Lesson 8

Question 1

Speak about the anticoagulant heparin

Answer 1

There are other compounds which act as anticoagulants, like heparin. There are heparin-like molecules expressed on the surfaces of endothelial cells which are able to bind and activate other molecules like antithrombin III and favour its action. Antithrombin III is cable of limiting the hemostasis since it is able to inhibit thrombin, and also interact with other factors belonging to the coagulation cascade: factor IX, X and XI, among which the most important is factor X since it is the link between the extrinsic and intrinsic pathway. Antithrombin III is naturally present in our body but with heparin we highly increase its activity. this activity is being able to capture the proteases which is fundamental for the coagulation cascade and prevent their action. Antithrombin III can be considered as a “suicide trap” for some proteases, covalently binding them, thus preventing the protease from further participation in the coagulation cascade. Heparin, acting as a cofactor for this reaction, accelerates the reaction by 1,000-fold. So, heparin has two important physiologic functions:

1. it serves as a catalytic surface to which both antithrombin III and the proteases bind
2. it induces a conformational change in antithrombin III that makes the reactive site of this molecule more accessible to the attacking protease, binding them and blocking their action

Heparin is a sulphated mucopolysaccharide stored in the secretory granules of mast cells. It is highly negatively charged, meaning that it cannot pass through the cell membrane. On the market there are many commercial preparations of heparin. These preparations are usually extracted by an animal source and used as they are after being differentiated through electrophoresis to divide them based on the molecular weight. They are different structurally and have different functions too. The most famous are:

• unfractionated heparin which has a weight raging from 5 to 30 kDa. It has 45 to 65 saccharide units, and since these are large units they can bind antithrombin III and thrombin at the same time, creating a very stable complex. In this context thrombin is inhibited. Moreover this molecule is able to bind to thrombin and factor Xa at the same time, inhibiting them. In this case factor Xa also creates a complex with antithrombin making the complex stable. it can also bind factor IX and XI. Clinically this molecule is used for people who have thromboembolic diseases. Due to the negative charges they cannot be administered orally so they need to be injected. The unfractionated forms are used in the hospital and administered intravenously but for prophylaxes they are administered subcutaneously, and in this case the patient himself applies it. For what concerns the side effects, for this type of molecule drugs are more prone to side effects, the most important one being bleeding, this is why when a patient is using this drug blood tests are done, to prevent the possibility of reaching a specific concentration and cause bleeding.
• low-molecular-weight (LMW) heparin whose weight goes from 1 to 5 kDa. It has the same initial portion of the other preparation, and that is the portion which binds to antithrombin III, this portion is called pentasaccharide and is fundamental for the interaction with ATIII. this pentasaccharide is enough to bind at the same time to factor 10a and ATIII forming a complex. however since LMW are smaller, they lack the terminal portion of saccharide which is fundamental to form the complex between ATIII and thrombin, so they cannot form this complex. At the end of the day LMW has a more selective activity towards factor Xa. Also they have a 3 fold higher ability to block factor Xa compared to thrombin. At the end of the Xa pathway thrombin is inhibited anyway but this compound does not block it much directly. So the binding of ATII to the LMW makes the binding with factor Xa easier, infact the complex ATIII and LMW is called “suicide trap” for this protease. Lets see the application of this molecule at a clinical level. Usually LMW heparins are the ones of choice for the prophylaxes, so for example if we have a broken leg and it had to be immobilized for some time to prevent the formation of thrombi, heparin LMW is used. It is not necessary to run blood tests for patients taking LMW heparin because there is half of the effect compared to the other molecule, and thus a lot less side effects.

Question 2

What are the newer generation anticoagulant drugs which have been deisgned starting from heparin?

