This research was funded by the Science and Technology Bureau of Xiamen City (No. 3502Z20227197), the Science and Technology Bureau of Fujian Province (No. 2019J01070; No. 2022J01311), and High-level Talents Innovation and Entrepreneurship Project of Quanzhou Science and Technology Plan (No. 2022C015R). We thank Fucai Wang (Huaqiao University) and Lei Tong (Huaqiao University) for their technical assistance.

1. Peanut worm homogenate preparation
<ol>
	<li>Add 50 g of peanut worms and 150 mL of saline solution into the homogenizer.</li>
	<li>Homogenize at 24000 rpm for 60 s. 
	NOTE: Repeat 3 times.</li>
	<li>Centrifuge the homogenate at 9710 x g for 30 min at 4 &#176;C.</li>
	<li>Collect the supernatant as the peanut worm homogenate for further study.</li>
</ol>
2. Fibrinogen-PAGE gel preparation
<ol>
	<li>Weigh 0.01 g of fibrinogen into a 50 mL glass beaker.</li>
	<li>Add 1.9 mL of DDH2O, 1.3 mL of Tris-HCl (1.5 M, pH 8.8), and 0.05 mL of SDS (10%, w/v) into the beaker; mix well by pipetting.</li>
	<li>Place the beaker at 37 &#176;C for 30 min. 
	NOTE: This step is to dissolve the fibrinogen.</li>
	<li>Add 1.7 mL of acrylamide (30%, w/v), 0.05 mL of ammonium persulfate (APS; 10%, w/v), 0.002 mL of TEMED; mix well by pipetting. 
	NOTE: This is separating gel.</li>
	<li>Pour the separating gel into a 10-well gel mold.</li>
	<li>Add 2 mL of DDH2O into the gel mold; wait for 30 min.</li>
	<li>Remove the water thoroughly by turning the mold upside down.</li>
	<li>Add 1.4 mL of DDH2O, 0.25 mL of Tris-HCl (1.0 M, pH 6.8), 0.02 mL of SDS (10%, w/v), 0.33 mL of acrylamide (30%, w/v), 0.02 mL of APS (10%, w/v), 0.002 mL of tetramethylethylenediamine (TEMED) into the beaker; mix well by pipetting. 
	NOTE: This is loading gel.</li>
	<li>Pour the loading gel into the same gel mold as step 2.5; insert the comb immediately. 
	NOTE: The fibrinogen-PAGE gel used here was composed of the fibrinogen (0.2%) containing separating gel and loading gel. The concentration of fibrinogen can be adjusted as needed.</li>
</ol>
3. Electrophoresis
<ol>
	<li>Place the prepared fibrinogen-PAGE gel together with the glue mold into the electrophoresis tank; pour 1300 mL of electrophoresis solution (1.963 g of Tris, 12.22 g of glycine, 0.65 g of SDS, and 1300 mL of DDH2O) into the tank; take out the comb.</li>
	<li>Add 5 &#181;L of 5x non-denaturing loading buffer to the 20 &#181;L of sFE and 20 &#181;L of&#160;peanut worm homogenate; mix well by pipetting; load them into the prepared fibrinogen-PAGE gel. 
	NOTE: Do not boil the mixture. sFE is a fibrino(geno)lytic enzyme identified previously6,7,8 and is used as a positive control in this study. Peanut worm homogenate is the sample to be detected. Non-denaturing loading buffer without bromophenol blue, &#946;-mercaptoethanol, and SDS compared with the SDS-PAGE loading buffer. Each sample lane is separated with the 1x SDS-PAGE loading buffer to clearly detect the movement of the protein.</li>
	<li>Perform electrophoresis at 80 V for 30 min, or 120 V for 20 min.</li>
	<li>Remove the fibrinogen-PAGE gel from the glue mold.</li>
</ol>
4. Renaturation
<ol>
	<li>Transfer the fibrinogen-PAGE gel to a plastic box.</li>
	<li>Add 100 mL of Tris-HCl (0.05 M, pH 7.4) and 2 mL of Triton X-100 into the box. Place it on a shaker at 60-70 rpm for 10 min.</li>
	<li>Remove the wash buffer by pipetting. 
	NOTE: Repeat three times.</li>
</ol>
5. Incubation
<ol>
	<li>Add 50 mL of Tris-HCl (0.05 M, pH 7.4) into the plastic box.</li>
	<li>Incubate at 37 &#176;C for 4 h. 
	NOTE: Incubation time can be adjusted according to the strength of fibrinogenolytic activity.</li>
</ol>
6. Staining
<ol>
	<li>Remove the incubation buffer by pipetting.</li>
	<li>Add 50 mL of Coomassie Brillant Blue Staining solution into the box and place the box on a shaker at 60-70 rpm for 30 min.</li>
</ol>
7. Decolorization
<ol>
	<li>Remove the staining solution by pipetting.</li>
	<li>Add 50 mL of DDH2O into the box.</li>
	<li>Place the box on a shaker at 60-70 rpm for 30 min. 
	NOTE: DD H2O was used as the destaining solution here. The shaking time can be adjusted according to the strength of fibrinogenolytic activity.</li>
</ol>

