CME/SAM. Influence of Coagulation and Anticoagulant Factors on Global Coagulation Assays in Healthy Adults

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1 Coagulation and Transfusion Medicine / Global Coagulation Assays and Coagulation Proteins Influence of Coagulation and Anticoagulant Factors on Global Coagulation Assays in Healthy Adults Seon Young Kim, MD, 1 Ji-Eun Kim, MS, 1,2 Hyun Kyung Kim, MD, PhD, 1,2 Inho Kim, MD, PhD, 2,3 Sung-Soo Yoon, MD, PhD, 2,3 and Seonyang Park, MD, PhD 3 Key Words: Global coagulation assay; Prothrombin time; Activated partial thromboplastin time; Thrombin generation assay; Coagulation factor; Anticoagulant factor; Vitamin K DOI: 10.9/AJC5C4AGFRDKMX CME/SAM Upon completion of this activity you will be able to: outline the actions of coagulation factors and anticoagulation factors in the coagulation process and their influence on the results of global coagulation tests. describe the principles and parameters of the thrombin generation assay. list various factors influencing coagulation protein levels. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 398. Exam is located at Abstract It remains unclear how coagulation and anticoagulant factors influence global coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (aptt), and thrombin generation assay (TGA). We measured PT, aptt, coagulation factor and protein levels, and TGA parameters (lag time, endogenous thrombin potential [ETP], and peak thrombin) in 252 apparently healthy adults. Vitamin K dependent coagulation and anticoagulant factors were significantly correlated with blood lipids. PT was determined by factor (F) V and ; aptt was dependent on antithrombin, protein C,, and. Lag time was mainly determined by,, and protein S and peak thrombin by and. Antithrombin (for ETP and lag time) and protein S (for lag time) contributed significantly to TGA inhibition. This knowledge about determinants of global coagulation assays may help interpret the results of coagulation assays and contribute to the future development of diagnostic tools. The synchronized plasma levels of vitamin K dependent proteins with opposite functionalities may compensate a propensity to hyper- or hypocoagulability in a normal population. Normal hemostasis is a state of equilibrium between procoagulant and anticoagulant factors in circulating blood. An imbalance in these hemostatic systems produces either hemorrhagic or thrombotic conditions. For the early detection and management of coagulopathy, global coagulation tests that can detect delicate hemostatic changes are essential. Prothrombin time (PT) and activated partial thromboplastin time (aptt) are widely used in clinical laboratories as routine screening tests of the coagulation system. However, these tests are not strongly predictive of hemorrhagic risk and vary among individuals. 1 There is increasing evidence that the evaluation of thrombin generation gives useful information regarding coagulation status. 2-5 In the thrombin generation assay (TGA) using an automated calibrated thrombogram, thrombin generation curves can be operationally characterized as displaying initiation, propagation, and termination phases. After stimulation with tissue factor (TF), the consequent formation of endogenous thrombin potential (ETP) is measured in plasma. The ETP has been shown to be a good indicator of prothrombotic 4,6-8 and hemorrhagic tendencies. 3,9,10 Several studies have shown that elevated ETP is associated with a risk for venous thromboembolism 2,4,8,11 ; elevated levels of factor (F) XI,,,, prothrombin, or fibrinogen are seen to be correlated with a risk for venous thromboembolism risk Although elevated procoagulant levels were shown to correlate with abnormal in vitro thrombin generation, their individual relationships with in vivo thrombin generation remain unclear. The subclassification of individuals with elevated thrombin generation based on the underlying changes in different coagulation factors and 3 Am J Clin Pathol 2013;139: DOI: 10.9/AJC5C4AGFRDKMX

