Haemostasis 1 - Clots, Thrombi & Antiplatelets
In this video I’m going to talk about haemostasis-in particular, I’m going to talk about the formation of clots, thrombi, and antiplatelet drugs.
Well, first of all, I want to make a couple of definitions.
The first one is a clot-now, a clot is good. A clot stops bleeding, for instance when you injure yourself and a clot stops bleeding. A thrombus, on the other hand, is bad-it’s also a clot but it happens in a blood vessel, so it actually blocks the vessels.
So, let’s look at how this happens.
I’ll draw a blood vessel here.
And in this blood vessel of course we have some red blood cells. Now, this is a bit of damage to the endothelium. The endothelial cells are the cells who make up the blood vessel walls. So, if we get some damage to those, for instance a break within the vessel wall, then you get a bit of collagen exposed here. Collagen is a protein which is integral to the structure of vessel walls. Now, when you have some collagen exposed like this he gets some platelets stick to it, and these little platelets are just floating around in the blood may start to stick too like this.
Now, when that happens, the platelets get activated and they release a chemical into the blood stream which calls on more platelets. This chemical is called Thromboxane A2, You might have heard this before in the arachidonic acid pathway. If you haven’t, have a look at the video on arachidonic acid, and of course it is on hand written tutorials.
Now this calls more platelets onto the scene. Then, on top of the platelets, we get a bunch of proteins called fibrin. And they are basically big fiber strands that help make the blood clot and they stick the platelets together. Then, this fibrin will trap a couple of red blood cells in it, and then, on top of that, we will get even more fibrin deposited. And then this process kind of abates. And this is what causes a clot-or a thrombus. Now this whole process can be avoided if we stop the Thromboxane production by platelets, and this is done with a COX inhibitor, or a cyclooxygenase inhibitor. Again, have a look at the arachidonic acid and Elicosanoids tutorial at handwrittentutorials.com for more information. An example of COX inhibitor is aspirin. And that blocks Thromboxane A2 production, and this means that the platelets won’t stick together and the platelet won’t be formed. In this way, you can actually stop thrombin forming-but the side effect is that you can also get excessive bleeding. And that’s an overview of clots, thrombi and anti platelets. We’ll have a look at how the production of fibrin is controlled in the next tutorial..
Haemostasis 2 - Coagulation Cascade
In this tutorial we are going to discuss the coagulation cascade.
This is a sequence of interactions between proteins to cause fibrin deposition at the location of tissue injury.
There are two pathways-the intrinsic and extrinsic pathway.
We will look at the intrinsic pathway first.
The intrinsic pathway is less important for initiating coagulation than the extrinsic pathway. However, the intrinsic pathway IS very important for the amplification of the cascade.
So, let’s look at what’s involved.
These pathways consist of a number of proteins called factors, activating one another. These are usually denoted by using Roman numerals. And the first factor of the intrinsic pathway is factor XII.
The intrinsic pathway is also known as the contact pathway. This is because factor XII can be converted to its active form –factor XIIa- when it comes in contact with negatively charged surfaces.
Most commonly, these surfaces clasp within a pathology lab
So, as you can see, the active versions of these factors are denoted by the letter “a”- in this case, the active form of factor XII is factor XIIa.
Now, factor XIIa causes the conversion of factor XI to factor XIa. Factor XIa then in turn converts factor IX into factor IXa. Factor IXa then converts factor X into factor Xa. But it does links with the help of factor VIIIa, which is being created from factor VIII.
Factor Xa can then convert prothrombin to thrombin-and it does this with the help of factor Va, which of course has been created from factor V. Thrombin then converts fibrinogen to fibrin.
Fibrin forms a mesh at the injury site to help produce the blood clot.
Now, let’s look at the extrinsic pathway. This pathway is much simpler, and it is also a pathway which is most important for initializing the coagulation cascade
It begins when a protein called tissue factor is released from damaged tissue. Factor VII is converted to Factor VIIa and then tissue factor and factor VIIa combine to convert factor X to Xa
The pathway then continues on like the intrinsic pathway until fibrin is produced
One last thing we need to consider, is the positive feedback effect that thrombin has on the cascade. Thrombin has a role in accelerating the production of factor XIa, Factor VIIIa, and Factor Va. In this way, the cascade is amplified to produce the necessary fibrin in a shorter amount of time.