Answer 2

Since we are talking about a cascade every step can be amplified so factor X is able to produce a lot of molecule of thrombin so if we block it there is a strong decrease in concentration of thrombin. There has been a development of other drugs that can work on factor X to prevent the amplification cascade effect.

there 2 type of new drugs:

1. fondaparinux: A farther development of LMW heparin formed by pentasaccharide units alone. This is useful because these units are enough to induce the conformational modification in the ATIII molecule to complex with factor Xa and block it. This is also a way to block heparin in an indirect way. Fondaparinux binds to antithrombin III inducing the conformational change required for conjugation to factor Xa. since this drug has charge it is administred through subcutaneous injection.
2. We also have drugs like Rivaroxaban and apixaban that bind directly to factor Xa inactivating it directly without the intervention of ATIII. They have been produced in order to interact with the molecule to its active site and block the enzyme. This time they are not charged and are administred orally. They are able to do this because they bind to Xa not when it is free in the plasma but when it is complexed with factor Va and bound to the membrane, in this situation factor Xa is blocked so it stuck to the membrane and can be attacked.

Question 3

Which are other anticoagulants which work directly on thrombin?

Answer 3

There are also anticoagulants that work directly on thrombin by blocking its activity in a specific way, lets remember that thrombin is a molecule that has a role both in the coagulation cascade and platelet aggregation. Thrombin is infact able to produce fibrin and also to activate factor XIII which in turn is able to form the cross link fibrin polymer and stabilize the clot. Also, thrombin has receptors on platelets and can induce their aggregation. So by blocking thrombin we can block part of the cascade. Thrombin has many functions, for examples amplifying the coagulation cascade by activating factor V.

Thrombin has an heparin binding size, an active site which is **the catalytic site and the exosite, an important site for substrate recognition. The interaction with these sites allows for the correct orientation of the substrate.

In order to block thrombin activities we have two different type of drugs:

1. Small molecule inhibitors like argatroban that bind only to the active site of thrombin. These molecules are specific for thrombin so they do not bind to factor Xa. These small molecules are excreted by biliary secretion so we need to pay attention to patients which have problems with their liver, and can be orally administered
2. Recombinant polypeptides derived from a protein called hirudin which has been obtained by leeches (animals that suck blood and use this protein to make it more fluid). We have been able to develop different drugs derived from hirudin, like Lepirudin, 65 aa long, which binds irreversibly to both free and fibrin-bound thrombin, and Bivalirudin, 20 aa long, which has a short half-life and can be used in a more controlled way. Sometimes leeches themselves are still used, for example when reattaching a limb and prevent formation of clots. These drugs are excreted renally, thus care is needed in patients with renal insufficiency and cannot be administered orally so they are administered intravenously.

so for people with kidney problems we use compounds of the first class, while for people with liver problems we use compounds from class 2

Among all other things, thrombin is also able to activate the production of protein C which has an anticoagulant effect by working on factor Va and VIIIa. This is not only effect of protein C infact this protein is able to form a complex with another protein called protein S and control hemostasis, on top of that it is also able to reduce the plasminogen activator inhibitor 1 PAI1, a compound able to control the production of fibrin, and if we inhibit the inhibitor we stimulate fibrin. Moreover it has an important role in the control of the inflammation because protein C is able to inhibit TNF-alpha which we know that it is important in the inflammation process.

Let’s say that we have a clinical situation in which inflammation and coagulation are present at the same time, for example a septic shock. In this case we can use as a drug a genetically engineered recombinant activated protein C (r-APC), that can be used to cure this problem by decreasing mortality in a significant way.

Question 4

Speak about trombolytic agents

Answer 4

The compounds that we have previously seen prevent the formation of thrombin, however, if we are in an emergency situation, for example let’s sat that a patient has an ischemic attack and has already developed a thrombus which is blocking an artery, we need a drug that can degrade the thrombus, thus a thrombolytic agent.

These drugs lyse the clots which are already formed and restore the patency of the lumen of the vessels consenting the blood flow. How do they work? They work on the transformation of plasminogen into plasmin, using the fact that plasmin is a protease able to degrade fibrin so it is involved in the process called fibrinolysis. Also, these drugs use molecules called plasminogen activators which are capable of transforming plasminogen into plasmin, so that we have fibrinolysis. This compound is useful to control the size of the clot when there is a damage in the endothelial tissue, but there are no clots in other areas. For example, this compound might be used to block the formation of the atherosclerotic plaques. It is important to note that these drugs only degrade freshly formed thrombi, not the ones which were already present in the body.

