Science Buddies: "Ask an Expert"

I am trying to design an experiment that has to do with blockage or coagulation in blood vessels and arteries, but so far I have no idea what to ask for my experiment. So far I have research the types of hemophilia type A & B, as well as Atherosclerosis. They have helped, but not as much as I wished. I would really appreciate it, if you could give me some ideas and help me with my research. Thank you, I appreciate it very much.

The two disorders you looked up, while dealing with clotting, are almost polar opposites. Hemophilia as you discovered is the lack or inhibition of a clotting factor, while atherosclerosis is primarily a narrowing of the coronary arteries by lesions that then rupture and encourage clots. As a data gathering expedition both are good research, it is important to fully understand all the conditions related to your topic. But as far as a project goes there are two very different routes you can take.

If you choose to pursue the hemophilia angle you can examine what these clotting factors do, and then look at ways different therapies work. Factor VIII replacement for instance. Social and scientific issues related to this may be the emergence of HIV in the 1980's, and the way in which it affected the hemophiliac population much more than the healthy population. Blood supply safety would be another interesting way to look at this.

On the other hand Heart Disease is one of the biggest killers in the United States. Investigating this has massive implications in science and public health. One interesting point may be to collect data from drug manufactures and determine if the medications that are used to prevent or treat heart disease work as if they intended. For instance statin drugs which are prescribed to lower cholesterol has been shown to not really lower blood cholesterol levels all that much. But at the same time there has been a decrease in heart attacks in the patient population being treated with statins. So if it doesn't lower cholesterol, which is what was thought to be the major heart disease risk factor, what does it do that lowers the risk of heart disease?

I was researching more about coagulation. I found out what fibrin and fibrinogen, which is factor I, in the process of coagulation. I want to find out how we can change fibrin, in order to stop the clot, and let the blood flow through. Is there a way I can do that, without taking the whole fibrin out and replacing it.

This is a very interesting and sophisticated idea, and one I happen to know a lot about, but I don't think you'll be able to test it with the resources available to you in high school or lab or with the time you have. Just for your information, in my old lab we were very interested in. During you research you probably learned that thrombin (also called factor II) cleaves fibrinogen (soluble) to turn it into fibrin (insoluble, and one of the primary factors in a clot). In my lab we engineered a form of fibrinogen that could not be cleaved by thrombin and we made mice that express this mutant form of fibrinogen. As expected, the blood from these mice did not clot normally and acted similarly as blood from mice without any fibrinogen. The idea of modifying fibrinogen to let blood flow freely, presumably as a treatment for blood clots and heart disease, is one that has been considered in the medical field. The problem of course is balancing breaking up a clot with leading to too much bleeding. People with harmful clots are often administered "clot busting" drugs to do just this, but the doses and location of administration have to be carefully monitored so as not to lead to too much bleeding. Again, I'm not sure how you'd be able to study this in your high school lab, but I definitely think it's great that you're thinking along these lines. This is exactly how scientists think.

You seem to be really interested in blood coagulation (which I think is great, since so am I), so I recommend you look through the project ideas here to see if one interests you, at least as a starting point. One science fair project idea on this website explores how anticoagulants work. (https://www.sciencebuddies.org/science- ... ml#summary). Perhaps you could use this as a starting point and modify it to fit your interests.

Please post again with more ideas and any further questions.

Could you please explain to me in more broad and specific terms what coagulation blood is? I would really appreciate it.

Hi Lupee,

In the simplest terms, coagulation is the blood clotting. This is an absolutely necessary process that occurs after you get a cut, for example. Without coagulation (clotting) you would just continue to bleed out of even the most minor cuts and you wouldn't be able to recover from even minor injuries. As with many things in the body though, blood coagulation needs to be tightly regulated, because while you would die from blood loss without a clot, clots in the wrong place also pose a danger. If you get a blood clot in your vessels that does not go away, it can lead to heart attack, stroke, and other life-threatening problems. The way our bodies control coagulation to prevent both blood loss and blockages in the arteries and vessels is through a cascade. Injuries to your blood vessels (for example a cut) start the cascade that eventually leads to the formation of a clot. As you read previously, this involves the conversion of fibrinogen into fibrin, as well as several other factors. Almost immediately after initiation of the clotting cascade occurs, another cascade is also activated which will dissolve the clot (plasminogen is converted into plasmin, which basically chops up fibrin to dissolve the clot).