And that’s an overview of the coagulation cascade.
In the next haemostasis handwritten tutorial, we will look at the drugs who can modify formation of blood clots and thrombin.
Haemostasis 3 - Anticoagulants & Thrombolytics
This tutorial is the third in the haemostasis series. And in this video we will be looking at anticoagulant and thrombolytic drugs.
So, in our last tutorial we talked about the coagulation cascade, and how factor X is converted into Factor Xa. Factor Xa catalyzes the conversion of prothrombin into thrombin, which in turn catalyzes the conversion of fibrinogen into fibrin. We are also aware of the fact that factor X can be converted into factor Xa by the intrinsic pathway or the extrinsic pathway.
Now, let’s have a look at how some drugs act on this pathway. But, first of all, we are writing another couple of elements. The first one is an endogenous, meaning “present in the body”, compound known as antithrombin III, and it is an inhibitor of Xa. Fibrin can also be broken down in the body into fibrin degradation products. And this is catalyzed by an enzyme called plasmin, which is created from a protein called plasminogen.
So, we have a couple of very commonly used drugs, heparin, or its cousin Low Molecular Weight Heparin, or LMWH for short. And these two drugs are activators of antithrombin III, thereby inhibiting factor Xa, and stopping the coagulation cascade. If a thrombus is formed, then we can use the drugs tissue plasminogen activator or TPA, or streptokinase, which are drugs which activate the conversion of plasminogen into plasmin. Now, antithrombin III actually inhibits thrombin production as well, so its effects are twofold.
Now, let’s have a look at another mechanism.
In the body, glutamic acid plus oxygen and CO2 can be converted into a weird amino acid called gamma-carboxyglutamic acid. And this strange amino acid is a component, in factors II, VII, IX, and X.
Now, this conversion process requires reduced vitamin K, which then becomes oxidized vitamin K, so it helps reduce the glutamic acid reaction. Oxidized Vitamin K then gets converted back into Vitamin K and then again into reduced Vitamin K. And this occurs by the action of an enzyme called Vitamin K reductors, which act on both steps.
Now, a very commonly used drug called warfarin is a vitamin K reductor’s inhibitor. Therefore, none reduced Vitamin K is produced and no gamma-carboxyglutamic acid is produced. Therefore, factors II, VII, IX and X can’t be produced, and the coagulation cascade doesn’t work.
And that’s an overview of haemostasis, anticoagulants, and thrombolytics.
The purpose of this short video is to introduce the viewer to the three-dimensional structural aspects of the heparin molecule.
Heparin is probably most familiar from its uses in the clinic as an anticoagulant. These molecules, and related sulfated materials, are important for a large number of normal and pathological structures. In order to understand the function of heparin-like materials, it is important to understand the three-dimensional aspects of its structure.
Shown here is a heparin dodesaccharide, a twelve-unit heparin fragment. Heparin is a heterogeneous, sulfated polysaccharide, belonging to the larger family of Glycosaminoglycans, or GAGs for short, and represents an important class of macro molecules. Currently, heparin has been shown to interact with over fifty different proteins from many functional categories. Structurally, heparin is a linear, psuedo-helical, alternating copolymer, with a period of approximately four residues that consists of repeating units of pyranosyluronic acid, and 2-amino-2-deoxyglucopyranose residues, which are the derivatives of six carbon sugars, and are all found in their cyclic, hemiacetal form.
The pyranosyluronic residues, now shown in red, can either be glucuronic, or iduronic acid.
Furthermore, these residues can have multiple sulfation patterns, as will be described later in more detail.
In this case, all the sugars in red are identical iduronic acid residues.
The remaining residues, now shown in blue, are all glucosamine. The individual glucosamine residues are capable of multiple sulfation and acetylation patterns. In this case, however, all the glucosamine residues are identical. Here you can see how the individual residues are all connected via the so-called 1-4 glycosidic linkages, as highlighted in yellow. These linkages give heparin chains the flexibility that may be important during protein binding.
