Key role of integrin αIIbβ3 signaling to Syk kinase in tissue factor-induced thrombin generation

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

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The Role of Factor XI in Thrombin Generation Induced by Low Concentrations of Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615964 ◽

2001 ◽

Vol 85 (06) ◽

pp. 1060-1065 ◽

Cited By ~ 66

Author(s):

Irene Keularts ◽

Ariella Zivelin ◽

Uri Seligsohn ◽

H. Coenraad Hemker ◽

Suzette Béguin

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Contact Activation ◽

Factor Xi ◽

Platelet Poor Plasma ◽

Low Concentrations ◽

Time Determination ◽

Thrombin Formation

SummaryThrombin generation has been studied in the plasma of severely factor XI deficient patients under conditions in which contact activation did not play a role. In platelet-rich as well as platelet-poor plasma, thrombin generation was dependent upon the presence of factor XI at tissue factor concentrations of between 1 and 20 pg/ml i.e. ~ 0.01 to 0.20% of the concentration normally present in the thromboplastin time determination. The requirement for factor XI is low; significant thrombin generation was seen at 1% factor XI; at 10%, thrombin formation was nearly normalised. A suspension of normal platelets in severely factor XI deficient plasma did not increase thrombin generation. This implies that there is no significant factor XI activity carried by normal platelets, although the presence of factor XI and factor XI inhibitors in platelets cannot be ruled out.

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Significant Correlations between Tissue Factor and Thrombin Markers in Trauma and Septic Patients with Disseminated Intravascular Coagulation

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615025 ◽

1998 ◽

Vol 79 (06) ◽

pp. 1111-1115 ◽

Cited By ~ 96

Author(s):

Satoshi Nanzaki ◽

Shigeyuki Sasaki ◽

Osamu Kemmotsu ◽

Satoshi Gando

Keyword(s):

Tissue Factor ◽

Disseminated Intravascular Coagulation ◽

Thrombin Generation ◽

Trauma Patients ◽

Elevated Plasma ◽

Antigen Concentration ◽

Intravascular Coagulation ◽

Factor Antigen ◽

D Dimer

SummaryTo determine the role of plasma tissue factor on disseminated intravascular coagulation (DIC) in trauma and septic patients, and also to investigate the relationships between tissue factor and various thrombin markers, we made a prospective cohort study. Forty trauma patients and 20 patients with sepsis were classified into subgroups according to the complication of DIC. Plasma tissue factor antigen concentration (tissue factor), prothrombin fragment F1+2 (PF1+2), thrombin antithrombin complex (TAT), fibrinopeptide A (FPA), and D-dimer were measured on the day of admission (day 0), and on days 1, 2, 3, and 4 after admission. The levels of plasma tissue factor in the DIC group were more elevated than those of the non-DIC group in both the trauma and the septic patients. In patients with sepsis, tissue factor levels on days 0 through 4 in the non-DIC group showed markedly higher values than those in the control patients (135 ± 8 pg/ml). Significant correlations between tissue factor and PF1+2, TAT, FPA, and D-dimer were observed in the DIC patients, however, no such correlations were found in the non-DIC patients. These results suggest that elevated plasma tissue factor in patients with trauma and sepsis gives rise to thrombin generation, followed by intravascular coagulation.

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Role of tissue factor expression in thrombin generation by canine tumor cells

American Journal of Veterinary Research ◽

10.2460/ajvr.77.4.404 ◽

2016 ◽

Vol 77 (4) ◽

pp. 404-412 ◽

Cited By ~ 5

Author(s):

Erika J. Gruber ◽

James L. Catalfamo ◽

Tracy Stokol

Keyword(s):

Tissue Factor ◽

Tumor Cells ◽

Thrombin Generation ◽

Tissue Factor Expression ◽

Factor Expression ◽

Canine Tumor

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The Concentration of Exogenously Added Lipids and Endogenously Available Microparticles Are Major Determinants of Thrombin Generation in Plasma.

Blood ◽

10.1182/blood.v108.11.4000.4000 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4000-4000

Author(s):

C. Kluft ◽

P. Meijer ◽

R. Kret ◽

V. Kaufmann ◽

J. Mager

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Area Under The Curve ◽

Coagulation Factors ◽

Factor Ix ◽

Fixed Amount ◽

High Lipid ◽

Factor Ii ◽

The Difference

Abstract Thrombin generation tests are started by adding coagulation activator (e.g. tissue factor or contact activator) together with lipids. We evaluated the role of lipids in tests started with a fixed amount of tissue factor (7.16 pM) and addition of either 3.2 (high) or 0.32 (low) μM of lipids (Technothrombin ® TGA assays from Technoclone, Vienna) in both normal plasma and plasma that was ultracentrifuged (30 minutes at 15,000 g) to remove microparticles (MPs). The tests were performed in plasma samples of groups of apparently healthy individuals. It was observed in 54 healthy volunteers that starting with high or low lipids substantially influenced the total amount of thrombin generated expressed by the area under the curve (AUC) (AUC median 2492, IQR 716 versus AUC median 1154, IQR 652 nM*min, respectively), the rate of thrombin formation or velocity index (VI) (median 53.4, IQR 43.6 and median 11.7, IQR 10.6 nM/min, respectively), and the lag time to the start of thrombin generation (median 10.3, IQR 2.4 versus median 17.5, IQR 5.0 minutes, respectively). It can be concluded that the VI is the most sensitive variable showing approximately a factor of 5 difference between high and low lipid. The difference of adding high or low lipid on VI was primarily dependent upon the lipid concentration and to a limited extend influenced (univariate) by factor II levels (12%) and factor IX levels (10 %), taking into account practically all known coagulation factors (fibrinogen, II, V, VII, VIII, IX, X, XI, XII, PC, PS, PZ, TFPI, PCI) determined in the 54 plasma’s as potential determinant. In plasma of 36 volunteers microparticles were removed and VI dropped to 19% in comparison to the untreated plasma when tested with the addition of high lipid and to 3.1% with the addition of low lipid. Re-addition of MPs to a specific plasma restored VI dose dependently with an optimum at 2x104 MPs/ml. The same level of VI (63.8 and 62.0 nM/min, respectively) was reached with high and low lipid addition when 2.104 MPs/ml were added. It is concluded that endogenous MPs play an important role in thrombin generation tests, in particular but not exclusively when the test is performed with low levels of added lipids.