Unveiling Fibrinogenolytic Agents in Peanut Worms with Fibrinogen-PAGE

<ol>
	<li>Bikdeli, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COVID-19+and+thrombotic+or+thromboembolic+disease:+Implications+for+prevention,+antithrombotic+therapy,+and+follow-up.">COVID-19 and thrombotic or thromboembolic disease: Implications for prevention, antithrombotic therapy, and follow-up.</a> J Am Coll Cardiol. 75 (23), 2950-2973 (2020).</li><li>Tsivgoulis, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombolysis+for+acute+ischaemic+stroke:+current+status+and+future+perspectives.">Thrombolysis for acute ischaemic stroke: current status and future perspectives.</a> Lancet Neurol. 22 (5), 418-429 (2023).</li><li>Tang, M., Hu, C., Lin, H., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinolytic+drugs+induced+hemorrhage:+mechanisms+and+solutions.">Fibrinolytic drugs induced hemorrhage: mechanisms and solutions.</a> Blood Coagul Fibrinolysis. 34 (5), 263-271 (2023).</li><li>Lu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+characterization,+and+chemical+modification+of+Bacillus+velezensis+SN-14+fibrinolytic+enzyme.">Purification, characterization, and chemical modification of Bacillus velezensis SN-14 fibrinolytic enzyme.</a> Int J Biol Macromol. 177, 601-609 (2021).</li><li>Abu-Tahon, M. A., Abdel-Majeed, A. M., Ghareib, M., Housseiny, M. M., Abdallah, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombolytic+and+anticoagulant+efficiencies+of+purified+fibrinolytic+enzyme+produced+from+Cochliobolus+hawaiiensis+under+solid-state+fermentation.">Thrombolytic and anticoagulant efficiencies of purified fibrinolytic enzyme produced from Cochliobolus hawaiiensis under solid-state fermentation.</a> Biotechnol Appl Biochem. 70 (6), 1954-1971 (2023).</li><li>Lin, W., Lin, H., Xin, P., Yan, H., Kang, B., Tang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comprehensive+approach+to+analyze+the+cell+components+of+cerebral+blood+clots.">A Comprehensive approach to analyze the cell components of cerebral blood clots.</a> J Vis Exp. (197), e65791 (2023).</li><li>Tang, M., Lin, H., Hu, C., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+a+fibrinolytic+enzyme+from+Sipunculus+nudus.">Affinity purification of a fibrinolytic enzyme from Sipunculus nudus.</a> J Vis Exp. (196), e65631 (2023).</li><li>. Plasmin affinity purification method based on agarose gel and plasmin and application thereof Available from: <a target="_blank" href="https://patents.google.com/patent/CN116103270A/en?oq=CN202310108470">https://patents.google.com/patent/CN116103270A/en?oq=CN202310108470</a> (2023)</li><li>Walton, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+fibrin+plate+method+for+the+assay+of+plasminogen+activators.">An improved fibrin plate method for the assay of plasminogen activators.</a> Clinica Chimica Acta. 13 (5), 680-684 (1966).</li><li>Ngere, J. B., Ebrahimi, K. H., Williams, R., Pires, E., Walsby-Tickle, J., McCullagh, J. S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-exchange+chromatography+coupled+to+mass+spectrometry+in+life+science,+environmental,+and+medical+research.">Ion-exchange chromatography coupled to mass spectrometry in life science, environmental, and medical research.</a> Anal Chem. 95 (1), 152-166 (2023).</li><li>Kato, S., Takeuchi, K., Iwaki, M., Miyazaki, K., Honda, K., Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitin-+and+streptavidin-mediated+affinity+purification+systems:+A+screening+platform+for+enzyme+discovery.">Chitin- and streptavidin-mediated affinity purification systems: A screening platform for enzyme discovery.</a> Angew Chem Int Ed Engl. 62 (31), e202303764 (2023).</li><li>Sharma, N., Sharma, R., Rajput, Y. S., Mann, B., Singh, R., Gandhi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+methods+for+milk+proteins+on+polyacrylamide+gel+electrophoresis:+Critical+analysis+and+options+for+better+resolution.">Separation methods for milk proteins on polyacrylamide gel electrophoresis: Critical analysis and options for better resolution.</a> International Dairy Journal. 114, 104920 (2021).</li><li>. Preparation and application of natural fibrinolytic enzyme from peanut worm Available from: <a target="_blank" href="https://patents.google.com/patent/CN109295042A/en">https://patents.google.com/patent/CN109295042A/en</a> (2019)</li></ol>

