dimer were evaluated as markers of hemostatic activation.Results: We demonstrate that the levels of type-2 PS-positive red cells are significantly increased in HbSS patients (1.37 ± 0.97%, p<0.01) compared to children with HbSC disease (0.32 ± 0.21%) and age- and race-matched controls (0.15 ± 0.15%, n=19). WBTF and D-dimer showed significant associations with both type-1 and -2 red cells with no significant differences in the strength of their association. However, significantly greater correlations were noted between type-2 PS red cells and hemolytic markers compared to those noted with type-1 (Steiger's Z=3.05 to 4.59, p<0.01). In addition our in vitro studies demonstrate increased osmotic fragility of these red cells.Table 1Association of PS-positive RBCs with markers of hemolysis and hemostatic activationBiomarkerType-1 PS-positive RBCsType-2 PS-positive RBCsMarkers of HemolysisLDHr = 0.44, p<0.002r = 0.63, p<0.00001% Reticulocyter = 0.43, p=0.002r = 0.66, p<0.00001Hemoglobinr =-0.35, p=0.014r =-0.63, p<0.00001Markers of Hemostatic ActivationWBTFr = 0.41, p=0.008r = 0.56, p<0.0002F1+2r = 0.26, p=0.07r = 0.31, p<0.03D-dimerr = 0.46, p<0.001r = 0.56, p<0.0005Conclusions: Type-2 PS-positive red cells are elevated in SCD and the number of these cells correlates significantly with both markers of hemolysis and hemostasis. These findings provide a patho-physiologic link between the intravascular hemolytic milieu of SCD and the hemostatic perturbations previously noted in this disease. DisclosuresNo relevant conflicts of interest to declare.

Retinoic acid inhibits tissue factor and HMGB1 via modulation of AMPK activity in TNF-α activated endothelial cells and LPS-injected mice.

Retinoic acid (RA) is the active vitamin A derivative and has diverse immunomodulatory actions. We hypothesized that RA reduces prothrombotic mediators such as tissue factor (TF) in endothelial cells during inflammatory conditions via an AMPK-dependent pathway, which attenuates cardiovascular complications. RA significantly increased AMPK and Akt phosphorylation in a time- and concentration-dependent manner in endothelial cells (EC). RA downregulated TF expression at the transcriptional and translational levels in TNF-α activated ECs, which was reversed by the silencing of AMPK and transfection of DN-AMPK. Interestingly, the PI3-kinase inhibitor LY294002 reversed the RA effect on TF expression. Increased AMPK phosphorylation by RA was inhibited by LY294002. However, increased Akt phosphorylation was not reduced by compound C, indicating that PI3K/Akt signaling modulates AMPK activity. In addition, RA reduced HMGB1 release in TNF-α activated ECs, which was reversed by both LY294001 and siAMPK. Importantly, administration of RA (1mg/kg) significantly reduced blood TF activity, circulating HMGB1 and PAI-1 levels and expression of hepatic TF mRNA as well as fibrin deposition in LPS (5mg/kg)-injected mice. Taken together, the activation of PI3K/Akt by RA modulates AMPK activity in ECs and plays a crucial role in the inhibition of coagulatory factors such as TF, PAI-1, and HMGB1 in inflammatory conditions.

Ultrasensitive detection based on gold nanoparticles for the platelet-associated tissue factor in patients with thrombotic diseases

The platelet-associated tissue factor (TF) is a physiological index related closely to thrombotic diseases discovered recently. A novel plasmonic enzyme-linked immunosorbent assay (ELISA) strategy has been developed for colorimetric amplification detection of the platelet-associated TF in this report. The detection mechanism is based on the fact that acetylcholinesterase (AChE) can catalyze acetylthiocholine chloride (ATC) to produce thiocholine which can cause the aggregation of gold nanoparticles (AuNPs) through forming Au–S bonds, and thus control the color and absorption spectra changes of AuNPs. This approach gave a detection limit narrowed down to 10−14 g mL−1, a value that is much lower than that of conventional ELISA procedures. The results of plasmonic ELISA in platelet-associated TF assays of blood specimens from 40 patients revealed that this assay can be used for the quantitative detection of the platelet-associated TF in human blood. This method provided a valuable tool for the ultrasensitive detection of the platelet-associated TF that shows potential for diagnosis and therapeutic monitoring of thrombotic diseases.

