Failure of corn trypsin inhibitor to affect the thrombin generation assay in plasma from severe hemophiliacs

2014 ◽  
Vol 12 (9) ◽  
pp. 1558-1561 ◽  
Author(s):  
B. M. Mohammed ◽  
E. J. Martin ◽  
V. Salinas ◽  
R. Carmona ◽  
G. Young ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Thrombin Generation ◽  
Thrombin Generation Assay ◽  
Corn Trypsin Inhibitor

scholarly journals Novel Thermostable Inhibitor of Contact Activation Tica Effectively Blocks Contact Activation in Low Tissue Factor Thrombin Generation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1146-1146
Author(s):  
Tom Van De Berg ◽  
Dennis P.L. Suylen ◽  
M.G.L. Christella D. Thomassen ◽  
Rene van Oerle ◽  
Henri M.H. Spronk ◽  
...  
Keyword(s):  
Shelf Life ◽  
Tissue Factor ◽  
Trypsin Inhibitor ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Blood Collection ◽  
Contact Activation ◽  
Current Standard ◽  
Blood Drawing ◽  
Corn Trypsin Inhibitor

Background: Thrombin generation and other clotting assays suffer from a wide variation of pre-analytical variables. One of those pre-analytical variables is contact activation through blood withdrawal methods, different syringes, differences in blood coagulation tubes, blood transport and sample handling. It has been shown that the addition of contact activation inhibitors in low tissue factor activated thrombin generation leads to a correction of the, in these circumstances significant, increase in thrombin generation due to contact activation. We compare the novel 'thermostable inhibitor of contact activation' (TICA) to the current standard 'corn trypsin inhibitor' (CTI). Aim: Comparing the effectiveness of novel contact activation inhibitor TICA to the current standard CTI in low tissue factor-induced thrombin generation and recalcification in sodium citrate anticoagulated platelet poor plasma (PPP) and platelet rich plasma (PRP). Methods: We compared TICA, Corn trypsin inhibitor and plasma without contact activation inhibitors in low tissue factor PPP thrombin generation and in PRP recalcification thrombin generation, the latter the most sensitive condition for contact activation. In addition, we compared low tissue factor activated thrombin generation in plasma from severe hemophilia A patients with and without TICA during and after blood drawing. Thermostability - as a measure of shelf life - was measured and compared to CTI. Results: TICA is able to fully block contact activation in PRP recalcification experiments and is comparable to CTI in doing so. TICA significantly lowers low tissue factor induced thrombin generation by blocking contact activation. Pre-loading vacuum blood collection tubes with contact activation inhibitors is superior in inhibiting contact activation compared to addition of the inhibitor during the thrombin generation assay itself. TICA did not alter coagulation activity when added to FXIIa deficient plasma in thrombin generation. In contrast to CTI TICA is heat stable which will be of benefit to shelf life of pre-loaded blood drawing tubes. Conclusion: TICA is able to fully block contact activation and has several advantages over CTI. Disclosures No relevant conflicts of interest to declare.

Monitoring thrombin generation: Is addition of corn trypsin inhibitor needed?

2009 ◽  
Vol 101 (06) ◽  
pp. 1156-1162 ◽  
Author(s):  
Arne Dielis ◽  
Marina Panova-Noeva ◽  
René van Oerle ◽  
José Govers-Riemslag ◽  
Karly Hamulyák ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Thrombin Generation ◽  
Lag Time ◽  
Peak Height ◽  
Blood Collection ◽  
Contact Activation ◽  
In Vitro Measurements ◽  
Analytical Variable ◽  
Corn Trypsin Inhibitor

SummaryThrombin generation monitoring has the potential to be used as a clinical diagnostic tool in the near future. However, robust pre-analytical conditions may be required, and one factor that has been reported is in-vitro contact activation that might influence in-vitro measurements of thrombin generation and thereby act as an unpredictable pre-analytical variable. The aim of the current study was to investigate the influence of contact activation and the necessity of corn trypsin inhibitor (CTI) to abolish contact activation in thrombin generation measurements at low tissue factor (TF) concentrations. Thrombin generation was performed using the calibrated automated thrombinoscopy (CAT), thereby determining the endogenous thrombin potential (ETP), peak height, and the lag time, in plasma obtained from healthy volunteers. Addition of CTI after plasma preparation had no significant influence on thrombin generation triggered with 0.5 pM TF or higher, as demonstrated by unaltered ETP and lag time values between analyses with and without CTI. Addition of CTI before blood collection reduced thrombin generation triggered with 0.5 pM TF: both the ETP and peak height were significantly reduced compared to no CTI addition. In contrast, thrombin generation remained unaltered at a 1 pM TF trigger or above. This study demonstrates that addition of CTI after plasma separation is not necessary when triggering with TF concentrations of 0.5 pM and higher. Furthermore, it was demonstrated that it is not needed to pre-fill blood collecting tubes with CTI when measuring thrombin generation at TF concentrations of ≥1 pM.

