the Coagulation Cascade

[deleted-154507]

While looking at the coagulation cascade I came across the extrinsic and intrinsic pathway. I know that they are used to activate factor X, but what are they, what is the difference between the extrinsic and intrinsic pathway?

thanks,
Lidia

[SciB]

Hi Lidia,

Blood clotting is a really complex process as you are finding out! I assume you have been reading the Wiki entry for blood coagulation which mentions the extrinsic and intrinsic pathway parts of clotting. I don't know why these steps have different names. What the wiki calls 'extrinsic' is the main blood clotting process where thrombin is formed and converts fibrinogen to fibrin--the fibrous protein that holds the adhering platelets together in a clot. The 'intrinsic' pathway appears to be a minor series of steps that produces some other active factors that are not that important.

For your purposes in investigating ways to break up an unwanted clot, I would concentrate on the thrombin---fibrinogen---fibrin pathway. This pathway has many factors, and hereditary defects in these proteins can cause serious diseases such as hemophilia.

Like most things in your body, the blood clotting system is both good and bad. We would bleed to death without it, but if a blood clot blocks a cardiac or cerebral artery, we can die from heart attack or stroke. Good health is maintained by having a fully active feedback control system that prevents abnormal functioning of a pathway. If the control system starts to break down then bad things can happen and the physician has to step in and try to restore control.

Now that you have a good understanding of the coagulation system, what have you decided to investigate in your project? You have probably read about plasmin which is the body's own clot-dissolver. Plasmin, or rather the inactive form plasminogen, is a blood protein that responds to blood clots and some specific factors to break up the fibrin protein that holds the clot together thus dissolving it. It is this treatment that is sometimes used by doctors to help a person suffering a stroke, but it has to be started as early as possible to prevent damage.

I hope I have cleared up some of your confusion about coagulation. Let us know what your project is going to involve and we will help you with the details. And, of course, when you have more questions, we are here to try and answer them.

All the best,

Sybee

[deleted-154507]

Hello sybee,

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.

After ordering the two proteins plasmin and fibrin, we plan to put/use one half of a milligram, which will be diluted, 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 using the Gel Doc™ EZ System 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, which will hopefully be where the plasmin chops up the fibrin.
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, run it, and take a picture of it. After the 30 minute interval, 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.

I will really appreciate any suggestions or comments on my experiment.

Thank You,
Lidia

[SciB]

Hi Lidia,

From your description of the experimental plan, I'd say you have everything under control. This will be a good lab learning experience, because running proteins on SDS-PAGE gels, staining and photographing them is one of the most frequently used molecular biology methods.

It would be really cool if you could do the experiment as you had planned using an actual blood clot. You could try it. I think if you had two identical blood clots and incubated one with phosphate-buffered saline (PBS) and the other with PBS containing plasmin for 30 minutes or an hour at 37 degrees C, you would be able to digest away some of the fibrin in the clot. You could test this by centrifuging the tubes containing the clots and running the supernatant fluids on your SDS-PAGE. I predict that after incubating a clot with plasmin you will see a band of degraded fibrin of about the same size as the band you get from just incubating plasmin with fibrin. Incubating the clot with PBS alone should not give a degraded fibrin band, or a faint one at the most.

Good luck with your experiments and do let us know how they turn out!

All the best,

Sybee

[deleted-154507]

While doing my experiment I came across a problem. In the experiment I need to order the proteins plasmin and fibrin. I found Plasmin, but only fibrinoge, which is fibrin's inactive form. In the website they are also selling thrombin, which is what makes fibrinogen change into fibrin. If I order fibrinogen and thrombin and put them together in a test tube would it work? would fibrinogen turn into fibrin simply by combining them or would I need to do somothing else? Is it even possible?

I would really appreciate your help,
Lidia

[deleted-140482]

Hi Lidia,

You can combine your fibrinogen and thrombin to create fibrin without a problem, but you'll need to make sure you include calcium in your reaction mixture (usually as CaCl2). Also, you'll want to do all of this in an appropriate buffer system (usually Hepes Buffered Saline or Tris Buffered Saline), and you'll have to be careful with your concentrations, since your goal is to convert as much of the fibrinogen into fibrin as possible. I suggest spending some time on Google or PubMed to find some specific protocols.

One other thing of note, make sure that wherever you get your fibrinogen and thrombin from, they come from the same species. They don't all work interchangeably, so if you have human thrombin and mouse fibrinogen you might not get very good cleavage.

Alternatively, you could do the same basic experiment with plasma like Sybee suggested. You can take diluted plasma, add thrombin and calcium at 37 degrees Celsius and get a good clot formed in only about 20 seconds or so.

JMP

[deleted-154507]

JMP,

Thank you so much, you are so helpful. We ordered our proteins from the website you had suggested to my partner which was "Hematech" and they are all from mouse. Unfortunatelly due to safety we cant work with actual blood plasma yet, but hopefully we will later on.
I appreciate your time and help.

Lidia