Divergent Effect of Insulin On Whole-Blood Tissue Factor Procoagulant Activity in the Presence and Absence of Lipopolysaccharide

Abstract 636 Background:Individuals with diabetes mellitus (DM) have accelerated atherosclerosis and are prone to acute vascular events. Both hyperglycemia (HG) and hyperinsulinemia (HI) are independent risk factors for mortality in Type 2 DM (T2DM). Tissue factor (TF) is the principal initiator of blood coagulation. There is a circulating pool of TF in blood, which is thrombogenic. We have shown that membrane-bound TF levels are elevated in T2DM (J Clin Endocrinol Metab 2007, 92; 4352–8), and T1DM (Diabetes Care, 2012;35:1322–7). Infusion studies in normal healthy subjects showed that HG (∼200 mg/dl), HI and combined HG+HI elevated whole blood TF-procoagulant activity (TF-PCA) by 2, 6 and 9-fold, respectively (Diabetes 2006, 55; 202–8). In T2DM patients, the elevation by HI was 30%, and by HG+HI was 80% (J Clin Endocrinol Metab 2007, 92; 4352–8). However, in contrast to the findings in T2DM, our studies in T1DM patients (Diabetes Care, 2012;35:1322–7) showed that neither acutely raising blood glucose or insulin for 24 h, nor raising blood glucose together with insulin for up to 24 h increased TF-PCA. In fact, TF-PCA levels decreased during combined HG and HI and selective HG. To better understand the roles of HG and HI on TF-PCA, we studied their effects in vitro in whole blood from healthy individuals under both unstimulated and lipopolysaccharide (LPS)-stimulated conditions; the latter was done because patients with T2DM have evidence of platelet/monocyte activation as well as elevated plasma LPS levels. Methods:Glucose (200 mg/dL, HG) and insulin (1, 10 or 100 nM, HI) were added either alone or in combination (HG+HI) to blood from 6 healthy subjects and incubated for 4 hr at 37°C. These studies were performed in the presence or absence of 1 mg/ml LPS. Membrane bound TF-PCA was measured in whole blood lysates by a two-stage clotting assay (Key et al, Blood 1998, 91; 4216–23). Results:Basal glucose and insulin levels were 75±4 mg/dL and 99±24 pmol/L (n=6, mean±SE), respectively. With added glucose (HG) TF-PCA increased from baseline (22±5 U/ml mean ± SE) to 41±7 U/ml (p=0.006) at 2 hr and to 57±10 U/ml (p=0.0005) at 4 hr. Similarly, at 2 hr there was an increase with insulin 10 nM (33±8, p=0.033) and 100 nM (44±12, p=0.04). A combination of HG with increasing insulin concentrations (1, 10 or 100 nM) increased TF-PCA at 2 hr to 42±6 (p=0.04), 50±9 (p=0.02), and 63±12 (p=0.002), respectively. At 4 hr the corresponding levels were 71±16 (p=0.031), 69±15 (p=0.003), and 93±20 (p=0.004), respectively. Thus, HG, HI and HG+HI increased TF-PCA levels with highest levels being with the combination. This is in line with the findings from our infusion studies in healthy subjects. Studies in the presence of LPS:LPS alone induced a marked increase in TF-PCA from 22±5 U/ml to 826±93 (p<0.001) by 2 hr and to 591±113 (p<0.01) by 4 hr. The small increase with HG in presence of LPS was not statistically significant. With added insulin TF-PCA at 2 hr was lower than with LPS alone (826±93 U/ml) with 10 nM insulin (587±119, p=0.037) and 100 nM insulin (552±207, p=0.025). This was noted at 4 hr with 10 nM insulin (591±113 vs 472±83, p=0.014). This is in contrast to the findings in the absence of LPS-stimulation where HI stimulated TF-PCA. Notably, in the presence of LPS, the combination of HG with increasing HI caused a marked increase in TF-PCA at 2 hr over that with LPS alone (826±93 U/ml) at 1 nM insulin (2369±234, p=0.003) and 100 nM insulin (1992±288, p=0.009). This persisted at 4 hr (LPS: 591±113 vs 2595±49, p=0.006; and vs 1936±347, p= 0.005). TF-PCA with HG + HI at 2 and 4 hr was higher than with LPS plus glucose or insulin alone. Conclusions:In non-diabetic subjects HG and HI individually and in combination, increased whole blood TF-PCA. However, insulin inhibited the marked increase in TF-PCA induced by LPS. These findings suggest that the insulin effect is influenced by the cellular activation state. This may be relevant because platelets/monocytes are activated and endogenous LPS levels are elevated in DM. In LPS-stimulated samples, combined HG + HI induced the highest TF-PCA levels suggesting that HG may override the inhibitory effect of insulin in the presence of LPS. Further studies, including in patients with T1DM and T2DM, are needed to understand the effects of HG and HI and the cellular activation state on TF expression. Disclosures:No relevant conflicts of interest to declare.

