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

Blood ◽  
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.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor X ◽  
Dose Dependent

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.

scholarly journals Plasma Kallikrein Contributes to Coagulation in the Absence of Factor XI by Activating Factor IX

2020 ◽  
Vol 40 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Mayken Visser ◽  
René van Oerle ◽  
Hugo ten Cate ◽  
Volker Laux ◽  
Nigel Mackman ◽  
...  
Keyword(s):  
Complex Formation ◽  
Ellagic Acid ◽  
Thrombin Generation ◽  
Factor Ix ◽  
Coagulation System ◽  
Plasma Kallikrein ◽  
Factor Xia ◽  
The Difference ◽  
Long Chain

Objectives: FXIa (factor XIa) induces clot formation, and human congenital FXI deficiency protects against venous thromboembolism and stroke. In contrast, the role of FXI in hemostasis is rather small, especially compared with FIX deficiency. Little is known about the cause of the difference in phenotypes associated with FIX deficiency and FXI deficiency. We speculated that activation of FIX via the intrinsic coagulation is not solely dependent on FXI(a; activated FXI) and aimed at identifying an FXI-independent FIX activation pathway. Approach and Results: We observed that ellagic acid and long-chain polyphosphates activated the coagulation system in FXI-deficient plasma, as could be demonstrated by measurement of thrombin generation, FIXa-AT (antithrombin), and FXa-AT complex levels, suggesting an FXI bypass route of FIX activation. Addition of a specific PKa (plasma kallikrein) inhibitor to FXI-deficient plasma decreased thrombin generation, prolonged activated partial thromboplastin time, and diminished FIXa-AT and FXa-AT complex formation, indicating that PKa plays a role in the FXI bypass route of FIX activation. In addition, FIXa-AT complex formation was significantly increased in F11 −/− mice treated with ellagic acid or long-chain polyphosphates compared with controls and this increase was significantly reduced by inhibition of PKa. Conclusions: We demonstrated that activation of FXII leads to thrombin generation via FIX activation by PKa in the absence of FXI. These findings may, in part, explain the different phenotypes associated with FIX and FXI deficiencies.

scholarly journals Differences in coagulopathy indices in patients with severe versus non-severe COVID-19: a meta-analysis of 35 studies and 6427 patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alberto Polimeni ◽  
Isabella Leo ◽  
Carmen Spaccarotella ◽  
Annalisa Mongiardo ◽  
Sabato Sorrentino ◽  
...  
Keyword(s):  
Platelet Count ◽  
Meta Analysis ◽  
Severe Disease ◽  
Coagulation Factors ◽  
Primary Analysis ◽  
Severe Group ◽  
Intravascular Coagulopathy ◽  
The Difference ◽  
D Dimer

AbstractCoronavirus disease 2019 (COVID-19) is a highly contagious disease that appeared in China in December 2019 and spread rapidly around the world. Several patients with severe COVID-19 infection can develop a coagulopathy according to the ISTH criteria for disseminated intravascular coagulopathy (DIC) with fulminant activation of coagulation, resulting in widespread microvascular thrombosis and consumption of coagulation factors. We conducted a meta-analysis in order to explore differences in coagulopathy indices in patients with severe and non-severe COVID-19. An electronic search was performed within PubMed, Google Scholar and Scopus electronic databases between December 2019 (first confirmed Covid-19 case) up to April 6th, 2020. The primary endpoint was the difference of D-dimer values between Non-Severe vs Severe disease and Survivors vs Non-Survivors. Furthermore, results on additional coagulation parameters (platelet count, prothrombin time, activated partial thromboplastin time) were also analyzed. The primary analysis showed that mean d-dimer was significantly lower in COVID-19 patients with non-severe disease than in those with severe (SMD − 2.15 [− 2.73 to − 1.56], I2 98%, P < 0.0001). Similarly, we found a lower mean d-dimer in Survivors compared to Non-Survivors (SMD − 2.91 [− 3.87 to − 1.96], I2 98%, P < 0.0001). Additional analysis of platelet count showed higher levels of mean PLT in Non-Severe patients than those observed in the Severe group (SMD 0.77 [0.32 to 1.22], I2 96%, P < 0.001). Of note, a similar result was observed even when Survivors were compared to Non-Survivors (SMD 1.84 [1.16 to 2.53], I2 97%, P < 0.0001). Interestingly, shorter mean PT was found in both Non-Severe (SMD − 1.34 [− 2.06 to − 0.62], I2 98%, P < 0.0002) and Survivors groups (SMD − 1.61 [− 2.69 to − 0.54], I2 98%, P < 0.003) compared to Severe and Non-Survivor patients. In conclusion, the results of the present meta-analysis demonstrate that Severe COVID-19 infection is associated with higher D-dimer values, lower platelet count and prolonged PT. This data suggests a possible role of disseminated intravascular coagulation in the pathogenesis of COVID-19 disease complications.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IXa in Activated Prothrombin Complex Concentrates Is the Dominant Contributor to Enhanced Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
State Level ◽  
Peak Level ◽  
Coagulation Factors ◽  
Major Effect ◽  
Factor Ix ◽  
Factor X ◽  
Factor Ixa ◽  
Activated Prothrombin Complex Concentrates

