The overall goal of the following procedure was to develop a microplate assay that measures the activity of a coagulation protein factor V during the formation of a fibrin clot in human plasma. In response to injury, the blood coagulation process is activated and results in a series of Xymogen to protease conversion reactions that ultimately lead to the generation of the clotting enzyme thrombin, thrombin cleves soluble fibrinogen to fibrin monomers, which then associate to form an insoluble fibrin clot. Normally, blood remains as a freely flowing liquid during the healthy or uninjured state, and the amount of fibrin formed is equal to the amount of fibrin degraded or broken down.
However, in certain disease situations, too much clotting leads to heart disease and stroke, whereas too little clotting leads to bleeding tendencies, which are often seen in the hemophilias. Factor V is a protein in plasma that in its activated form factor five A is a critical accelerator of thrombin generation during fibrin clot formation as part of the prothrombin enzyme complex, prothrombin is made up of four components factor 10 A, the protease factor five A, the co-factor lipid, the surface and calcium. The meline factor five A increases the catalytic efficiency or kcat of factor 10 A towards its substrate prothrombin by approximately 3000 fold, thereby profoundly enhancing thrombin generation and fibrin clot formation.
The microplate platform is preferred for measuring enzyme catalyzed events because of convenience, time cost, small sample volumes, continuous monitoring and high throughput. The fibrin clot formation event may be monitored with a microplate reader by measuring the increase in light absorbance of plasma as it changes from a clear yellow appearance with no fibrin clot present and minimal light absorbance to an opaque cloudy appearance with a fibrin clot and maximal light absorbance. The main advantages of this technique over existing methods such as manual tilt tube assays or assays with automated analyzers is the method is fast, inexpensive, and has small sample volume requirements and has high throughput.
The assay measures the activity of unintentionally activated and thrombin activated factor five to obtain a complete quantitative assessment of its total functional activity. Demonstrating that technique will be Derek Tilley, a master student in the laboratory. In order to measure the factor five activity in a plasma sample, it is necessary to construct factor five one stage activity, standard curves of the time of fibrin clot formation in panel A and the initial rate of fibrin clot formation in panel B using normal human plasma or NHP as a standard which contains by definition one unit of factor five Activity per ML thaw the Factor five deficient plasma, normal human plasma, and sample plasma unknowns and thromboplastin at 37 degrees for 10 minutes.
Gently mix the factor five deficient plasma and thrombo plasty transfer to separate plastic washing trays and incubate on ice. Store the normal human plasma and sample plasma unknowns on ice after gently Mixing. Store the Calcium chloride 25 millimolar in distilled Water in a separate washing tray at room temperature for the factor five one Stage activity standard curves.
Prepare 200 microliters each of twofold serial dilution of normal human plasma from 0 2 4 8 16 32 64, 1 28, 2 56, and 512 fold in HBS pH 7.4 using 1.5 ml micro centrifuge tubes, vortex well and incubate on ice. For the plasma sample unknowns, prepare 200 microliters each of 40 fold dilutions of normal human plasma and unknown sample plasmas in HBS pH 7.4 using 1.5 ML micro centrifuge tubes. Place five microwell strips into the template holder of the microplate reader.
Turn on the Microplate reader and access Softmax Pro microplate application software using a multi-channel pipette. Make simultaneous additions of 50 microliters of factor five deficient plasma to all sample microplate wells using a single pipette add 50 microliters of the prepared 10 serial dilutions of normal human plasma and the 40 fold dilutions of normal human plasma and sample plasma unknowns to separate MICROPLATE wells using a multi-channel pipette, add 50 microliters of thrombo plasty to all sample wells. Incubate in the Microplate reader at 25 degrees Celsius for one minute with shaking during the 15 to 25 seconds of the one minute incubation.
Using the computer interfaced with the MICROPLATE reader access Softmax Pro Microplate application software. Set the Microplate reader to absorbance wavelength to 405 nanometers mode to kinetic temperature to 25 degrees Celsius and program the Microplate reader to shake for the first five seconds after calcium chloride addition and measure absorbance at 405 nanometers every five seconds for six minutes. Thereafter, the five second shaking interval is not included in the calculated clot times using two multi-channel pipettes simultaneously add 50 microliters of calcium chloride to the two microplate strips with samples.