2 Coagulation and Transfusion Medicine / Original Article anticoagulant factors may help in understanding the clinical risk of elevated thrombin generation. In previous studies of normal coagulation, the normal ranges of procoagulant and anticoagulant factors have been generally found to be between 50% and % However, a number of studies have shown that variations of coagulation proteins in this range are associated with thrombotic risk An individual without apparent coagulopathy whose coagulation factors fall within the reference range may have a potential risk for thrombosis or hemorrhage because of an imbalance between coagulation and anticoagulation factors. Given that global coagulation assays such as PT, aptt, and TGA are widely used for the detection of hemostatic dysfunction, it is necessary to know how individual levels of coagulation and anticoagulant factors influence global coagulation test results. Sporadic studies have reported the coagulatory determinants of aptt 27 and TGA 18 in normal populations. However, to our knowledge, no report has compared the influence of coagulation and anticoagulant factors on 3 global coagulation tests (PT, aptt, TGA) through their simultaneous measurement in the same healthy population. In this study, we investigated the influence of coagulation and anticoagulant factors on 3 global coagulation tests, including PT, aptt, and TGA, in a population of normal healthy adults. Materials and Methods Study Population and Specimen Collection A total of 252 consecutive apparently healthy adults for whom coagulation screening tests were requested in routine health visits were included in the present study. The mean age of the subjects was 54 years (range, years). All of the subjects were Korean; 151 were male and 101 were female subjects. Mean ages of the men and women were similar (54.7 and 52.9 years, respectively; P =.245). Peripheral venous blood samples were collected in commercially available tubes containing mol/l sodium citrate (Becton Dickinson, San Jose, CA). Plasma was separated by centrifugation of whole blood at 1,550g for 15 minutes within 2 hours after blood collection. The aliquots of plasma were stored at 80 C. This study was reviewed and approved by the institutional review board of Seoul National University College of Medicine (Seoul, Korea). Thrombin Generation Assay and Other Coagulation Tests TF-triggered thrombin generation in platelet-poor plasma was measured using the calibrated automated thrombogram method (Thrombinoscope, Maastricht, The Netherlands) as described previously. 28 Briefly, 20 μl of reagent containing TF at a final concentration of 5 pmol/l or 1 pmol/l (PPP Reagent 5 pmol/l or PPP Reagent Low, respectively; Thrombinoscope BV), along with phospholipids or thrombin calibrators, was dispensed into each well of round-bottomed 96-well plates, and then 80 μl of test plasma specimen was added. After the addition of 20 μl of fluorogenic substrate in HEPES buffer containing calcium chloride, the fluorescent signal was read in a Fluoroskan Ascent fluorometer (Thermo Labsystems, Helsinki, Finland) and thrombin generation curves were calculated using the Thrombinoscope software (Thrombinoscope). Thrombin generation curves were evaluated using parameters that describe the initiation, propagation, and termination phases of thrombin generation, namely, lag time, ETP, and peak thrombin height (peak thrombin). The lag time is defined as the time to reach one sixth of the peak height and is a measure of the initiation phase. It is equivalent to the clotting time. The peak height is defined as the maximum thrombin concentration produced. ETP is the area under the thrombin generation curve and represents the total amount of generated thrombin. Coagulation tests, including PT, aptt, and factor assays, were performed on an automated coagulation analyzer, ACL TOP (Beckman Coulter, Fullerton, CA). PT was measured using the HemosIL RecombiPlasTin reagent (Instrumentation Laboratory, Milan, Italy), and aptt was measured using the SynthASil reagent (Instrumentation Laboratory). Fibrinogen was measured using the HemosIL Fibrinogen-C XL reagent (Instrumentation Laboratory) based on the Clauss method. Coagulation factors were tested using a PT-based