The authors have no conflicts of interest to disclose.

sFE is a novel fibrino(geno)lytic enzyme isolated from peanut worms by our team previously3,6,8,13. However, the identification processes of sFE were time- and labor-consuming, involving the fibrinogenolytic activity detection, protein components separation, and activity confirmation stages. As a simple method, fibrinogen plate assay is mostly used in the preliminary screening stage to check the fibrinogenolytic activity of the samples. However, the fibrinogen plate assay method cannot separate which component is active. Therefore, chromatography and native/SDS-PAGE have to be used to separate their components after activity detection. Then, reaction products are resolved again by the SDS-PAGE to confirm the degradation of substrate fibrinogen. In this work, the substrate fibrinogen was added to PAGE gel to detect the fibrinogenolytic agent. After electrophoresis, renaturation, incubation, staining, and decolorization, the molecular weight and fibrinogenolytic activity of the active fibrinogenolytic agents were clearly displayed with the same gel. By this method, we successfully detected more than one active fibrinogenolytic agent from&#160;peanut worm homogenate within 6 h.
To achieve the optimum results, more attention should be paid to the fibrinogen-PAGE gel preparation, renaturation, and incubation. Generally speaking, the lower the concentration of fibrinogen, the more colorless the fibrinogenolytic zone, which indicates fibrinogenolytic activity of the agents. However, the lower the concentration of fibrinogen, the more colorless the background, and the more difficult it is to distinguish the fibrinogenolytic zone from the background. The renaturation time will affect the fibrinogenolytic activity of components significantly. If this time is too short to wash off the SDS in the PAGE gel, the fibrinogenolytic activity of components will be inhibited by remanent SDS. Similarly, If the incubation time is too short, the fibrinogenolytic reaction is not thorough. While the incubation time is too long, the marker used to indicate the molecular weight will be washed away. In this study, although those conditions have been optimized, these conditions were set to separate and display the fibrinogenolytic agents from peanut worms. So, personalized conditions of those critical steps should be set according to specific fibrinogenolytic agents.
It is important to mention that this fibrinogen-PAGE is only suitable for screening the fibrinogenolytic agents with fibrinogen lytic activity but not for the agents with fibrinogen lytic activity or plasminogen activation activity. If we want to display those activities, the method can be optimized by replacing the fibrinogen with fibrin or plasminogen. Another point worth noting is that the active fibrinogenolytic agents tested in this study were monomers, which were not affected under the SDS electrophoresis condition. Therefore, if the tested agents are polymers, this method should be adjusted to a native-PAGE condition that can not display the molecular weight. Therefore, the fibrinogen-PAGE method introduced here was only a general basic version, which can be optimized to screen the other fibrinogenolytic agents quickly.

