1Applied Bioscience Program, Faculty of Science, University of Ontario Institute of Technology, 2Nursing Program, Faculty of Health Sciences, University of Ontario Institute of Technology, 3Medical Laboratory Science Program, Faculty of Health Sciences, University of Ontario Institute of Technology
Tilley, D., Levit, I., Samis, J. A. Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay. J. Vis. Exp. (67), e3822, doi:10.3791/3822 (2012).
In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.
1. Preparation of FV-deficient Human Plasma
2. Preparation of Thromboplastin
3. Preparation of FV 1-stage Microplate Coagulation Assay Activity Standard Curves
4. Assaying Human Plasma Samples with the FV 1-stage and 2-stage Microplate Coagulation Assay
Representative Results - Preparation of FV 1-stage microplate coagulation assay activity standard curves
A representative example of fibrin clot formation in the FV assay over time generated by the microplate reader is illustrated in Figure 1. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation. Inspection of the wells upon assay completion confirmed that clot formation occurred. All reactions reached approximately the same extent of clot formation with a general change in absorbance at 405nm of 0.35 - 0.45 Units between the starting absorbance before and the maximal absorbance after thromboplastin and calcium chloride addition. Representative examples of FV 1-stage activity standard curves of Log Clot Time (in seconds) vs. Log FV Activity (Units/ml) and Log Initial Rate of Clot Formation (in mUnits/min) vs. Log FV Activity (Units/ml) for serial dilutions of NHP is shown in Figure 2A and Figure 2B, respectively. Fitting the Log-Log plot of Clot Time vs. FV 1-Activity indicated a strong relationship between these variables after linear regression analysis (Figure 2A; R2 = 0.980). Fitting the Log-Log plot of the Initial Rate of Clot Formation vs. FV Activity also indicated a strong relationship between these variables after linear regression analysis (Figure 2B; R2 = 0.983). The relationship of both Log Clot Time and Log Initial Rate of Clot Formation vs. Log FV Activity remained linear for NHP diluted up to 512-fold. Since FV circulates in NHP at approx 12-40nM 16, the microplate assay is sensitive to approx 24-80pM FV in NHP. Given that the dissociation constant of the interaction of FVa-FXa-lipid in prothrombinase is approximately 1nM 17, the FV microplate assay is entirely suitable for measurement of FV levels in the physiologically relevant nM range for FVa function in prothrombinase.
Using the FV microplate assay, it was also determined that the normal range of FV activity in the FV 1-stage activity assay of 15 healthy control plasmas (Male and Female, Age 18-20yrs) was (Mean ± Standard deviation; Range): 0.96 ± 0.14U/ml; 0.68-1.11U/ml. This agrees well with the normal healthy FV activity and range (0.66-1.14U/ml) reported by Cutler et al. for the FV 1-stage activity determined with an automated analyzer 7. The intra-assay variability of the time, extent, and initial rate of clot formation in the FV 1-stage assay in 6 wells on 8 different days was 3.4%, 4.4%, and 3.1%, respectively. The inter-assay variability of the time, extent, and initial rate of clot formation in the FV 1-stage assay in 6 wells of 8 different experiments on 8 different days was 7.1%, 7.8%, and 9.2%, respectively. Thus, the intra- and inter-assay variability of these three measured variables was at a low and acceptable level for robust assay performance within and between microplate assay of multiple samples simultaneously (Up to 12).
Representative Results - Assaying human plasma samples with the FV 1-stage and 2-stage microplate coagulation assay
The standard curve of Log Clot Time vs. Log FV Activity (Figure 2A) was used to measure the FV activity in NHP and 9 DIC patient plasmas which were not intentionally activated with added thrombin (FV 1-stage activity) or were intentionally activated with added thrombin (FV 2-stage activity) and the results are shown in Table 1. All 9 DIC patient plasmas exhibited FV 1-stage activities and initial rates of clot formation that were decreased on average, by 54% and 18%, respectively, from NHP. The extents of clot formation in the FV 1-stage assay in the DIC patients were not largely different from NHP, and increased on average, by 13% from NHP.
Activation of NHP with thrombin generated an approximate 8-fold increase in FV 2-stage activity above the FV 1-stage activity (Table 1). This indicates that the FV in NHP was mainly present in its unactivated form and agrees with the previously reported results using manual tilt-tube FV assays 3, 4 and automated FV assays 5-7. The FV 2-stage and total activity were also decreased in the DIC patients on average, by 44% and 42%, respectively, from NHP. The initial rates and extents of clot formation in the FV 2-stage assay in the DIC patients were not significantly different from NHP, and varied on average, by approx 9% and 4%, respectively, from that observed with NHP. These results indicated that compared with the FV in NHP, the FV in the DIC patient plasmas resulted in a prolonged time and decreased rate of fibrin clot formation and that the patient FV was on average, only 56% as activatable with thrombin as well.