The first drug of this class was put on the market before genetic engendering became famous. It derives from a bacteria called beta-haemolytic streptococcus which has an haemolytic function because it produces a protein called streptokinase. This protein’s function involves two different steps:

1. Complexation: a reaction in which the streptokinase binds to a plasminogen molecule forming a complex, this reaction induces a conformational change in the plasminogen molecule that makes it expose its active site.
2. Cleavage: streptokinase complexed plasminogen can now proteolytically cleave other plasminogen molecules to plasmin

This compound has two important limitations: since it is not a protein that is produced by our body, our immune system reacts and starts to produce antibodies, so if the patient needs this drug a second time, he cannot have it because of the risk of a shock reaction. Moreover, its thrombolytic activity is not specific, so it can result in systemic fibrinolysis.

To overcame this limitation, genetic engineering was used to produce the endogenous factor t-PA which is normally involved in the degradation of fibrin by activating plasminogen and turning into plasmin. This factor also has a high specificity so it can target the thrombi that have to be destroyed. This drug is found on the market under the name of alteplase. Here we can see the structure of t-PA which has a portion called finger that can bind to fibrin and be stimulated by it, this portion is also important for plasma clearance ( the overall ability of the body to eliminate a drug) and to determine the half life of the molecule. Then we have the EGF portion which is also important for the plasma clearance and two other portions called Kringle which is a German word to describe its “sweet bread shape”. Kringle I and II are also important for the clearance, especially kringle II. The very last portion is the c terminal portion where we have the serine protease enzymatic activity, so this is the active portion of the factor and it is also important because it is fundamental for the inhibition of PAI-1 (t-PA inhibitor-1).

Starting from alteplase we engineered other 3 molecules: reteplase (r-tPA), lanoteplase (n-tPA) and tenecteplase (TNK-tPA) which have been developed starting from the initial molecule. These modifications are useful to change the original characteristics of the drug .

So, in TNK there are modification in the portion of the serine protease site and also in other areas, the effect is a longer half life because of a reduced clearance and an increased specificity for fibrin. Reteplase is smaller because the Kringle I, EGF and finger portions have been cleaved, while for lanoteplase only the finger and EGF portions have been cut.

Question 5

how does the heart contract and conduct? what cells are involved? how does conduction work?

Answer 5

The heart is both a mechanical and electrical organ. The conduction of our heart is very important, but the contraction is also fundamental, so we need both: the mechanical component (contraction) to pump the blood in our body and the electrical component (conduction) to give a rhythm to the pumping. When discussing heart pathologies we might have problems with either of these two functions, if we have problems with the mechanical part we have heart failure, in this case the contraction is not working but the rhythm is still there, on the other hand if we have alterations in the rhythm, so with the electrical function, we have cardiac arrythmia, where the blood is being pumped with no rhythm.

At the beginning, the contraction is started because of the electrical stimulus starting in the sinoatrial (SA) node, so at first we have the contraction of both of the atriums, then the atriums relax and the electric conduction travels down in the ventriculi which then contract and subsequently relax. This is the entire heart contraction cycle.

An important thig that is often given for granted is that the cardiac action potential is a spontaneous event that proceeds based on the characteristic responses of ion channels to changes in membrane voltage. The heart infact, has two types of myocytes:

• one of them has a spontaneous activation of the action potential, meaning they posses automaticity, the ability to depolarize above a threshold voltage in a rhythmic fashion, these cells are called pacemaker cells, The primary pacemaker cells are found in the sinoatrial node, these are the proper pacemaker cells that give the heart its rhythm. If there are problems some other cells which also own automaticity might take over. In particular we have the atrioventricular (AV) node which acts as the secondary pacemaker, and also further down we find the bundle of His and the Purkinje fibers, all capable of automatisms, so able to depolarize spontaneously in a rhythmic way. The pace of our heart is given by sinoatrial nodal cells, which have a spontaneous rhythm which have a rate between 60 and 100 beats per minute; during these beats the heart makes an entire cycle. Why are the SA node cells more import for our heartbeat compared to the others? because they are the ones that cause the faster rhythm (60-100 times per minute). While the AV node has a lower spontaneous activity making the heart beat 50-60 times per minute, and the Purkinje fibers have an even lower activity: 30-40 times per minute. The other cells are infact called “latent pacemakers” because their activity is overridden by the SA node cells.
• the other type, the non pacemaker-cells, cannot spontaneously contract, these are found in the atrium and ventriculus and are responsible for the contraction of the heart, so differently from the other cells they have the ability to contract.