Tissue factor antisense deoxyoligonucleotide prevents monocrotaline/LPS hepatotoxicity in mice

Tissue factor (TF) is a membranous glycoprotein that functions as a receptor for coagulation factor VII/VIIa and activates the coagulation system when blood vessels or tissues are damaged. TF was upregulated in our monocrotaline (MCT)/lipopolysaccharide (LPS) hepatotoxicity model. We tested the hypothesis that TF-dependent fibrin deposition and lipid peroxidation in the form of oxidized low-density-lipoprotein (ox-LDL) accumulation contribute to liver inflammation induced by MCT/LPS in mice. In the present study, we blocked TF using antisense oligodeoxynucleotides against mouse TF (TF-ASO). TF-ASO (5.6 mg kg(-1) ) was given i.v. to ND4 male mice 30 min after administration of MCT (200 mg kg(-1) ) p.o. followed after 3.5 h by LPS i.p. (6 mg kg(-1) ). Blood alanine aminotransferase (ALT), TF, ox-LDL, platelets, hematocrit and keratinocyte-derived chemokine (KC) levels were evaluated in different treatment groups. Fibrin deposition and ox-LDL accumulation were also analyzed in the liver sections using immunofluorescent staining. The results showed that TF-ASO significantly restored blood ALT, hematocrit and KC levels, distorted after MCT/LPS co-treatment, as well as preventing the accumulation of ox-LDL and the deposition of fibrin in the liver tissues, and thereby inhibited liver injury caused by MCT/LPS. In a separate experiment, TF-ASO administration significantly prolonged animal survival. The current study demonstrates that TF is associated with MCT/LPS-induced liver injury. Administration of TF-ASO successfully prevented this type of liver injury.

Tissue factor-positive monocytes in children with sickle cell disease: correlation with biomarkers of haemolysis.

Tissue Factor (TF) initiates thrombin generation, and whole blood TF (WBTF) is elevated in sickle cell disease (SCD). We sought to identify the presence of TF-positive monocytes in SCD and their relationship with the other coagulation markers including WBTF, microparticle-associated TF, thrombin-antithrombin (TAT) complexes and D-dimer. Whether major SCD-related pathobiological processes, including haemolysis, inflammation and endothelial activation, contribute to the coagulation abnormalities was also studied. The cohort comprised children with SCD (18 HbSS, 12 HbSC, mean age 3·6 years). We demonstrated elevated levels of TF-positive monocytes in HbSS, which correlated with WBTF, TAT and D-dimer (P = 0·02 to P = 0·0003). While TF-positive monocytes, WBTF, TAT and D-dimer correlated with several biomarkers of haemolysis, inflammation and endothelial activation in univariate analyses, in multiple regression models the haemolytic markers (reticulocytes and lactate dehydrogenase) contributed exclusively to the association with all four coagulant markers evaluated. The demonstration that haemolysis is the predominant operative pathology in the associated perturbations of coagulation in HbSS at a young age provides additional evidence for the early use of therapeutic agents, such as hydroxycarbamide to reduce the haemolytic component of this disease.