clotting assay with the HemosIL RecombiPlasTin reagent (for,,, and ) and an aptt-based clotting assay using the SynthASil reagent (for,,, and ). Antithrombin, protein C, and protein S activity were determined with chromogenic assays (HemosIL liquid antithrombin and HemosIL Protein C; Instrumentation Laboratory) and an immunoassay (HemosIL Free Protein S, Instrumentation Laboratory). Statistical Analysis Data were compared using the Mann-Whitney U test and Kruskal-Wallis analysis for continuous variables and the χ 2 test for categorical variables. The coefficient of variation was calculated as the ratio of the standard deviation to the mean so as to assess interindividual variability. Correlations are expressed as Pearson coefficients. The relative effects of coagulation and anticoagulant factors on thrombin generation, PT, and aptt were assessed using multiple linear regression analysis. For each model, the adjusted R 2 and the standardized regression coefficients (β) of the independent variables were calculated. All analyses were carried out using SS 12.0 software (SS, Chicago, IL). A probability value (P) of less than.05 was considered significant. Am J Clin Pathol 2013;139: DOI: 10.9/AJC5C4AGFRDKMX 371

3 Kim et al / Global Coagulation Assays and Coagulation Proteins Results Coagulation Protein Levels and Thrombin Generation Parameters in a Healthy Population The mean and 2.5 to 97.5 percentile values of each coagulation and anticoagulant factor and the 3 global coagulation tests (PT, aptt, and TGA) are summarized in Table 1. Women had higher levels of and antithrombin and lower levels of and protein S. Women had lower peak thrombin and ETP values than men on stimulation with 1 pmol/l TF. The reference ranges of coagulation and anticoagulant factors from our study were generally similar to those of previous studies. 29,30 Significantly higher lower limits of were found in our study compared with reference intervals from the previous study (77%-121%), but the median value (114%) was similar to those reported previously (95%- %). 30 Compared with 1 pmol/l TF, TGA results with 5 pmol/l TF demonstrated shortened lag time and time to peak as well as higher peak thrombin and ETP, consistent with previous studies. 18,20 With 1 pmol/l TF, the lag time varied 2-fold (range, min) and peak thrombin varied over a 4-fold range in this healthy population. The interindividual variabilities in lag time, peak thrombin, and ETP with 1 pmol/l TF were 23.7%, 34.1%, and 23.1%, respectively, consistent with previously reported findings. 20,31-33 Correlation Among Levels of Coagulation Factors, Anticoagulant Factors, and Other Laboratory Results No significant correlation was seen between PT and any of the TGA parameters. With 1 pmol/l TF, lag time showed a positive correlation with aptt (r = 0.161), whereas peak thrombin and ETP showed inverse correlations with aptt (r = and 0.277, respectively). With 5 pmol/l TF, peak thrombin and ETP showed inverse correlations with aptt (r = 0.2 and 0.129, respectively). Weak to moderate correlations were observed between various coagulation factors and anticoagulant factors Table 2. Interestingly, the levels of vitamin K dependent anticoagulant factors such as protein C and protein S showed strong correlations with those of vitamin K dependent coagulation Table 1 Factor Levels and Global Coagulation Test Results in Our Healthy Population All Samples (n = 252) Males (n = 151) Females (n = 101) 2.5th-97.5th Interindi- 2.5th-97.5th Interindi- 2.5th-97.5th Interindi- Mean ± SD Percentile vidual CV Mean ± SD Percentile vidual CV Mean ± SD Percentile vidual CV Coagulation and anticoagulant factors Fibrinogen, ± ± ± mg/dl, % ± ± ± 12.5 a , % ± ± ± , % 98.7 ± ± ± , % ± ± ± , % ± ± ± 16.7 b , % ± ± ± , % ± ± ± , % 81.8 ± ± ± , % 96.7 ± ± ± 9.4 b Protein C, % ± ± ± Protein S, % ± ± ± 34.5 c PT Time, s 11.0 ± ± ± Percent ± ± ± INR 0.97 ± ± ± aptt, s 30.0 ± ± ± pmol/l TF Lag time, min 3.9 ± ± ± Peak thrombin, ± ± ± nmol ETP, nmol/min 1,785.4 ± ,173-2, ,802.7 ± ,173-2, ,759.5 ± 2.4 1,166-2, pmol/l TF Lag time, min 9.3 ± ± ± Peak thrombin, ± ± ± 50.0 c nmol/l ETP, nmol/min 1,359.4 ± , ,401.0 ± , ,297.5 ± b 637-1, aptt, activated partial thromboplastin time;, antithrombin; CV, coefficient of variation; F, factor; ETP, endogenous thrombin potential; INR, international normalized ratio; PT, prothrombin time; TF, tissue factor. a P <.05, females vs males. b P <.01, females vs males. c P <.001, females vs males. 372 Am J Clin Pathol 2013;139: DOI: 10.9/AJC5C4AGFRDKMX