Background. Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa and factor X 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 treating this bleeding in hemophilia A patients with inhibitors is to give an activated prothrombin complex concentrate (APCC; FEIBA) (disfavored in NHF MASAC #255). APCC contains a number of coaguation factors including prothrombin, factor X (FX), and factor IX (FIX). APCC also contains activated factor X (FXa) and factor IX (FIXa). Previous work has shown that when APCCs are added to hemophilia A plasma containing emicizumab there is a significant increase in thrombin generation [J Thromb Haemost 2018;16:1580-1591]. The goal of this work was to study thrombin generation in hemophilia A plasma with emicizumab and to examine the role of the zymogen and activated components of an APCC in the increased thrombin generation. Methods. In hemophilia A plasma, thrombin generation assays were done 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). The components of APCC were studied at concentrations that should mimic the levels seen in the plasma of a patient given a dose of 50 U/kg: prothrombin 1800 nM; FX 130 nM; FIX 90 nM; and FIXa 0.4 nM. Results. When initiated with low TF, hemophilia A plasma alone had essentially no thrombin generation. As expected, adding emicizumab enhanced thrombin generation. The addition of zymogen coagulation factors, prothrombin, FIX, and FX, separately or together gave a small increase in thrombin generation. However, addition of FIXa to emicizumab gave a large increase in peak thrombin. In hemophilia A plasma with emicizumab and FIXa, addition of prothrombin further increased thrombin generation and specifically increased the peak level of thrombin. Further addition of FX or FIX, separately or together, only minimally increased thrombin generation. Discussion. The strong contribution of factor IXa to the effects of APCCs on thrombin generation in hemophilia A plasma depends on the presence of emicizumab. In the absence of emicizumab, a study of the individual components of APCC showed that a combination of FXa and prothrombin at levels found in APCCs had the major effect on thrombin generation [Haemophilia. 2016;22:615-24]. That study found that FIXa did not increase thrombin generation. However, in the presence of emicizumab, despite the weak solution phase affinity of the bifunctional antibody for FIXa, small amounts of FIXa were the most significant contributor to thrombin generation. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

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

2001 ◽  
Vol 85 (06) ◽  
pp. 1060-1065 ◽  
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.

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

1998 ◽  
Vol 79 (06) ◽  
pp. 1111-1115 ◽  
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.