Arterial smooth muscle injury causes blood tissue factor elevation predicting restenosis after pci

Objective. The predictability of the whole-blood tissue factor levels for restenosis after coronary angioplasty is uncertain. We first probed in depth the association between plasma tissue factor concentrations and the development of restenosis after coronary intervention with an animal pathological model. Methods and results. Thirty pigs were used and their coronary arteries were injured for the dilatation of balloons. Morphological measurements include neointimal area, injury score and the extent of area stenosis. Whole-blood tissue factor levels were measured before and after intervention. The circulating tissue factor levels increased significantly after intervention (baseline value, 328.54 ± 47.46 pg/ml; at 30th minute, 618.96 ± 119.08 pg/ml; at 24th hour, 639.34 ± 116.21 pg/ml) (p < 0.01), and the degrees of tissue factor changes correlated positively to the neointimal hyperplasty (r30th min = 0.751, r24th hour = 0.72, p < 0.01). There was no significant difference with the baseline whole blood tissue factor (TF) levels between restenotic and non-restenotic cases (330.83 ± 47.32 vs. 325.1 ± 49.57 pg/ml) (p > 0.05). The injury of media led to the most distinctive changes of blood tissue factor (p < 0.01). Conclusion. Higher values of whole-blood tissue factor may be a predictor of restenosis, and the damaged media might be the main reason of the tissue factor increase.

Circulating Tissue Factor Procoagulant Activity (TF-PCA) Is Elevated in Type 1 Diabetes Mellitus: Differential Regulation of TF-PCA in Type 1 and Type 2 Diabetes

Abstract 540 Background:Type 1 diabetes mellitus (T1DM) is a hypercoagulable state associated with increased acute cardiovascular events. Potential risk factors for this include alterations in coagulation and fibrinolytic systems. Tissue factor (TF) is the principal initiator of blood coagulation. Several studies show that there is a circulating pool of TF in blood, which is thrombogenic, and elevated in thrombotic states. We have shown (J Clin Endo Metab 2007, 92:4352-8) that circulating TF procoagulant activity (TF-PCA) is elevated in patients with Type 2 DM (T2DM) and increases further with acute combined hyperglycemia-hyperinsulinemia and selective hyperinsulinemia. There is currently no information on circulating TF-PCA levels and TF responses to hyperglycemia and/or hyperinsulinemia in patients with T1DM who are at comparable risk for cardiovascular events as T2DM patients. Objective:To investigate circulating TF-PCA and other coagulation factors under basal conditions and in response to acute selective hyperglycemia, selective hyperinsulinemia and combined hyperglycemia and hyperinsulinemia in T1DM. Methods:Three study protocols were used: 1) acute correction of hyperglycemia (with IV insulin) followed by 24 h of hyperglycemia, 2) 24 h of selective hyperinsulinemia and 3) 24 h of combined hyperinsulinemia and hyperglycemia. Studies were performed in 9 T1DM patients and 7 non-diabetic subjects. T1DM patients were on a basal/bolus insulin regimen (insulin glargine, 15–70 units at night) or Novolog 70/30 mix twice daily (45-50 units). Circulating membrane bound TF-PCA was measured in whole blood lysates by a two-stage clotting assay (Key et al, Blood; 1998:91). Results:Basal TF-PCA (64.7 ± 6.0 vs. 24.6 ± 1.2 U/ml, p < 0.001) and plasma factor VIIa (104 ± 24 vs. 38 ± 8 mU/ml, p < 0.03), the activated form of factor VII, were higher in T1DM (n=9) than in non-diabetic controls (n= 7) indicating a chronic procoagulant state. Plasma FVIIc, FVIII, thrombin-antithrombin complexes (TAT) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were not significantly different between patients and controls. When control subjects and T1DM patients were combined, HbA1C correlated with TF-PCA (r=0.71, p=0.0001, n=23). Plasma adiponectin was elevated in T1DM patients compared to control subjects (12.4 ± 1.9 vs 6.7 ± 2.0 μg/ml, p < 0.05).Acutely normalizing hyperglycemia in T1DM patients over 3–15 h did not decrease TF-PCA. There were also no changes in plasma FVIIc and FVIII. To explore effects of acutely raising plasma glucose, glucose was raised from 103 ± 8 to ∼ 300 mg/dl by infusion of 20% dextrose and maintained for 24 hours. Insulin concentrations were kept at basal concentrations (6 and 13 μU/ml) by IV infusion. In our previous studies in non-diabetic subjects (Diabetes 2006, 55,202-8) and in T2DM patients (J Clin Endo Metab 2007, 92,4352-8), raising glucose and insulin together produced a marked increase in circulating TF-PCA. We therefore raised glucose to ∼ 250 mg/dl and insulin to ∼ 100 μU/ml together in 8 T1DM patients. Raising glucose levels alone or in combination with insulin decreased circulating TF-PCA by 26% (p < 0.02) and 37% (p < 0.01), respectively, which is in striking contrast to the elevations noted in non-diabetic controls and T2DM patients. To explore effects of selective hyperinsulinemia, plasma insulin levels were raised in 3 T1DM patients by IV infusion of regular insulin from 15 ± 0.2 to ∼ 75 μU/ml and maintained for 24 hours while plasma glucose was kept at ∼ 100 mg by infusion of 20% dextrose. Again, in contrast to our studies in T2DM patients and healthy subjects we found no increase in TF-PCA Conclusions:Circulating TF-PCA and FVIIa levels are elevated in T1DM patients indicating a potential prothrombotic state. The studies on acutely induced hyperinsulinemia and hyperglycemia indicate that the regulation of TF expression is different in T1DM and T2DM. This may be due to multiple mechanisms, including a differential effect of insulin on monocytes TF expression in T1DM and T2DM, and due to differences in plasma adiponectin, which has been shown to inhibit TF expression and is elevated in T1DM. Additional studies are needed to obtain insights into the mechanisms regulating the differential expression of TF in the two forms of diabetes. Disclosures:No relevant conflicts of interest to declare.