scholarly journals In Covid-19 Infection, Plasma Extracellular Vesicle Tissue Factor Activity Does Not Correlate with D-Dimer Levels

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1045-1045
Author(s):  
Thomas J. Girard ◽  
Irem Eldem ◽  
Kenneth E Remy ◽  
Monty Mazer ◽  
Jorge Di Paola
Keyword(s):  
Tissue Factor ◽  
Disease Severity ◽  
Trypsin Inhibitor ◽  
Thrombin Generation ◽  
Sample Collection ◽  
Contact Activation ◽  
Heparin Treatment ◽  
Significant Difference ◽  
D Dimer ◽  
Corn Trypsin Inhibitor

Abstract Objective: Identify a plasma-based activity, or biomarker, that defines the mechanism(s) by which Covid-19 disease triggers excessive coagulation. Introduction: While acute respiratory syndrome is the fundamental feature of severe Covid-19 disease, having a high level of the coagulation biomarker D-dimer upon admission is associated with increased thrombosis and mortality. As such, hospitalized patients are often placed on anticoagulant heparins. How Covid-19 triggers excessive coagulation is unresolved. Sars-CoV-2 infection could expose existing tissue factor (TF) to blood or, via cytokines, induce TF expression on cells that are in direct contact with blood. Extracellular vesicles (EV) are lipid bound microparticles released by all types of healthy and damaged cell and Covid-19 patient plasma EV TF activity has been recently reported. Cellular activation and damage due to SARS-CoV-2 could also release polyanionic nucleic acids and polyphosphates and generate neutrophil extracellular traps as contact surfaces for clot formation. Methods: Study 1. We attempted to identify excessive coagulation pathway activities in Covid-19 plasma-based, Ca++-induced thrombin generation assays. Assays were performed in the absence and presence of selective extrinsic (TF) and intrinsic (contact activation) pathway inhibitors (n=296 plasma samples). D-dimer levels were also determined. In a smaller study, Covid-19 patient samples were collected directly into citrate or citrate plus corn trypsin inhibitor, then processed for analysis. Study 2. We conducted studies to evaluate the extent to which EV TF activity contributes to the Covid-19-associated coagulopathies. Plasma EVs were isolated and EV TF activity determined by the difference in FXa activity in the absence vs presence of anti-TF antibody. D-dimer and tissue factor pathway inhibitor a (TFPIa) antigen levels were measured. Data from 232 samples collected from 96 Covid-19 positive patients and 18 samples from 14 healthy controls were analyzed. For each study analysis, patient samples were organized into groups based on the disease severity outcomes as follows: hospitalization (Hospitalization; n=37); intensive care (ICU; n=16); mechanical ventilation (Ventilation; n=22); or fatality (Deceased; n=22). Result: Study 1. Covid-19 samples showed considerable thrombin generation variability with some samples failing to generate thrombin; pathway selective inhibitors reduced thrombin generation while heparinase treatment increased thrombin generation. Upon analysis, thrombin generation parameters showed no significant correlations to either D-dimer levels or disease severity. Instead, plasma prepared from blood collected directly into corn trypsin inhibitor revealed that contact activation that occurred post-sample collection dominates procoagulant activity. Study 2. Figure 1, shows EV TF activities, D-dimer and TFPIα levels obtained for Covid-19 samples, with data segregated based on disease severity outcomes. Statistically significant difference versus the Hospitalized group are shown. TFPIa levels were highest in heparin IV patients (24.4+1.5 nM) vs Heparin-SQ (12.8+0.9 nM) vs enoxaparin (10.8 +0.7 nM) (p value:<0.0001). It is known that heparin treatment increases circulating TFPIα, however an increase in TFPIα might also further increase circulating TF/FVIIa/XaTFPI inhibitory complex, which would dissociate in citrated plasma, and might account for the increase in EV TF in other studies. Conclusions: Contact activation that occurs post-sample collection is sufficient to obscure endogenous triggers of coagulation, if present, in Covid-19 patients' plasma. D-dimer and TFPIα strongly correlate with disease severity although the latter is likely affected by heparin treatment. The most severe Covid-19 patients with high D-dimer did not show detectible plasma EV TF activity. Plasma EV TF activity does not appear to adequately represent the mechanism responsible for elevated D-dimer levels in Covid-19 cases. Figure 1 Figure 1. Disclosures Di Paola: CSL Behring: Consultancy, Honoraria.