4 Coagulation and Transfusion Medicine / Original Article factors including,, and. Correlations among vitamin K dependent coagulation factors (,,, and ) were higher than among other non vitamin K dependent coagulation factors.,,, and were positively correlated to age (r = to approximately 0.263) Table 3. and antithrombin levels tended to decrease with age (r = and r = 0.131, respectively). The various laboratory test results revealed weak correlations with several coagulation proteins. Of note, cholesterol and triglyceride levels showed significant correlations with vitamin K dependent coagulation proteins including,,,, protein C, and protein S (r = ). Effects of Coagulation and Anticoagulant Factors on PT and aptt The concentrations of and decreased significantly with prolonged PT Figure 1A. On the other hand,,,,,, and decreased with prolonged aptt Figure 1B. In a multiple linear regression analysis Table 4, the β value of for the dependent variable PT was This value means that when increases by 1 standard deviation (18.3), PT shortens by The strongest determinants of PT results were and. was also a determinant of PT. The aptt value was mainly dependent on and levels and, to a lesser extent, negatively determined by antithrombin and protein C. Table 2 Correlations Between Coagulation and Anticoagulant Factors in Our Healthy Population (n = 252) a Fibrinogen b c b c b b b b b b b b b b b b b b b b b b b b b b c b b b b c b b b c b b c c b b b b b b b b b c b c b b b 0.1 c c b, antithrombin; F, factor;, protein C;, protein S. a Data presented are Pearson correlation coefficients. b P <.001. c P <.05. Table 3 Correlation of Coagulation and Anticoagulant Factors With Other Parameters in Our Healthy Population (n = 252) a Fibrinogen Age b b c b b WBC c b c PLT c b c c b b c c 0.0 ESR c b c b b CRP 0.3 c BUN b b c c b b Cr b c b Ca b b b c c c b b c P b b b c Glu b b b 0.2 c c c b Protein b b b b Alb c b b b c c AST b b b b ALT b b b ALP c b b b b Chol c b b c b c b TG c c c c c c LDL c b c c b c Alb, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase;, antithrombin; BUN, blood urea nitrogen; Ca, calcium; Chol, cholesterol; Cr, creatinine; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; F, factor; Glu, glucose; LDL, low-density lipoprotein; P, phosphorus;, protein C; PLT, platelet; Protein, serum total protein;, protein S; TG, triglyceride. a Data are presented as Pearson correlation coefficients. b P <.05. c P <.001. Am J Clin Pathol 2013;139: DOI: 10.9/AJC5C4AGFRDKMX 373

5 Kim et al / Global Coagulation Assays and Coagulation Proteins Effects of Coagulation and Anticoagulant Factors on the Thrombin Generation Assay Lag times were progressively prolonged with increasing concentrations of anticoagulant factors such as antithrombin and protein S Figure 1C and Figure 1D. In a multiple linear regression analysis, the significant negative determinants of lag time with 5 pmol/l TF were and, and the significant positive determinants of the same were antithrombin and protein S (Table 4). With 1 pmol/l TF, the only anticoagulant that emerged as a significant positive A 1 B PT aptt C 1 D Lag Time (5 pmol/l TF) Lag Time (1 pmol/l TF) Figure 1 Changes in the mean levels of coagulation factors and anticoagulant factors based on prothrombin time (PT), activated partial thromboplastin time (aptt), and lag time. On the x-axis, each point represents the mean value of coagulation factor levels in the intervening range indicated ( 2 SD indicates the range of mean 2.5 SD to mean 1.5 SD; 1 SD, mean 1.5 SD to mean 0.5 SD; mean, mean 0.5 SD to mean SD; 1 SD, mean SD to mean SD; 2 SD, mean SD to mean SD) of PT (A), aptt (B), lag time at 5 pmol/l tissue factor (TF) (C), and lag time at 1 pmol/l TF (D). Solid lines indicate P <.05 by test for trend, with meaningful directional changes compatible with test results., antithrombin; F, factor;, protein C;, protein S. 374 Am J Clin Pathol 2013;139: DOI: 10.9/AJC5C4AGFRDKMX