In recent years, due to the continued rise of thrombotic diseases, thrombotic diseases have become a new major global health problem1. At present, antithrombotic drugs are classified into three groups: anti-platelet aggregation drugs, anticoagulants, and thrombolytic drugs. Among them, thrombolytic drugs, such as urokinase (UK), tissue plasminogen activator (tPA), etc., are the only clinically used drugs that can hydrolyze thrombus2. Meanwhile, more safe and effective thrombolytic drugs are being developed by the identification of novel thrombolytic agents3.
However, the identification of novel thrombolytic agents is time-consuming and labor-intensive, which mainly involves the separation/purification and characterization/checking stages. The former is to separate each component, and the latter is to display their fibrino(geno)lytic activities4,5. In previous studies, despite the fact we have successfully isolated a novel fibrino(geno)lytic enzyme (sFE) from the peanut worm (Sipunculus nudus) by affinity chromatography and fibrin(ogen) plate assay6,7,8, these processes are such time and labor-consuming. First, it is necessary to determine whether the peanut worm homogenates have fibrino(geno)lytic activity by the fibrin(ogen) plate method and reaction products based on PAGE9. Then, a series of chromatography, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, and other methods, have to be carried out for separation and purification10,11. Subsequently, the fibrin plate assay is performed again to check the fibrinogenolytic activity of each separated component. Finally, native-PAGE and sodium dodecyl sulfate (SDS)-PAGE are performed to determine the molecular weight of the active fibrinogenolytic agents12. Therefore, it is critical to rapidly separate and display the active fibrinogenolytic agents.
To rapidly separate and display the active fibrinogenolytic agents in the peanut worm homogenates, a new fibrinogen-PAGE method was developed by combining the PAGE and fibrinogen plate methods, i.e., the substrates of fibrinogenolytic agents fibrinogen were added to the native-PAGE gel. After native-PAGE, the components were separated by their molecular weight. Meanwhile, each active fibrinogenolytic component can be displayed by staining. By this method, we successfully detected more than one active fibrinogenolytic agent of peanut worm&#160;homogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