Erythrocyte Participation in Thrombin Generation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 374-374
Author(s):  
Matthew Whelihan ◽  
Kenneth G. Mann
Keyword(s):  
Board Of Directors ◽  
Trypsin Inhibitor ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Maximum Level ◽  
Lag Phase ◽  
Advisory Committees ◽  
Contact Pathway ◽  
Corn Trypsin Inhibitor ◽  
Prothrombin Activation

Abstract Abstract 374 Introduction: The potential contributions of erythrocytes (RBCs) to coagulation biochemistry have been controversial. We evaluated the potential for RBCs to participate in thrombin generation using minimally altered whole blood. Methods: Platelet poor plasma (PPP), platelet rich plasma (PRP) adjusted to a physiologic concentration with PPP, washed RBCs (PLTs <0.5%) and washed PLTs were prepared from contact pathway inhibited (corn trypsin inhibitor, CTI) whole blood in the absence of other anticoagulants. RBCs and PLTs were evaluated for phosphatidylserine (PS) exposure by FACS using FITC-labeled bovine lactadherin and their ability to support prothrombinase (1.4μM prothrombin, 20nM factor Va, 200pM factor Xa (FXa), 3.4μM antithrombin (AT) at physiologic concentrations of RBCs and PLTs). CTI whole blood and prepared sub fractions (PRP, PPP, PRP+RBC, PPP+RBC) were subjected to a 5pM tissue factor (TF) stimulus and samples analyzed by Western blotting and α-thrombin (αIIa) antithrombin (αTAT) ELISA (Rand et al. Blood, 1996). Results: CTI whole blood (N=3 donors mean±SD) clotted in 4±1.5 min whereas the matching PRP clotted in 8±2 min. In αTAT ELISA analyses, PRP showed a corresponding increase in lag phase and a 50% decrease in the maximum rate (26±9nM/min vs 58±7nM/min) and extent (263±62nM vs 476±86nM) of αTAT formation compared to whole blood. When PRP was reconstituted with physiologic levels of washed RBCs, the rate and extent of αTAT formation as well as the lag phase were restored to that observed in CTI whole blood. Addition of buffy coat to PRP, to test white blood cell contributions, had no effects on any of these parameters. Western blotting showed a significant decrease in prothrombin consumption in the PRP experiments compared to whole blood and PRP with washed RBCs. When RBCs were added to PPP (N=1), there was no significant prolongation of the lag phase and the rate of αTAT formation (21nM/min) was half of that seen in the corresponding whole blood (41nM/min). However, the maximum αTAT generated in RBCs+PPP (267nM) vs whole blood (343nM) differed by only 22%. Collectively, these data indicate a significant role for RBCs in the propagation of thrombin generation in CTI whole blood. FACS analysis of PS exposure on RBCs showed the following: untreated RBCs showed minimal binding to lactadherin (1.3%) compared to the positive control (99.7%; RBCs treated with 10mM N-ethylmaleimide (NEM) and 4μM ionophore A23187); pretreatment of the RBC population with 10nM αIIa or FXa showed a 5-fold increase in lactadherin binding over the untreated control indicating the presence of surface conditions capable of supporting prothrombinase. This proportion (5%) of PS-expressing RBCs represents a similar proportion of PLTs to RBCs (4%) in whole blood. Prothrombin activation (N=2)(1.4μM II, 20nM FVa, 200pM FXa, 3.4μM AT) on untreated RBCs exhibited a 3.5±0.5 minute lag phase followed by αTAT production which reached a rate of 18nM/min and maximum level of 180nM. In experiments performed in the absence of AT, pretreatment of RBCs with either 10nM αIIa or FXa shortened the lag phase by 1±0.25min. PAR-1 (TFLLRN) activated PLTs or activated PLTs+RBCs showed no lag phase, and no difference in the rate of αTAT production (25nM/min) or maximum level (200nM) of αTAT generated. Conclusions: In contact pathway-inhibited whole blood initiated with TF, RBCs appear essential for normal thrombin generation. Our approach rapidly fractionates CTI blood in the absence of other anticoagulants and yields populations of minimally altered RBCs that when subjected to a Tf stimulus produce thrombin more rapidly than PRP. Physiologic levels of washed RBCs pretreated with αIIa or FXa support a level of prothrombin activation similar to that observed with washed activated PLTs. These findings suggest that RBCs participate in thrombin generation and produce a PS-equivalent membrane when treated with αIIa or FXa. Disclosures: Mann: Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Inhibition of Thrombin Generation in Plasma by Inhibitors of Factor Xa

1997 ◽  
Vol 78 (04) ◽  
pp. 1215-1220 ◽  
Author(s):  
D Prasa ◽  
L Svendsen ◽  
J Stürzebecher
Keyword(s):  
Thrombin Generation ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Thrombin Generation Assay ◽  
Ic50 Values ◽  
Fxa Inhibitor ◽  
20 Nm ◽  
Tick Anticoagulant Peptide ◽  
Inhibition Of Thrombin

SummaryA series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations.The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

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