6 Coagulation and Transfusion Medicine / Original Article determinant of lag time was protein S. was a positive determinant, revealing an inverse influence compared with other coagulation factors. As expected, apparent increases in,, and were seen as peak thrombin increased with both 1 pmol/l and 5 pmol/l TF Figure 2A and Figure 2B. Antithrombin levels decreased as peak thrombin and ETP increased. In a multiple linear regression analysis, the significant positive determinants of peak thrombin were and with both 5 pmol/l and 1 pmol/l TF (Table 4). was a positive determinant of peak thrombin with 5 pmol/l TF. and had a negative influence on peak thrombin. Antithrombin was a significant negative determinant of peak thrombin with 5 pmol/l TF. The significant determinants of ETP were,, and antithrombin with 5 pmol/l TF and and antithrombin with 1 pmol/l TF. Discussion PT and aptt are routine coagulation screening tests that estimate deficiencies in coagulation factors. Because the normal values of these global tests showed wide interindividual variations in our study, it is assumed that wide variations in coagulation protein levels affect the PT and aptt results. Although several reports have described the determinants of PT and aptt in pathologic conditions, such as oral anticoagulation 34 or hemophilia, 1 the dependence of these tests on the levels of coagulation and anticoagulant factors in a healthy population has not been studied in detail. By simultaneously measuring coagulation and anticoagulant factors, as well as global coagulation assays in plasma specimens from normal healthy persons, our study demonstrated that the PT value was significantly determined by and and that aptt was mainly dependent on and. Moreover, the levels of anticoagulant factors such as antithrombin and protein C were shown to be significant determinants of the aptt value. Given that the clotting process is initiated by the binding of TF to (a), the level may be important in determining the PT value. Interestingly, we observed that levels also significantly influenced PT values. The endpoint of PT is defined as the time at which only around 5% of all physiologically relevant thrombin is formed. 35 To generate an initial small amount of thrombin, the level of appears to be important in the PT system. Although the principle is not known in detail, the knowledge that and have significant effects on PT is of note for the interpretation of PT values in a normal population. As expected, and levels were identified as major determinants of aptt in our results. This is in agreement with observations from a previous study that and were the 2 strongest contributors determining the aptt level. 27 Because is known to be a risk factor for thrombosis, 36 aptt may be used as a supplementary indicator of thrombosis risk. In our study, was also a major determinant of aptt. If the reagent used in the aptt test first activates a large amount of, this activated can drive the subsequent coagulation cascade until a small amount of thrombin is formed. Because is not the main contributor to the physiologic coagulation process, 1 the dependency of aptt on the Table 4 Determinants of Global Coagulation Tests Using Multivariate Regression Analysis a Lag Time Peak Thrombin ETP PT aptt 5 pmol/l TF 1 pmol/l TF 5 pmol/l TF 1 pmol/l TF 5 pmol/l TF 1 pmol/l TF SD β β β β β Β β β Adjusted R Fibrinogen b b b c b b c b c b b b b b b b b b c b b c b b b b 0.1 b b c aptt, activated partial thromboplastin time;, antithrombin; ETP, endogenous thrombin potential; F, factor;, protein C;, protein S; PT, prothrombin time; TF, tissue factor. a Data are presented as standardized regression coefficients (β). b P <.05. c P <.001. Am J Clin Pathol 2013;139: DOI: 10.9/AJC5C4AGFRDKMX 375