Figure 1. Clot formation in normal pooled human reference plasma measured with the kinetic microplate FV 1-stage coagulation assay. Fibrin clot formation in NHP was continuously monitored at 405nm using a kinetic microplate reader. The plot is the microplate reader output of a 6 min reaction of 32-fold diluted NHP, FV-deficient plasma, thromboplastin, and calcium chloride in a microplate well. The vertical axis represents the change in absorbance at 405nm that occurred as a result of fibrin formation in plasma. The time of fibrin formation was defined as the time to reach the half maximal increase in absorbance or the midpoint of curve (36.40 sec). The initial rate of clot formation was defined as the rate of change of absorbance at 405nm over the first 5 time points of the linear increase of the absorbance portion of the curve (611.88 mUnits/min). The extent of clot formation was defined as the difference between the maximum and minimum absorbance at 405 nm (0.35 Units).
Figure 2. Standard curves of time and initial rate of clot formation vs. Factor V activity in normal pooled human reference plasma using the FV 1-stage microplate assay. NHP was serially diluted (0- to 512-fold in HBS) and assayed with the FV 1-stage microplate assay as described in the protocol. Log-Log plots of the time and initial rate of clot formation vs. FV activity in the FV 1-stage microplate assay are shown after linear regression modeling of the data in panels A and B, respectively.
|Sample||1-stage assay Activity (Units/ml)||1-stage assay Extent (Units)||1-stage assay Initial Rate (mUnits/min)||2-stage assay Activity (Units/ml)||2-stage assay Extent (Units)||2-stage assay Initial Rate (mUnits/min)||Total activity (Units/ml)|
Table 1 FV Activity in NHP and in 9 Patients That Developed Disseminated Intravascular Coagulation (DIC). The FV 1-stage, 2-stage, and total activity in NHP and 9 DIC patient plasmas were determined from the FV 1-stage microplate assay standard curve of time of clot formation vs. FV activity (Figure 2A) and are given in Units/ml. The initial rates (Initial rate of increase in A405nm over first five time points in mUnits/min) and extents (Maximum A405nm - Minimum A405nm) of clot formation in the FV 1- and 2-stage microplate assay are also shown.
The kinetic microplate assay method outlines the development of a novel, rapid, inexpensive, and convenient technique for measurement of FV coagulation activity in samples of human plasma which have not (FV 1-stage assay) or have been intentionally activated with added thrombin (FV 2-stage assay). The assay utilizes a kinetic microplate reader and all the materials and reagents required are commercially available or may be made in-house. The assay continuously monitors the change in the light scatter of plasma during fibrin clot formation at 405nm. The microplate assay has the advantage of using small plasma sample volumes (μl) and is amenable for analysis of multiple samples simultaneously (up to 12). These assay attributes are advantageous when expensive equipment and reagents are used (Automated analyzers and Factor-deficient plasmas); only small sample volumes are available (Patient plasmas) or analyzing a large number of samples (During FV purification from plasma).
The FV microplate assay has the added advantages that it is convenient, fast, and does not require the isolation and purification of the required constituent components. Compared with manual tilt-tube 3, 4 and automated 5-7 FV assays that have been reported, the FV microplate assay has comparable useful range of clot times (25-75 sec) and corresponding FV levels in the sample (0.5-0.005 Units/ml), and sensitivity/detection limits (20-80pM). Compared with manual tilt-tube FV assays which suffer from a subjective visual assessment of clot formation 3, 4, the FV microplate assay permits objective and quantitative measurement of the clot time, initial rate, and extent of fibrin clot formation. Compared with automated FV assays which suffer from the requirement of expensive analyzers and reagents 5-7, the FV microplate assay reagents and materials are inexpensive and all the required materials may be purchased commercially or made in-house. Manual tilt-tube 3, 4 and automated 5-7 FV assays generally only provide the time for clot formation; not the time, initial rate, and extent of fibrin clot formation which are all accurately measured spectrophotometrically with the FV microplate assay. Finally, manual tilt-tube 3,4 and automated 5-7 FV assays suffer from only being able to measure the FV activity in plasma samples one at a time; whereas the FV microplate assay is amenable to high-throughput and 12 samples may be assayed simultaneously.