<table><tbody><tr><td>1&amp;nbsp; M Tris-HCl (pH 6.8)</td><td>Solarbio</td><td>T1020</td><td></td></tr><tr><td>1.5 M Tris-HCl (pH 8.8)</td><td>Solarbio</td><td>T1010</td><td></td></tr><tr><td>30% Acrylamide/Bis-acrylamide</td><td>Biosharp</td><td>BL513B</td><td></td></tr><tr><td>Ammonium persulfate</td><td>XiLONG SCIENTIFIC</td><td>7727-54-0</td><td></td></tr><tr><td>Beaker</td><td>PYREX</td><td>2-9425-02</td><td></td></tr><tr><td>Centrifuge Tube (1.5 mL)</td><td>Biosharp</td><td>BS-15-M</td><td></td></tr><tr><td>Constant Temperature Incubator</td><td>JINGHONG</td><td>JHS-400</td><td></td></tr><tr><td>Coomas Brillant Blue Stainning solution</td><td>Beyotime</td><td>P0017F</td><td></td></tr><tr><td>Electronic Analytical Balance</td><td>DENVER</td><td>TP-213</td><td></td></tr><tr><td>Fibrinogen</td><td>Solarbio</td><td>F8051</td><td></td></tr><tr><td>Gel loading pipette tips, Bulk</td><td>Biosharp</td><td>BS-200-GTB</td><td></td></tr><tr><td>Homogenizer</td><td>AHS</td><td>ATS-1500</td><td></td></tr><tr><td>Horizontal rotation oscillator</td><td>NuoMi</td><td>NMSP-600</td><td></td></tr><tr><td>Milli-Q Reference</td><td>Millipore</td><td>Z00QSV0CN</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell</td><td>BIO-RAD</td><td>165-8000~165-8007</td><td></td></tr><tr><td>N,N,N&#039;,N&#039;-Tetramethylethylenediamine</td><td>Sigma</td><td>T9281</td><td></td></tr><tr><td>Pipette Tip (1 mL)</td><td>Axygene</td><td>T-1000XT-C</td><td></td></tr><tr><td>Pipette Tip (10 &amp;micro;L)</td><td>Axygene</td><td>T-10XT-C</td><td></td></tr><tr><td>Pipette Tip (200 &amp;micro;L)</td><td>Axygene</td><td>T-200XT-C</td><td></td></tr><tr><td>Pipettor (1 mL)</td><td>Thermo Fisher Scientific</td><td>ZY18723</td><td></td></tr><tr><td>Pipettor (10&amp;micro;L)</td><td>Thermo Fisher Scientific</td><td>ZX98775</td><td></td></tr><tr><td>Pipettor (200 &amp;micro;L)</td><td>Thermo Fisher Scientific</td><td>ZY20280</td><td></td></tr><tr><td>Pipettor (50 &amp;micro;L)</td><td>Thermo Fisher Scientific</td><td>ZY15331</td><td></td></tr><tr><td>Refrigerated Centrifuge</td><td>cence</td><td>H1650R</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>V900859</td><td></td></tr><tr><td>Tris</td><td>Solarbio</td><td>T8060</td><td></td></tr><tr><td>Tris-HCl</td><td>Solarbio</td><td>T8230</td><td></td></tr><tr><td>Triton X-100</td><td>Solarbio</td><td>T8200</td><td></td></tr></tbody></table>

rapid separation and display of active fibrinogenolytic agents in sipunculus nudus through fibrinogen-polyacrylamide gel electrophoresis

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus)&#160;in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

After electrophoresis, all the bands of the marker were clearly displayed. The 1x SDS-PAGE loading lanes only showed 10 kDa bands (bromophenol blue). The sFE and peanut worm samples did not show any observable bands (Figure 1). Although the bands of the samples are not visible, the performance of the marker and bromophenol blue indicated that the electrophoresis was successful, and the samples were separated according to their molecular weight.
Although the whole gel turned blue due to the staining, the decolorization made the area where fibrinogen was degraded by fibrinogenolytic agents transparent. Compared with the marker, the sFE indicated an active fibrinogenolytic band near 25 kDa. The peanut worm homogenate displayed 25 kDa, 33 kDa, 43 kDa, and a series of bands bigger than 70 kDa (Figure 2). The results indicated that sFE only contained a 25 kDa fibrinogenolytic component, but the peanut worm homogenate contained sFE components and other fibrinogenolytic agents. Supplementary Figure 1 is an example of a gel image showing the marker in deep blue and the target samples as bright bands.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66536/66536fig01.jpg" /> 
Figure 1: Gel after electrophoresis. M: Marker; Lane 1: 1x SDS-PAGE loading buffer; Lane 2: sFE; Lane 3: 1x SDS-PAGE loading buffer; Lane 4: peanut worm homogenate; Lane 5: 1x SDS-PAGE loading buffer. <a href="https://www.jove.com/files/ftp_upload/66536/66536fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66536/66536fig02.jpg" /> 
Figure 2: Gel after decolorization. M: Marker; Lane 1: 1x SDS-PAGE loading buffer; Lane 2: sFE; Lane 3: 1x SDS-PAGE loading buffer; Lane 4: peanut worm homogenate; Lane 5: 1x SDS-PAGE loading buffer. <a href="https://www.jove.com/files/ftp_upload/66536/66536fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Gel image showing the marker in deep blue and the target samples as bright bands. M: Marker; Lane 1: 1x SDS-PAGE loading buffer; Lane 2: sFE; Lane 3: 1x SDS-PAGE loading buffer; Lane 4: peanut worm homogenate; Lane 5: 1x SDS-PAGE loading buffer. <a href="https://www.jove.com/files/ftp_upload/66536/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>

