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CARDIOVASCULAR ANESTHESIA

The Impact of Tissue Factor Pathway Inhibitor on Coagulation Kinetics Determined by Thrombelastography

Paul Audu, MD^*, Vance G. Nielsen, MD, Valerie Armstead, MD^*, Garry Powell, BS^*, Jerry Kim, MD^*, Larry Kim, MD^*, and Munira Mehta, MBBS^*

From the *Department of Anesthesiology, Thomas Jefferson University, Philadelphia, Pennsylvania; and Department of Anesthesiology, The University of Alabama at Birmingham, Birmingham, Alabama.

Address correspondence and reprint requests to Vance G. Nielsen, MD, Department of Anesthesiology, The University of Alabama at Birmingham, 901 South 19th Street, Basic Medical Research II, Room 206, Birmingham, AL 35249-6810. Address e-mail to vnielsen{at}uab.edu.

	Abstract

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BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a 40-kDa, endogenous protein that inhibits tissue factor (TF)-initiated coagulation by bonding with activated factor X (FXa). The TFPI/FXa complex then subsequently binds with TF/activated factor VII (FVIIa) complex, ultimately inhibiting thrombin generation. Heparin administration causes endothelial release of TFPI concentrations up to sixfold normal values. Thrombelastography (TEG®) is often used to monitor hemostasis in the perioperative period, and TFPI could potentially affect the diagnostic interpretation of TEG-based data, given its inhibition of both common and TF coagulation pathways. Thus, in this study we characterized the effect of TFPI on coagulation kinetics via TEG.

METHODS: Whole blood, Factor VII-deficient plasma, and normal plasma were exposed in vitro to various concentrations of TFPI, after which unmodified, celite-activated, and TF-activated TEG were performed.

RESULTS: The addition of 87.5 ng/mL TFPI (twice normal concentration) was required to prolong clot propagation in whole blood, with propagation and strength only significantly affected by the addition of 175 ng/mL concentrations. Experiments with Factor VII-deficient plasma demonstrated that TFPI-mediated suppression of coagulation kinetics at these concentrations was secondary to FXa inhibition. Celite activation markedly attenuated TFPI-mediated effects on coagulation kinetics, whereas TF activation accentuated TFPI-mediated prolongation of clot initiation and diminution of propagation.

CONCLUSIONS: In settings involving heparin administration (e.g., cardiopulmonary bypass), TFPI-mediated inhibition of coagulation should be considered during TEG-based hemostatic monitoring.

	Introduction

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Tissue factor pathway inhibitor (TFPI) is a 40-kDa, endogenous protein that inhibits tissue factor (TF)-initiated coagulation by bonding with activated factor X (FXa), with the TFPI/FXa complex then subsequently binding the TF/activated factor VII (FVIIa) complex (1). The circulating concentration of TFPI is normally 70–150 ng/mL (1.8–3.8 nM), but it can be increased 3–6-fold by heparin-mediated endothelial release (2–6). Of interest, increased TFPI concentrations were associated with postoperative bleeding after low-molecular-weight heparin prophylaxis after orthopedic or general surgery (7). Thus, it is likely that physicians will encounter patients perioperatively with increased TFPI concentrations.

Thrombelastography (TEG®) is often used to monitor hemostasis in the perioperative period, but the effects of TFPI on TEG have not been described. Given that TFPI inhibits both common pathway (FXa) and TF-initiated pathway (TF/FVIIa) proteins, it is likely that TFPI could significantly affect TEG-derived variables, and perhaps, result in an incorrect interpretation that a coagulation factor deficiency was present. Thus, the purpose of this study was to characterize the effect of TFPI on coagulation kinetics via TEG.