One of the properties that make heparin so unusual is that it has the highest negative charge density of any known biological macromolecule. This is due to the high density of sulfate groups (shown in yellow) and carboxylate groups (shown in red). Each color cluster contains a -1 charge, while the entire heparin chain has an overall -24 charge, and it is only forty Angstroms long.
Because of this high charge density, it was initially believed that heparin interactions were purely ionic in nature. While ionic interactions certainly plays a role in its interactions, there is increasing evidence that indicates many of the heparin protein interactions have specific structural requirements such as observed in its binding to anti-thrombin 3.
Although heparin is often found in the extended helical shape (shown here) heterogeneity on the residue level creates many structural variations. In order to understand its variations, we need to now take a closer look at individual residues.
The first residue we will examine is one of the glucosamine residues, and it's readily identified by the nitrogen attached to the #2 carbon. Notable in this particular residue is the two N-sulfation and the six O-sulfation. However, this residue type has many possible structural variations. To better understand these variations, we will now take a closer look at this residue.
As shown here, the residue is 2-N sulfated. However, also very common is the substitution shown next with an acetyl group for a sulfate on the nitrogen.
This substitution has the interesting effect of causing a change in the preferred conformation of the neighboring, iduronic acid residue. As we will see later, in the change of the iduronic acid conformation, can greatly affect the way it engages some protein interactions.
Returning now to the original N-sulphated form, we will take a closer look at the O-sulfation possibilities for this residue. This particular residue has O-sulfation on the #6 carbon. Also possible is the 3-O sulfation alone. A rare combination is the one shown now- it has the N-sulfation shown previously, along with 3- and 6-O sulfation. While this combination is rare, it is one of the key requirements for binding with antitrhombin-3, and the basis for the anticoagulation drug Arixtra. Finally, this residue can have no 3- or 6-O sulfation at all. Keep in mind that at physiological pH, each one of the O-sulfate groups carries a -1 charge, while the alternative, N-substitute alcohol group is neutral.
From these examples it is very easy to see how the different substitution patterns strongly affect both the accessible surface and the charge of the individual residues. Also of interest in this residue, is the way in which the two sulphate groups are connected to the sugar ring, as can be seen here with the # 2 carbon is actually located in the sugar ring itself, while the #6 carbon is tethered outside the ring, giving the sulphate group additional flexibility.
Now, you will see that the rest of the atoms have been removed, leaving only the sugar ring (in blue). In this view, is easier to see the resemblance of a home recliner, that it is typical of the chair conformation.
As stated earlier, the glucosamine residues, while having different substitution patterns are found almost exclusively in the chair conformation just seen. This is in contrast to the next residue we will examine-the iduronic acid residue.
Depending on the substitution pattern and protein interactions, this residue can be found in either of two conformations. We will explore both conformations, but the one shown here is positioned in the chair conformation. The key features of this residue are the 2-O sulfation, which is located in the so-called axial position or “down” in this case. And the carboxyl group, located on the #6 carbon.
This residue can also be found (as shown) now lacking the 2-O sulfation groups. With the other atoms hidden, again is it easy to see the chair conformation.
In general, it is true that the chair conformation just shown is the most stable form for a 6-Carbon sugar. However, due to the bulky nature of the 2-O sulphate groups, this residue is also stable in a much different conformation.
As we approach the next residue, keep in mind that it has exactly the same chemical composition-only the conformation, or shape, is different. This iduronic acid residue is found in what it is called a “twist” or “Skew-Boat” conformation. This is different from the traditional boat conformation in that the #1 carbon, as illustrated in green, is found above the plane of the ring, as opposed to below it. Now, shown for comparison, is the actual boat conformation. Of key importance is the location of the 2-O sulfate groups. In the previous case, these residues were located axially, while in this case it is in the more energetically favorable position-in the equatorial position, straight out to the right.
Depending on the conformation, this sulfate can be in dramatically different positions. Realizing that approximately half of the residues and heparin fragments are iduronic acid, this flexibility allows for a great variety of possible three-dimensional structures.
This concludes the short introduction to the three-dimensional aspects of heparin structure. Hopefully, the diversity of structures found in heparin and related molecules can be better understood in the future. This may allow us to understand its many roles in normal and pathological biological processes, as well as help us develop new therapeutic agents.