Spatial Separation of TF-Carriers Modulates Procoagulant Activity of Circulating TF

Abstract 2265 Background:Tissue factor (TF) is essential for the hemostatic initiation of the extrinsic pathway of blood coagulation. We found that cancer and benign tumor cells have different TF-dependent procoagulant activities (PCA). Intravascular microparticle-, cancer-, or other cell-associated TF may initiate thrombosis but circulating TF levels are not necessarily concordant with thrombosis. We investigated the role of TF carrier burden on the procoagulant activity of circulating TF. Methods:We utilized human monocytic-like U937 cells and thromboplastin-coated polymer microspheres as TF carriers. U937 cells were stimulated with endotoxin to induce surface expression of TF. Particle effect on clotting was measured in a closed transport system using a coagulometer. Occlusive thrombus formation was measured in an open transport system using a flow chamber under a constant pressure gradient. Zymogen activation was recorded with a chromogenic substrate in a closed transport system. Results:This study was designed to assess whether the spatial separation of intravascular TF-carriers in blood, demonstrated with TF-inducible human monocytic cell line U937 or TF-coated polymer microspheres, affected procoagulant activity and hence thrombogenic potential. Experiments were performed to characterize the effects of TF carrier number on the kinetics of clot formation in both open and closed systems. The procoagulant activity of TF carriers was found to correlate with spatial separation in both closed, well-mixed systems as well as open, flowing systems. TF carriers enhanced the amidolytic activity of FVIIa towards the chromogenic substrate, S-2366, as a function of carrier count. Initiation time correlated with spatial separation, whereas enzyme reaction rates correlated with inverse spatial separation. Conclusions:These findings suggest that transport of coagulation factors to the surface of a circulating TF carrier may constrain the enzymatic rate of TF-dependent coagulation reactions, and therefore modulate circulating TF procoagulant activity. By shortening the separation of TF carriers in suspension, the proximity of plasma coagulation factors to a TF surface is increased, and we measured a resulting increase in procoagulant activity for these carriers. Our results suggest that patient-specific sensitivity to circulating TF may depend upon plasma concentrations of TF-dependent coagulation factors (FVII, FX and FII) as the concentration of factors in plasma influences their transport rate. Further, the distribution of circulating TF within the vasculature may influence procoagulant activity of circulating TF by altering the proximity of TF-carriers to plasma coagulation factors. Thus, clinical relevance of circulating TF may not depend on whole blood TF concentration, but rather on the distribution of circulating TF over different carriers (tumor cells versus tumor cell-derived microparticles) or patient-specific concentration of plasma coagulation factors in the extrinsic pathway (i.e. FVII) downstream of TF. Disclosures:Gruber:Aronora, LLC: Consultancy, Equity Ownership.