I hope this helps give you a broader understanding of coagulation. If you have any more specific questions about coagulation, feel free to post them.
JMP

Is it possible to extract enzymes alive, such as plasmin which is used to break down fibrin?

It is possible to purify plasmin, although it is generally not easy. Luckily, other people do the purification for us and we can buy it from them.

For example:
http://www.haemtech.com/Enzymes/Plasmin.htm

What's the difference between the coagulation cascade and the plasmin cascade? I knew about the coagulation cascade, but never heard about the plasmin cascade. I would really be interested in knowing more about it. Thank you.

Dear JMP,

I appreciate the help you have provided my partner and I. Through the previous responses we have acquired a lot of knowledge, as well as some information regarding your research about blood clots. We would like to learn more about your results concerning plasmin and fibrin. Would you be willing to help us with our experiment and all the questions and difficulties concerning our experiment. Your help would really be appreciated.

Sincerely,
Guadalupe López
Lidia Monterroso

Hi Lupee,

I'm certainly happy to help give advice on whatever experiments you decide to perform. Do you have a specific science fair experiment in mind? I'm not sure what information you want on plasmin and fibrin. You ask about the coagulation cascade v. the plasmin cascade. I would generally consider what you are calling the plasmin cascade as the fibrinolytic cascade (i.e. lysis of fibrin) and together, coagulation and fibrinolysis make up the hemostatic cascade. The Wikipedia page on fibrinolysis seems to be fairly informative, so you might try looking there to get some more general information. http://en.wikipedia.org/wiki/Fibrinolysis

A google image search for hemostatic cascade or fibrinolysis/fibrinolytic cascade will also give you some diagrams of the overall cascade that may be useful to you.

I hope this information is helpful to you, and again, if you can give us a better idea of what your planning experiment is, we can better help with more detailed answers and concrete ideas.

Thanks,
JMP

Dear JMP,

After ordering the two proteins plasmin and fibrin, we plan to put/use one half of a milligram of both proteins and run them through the gel, perform SDS PAGE on them, molecular weight marker will be used. This will be our control. The gel will be ran for as long as needed. May or may not have any dye to measure how long it has traveled. After we turn off the apparatus we will take a picture of the gel in order to keep it on file.
For the most challenging part of our experiment we plan on using one half of a milligram of fibrin and half a milligram of plasmin. This time it will be mixed up and we will have them both together on the same test tube. We will let them rest for a certain time.
There will be intervals, a 7 minute interval, a 30 minute interval, and a hour interval on how long plasmin and fibrin will be together in the test tube. In order to save money on the gels we will only use one. We will start with the 7 minute interval, after the 30 minutes, we will take a small amount from the same test tube and inject that amount into the previous gel. (The previous substances and positions of the other, 7 minute interval, will be altered, but since they are recorded it will not change the data.) After using the Gel Doc™ EZ System, and 1 hour interval has passed, the same will be done. The results will be recorded on a chart, and probably graphed. The results will definitely be analyzed.

After designing our experiment we came across a barrier. We need a qualified scientist because the possibilities are that we will be working with potentially hazardous biological agents and DEA-controlled substances, and we need one because they are necessary for the Intel-affiliated Contra Costa County Science & Engineering Fair (CCCSEF) applications. Would you be willing to be our mentor for this experiment?

We would really appreciate the help and time you would take to aid us.

Thank You,
Guadalupe López
Lidia Monterroso

I'm sorry, but we are not allowed to interact with students outside of the boards, so I cannot be your mentor on this project. You should look for a mentor near you. Check out our project guide on "How to Find a Mentor" posted here: https://www.sciencebuddies.org/science- ... tors.shtml

Good luck on your project, and if you have any more questions I can answer, I would be happy to help in that way.
JMP

Hi JMP,

Thank you for letting us know and if we have questions we'll gladly ask you. What do you think about our experiment?