How does conduction work in the heart? As we know ions are not distributed equally across cell membranes, infact there are transporters which drive K+ into cells while pumping Na+ and Ca2+ out. The final membrane potential depends on the number of channels of each type, their conductances, and the duration for which each channel remains open. In the myocyte, at rest the membrane is relatively permeable to K+, because some types of K+ selective channels are open, but it is not very permeable to Na+ or Ca2+. So that means that the resting membrane potential is close to the equilibrium potential for K+. As we know myocytes, like also neurons and muscle cells do not only have a resting potential but also an action potential; in heart it lasts for about half a second which is a long time, this is needed to let both the conduction and the contraction happen, infact we need time to allow the complete emptying of the heart chambers from the blood.

So, as we said , SA cells are important for the heart rhythm while the ventricular muscle cells, which being muscles also have an action potential, are important for the contraction and the emptying of the chambers.

Question 6

Speak about the electrical dysfunctions

Answer 6

What can possibly go wrong with this mechanism? Dysrhythmia is a synonym of arrythmia, and it means that the rhythm is no more kept. This disfunction can be categorized in two ways:

• According to the site of origin of the abnormality, so we can have atrial arrythmia, junctional arrythmia which is in between the other two type of chambers, or ventricular arrythmia
• According to the rate of contraction. So whether the rate is increased, we have tachycardia, if it is decreased we have bradycardia. There might be palpitations, meaning awareness of the heartbeat, or symptoms of cerebral hypoperfusion which is **faintness or loss of consciousness, so low blood concentration in the brain is also important for the heart rate.

The most common types of tachyarrhythmias are atrial fibrillation, where we have a completely irregular rhythm, and supraventricular tachycardia, where the heartbeat is rapid but regular. Other issues might include Ectopic beats in which there are some more beats during a cycle, or the less common ventricular prolems which are also more serius and can potentially lead to death they include: ventricular tachycardia, and ventricular fibrillation, where the electrical activity in the ventricles is completely chaotic and cardiac output ceases, which might cause a stop in blood ejection. Lastly, Bradyarrhythmias include various kinds of heart blocks (e.g. at the AV or SA node) which might lead to the complete cessation of electrical activity.

why do we have these problems? there are two main reasons: problems with the formation of the impulse, so something is going on in the SA node, or problems with the impulse conduction, which is related to all other cells involved in the conduction and we have arrythmias.

Question 7

Speak about the sinoatrial node cells action potentialand the atrioventricular cells action potential

Answer 7

Let’s now look into the sinoatrial node’s cells action potential remembering that they manage the heartbeat which changes based on what is happening, like if we are relaxed or running.

The action podetial can be divided in three phases:

• phase 4: also called slow depolarization phase because we have an initial increase in the membrane potential, which is quite slow. This is due to the so called *I*f current, also called “pacemaker current” or “funny current”, the name derives from the fact that this current is due to cation channels called “funny channels” which are not selective for one ion but allow the passage of different cations. So, it is a nonselective current. This current is important for automaticity because it is spontaneous. So, we have this firs current due to the closing of potassium channels and the opening of “funny” channels that allow sodium (Na+) and potassium (K+) ions to enter the cell. Additionally, T-type calcium channels open transiently, allowing calcium (Ca²⁺) ions into the cell.
• phase 0: we have an action potential upstroke, which is due to a current called inward calcium current Ica. Once the membrane potential reaches the threshold, usually around -40 mV, calcium channels open, leading to a rapid influx of Ca²⁺ ions. This rapid entry of Ca²⁺ ions causes the membrane potential to become positive, creating the upstroke of the action potential, which is slower compared to the rapid sodium-driven upstroke seen in other cardiac cells.
• phase 3: it is the repolarization phase, due to an outward potassium current Ik, infact, after the upstroke, the calcium channels start to close, and potassium channels open, allowing K⁺ ions to exit the cell. This efflux of K⁺ leads to a decrease in membrane potential, repolarizing the cell back towards its resting membrane potential. After this phase the SA node goes back to phase 4.