The role of carrier number on the procoagulant activity of tissue factor in blood and plasma

Tissue factor (TF) is a transmembrane glycoprotein cofactor of activated blood coagulation factor VII (FVIIa) that is required for hemostatic thrombin generation at sites of blood vessel injury. Membrane-associated TF detected in circulating blood of healthy subjects, referred to as intravascular or circulating TF has been shown to contribute to experimental thrombus propagation at sites of localized vessel injury. Certain disease states, such as metastatic cancer, are associated with increased levels of intravascular TF and an elevated risk of venous thromboembolism. However, the physiological relevance of circulating TF to hemostasis or thrombosis, as well as cancer metastasis, is ill-defined. This study was designed to assess whether the spatial separation of intravascular TF carriers in blood, demonstrated with TF-inducible human monocytic cell line U937 or TF-coated polymer microspheres, affected procoagulant activity and hence thrombogenic potential. Experiments were performed to characterize the effects of TF-carrier number on the kinetics of clot formation in both open and closed systems. The procoagulant activity of TF carriers was found to correlate with spatial separation in both closed, well-mixed systems and open, flowing systems. TF carriers enhanced the amidolytic activity of FVIIa toward the chromogenic substrate, S-2366, as a function of carrier count. These results suggest that TF-initiated coagulation by circulating TF is kinetically limited by mass transport of TF-dependent coagulation factors to the TF-bearing surface, a constraint that may be unique to circulating TF. Spatial separation of circulating TF carriers is therefore a critical determinant of the procoagulant activity of circulating TF.

Xuezhikang, Extract of Red Yeast Rice, Inhibited Tissue Factor and Hypercoagulable State Through Suppressing Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Extracellular Signal-regulated Kinase Activation

Xuezhikang, extract of red yeast rice, is a traditional Chinese medicine with multiple cardioprotective effect. It contains a family of naturally occurring statins, such as lovastatin. Tissue factor (TF) is overexpressed in macrophages of lipid core plaques, which display high procoagulant activity and seem to be a potentially target for anti-atherothrombosis. Therefore, the purpose of this study was to explore the effect and possible molecular mechanisms of xuezhikang on inhibiting TF expression and hypercoagulable state and the differences compared with lovastatin. Our results showed that xuezhikang significantly suppressed oxidized low-density lipoprotein-induced TF expression in macrophages in a concentration-dependent manner. Xuezhikang reduced nicotinamide adenine dinucleotide phosphate oxidase activity by decreasing membrane translocation of p47 through inhibition of extracellular signal-regulated kinase 1/2 activation. Nicotinamide adenine dinucleotide phosphate inhibitor (diphenyleneiodonium) also inhibited the oxidized low-density lipoprotein-induced TF expression, similar to the effects of xuezhikang. Furthermore, consistent with the severity of aortic atherosclerosis, xuezhikang (300 mg·kg·d) significantly reduced blood coagulation activation and TF expression in high-cholesterol diet-induced atherosclerotic rats. In addition, xuezhikang was more potent than lovastatin on inhibiting the expression of TF and nicotinamide adenine dinucleotide phosphate oxidase activation. These observations provide evidences that inhibition of xuezhikang on hypercoagulation and TF expression may partly account for its cardioprotective benefits.

High intravascular tissue factor expression in dogs with idiopathic immune-mediated haemolytic anaemia

A high mortality occurs in dogs with idiopathic immune-mediated haemolytic anaemia (IMHA) during the first 2 weeks after the diagnosis. The aim of this study was to investigate the inflammatory response and coagulation abnormalities in dogs with IMHA in relation to the prognosis and to establish the contribution of whole blood tissue factor (TF) and IL-8 gene expressions. Gene expressions in dogs with IMHA were compared to healthy dogs, dogs with DIC, dogs with sepsis, and in two groups of dogs that underwent intensive care treatment but had no evidence for either DIC or sepsis. The whole blood TF and IL-8 expressions were up regulated in all non-IMHA groups. Similarly, the TF expression in IMHA dogs was high, but the intravascular IL-8 expression was not increased. The dogs with IMHA had a pronounced inflammatory response that included a high WBC, left shift and monocytosis in comparison to the other disease groups. Coagulation factor activities in IMHA dogs were decreased fitting consumptive coagulopathy and the acute phase proteins FVIII and fibrinogen were increased. The platelet parameters suggested platelet activation and high platelet turnover in IMHA dogs. The model that best explained mortality contained monocytosis, increased activated partial thromboplastin time and elevated creatinine. Whole blood TF gene expression is up regulated and may contribute to consumptive coagulopathy in dogs with IMHA. Increased TF expression by activated platelets is an alternative explanation and should be investigated.