Wait 15 seconds and immediately press the start icon in Softmax Pro from the computer to begin the assay. For each plasma sample analyzed, the time of clot formation is determined and defined as the time in seconds to reach the half maximal increase in absorbence. The initial rate of clot formation is defined as the rate of increase in absorbence over the first five time points of the linear portion of the increase in absorbence versus time.
The extent of clot formation is defined as the difference between the maximum and minimum absorbence achieved during the clot formation event at the end of the run. Use the ABSORBENCE versus time profiles in Softmax Pro to determine the time, initial rate and extent of clot formation for the serial dilution of normal human plasma. To construct the factor V one stage activity standard curves use the clot times for the 40 fold diluted normal human plasma and sample plasma unknowns to I interpolate the factor five activity in units per mill from the factor five one stage activity standard curve.
This represents the factor five one stage activity or that without intentional activation by thrombin. This is a representative example of fibrin clot formation in the factor five assay over time generated by the microplate reader. The profile represents fibrin clot formation using 32 fold diluted normal human plasma in a single microplate.
Well, the vertical axis denotes the change in absorbent at 405 nanometers that occurred as a result of fibrin clot formation. The time of clot formation was calculated as the midpoint of the absorbence versus time curve, 36.4 seconds. The initial rate of clot formation was calculated as 611.88 milli units per minute.
The extent of cloud formation was calculated as 0.35 units. The factor five assay accurately measures the time initial rate and extent of cloud formation. Inspection of the wells upon assay completion confirmed that clot formation occurred.
All reactions reached approximately the same extent of CloudFormation with a general change in absorbance at 405 nanometers of between 0.35 and 0.45 units between the starting absorbance before and the maximum absorbance after thrombo plasty and calcium chloride. Addition go representative examples of the factor five one stage activity, standard curve of clot time in seconds versus factor five activity in units per mil and the initial rate of claw formation in milli units per minute versus factor five. Activity in units per mil for normal human plasma are shown in panel A and panel B respectively.
Since factor five is normally activated with thrombin. The active co-factor factor five A a second assay after addition and incubation with thrombin is critical to accurately measure the factor five two stage activity of a plasma sample with intentional activation by added thrombin for the factor five two stage assay. Prepare 200 to 300 microliters of purified thrombin at her final concentration of a hundred nano molar in HBS pH 7.4 and incubate on ice dilute 120 microliters of the above normal human plasma and sample plasmas from 40 fold to 100 fold with 170 microliters of HBS pH 7.4 containing 2.8 millimolar.
Calcium chloride. Add 10 microliters of thrombin to achieve a 10 nanomolar final concentration To each sample.Vortex. Well to mix and incubate at 37 degrees For one minute, dilute the normal human plasma and sample plasmas to 500 fold by adding 400 microliters of HBS pH 7.4.
Determine the clot time for normal human plasma in each of the sample plasma unknowns assay from the absorbance versus time profiles in soft max Pro. Taking into account the 500 fold dilution, use the clot time for each sample to calculate the factor five two stage activity from the factor five one stage standard curve of clot time versus activity. This represents the factor five two stage activity or that with intentional activation by thrombin.
The total factor five activity may then be calculated as the factor five two stage activity minus the factor five one stage Activity. The standard Curve of log clot time versus log factor five activity was used to measure the factor five activity in normal human plasma and in plasmas from nine patients with disseminated intravascular coagulation or DIC, all nine DIC patients. Exhibited factor five one stage activities and initial rates of clot formation that were decreased on average by 54 and 18%The factor five two stage and total activity were decreased in the DIC patients on average by 44 and 42%compared to the normal human plasma.
The factor five and the DIC patients resulted in a prolonged time and decreased rate of fiber clot Formation. After watching this video, you should have a good Idea of how to implement this method in your laboratory. The method may be easily adapted to measure the activity of any coagulation factor in plasma, making it suitable for a broad range of research and clinical applications.
Ultimately, this information of factor V will positively impact healthcare environments through the earlier diagnosis and treatment of thrombotic or bleeding disorders such as DIC.