Let’s now take a look at the action potential of the ventricular muscle cells where we do not have automaticity. the membrane potential of the resting ventricular myocyte remains nearthe equilibrium potential for K+ **until the cell is stimulated by a wave of depolarization that is initiated by nearby pacemaker cells. Since their shape is different and more complex we have 5 different phases instead of 3.

• phase 4: the cells are at resting potential (K+ equilibrium potential)
• phase 0: There is a depolarization because the action potential has arrived. This phase is infact also called rapid depolarization and it is even more rapid then the SA node depolarization. This quick depolarization is due to voltage gated sodium channels. The sodium channels are open just for a couple of milliseconds because after that there is the desensitization so, even if we have another action potential they do not open anymore. This is good because it gives enough time for the complete contraction and ejection of blood from the ventriculi.
• phase 1: immediately after phase 0 we have an early phase of rapid repolarization. this is triggered by the inactivation of voltage gated sodium channels but also by the activation of some K channels.
• phase 2: also called the plateau phase, because the repolarization stops and there is a balance between an inward Ca2+ current which is important also because calcium is needed for contraction, and an outward K current, trying to repolarize the cell. So there is a stable positive potential value. Among the open calcium channels there are transient calcium channels Ca.T and long lasting calcium channels Ca.L which remain open for a longer amount of time.
• phase 3: this is a very rapid late phase of repolarization, which is very rapid compared to the early phase. This is due to the stop of the current through Ca.T and an opening of K+ channels so we have an outward K+ current.

And then the cycle begins again with phase 4.

Very often during clinical trials the overall electrical activity of the heart is measured, this can be done with an electrocardiogram ECG. The ECG measures the body surface potential induced by cardiac electrical activity. The P is the atrial depolarization, the complex of QRS represent ventricular depolarization, and the T represent ventricular repolarization. The QT interval is the span between the minimal depolarization of the ventricles to the repolarization of the ventricles while the ST interval goes from the end of the ventricular depolarization to their repolarization.

Question 8

Speak about altered automaticity

Answer 8

we have two different situations that can lead to defects in impulse formation, for now we will only look at one:

altered automaticity: which can happen spontaneously, for example at the gym or when we are stressed, in a physiological way by stimulation by the sympathetic system because of the b1 adrenergic receptors. Their activation produces the opening of a greater number of pacemaker channels, thus increasing the heart rate, causing a larger pacemaker current and hence a faster phase 4. The release of noradrenalin or adrenalin can infact increase the force of contraction and the rhythm. This is called a chronotropic effect and allows the heart to work more efficiently. On the other hand we also have situations in which the heart decreases its rhythm, like when we sleep, in this case the parasympathetic system is activated for example through the vagus nerve. this ability of the vagus nerve to control the heart rate is very efficient, this happens because we have a lot of fibers in the atrial where the SA is, and less in the ventricles.

we have non-physiological situations too: automaticity can be altered when latent pacemaker cells take over the SA node’s role as the pacemaker of the heart, when does this happen? for example when the SA nodal firing rate becomes pathologically slow or when the conduction of the SA impulse is impaired or when the opposite situation happens and the latent pacemaker cells develop an intrinsic rate of firing that is faster than the SA nodal rate this might be caused by ectopic beats due to ischemia, or electrolyte abnormalities, or heightened sympathetic tone.

Another cause for the take over of the latent pacemakers cell is when there is a direct tissue damage in the heart, for example after a myocardial infarction. Now, because of structural disruption of the cell membrane or the loss of gap junction connectivity the control over automaticity is lost and these cells take over.