	METHODS

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Whole Blood Experiments
All experiments involving whole blood were performed at Thomas Jefferson University. IRB approval was obtained before experimentation, and written informed consent was obtained from all participants. All subjects were healthy, had no history of a blood clotting abnormality, and were not receiving anticoagulant or antiplatelet medications. To clarify the role of blood collection on our observations, we first determined the effect of the "two-syringe" technique on TF activity and TEG variables. It has been purported that TF released from venipuncture could affect TEG variables (8–10). Concentration–response relationships of TFPI on TEG variables were then determined as subsequently described.

Two-Syringe Experiments
Subjects (n = 5) had a tourniquet applied to an upper arm, and phlebotomy was performed with a 23-G Vacutainer® blood collection set of plastic tubes (Becton Dickinson, Franklin Lakes, NJ). One 3-mL sample was obtained, with 360 µL immediately subject to unmodified TEG as subsequently described. The remainder was anticoagulated with sodium citrate (blood:sodium citrate, 9:1), centrifuged at 1500g for 15 min, and then stored at –70°C for assay of TF activity with a commercially available kit (American Diagnostica, Stamford, CT). The limits of detection of the assay were 1.88–30 pM TF. A second, 3-mL sample was immediately obtained with the same needle, with TEG and TF analyses performed just as with the first sample.

TEG TFPI Concentration–Response in Whole Blood
Subjects (n = 6) had a tourniquet applied to the upper arm, and phlebotomy was performed with a 23-G Vacutainer blood collection set. One 3-mL sample was obtained, with 400-µL aliquots added to plastic tubes preloaded with TFPI (American Diagnostica) just before TEG. Pilot studies demonstrated that the concentration of TFPI addition required to significantly prolong reaction time (R, defined as an amplitude of 2 mm, denoting clot initiation, min) with an n of 6 subjects (P < 0.05, ß 0.8) was 87.5 ng/mL (2.2 nM). Thus, the addition of TFPI concentrations of 0, 87.5, and 175 ng/mL (4.4 nM) were used in this series of experiments. Blood (360 µL) exposed to these concentrations of TFPI was placed in a disposable cup in a computer-controlled thrombelastograph® hemostasis system (Model 5000, Hemoscope Corp., Niles, IL) in <4 min after collection. The following standard variables were determined at 37°C: R, angle (, a measure of the speed of clot strength, degrees), and maximum amplitude (MA, a measure of clot strength, mm). Version 2.0 TEG software was used to collect data, and data were collected for 2.5 h per sample for this series of experiments.

Plasma-Based Experiments
All experiments involving plasma were performed at the University of Alabama at Birmingham. All experimental conditions were represented with n = 6, as this number of experiments required to obtain a ß 0.8 with an < 0.05 for most TEG variables, as demonstrated in previous in vitro investigations (11,12). Plasma, unlike whole blood from volunteers, is devoid of individual hemostatic variation mediated by platelets. As the precise activities of individual procoagulants and anticoagulants are known, normal, pooled plasma is used as a standard for most hematologic analyses performed in clinical laboratories. Lastly, as the products are commercially available, are noncellular, and can not be linked to individual donors, institutional ethical approval is not required as per the guidelines of the National Institutes of Health.

FVII-Deficient Plasma Experiments
Given that TFPI first inhibits FXa before inhibiting TF/FVIIa complex, the experiments using FVII-deficient plasma exposed to TFPI were required to determine whether the TFPI-mediated changes in TEG variables observed in whole blood at Thomas Jefferson University were primarily due to inhibition of FXa. Thus, citrated, FVII-deficient plasma (George King Bio-Medical, Overland Park, KS) was exposed to addition of 0, 87.5, or 175 TFPI ng/mL (TFPI was from the same lot as that used at Thomas Jefferson University) just before TEG analyses as subsequently described. The sample composition placed in the reaction cup was 330 µL plasma, 10 µL TFPI/0.9% NaCl, and 20 µL CaCl₂. TEG data were collected until MA was reached or 60 min had elapsed. In addition to the determination of R, , and MA, several other recently described (11,12) TEG variables were determined as subsequently described. Maximum elastic modulus (MG, dynes/cm²) was determined from MA, expressed by the following equation: G = (5000 x MA)/(100 – MA). Time to maximum rate of thrombus generation (TMG) is defined as the time interval (s) observed before maximum speed of clot growth. Maximum rate of thrombus generation (MTG) is the maximum velocity of clot growth observed (dynes/cm²/s). Lastly, total thrombus generation (TTG) is denoted as the total area under the velocity curve during clot growth (dynes/cm²), representing the amount of clot strength generated during clot growth. Version 4.2 TEG software was used to analyze this and all other plasma-based experiments.