The Impact of Factor IX Level on Thrombin Generation and Bleeding During Warfarin Anticoagulation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 541-541
Author(s):  
Yesim Dargaud ◽  
Maureane Hoffman ◽  
Claude Negrier ◽  
Leana Lefrapper ◽  
Dougald M. Monroe
Keyword(s):  
Tissue Factor ◽  
Vitamin K ◽  
Intracranial Haemorrhage ◽  
Thrombin Generation ◽  
Factor Ix ◽  
Target Range ◽  
Significant Difference ◽  
Generation Capacity ◽  
The Impact

Abstract Abstract 541 Bleeding occurs in from 10 – 16% of warfarin-treated patients. Having a PT-INR in the target range is associated with better outcomes. However, even patients with an INR in the target range of 2–3 can suffer bleeding, suggesting that INR does not perfectly reflect the therapeutic effect of warfarin. The goal of our studies was to determine whether the level of specific coagulation factors could predict the risk of bleeding while the INR was in the target range. We modeled warfarin anticoagulation in our previously published in vitro cell based-model by adjusting the levels of vitamin K-dependent factors to those of patients with an INR of 2–3. We then examined the effect of variations in the level of FIX. The cogulation reactions were initiated by monocyte-expressed tissue factor (assayed at 1pM). Variation in FIX had a marked effect on thrombin generation. However, in plasma with the same levels of factors, as expected, variations in FIX had no effect on the PT-INR. Thus, we hypothesized that a subject with a lower FIX level than average may have a lower level of thrombin generation than is indicated by the INR. The INR might, therefore, underestimate the level of anticoagulation in such a subject. If s/he is maintained in the “therapeutic range” as measured by the INR, s/he will actually be over-anticoagulated and prone to hemorrhage. A prospective, single centre clinical study has been carried out to test this hypothesis in warfarinized patients. Between October 2010 and June 2011, 312 consecutive patients admitted to the emergency department of Edouard Herriot Hospital in Lyon, with an INR between 1.8 and 3.2, were included in the study after obtaining informed consent. Twenty six patients were admitted for a bleeding episode, 18 for recurrent thrombosis and 268 for other medical reasons. Patients presenting with bleeding, 17 males and 9 females, were aged 74±14 years old compared to the rest of the patients aged 76±14. Among the 26 bleeders, 7 had a spontaneous intracranial haemorrhage, 2 had a trauma-induced intracranial haemorrhage, 12 presented a gastrointestinal bleeding and 5 exhibited muscle hematomas, severe epistaxis or urinary tract bleeding. PT-INR and vitamin K-dependent factor levels were determined in all patients. Thrombin generation capacity in platelet poor plasma was measured using Calibrated Automated Thrombin generation assay (Thrombinoscope bv, Maastricht, The Netherlands), with tissue factor 1pM and phospholipids PC:PS:PE 4μM. No statistically significant difference was observed in the PT-INR of bleeding patients (INR=2.4±0.4) and those having a thrombosis (INR=2.5±0.5) or patients admitted for other reasons (INR=2.6±0.2). Plasma prothrombin and factor × levels were also similar in all three groups. However, a statistically lower plasma factor IX activity was observed in bleeders (p=0.01, Mann Whitney test) compared to other groups, 47.6±20 IU/dL vs. 63±33 IU/dL. In all the warfarinized subjects with an INR between 1.8 and 3.2, no correlation was found between thrombin generation capacity and PT-INR results (p=0.85, Spearman correlation test). However, a statistically significant correlation was observed between thrombin generation capacity and factor IX levels (p=0.0002). In patients, presenting with warfarin-related haemorrhage, the endogenous thrombin potential (ETP) was significantly lower at 340±335 nM.min (p=0.05) then that of warfarinized subjects who did not suffer bleeding (ETP 406±215 nM.min). These data support our hypothesis based on our in vitro results and show that patients who bleed when their PT-INR is in the target range 2 – 3 might have defective thrombin generation related to a lower level of factor IX than expected. Thus, our results suggest that the appropriate target INR level might not be the same for all patients. Those with factor IX levels that differ significantly from the mean of the population might be managed best by selecting a target INR that is based on the level of thrombin generation. Of course, a “target range” for parameters of thrombin generation during warfarin therapy would need to be developed if the assay were to be used for this purpose. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Factor XI contributes to thrombin generation in the absence of factor XII

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Dmitri V. Kravtsov ◽  
Anton Matafonov ◽  
Erik I. Tucker ◽  
Mao-fu Sun ◽  
Peter N. Walsh ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor Xi ◽  
Factor Xii Deficiency ◽  
Low Concentrations

Abstract During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

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