7 Kim et al / Global Coagulation Assays and Coagulation Proteins value is not a desirable finding because it indicates poor correlation of aptt with the hemorrhagic potential. Until now, to our knowledge, the effect of anticoagulant protein levels on the aptt value has not been reported. The current study demonstrated that the aptt could be decreased by low levels of antithrombin and protein C. The aptt might be decreased in individuals who are heterozygous carriers of thrombophilia and contain mildly low levels of antithrombin or protein C. Future studies in a heterozygous carrier are required to confirm this. A 140 B Peak Thrombin (5 pmol/l TF) Peak Thrombin (1 pmol/l TF) C 140 D ETP (5 pmol/l TF) ETP Time (1 pmol/l TF) Figure 2 Changes in the mean concentrations of coagulation factors and anticoagulant factors based on peak height and endogenous thrombin potential (ETP) of thrombin generation assay. Each dot indicates the mean of coagulation protein levels in the intervening range indicated ( 2 SD indicates the range of mean 2.5 SD to mean 1.5 SD; 1 SD, mean 1.5 SD to mean 0.5 SD; mean, mean 0.5 SD to mean SD; 1 SD, mean SD to mean SD; 2 SD, mean SD to mean SD) of peak height at 5 pmol/l tissue factor (TF) (A), peak height at 1 pmol/l TF (B), ETP at 5 pmol/l TF (C), and ETP at 1 pmol/l TF (D). Solid lines indicate P <.05 by test for trend, with meaningful directional changes compatible with test results., antithrombin; F, factor;, protein C;, protein S. 376 Am J Clin Pathol 2013;139: DOI: 10.9/AJC5C4AGFRDKMX

8 Coagulation and Transfusion Medicine / Original Article In our TGA experiments, the TGA lag time with 5 pmol/l TF was negatively determined by and. Peak thrombin was significantly dependent on and levels with both 1 pmol/l and 5 pmol/l TF. In a previous study using an artificial plasma mixture spiked with coagulation factors, peak thrombin was found to be a sensitive parameter for the detection of and deficiency with 1 pmol/l TF but not with 5 pmol/l TF. 19 However, in our study, using normal plasma, both the 1 pmol/l and 5 pmol/l TF-stimulated peak thrombin levels were good representatives of and levels. This finding suggests the potential usefulness of peak thrombin as a screening test for hemophilia carriers; however, future studies are required to confirm this. Prothrombin was also a determinant of peak thrombin with 5 pmol/l TF but not 1 pmol/l TF. This is in agreement with a previous study that used 13.6 pmol/l TF. 18 Because a very small fraction of the prothrombin present in plasma is activated at low TF, the effect of prothrombin on peak thrombin seems to be significant only at high TF. In our study, the ETP values with both 1 pmol/l and 5 pmol/l TF were positively dependent only on but not on. Given that peak thrombin was determined by both and, ETP is considered a less sensitive parameter than peak thrombin. The high sensitivity of peak thrombin to and could be used for diagnostic purposes in the future. Antithrombin was an important determinant of ETP. Antithrombin is a serine protease inhibitor that regulates thrombin and a and, to a lesser extent,,,, and. It regulates the activities of coagulation factors that have already formed and therefore has a larger contribution to the ablation of the propagation phase 37 ; this can explain the importance of antithrombin as a determinant of ETP. The TGA lag time with 5 pmol/l TF was determined by the anticoagulant proteins antithrombin and protein S. The activated protein C/protein S system, which inactivates activated and, may play a role in regulating the initiation phase of coagulation. 37 Although the effect of protein S on lag time can be partially explained through this knowledge of the coagulation cascade, the detailed mechanism requires future investigation. Our study found significant positive correlations between several coagulation factors and anticoagulant factors, which are likely based on the specific conditions of the individual. Of note, vitamin K dependent coagulation factors (,,, ) significantly correlated with vitamin K dependent anticoagulant factors (protein C, protein S), suggesting that the vitamin K status determined the plasma concentrations of coagulation and anticoagulant factors in each individual. Moreover, the levels of vitamin K dependent proteins correlated with levels of blood lipids such as cholesterol and triglycerides. Consistent with our findings, one study reported significant correlations of vitamin K dependent coagulation factors (,, ) with cholesterol and triglycerides in healthy adults. 