Celite and TF-Activated TEG
To determine the effects of TFPI on plasma near-maximally activated via the contact pathway proteins (celite activation) or TF-mediated FVII activation, a final series of plasma-based experiments were performed. Normal, citrated plasma (George King Bio-Medical) was exposed to 0, 87.5, or 175 ng/mL TFPI before TEG analyses. The sample composition placed in the reaction cup was 330 µL plasma with or without TFPI, 10 µL of celite (final concentration 0.28 mg/mL) or TF (final concentration 0.01%, Instrumentation Laboratories, Lexington, MA), and 20 µL CaCl₂. TEG data were collected for 15 min as described previously (12), with TEG variables determined as mentioned previously.

Data Analyses
The data sets analyzed with parametric statistics were expressed as mean ± sd, whereas data analyzed with nonparametric statistics were expressed as median (first to third quartiles). Analyses of the effects of blood sampling on TF concentration and TEG variables were conducted with two-tailed Student’s t-tests. Analyses of the effects of different doses of TFPI on TEG variables in both whole blood and normal plasma were conducted with repeated measures one-way analysis of variance with the Holm–Sidak post hoc test for multiple comparisons. Analyses of the effects of different doses of TFPI in FVII-deficient plasma on TEG variables were conducted with Kruskal–Wallis one way analysis of variance with the Student–Newman–Keuls post hoc test for multiple comparisons. A P value of <0.05 was considered significant.

	RESULTS

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Two-Syringe Experiments
The TF in the first blood sample (18.6 ± 7.4 pM) was not significantly (P = 0.27) different from the TF values in the second blood sample (16.3 ± 3.9 pM). Similarly, values were not significantly different between the first and second blood samples for R (24.9 ± 1.3 vs 29.7 ± 11.5 min, P = 0.43), (28.3° ± 1.2° vs 25.8° ± 7.1°, P = 0.50), and MA (46.8 ± 6.5 vs 47.4 ± 7.4 mm, P = 0.53).

TEG TFPI Concentration–Response in Whole Blood
As depicted in Table 1, whole blood exposed to TFPI demonstrated a significant, concentration-dependent prolongation of R values. However, only blood exposed to the addition of 175 ng/mL TFPI was noted to have significantly decreased and MA values compared to samples exposed to 0 ng/mL TFPI.

FVII-Deficient Plasma Experiments
As listed in Table 2, FVII-deficient plasma demonstrated a significant, concentration-dependent prolongation of R and TMG values. Further, TFPI similarly decreased clot propagation by significantly decreasing and MTG. However, clot strength, denoted by MA, MG, and TTG, was not significantly affected by TFPI in the concentration range tested.

Celite and TF-Activated TEG
Celite-activated data are listed in Table 3. Plasma activated by celite demonstrated significantly prolonged clot initiation only in response to the addition of 175 ng/mL TFPI, denoted by increased R and TMG values. However, TFPI had no significant effect on either clot propagation or strength in plasma activated with celite. TF-activated data are displayed in Table 4. In sharp contrast to celite activation, plasma activated with TF demonstrated a significant prolongation of clot initiation (R, TMG) and diminished propagation (, MTG) in response to TFPI exposure. Clot strength (MA, MG, TTG) was only significantly diminished after exposure to an additional 175 ng/mL TFPI compared to samples exposed to 0 and 87.5 ng/mL TFPI.