The microplate assay was used to demonstrate that the FV in 9 DIC patient plasmas was less active than in NHP because of a combination of delayed clot times and lower initial rates of clot formation in the FV 1-stage assay. The initial rates of clot formation in the FV 2-stage assay in the DIC patient plasmas were not changed from NHP. The extents of clot formation in the DIC patient plasmas were also not changed from NHP in the FV 1-stage and 2-stage assays. Decreased FV 1-stage, 2-stage, and total activities may have been due to increased FV consumption 19 and/or inactivation 6 in accordance with other studies during the pathogenesis of this acquired blood disorder. Given the extent of clot formation in the DIC plasmas was the same as observed with NHP, this variable was most likely a result of the fibrinogen concentration in the FV-deficient plasma and not related to the characteristics of the patient plasmas.
The results from the microplate assay indicated that measurement of FV activity in samples of human plasma may be obtained from the time and initial rate of clot formation from the FV 1-stage assay and the time of clot formation and total activity from the FV 2-stage assay. It was not possible to obtain quantitative measurement of FV activity in a plasma sample based on the extent of clot formation in the FV 1- and 2-stage assays or the initial rate of clot formation in the FV 2-stage assay. Given all reactions reached approximately the same extent of clot formation of 0.35 - 0.45 Units between the initial absorbance before and the maximal absorbance after thromboplastin and calcium chloride addition, measurement of the extent of clot formation does not provide a quantitative assessment of FV activity in a given plasma sample.
The novel microplate assay will find use in research and clinical settings for measurement of FV activity in samples of human plasma and fractions during its purification from human and animal plasma. The microplate assay may be used to measure the time, initial rate, and extent of clot formation; parameters not generally monitored simultaneously in manual tilt-tube assays and automated coagulation analyzers. This information provides more of a complete quantitative assessment of the involvement of FV during the clot formation event in human plasma than has been previously reported 3-7. This information is especially important in both research and clinical laboratories when measuring significant changes in FV activity in samples of plasma or with purified FV or FVa. The FV microplate assay may also be used to characterize and measure compounds that may activate or inactivate FV and FVa, measure the FV activity in patients at risk for venous thrombosis as a result of the FV Leiden mutation 20, 21 and to monitor the activity of FV during its purification from plasma.
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor in the extrinsic, intrinsic, or common pathway using the appropriate factor-deficient plasma and initiating reagents for fibrin formation. The assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical laboratories. Ultimately, this information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for individuals afflicted with coagulation disorders, such as DIC.
No conflicts of interest declared.
The research was supported with Professional Development and Research Startup Funds from The University of Ontario Institute of Technology (UOIT) to Dr. John A. Samis and a Canadian Institutes of Health Research Health Professional Student Research Award to Irina Levit. The authors acknowledge Shannon Everett (Teaching and Learning Centre, UOIT) for her assistance preparing the video. The authors acknowledge Dr. Michael E. Nesheim (Department of Biochemistry, Queen's University, Kingston, ON) for his mentorship, insight, and helpful discussions. The authors also acknowledge Dr. Cheng Hock Toh (Roald Dahl Haemostasis and Thrombosis Centre, Royal Liverpool University Hospital, Liverpool, UK) for providing the DIC patient plasmas used in the study.
|Kinetic microplate reader||Molecular Devices||Spectra Max 190||SOFTmax PRO 4.3 LS software|
|Normal pooled human reference plasma||Precision Biologicals||CCN-10|
|Trisodium citrate||Becton Dickenson||B10242|
|FV-deficient human plasma4||Made from fresh human plasma and stored in aliquots at -70 °C.|
|3 and 60 ml syringes||Becton Dickenson||309585 and 136898|
|Butterfly apparatus||Vacutainer||367251||20 gauge|
|Thromboplastin||Trinity Biotech||T1106||Dialyzed vs. HBS, pH 7.4 and stored in aliquots at -70 °C.|
|Calcium chloride||Becton Dickenson||B10070|
|Human thrombin15||From human plasma; stored at -20 °C in 50% (v/v) glycerol.|
|Dialysis membrane||Fisher||21-152-6||6-8kDa molecular weight cutoff; 50mm x 3m.|
|Template holder and removable 8 well strips.||Nunc||14-245-63 and 469957|
|ELISA washing trays||BioRad||224-4872|
|1.5 ml microfuge and 15 ml screw capped tubes.||Sarstedt||72690 and 62554205|
|Multichannel pipette||Fisher||2703630||8 channel model.|