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Tissue Factor Bearing Microparticles in the Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v118.21.5174.5174 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5174-5174

Author(s):

Brady L Stein ◽

Brandon McMahon ◽

Ivy Weiss ◽

James Marvin ◽

Hau C. Kwaan

Keyword(s):

Sample Size ◽

Tissue Factor ◽

Thrombin Generation ◽

Myeloproliferative Neoplasms ◽

Flow Cytometric Analysis ◽

Jak2 V617f ◽

Lag Phase ◽

Thrombotic Risk ◽

Larger Sample

Abstract Abstract 5174 Background: Thrombosis is a well recognized complication in the myeloproliferative neoplasms (MPN), essential thrombocytosis (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). The mechanism for thrombosis is not well-established, nor are there relevant biomarkers to predict risk and/or recurrence. Circulating cellular microparticles (MP) containing procoagulant tissue factor (TF) have been shown to correlate with thrombotic risk in many forms of cancer and cardiovascular diseases. To investigate the role of MP in the MPN, we studied 16 patients (ET=5; PV=6; PMF=2; post-ETMF=1, and MPN NOS=2) and compared results to 15 healthy subjects. Methods: Citrated blood samples were collected from the 16 MPN patients and 15 controls. Platelet poor plasma (PPP) was obtained by centrifuging at 1,500 G for 20 minutes. 50 μL of PPP was added to 200 μL PBS (without Mg/Ca) and centrifuged at 20,000 G for 10 minutes. The sediment containing MP was resuspended in 100μL of buffer for labeling with TF, CD41a (platelets), CD14 (monocytes), CD66b (neutrophils), and CD33 (myeloid lineage). Following incubation, PBS was added to the suspension to a volume of 1ml for flow cytometric analysis (LSR Fortessa, FlowJo software). Electronic triggering was done on side-scatter, and acquisition regions were defined based on sizing beads (0.3 to 1.0 micron) along with annexin A5 positivity. Using MP sediment, TF activity was measured using chromogenic assays (Actichrome TF ELISA, American Diagnostica,) and thrombin generation (TGT) was assayed (Technothrombin TGA, diaPharma), with results expressed as lag phase, velocity-index, peak thrombin, and area under the curve (AUC). The Wilcoxon-Rank Sum test was used to compare group differences (MPN vs. control) in median values. Results: Among the MPN patients, 7(44%) were male, and the median age was 60 years. 11 (69%) were JAK2 V617F positive, and 3 (19%) had a prior history of thrombosis (2 hepatic vein thromboses, 1 myocardial infarction). At the time of collection, 14 (93%) were on aspirin, 1 (6%) was on Coumadin, and 5 (31%) were on Hydroxyurea. The median total MP number was increased in MPN patients vs. controls (243580 vs. 83120; p=0.0057). The median percentage of TF-bearing MP's was also significantly greater in MPN patients compared to controls (35.5% vs. 12%; p=0.0003). When comparing MPN patients to controls, these TF-bearing MP were derived from CD14 (31.5% vs. 2%; p<0.001) and CD41a (24% vs. 7%; p=0.0157), respectively, reflecting monocyte and platelet origins of the MPs. The TF-bearing MPs in MPN patients (N=10) were functionally active compared to controls (N=10) (median TF activity: 1.7 pM vs. 0.03 pM; p=0.0022). Thrombin generation assays were performed in 13 MPN patients and 9 controls, and were comparable: mean lag phase (14.4 vs. 10.15 minutes; p=0.26); mean velocity index (21.13 vs. 14.78; p=0.31); mean peak thrombin generation (111.66 nM vs. 120.41 nM; p=0.75); and mean AUC (3218.77 vs. 3807.41 p=0.37). Conclusion: Compared to controls, samples of JAK2 V617F-positive and negative MPN patients revealed a higher median total number of microparticles. Further, the proportion of TF-bearing MPs was higher in MPN patients, and of monocyte and platelet origin, suggesting their possible role in thrombotic complications. Though the MPs in MPN patients appear functional, based on higher TF activity, functional assays with thrombin generation testing failed to reveal a difference between MPN patients and controls. A lack of difference in TGT may suggest the presence of one or more inhibitors present in the MPN; the nature of this inhibitor is under investigation. Future studies, with a larger sample size and prospective follow-up are indicated to determine the role of MPs in predicting incident or recurrent thrombosis in the MPN. In addition, with a larger sample size, differences by MPN disease class and JAK2 V617F status will be uncovered. Disclosures: No relevant conflicts of interest to declare.

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