	DISCUSSION

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The present study is the first description of the effects of TFPI on coagulation kinetics in whole blood and plasma with clinically used TEG methods. The primary conclusion to be drawn from these data is that, at clinically encountered concentrations after heparin administration, TFPI significantly affects TEG data derived from either unmodified whole blood or celite-activated plasma samples by inhibition of FXa. The most likely explanation for this is that the stimulus for coagulation provided by the <20 pM TF noted in whole blood in this study is vastly overshadowed by contact activation from the plastic surfaces of the TEG reaction cup. This contention is supported by the data derived from experiments with FVII-deficient plasma, which responds poorly to TF (cannot form TF/FVIIa) but does respond normally to contact activation (11). In sum, 2–3-fold normal TFPI concentrations affect TEG variables in unmodified blood/plasma samples primarily by inhibition of FXa, not by inhibition of TF/FVIIa.

Increased circulating TFPI concentrations, up to sixfold of baseline values, have been associated with acute administration of both unfractionated and low-molecular-weight heparin (2–6). This large increase in TFPI concentration diminishes within 1–2 h, but elevated TFPI concentrations can persist for up to 24 h after bolus administration and during continuous infusion of heparin (2,3,5). Protamine administration rapidly decreased TFPI from the circulation to baseline values after administration of unfractionated heparin, whereas protamine administration only partially decreased circulating TFPI after low-molecular-weight heparin administration (2). Some clinical implications of these data include the impact of TFPI on TEG-based monitoring. If one is monitoring coagulation with TEG using a heparinase-containing sample during the conduct of cardiopulmonary bypass, elevated TFPI may prolong clot initiation of unmodified or kaolin/celite activated samples or decrease clot propagation in unmodified samples, giving the false impression that a coagulation factor deficiency is present (Tables 1 and 3). Similarly, if TF activation was used during TEG monitoring of patients undergoing cardiopulmonary bypass, an incorrect diagnosis of coagulation factor deficiency could be formulated, given the profound effect of TFPI on TF-initiated coagulation kinetics (Table 4).

In this study we also addressed whether endogenous TF activity released by blood collection affected TEG-derived data (8–10). Several investigators routinely discard 5–10 mL of blood before obtaining a sample by phlebotomy in anticipation of potential TF-mediated activation from damage to the vein entry site. The present study demonstrated no difference in TEG variables between the first and second 3-mL sampled specimens. Thus, it is reasonable to conclude that use of a two-syringe method is unwarranted when obtaining blood samples for TEG-based hemostatic analysis via phlebotomy.

In conclusion, the present study demonstrates that clinically relevant increases in TFPI concentration significantly affect TEG variables, reflective of either FXa inhibition after contact activation, or representative of FXa-TF/FVIIa inhibition after TF activation. In the settings involving heparin administration (e.g., orthopedic surgery, cardiopulmonary bypass), TEG-based monitoring can potentially be affected not only by antithrombin activation but also by increased TFPI activity in the circulation. Use of heparinase in vitro can be used to differentiate the effects of antithrombin on TEG variables in these settings. However, removal of heparin by either protamine administration or eventual clearance in vivo is required to diminish circulating TFPI activity to discern whether changes in clot kinetics are due to TFPI-mediated inhibition, loss of coagulation protein, or platelet function.

	ACKNOWLEDGMENTS

 
The authors express their gratitude to Dr. Yoogoo Kang for his input related to experimental design.

	Footnotes

 
Accepted for publication June 7, 2006.

There are no conflicts of interest; all funding for this project was intramural.

Supported by the Department of Anesthesiology of Thomas Jefferson University; and Department of Anesthesiology of The University of Alabama at Birmingham.

	REFERENCES

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Audu, P. Articles by Mehta, M. Search for Related Content PubMed PubMed Citation Articles by Audu, P. Articles by Mehta, M. Related Collections Cardiovascular Blood Monitoring (Non-cardiac)