Thank You,
Guadalupe López
Lidia Monterroso

I have a few technical questions about your experiment, but my biggest question is what is your hypothesis? The way you have stated things right now, you have told me what you are going to do, but not why. What are you testing (what is the question you are trying to solve) and what do you expect (your hypothesis)? Remember, when doing a science project you should be able to clearly identify a hypothesis, your variable, controls, etc. Once you have provided these things, it will be much easier to determine whether your experiment will test your hypothesis.

That said, it sounds like you are very interested in blood clotting and fibrinolysis, and I think it's great that you are putting so much time, effort, and thought into this. Keep up the great work!

Dear JMP,

My hypothesis is that by using plasmin, the normal protein that breaks down fibrin, which is what holds the clot together, I will be able to chop fibrin down, which would no longer hold the clot together, therefore dispersing it and letting the blood flow without any complications.

Sincerely,
Guadalupe Lopez

Sounds like you and your partner have a pretty good grasp on what you are doing. Technically I see one problem with your experiment. You propose to add protein onto a gel that you are already running, but this won't really work well. I think a better way to do this (and still conserve materials by only having one reaction going) will be to take an aliquot out of your reaction at each time point and stop the reaction by adding SDS Sample Buffer (this is the buffer you will have to put your sample into anyway in order to run it on the gel). This should stop the reaction. Then, once all of your time points are done, you can load all of them onto the gel at the same time and run it. In this way, you will got a complete picture that can readily compare each time point. Your mentor should also be able to help you with the technical details of this.

Again, it sounds like you and your partner generally have a good feel for what you are doing. Good luck, and let us know how it goes!
JMP

Dear JMP,

We came to the conclusion that we are using separate gels for each interval. And we are going to use mouse proteins to reduce any risks of viruses. I have a question concerning SDS PAGE, how many gels are used at a time because I hear about a stacking gel and a running gel and through all the research I've done I cannot see the difference between the two.

Thank you,
Guadalupe
Lidia

I can tell you've been doing your research, which is great. Generally speaking, only one gel is used at a time, but this gel is made up of two gels: the stacking gel and the running gel. When you use a polyacrylamide gel (polyacrylamide being what the PA are in PAGE), you first pick a percentage of polyacrylamide. This will be the percentage for your running gel. The stacking gel is generally a lower percentage that sits on top of your running gel, and what it essentially does is makes sure all of your samples line up perfectly. If you are pouring your own polyacrylamide gels, you would generally pour your running gel, let that set, and then pour the stacking gel on top of it, and let that set. I can give you a detailed protocol on how to do this, but if you are working with a mentor, then they should be able to help with this. On the other hand, if you are buying your gels then you don't need to worry about it, because the gels you buy will already have incorporated a stacking gel for you. You can just load your proteins and run the gel.

Hope that helps!

Dear JMP,

Thank you very much. This perfectly answered our question. We really appreciate it. We were looking into where to buy polyacrylamide gels, and we found a few websites but there might be better ones out there. Do you think you can recommend any reputable website where we can order polyacrylamide gels? We would really appreciate it.

Thank you,
Guadalupe
Lidia

Most labs probably buy their precast gels from either BioRad or Life Technologies, but I checked and Carolina Biological also sells a precast polyacrylamide gel (http://www.carolina.com/biotechnology-p ... lamide+gel). That might be easier, and you can buy them individually so you don't have to waste money by buying a whole box.

Dear JMP,

Thank you very mcuh for the link, it is very helpful. We also are struggling to know which buffer to use. We were researching about the buffers and we wanted to know what the difference is between sample and running buffer. We would really appreciate it.

Thank you,
Guadalupe
Lidia

Sample buffer is the buffer you will prepare your samples in before running them on a gel. This usually includes SDS, glycerol (it makes your sample heavy relative to the buffer), dye (usually bromophenol blue) so that you can see your samples and know how far they have traveled down the gel), and a buffer (often tris). If you are running a reducing gel, then it will also include beta-mercaptoethanol. You will dilute your sample in this buffer, if you are doing a reducing gel you would then boil your samples, and then load them into the wells on your gel.

The running buffer is the buffer that you will pour all around your gels while they are running. It allows the current to run, which is what makes your samples run down the gel. It usually consists of a buffer (again, mostly Tris), SDS, and glycine.

