regulation of Pathways

Steps in Amplification

How does a Blood Clot stay in Place?

It is VERY important to recall that each of the steps during the amplification stage of the cascade, is catalytic. in each picture one "molecule" is shown converting one other molecule. But since each is catalytic it can actually convert MANY of the those molecules. End result.... Each enzyme can concert 1000's of the next enzyme in the cascade. By the time we get to the "end", many thousands of Thrombins can convert 1000s of thousands of fibrinogen to fibrin. The cascade generate an amplification of the signal so that only a few of the initiation contacts are required to generate a protein fibrin protein nework large enough to ensure slot formation and stopping of further blood loss.

Now that we have an initiation (thromboplastin from cells activating factor VII) and an amplification process, we have another critical problem to fix. The site of injury is fixed: the coagulations factors are water soluble AND moving as the blood plasma flows. It doesn't do much good to activate the system and then have the blood clot show up somewhere downstream. That would actually generate more problems than it solves. This particular problem can be solved by making the water soluble proteins complex with the platelets which are clumping and getting anchored to the site of injury by the collagen.

Once factor IX start to convert to factor IXa (the active form) It joins a complex of proteins and phospholipids that contains:

• Factor IXa
• Factor VIIIa
• Ca²⁺
• phospholipids

The "water soluble" factors are drawn as ovals;
The membrane bound non-enzyme helper factors are the cylinders associated with the platelet membrane. The gray spheres represent the Ca²⁺ ions. (Rememeber from module 3 that Ca²⁺ associates with these proteins because of the vitamin K dependent γ carboxylation of glutamate - this form the calcium binding site.) This entire complex is associated with cell membranes as well as the intercellulr collagen. The hydrophobic effect ensures that is stays in place.

The amide hydrolysis of factor X occurs most efficiently in this complex. In blood clotting the phospholipids are an actual membranes... mostly belonging to platelets. This enzyme, helper protein, phospholipids complex is instrumental in three activities:

1. cleavage of an amide in factor X to convert it to the active factor Xa.
2. cleavage of an amide in factor VII to convert it to factor VIIa.
3. cleavage of a glycoprotein (protein with polysaccharide covalently attached) on the surface of platelet cells.

What's the the function of each of these activities?

1. activation of factor X to Xa is fairly obvious.. It is the next enzyme in the pathway. It must be activated to begin the activation of thrombin II to thrombin IIa.
2. It can also act on Factor VII. This is an accelerant for the cascade. Converting factor VII to factor VIIa makes it far more active in converting factor IX to factor IXa which in turn converts more of each factor X and IX. Recall from the previous page that forming the complex with Thromboplastin made factor VII slightly active in converting Factor IX to IXa. It will be FAR more active when its own amide bond is cleaved.
3. Causes platelets to clump together. Not only do they clump together they also tend to stick to collagen. A polysaccharide (sugar polymer) that usually part of connective tissues between cells. This polymer would not normally be present in blood but is observed only at the site of tissue damage. Since it is a polymer it serves to anchor the platelet clump to the site of injury.

It has been well established that factor IXa is the "active catalyst" here. What are all the other "cofactors" (phospholipids, Ca²⁺ and factor VIIIa) for?

So the platelet aggregate provides an anchor point. There's still two problems to solve then...

1. How to get water soluble proteins to associate with a membrane
2. How to make sure that those proteins that remain water soluble don't continue to flow downstream and create a clot in the wrong place.

Factor VIIIa and the Ca²⁺ provide the solution to to the anchoring. Factor VIIIa is a membrane bound protein that in and of itself does not have any catalytic activity, but does have a fairly high affinity for factor IXa. So using the same physical forces that maintain any quaternary structure Factor IXa associates with factor VIIIa.
The Ca²⁺ coordinates to the γ-carboxy glutamate side chains at we showed in module3. The Ca²⁺ can serve as a bridge between the proteins γ-carboxy-glutamates and the negative charges on the surface of the phospholipid layer of the membrane. So these too help to anchor Factor IXa to the membrane surface.

Proteins are inanimate objects... They do not actively seek out their binding sites, but associate with them once they have "blindly" bumped into them by random diffusion. Some of the "activated factors" may not associate with the membrane but continue downstream happily water soluble. This causes the obvious problem... If they are fully active they will start keep the clotting process going IN THE WRONG PLACE!

The factors IX and X though are not very active unless they are anchored to their appropriate factors which are anchored to the membrane. So indeed the phospholipids are "strictly" necessary to ensure the clotting process remain local!.

Quick Summary

1. intitiation depends on mutual binding of two proteins that are normally held in separate places
2. An amplification system is resposnible for a small amount of initiation complex resulting in the association of a large number fibrin molecules.
3. The entire amplification system is maintained in the appropriate place by membrane association to plateltes, which in turn are associated with intercellular collagen (exposed only where the injury took place).
4. Enzymes of the amplification system are only most active when anchored to the membrane.
5. The whole process is driven by the five physical forces that were discsused as those being responsible for protein folding in the first place.