Read this Scientific Article Protocol about Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis at JoVE.com

Author Spotlight: Time and Cost-Effective Fibrinogen-PAGE for Fibrinogenolytic Studies

Here, we present a fibrinogen-polyacrylamide gel electrophoresis (PAGE) protocol to rapidly separate and display the fibrinogenolytic agents of Sipunculus nudus.

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis

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This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

Medicine

Purifying a Fibrinolytic Enzyme from Sipunculus nudus via Affinity Chromatography

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and&#160;19,200 U/mL,&#160;respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.

Author Spotlight: Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus  

Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin ...

Combining the Chandler Loop and RT-FluFF for Ex Vivo Clot Lysis Monitoring

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates.
The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In Vitro Models

In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test ...

Bioengineering

Validating Xanthatin-Keap1 Interaction with Docking and CETSA

Protein Target Prediction and Validation of Small Molecule Compound

Proteins are fundamental to human physiology, with their targets being crucial in research and drug development. The identification and validation of crucial protein targets have become integral to drug development. Molecular docking is a computational tool widely utilized to investigate protein-ligand binding, especially in the context of drug and protein target interactions. For the experimental verification of the binding and to access the binding of the drug and its target directly, the cellular thermal shift assay (CETSA) method is used. This study aimed to integrate molecular docking with CETSA to predict and validate interactions between drugs and vital protein targets. Specifically, we predicted the interaction between xanthatin and Keap1 protein as well as its binding mode through molecular docking analysis, followed by verification of the interaction using the CETSA assay. Our results demonstrated that xanthatin could establish hydrogen bonds with specific amino acid residues of Keap1 protein and reduce the thermostability of Keap1 protein, indicating that xanthatin could directly interact with Keap1 protein.

Author Spotlight: Streamlining Protein Target Prediction and Validation via Molecular Docking and CETSA

The experiment used here shows a method of molecular docking combined with cellular thermal shift assay to predict and validate the interaction between small molecules and protein targets.

Proteins are fundamental to human physiology, with their targets being crucial in research and drug development. The identification and validation of ...

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
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Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
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2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
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Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
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Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
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You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
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Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...

Education

JoVE Science Education

Chemistry

Polyacrylamide Gel Electrophoresis: An Analytical Technique to Detect Heparan Sulfates of Varying Chain Lengths Isolated from Mouse Lung Tissue

Source: LaRiviere, W. B. et al., <a href="https://www.jove.com/v/62319">Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining</a>. J. Vis. Exp. (2021)
This video demonstrates the separation procedure of heparan sulfates isolated from mouse lung tissue using polyacrylamide gel electrophoresis.

Source: LaRiviere, W. B. et al., Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining. J. Vis. Exp. (2021)
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JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

UV-Visible Spectroscopy for Monitoring Fibrin Clot Formation

Source: Singh, P. K. et al., <a href="https://app.jove.com/v/58475">Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy.</a>&#160;J. Vis. Exp.&#160;(2018)
This video demonstrates the use of a UV-visible spectrophotometer for monitoring insoluble fibrin clot formation as a function of the change in the solution turbidity. The UV-visible spectrophotometer measures the solution&#8217;s absorbance over time, which represents the turbidity change due to the insoluble fibrin clot formation.

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis

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