You should readily find recipes online by searching for SDS sample buffer and SDS running buffer. Most recipes are fairly similar and occasional small changes won't make much difference.

JMP

Dear JMP,

Thank you very much for explaining to us the difference between running and sample buffer. We have a question dealing with calcium. When converting fibrinogen into fibrin we need calcium. But our question is if there is a specific type of calcium we need? I would really appreciate if you could explain this to us in detail.

Thank you,
Guadalupe
Lidia

Dear JMP,

My partner and I were wondering if we need mops or mes buffer? I was researching, and I found out that one of the different things is that you get different pH values.
I would really appreciate it if you would provide us with this information.

Thank you,
Guadalupe
Lidia

It's not clear to me from your question whether you are asking about buffers in which you intend to cleave fibrin or buffers in which you intend to run your gels. As you mentioned, different buffers buffer different ranges of pH. For cleavage of fibrin, you will probably want your buffer to be as close to physiological pH (7.4) as possible. I would recommend Hepes Buffered Saline (HBS), but Phosphate Buffered Saline or Tris Buffered Saline would also likely work. You just want something close to a pH of 7.4.

If you are asking about what buffer to run your gels in, depending on your precise gel system, different buffers will separate different molecular weights on a gel better, but it really depends on the details of your gel. If you are buying precast gels, then the manufacturer will usually have all of the information you need to determine what sort of running buffer is best on their website.

JMP

Dear JMP,

We attempted to create a blood clot, but we did not succeed. It took 2 days for us to complete the procedure. It was a double dilution for thrombin. In our procedure we added 2 µl of thrombin and 2 mL of HEPES buffer (which was a 1:100 dilution) in a 50 mL tube. After that we added 2 µl of the mixture into another 50 mL tube(which was a 1:10,000 dilution). After that we added 10 µl of that mixture in to the final clot tube which was composed of fibrinogen, NaCl and CaCl2. We want to know your input based on what we did and what mistakes we could have done.

Thank You,
Guadalupe
Lidia

It's hard for me to know for sure, but it sounds to me like your mixture is MUCH too dilute. I've never personally combined fibrinogen and thrombin to make a clot, but when adding thrombin to blood plasma, we use a total reaction volume of less than 100uL. For example, I might do a 1:10 final concentration of thrombin into plasma (2uL thrombin in 200mM CaCl2 in 10mM Tris) into 20uL plasma.

Did you get your concentrations of thrombin and fibrinogen from a paper? If not, I would look for concentrations that others have used and work from there.

JMP

Dear JMP,
It's been a while since we have talked. We are so happy to inform you that our project was successful. It went great at the science fair and due to our enthusiasm and confidence we were able to get the attention from representative from Chevron. We were the only ones who received a $500 scholarship from Chevron. And a lot of our accomplishments, we owe them to you. You helped us a lot, from learning what a blood clot is and where to find the materials to create one,to helping us with the buffers for electrophoresis. Since we are not allowed to know any of your personal information we unfortunately we cannot send you a thank you letter. That is why we are thanking you through Science Buddies.
My partner and I were pondering about how hard it would have been for us to find a good website to find the right proteins if you wouldn't have been there to help us. In our first attempt at making our blood clot we failed, but luckily we were able to make it in the second attempt. By the end of the experiment we were able to create 5 successful clots!
We found that the amount added to a clot really matters and our hypothesis about using only plasmin, no need of plasminogen activators or other proteins, was correct. In the end we came to the conclusion that adding 10 microliters only took 1 hour and 30 minutes to dissolve a good sized clot.
Again, thanks for everything, and we appreciate the time you took to answer our questions.

-Lidia Monterroso and Guadalupe López
Coagulated Blood

I'm so happy to hear that your science fair project went well, that you received recognition at your science fair, and most importantly that you enjoyed yourselves and learned. I'm glad I could be of help to you, but remember that you did all of the work yourselves, so congratulations on that. I truly appreciate the thank you though, and I hope that this experience has inspired you to continue learning and experimenting and maybe makes you want to consider doing future science fair projects or even a career in science.

Congratulations on your successful science fair project, and again, I'm happy to have been of help.

JMP