38 Vitamin K dependent coagulation proteins can bind to triglyceride-rich lipoproteins via hydrophobic interactions. Although we could not measure the concentration of circulating vitamin K in our healthy population, it is assumed that high levels of circulating lipids can increase the in vivo reservoir of lipid-soluble vitamin K and can eventually drive the synthetic cycle of vitamin K dependent coagulation proteins forward in the liver. This might be a part of a mechanism by which circulating lipids influence the levels of vitamin K dependent coagulation proteins. In addition, it is also possible that subclinical liver disease undetectable with routine liver function tests could be the common factors causing the correlation between the coagulation proteins and lipid levels. Of note, the levels of vitamin K dependent anticoagulation proteins were positively correlated with those of vitamin K dependent coagulation factors in our results. These synchronized plasma levels of proteins with opposite functionalities is likely to offset a propensity toward hypercoagulability or hypocoagulability in global coagulation assays. Future studies are required to demonstrate in detail the mechanisms of this potentially interesting finding. The current study has 3 potential limitations. First, the level of TF pathway inhibitor (TFPI) was not investigated despite the fact that TFPI plays an inhibitory role in the coagulation cascade through the inactivation of activated and. Second, to prevent contact activation, contact factor inhibitor can be used for the measurement of TGA at low concentrations of TF. 39 In our experiments, we did not use a contact factor inhibitor in TGA; therefore, the level of as a determinant of lag time with 1 pmol/l TF can be attributed to in vitro contact activation. Third, we could not investigate the effects of factors known to be correlated with hypercoagulable status such as menstrual status, hormonal treatment, or obesity. In summary, the current study demonstrated significant correlations among vitamin K dependent coagulation protein and blood lipids, suggesting that the plasma levels of vitamin K dependent coagulation proteins are controlled by the individual s lipid metabolism status in normal physiology. The global coagulation assays (PT, APTT, and TGA) were dependent on the levels of anticoagulant proteins as well as coagulation factors. In particular, the PT value was significantly determined by and, and aptt was dependent on and. Moreover, the levels of anticoagulant factors such as antithrombin and protein C were shown to be significant determinants of aptt. The TGA lag time was mainly determined by and. Peak thrombin was significantly dependent on and levels. ETP was dependent only on, suggesting that peak thrombin is a more sensitive parameter than ETP. Antithrombin (for ETP and lag time) and protein S (for lag Am J Clin Pathol 2013;139: DOI: 10.9/AJC5C4AGFRDKMX 377

9 Kim et al / Global Coagulation Assays and Coagulation Proteins time) were important contributors to TGA inhibition. It is expected that these findings will help in interpreting the results of global coagulation assays in a normal population and contribute to the development of alternative diagnostic tools in the future. To clearly determine the effects of specific factor deficiencies and excesses and to elucidate the detailed underlying mechanisms, further studies using factor-deficient plasma specimens would be required. From the 1 Department of Laboratory Medicine, 2 Cancer Research Institute, and 3 Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea. This work was supported by a National Research Foundation of Korea grant from the Korean Government ( ). Address reprint requests to Dr H.Y. Kim: Dept of Laboratory Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul -744, Korea; lukekhk@snu. ac.kr. 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