Lesson 7

Question 1

What is secondary hemostasis and what is the protein-protein hemostasis?

Answer 1

Secondary hemostasis happens kind of in parallel with first hemostasis. it is based on the coagulation cascade whose goal is to stabilize the clot and we have different factors belonging to the secondary hemostasis that can magnify the primary ones.

While in the primary hemostasis the main actors are platelets in the coagulation cascade we have a sequence of the enzymatic events which generate a large amount of fibrin. These factors are already present in our blood but are deactivated. They can quickly be activated by a specific reaction which then generate the cascade. This reaction happens in very specific sites where the damage is. All we need is a phospholipid based protein-protein complex which is formed thanks to an activated platelet. To form this complex, which then stimulates the cascade, the presence of negatively charged phospholipids, especially phosphatidylserine, is critical, as well as calcium. This complex is infact formed by proteic factors called factor Xa and factor Va, calcium ions and phospholipids form the platelets membranes or other cells, such as phosphatidylserine. Phosphatidylserin is normally found in the inner leaflet of the plasma membrane but after the receiving damage, it migrates on the other leaflet and activates the protein-protein complex.

Question 2

explain the coagulation cascade which happens during secondary hemostasis

Answer 2

The cascade that this complex generates has been described as composed by two branches: the intrinsic and extrinsic pathway but there is a common factor that unites the two pathways.

• the intrinsic pathway is called like this because it was studied in vitro and it triggered by internal damage of the endothelium, it has been seen that it starts with the activation of factor XII, a coagulation protein that is essential for surface-activated blood coagulation. Factor XII is normally inactive but can be activated by another protein. The activation of every factor happens thanks to the cleavage of a part of the protein. When factor XII gets activated it in turn activates factor XI which in turn activates factor IX. Then this factor, together with the activated factor VIII activates factor X which is the factor in common within the two pathways.
• the extrinsic pathway is triggered by the tissue injury itself, an external injury that causes blood to escape. When our tissue is damaged a tissue factor is is produced, this factor interacts with factor VIIa (activated version of factor VII) and then they can activate factor X, OR factor 7a alone is also able to activate factor IX and thus the intrinsic pathway.

After the activation of factor X we have the common pathway. This factor is now Xa and it collaborates with factor Va to transform prothrombin (inactive) into thrombin also called factor IIa when active. The activation of trombin is very important because it is involved in the production of fibrin. The inactive form of fibrin is fibrinogen which is activated by thrombin. Thrombin, on the other hand, is also able to activate factor XIII, which is important to form the final stable clot, infact factor XIII cross-links the fibrin polymers into a highly stable meshwork or clot. Moreover thrombin can also act with a positive feedback on factor Va, stimulating the synthesis of even more thrombin through factor X. Lastly, thrombin is also important for the activation of factor VII or factor VIII of the intrinsic or extrinsic pathway.

In total thrombin has 4 actions:

1. it converts fibrinogen into fibrin.
2. it activates factor XIII.
3. it acts with a positive feedback on factor Va
4. it activates the intrinsic or extrinsic pathway trough factor VII or VIII.

So thrombin is capable of amplifying both pathways and also simulate the continuous increase of fibrin.

Question 3

how does thrombin regulate hemostasis?

Answer 3

n addition to its procoagulant properties, thrombin also modulates the coagulation response by activating protease-activated receptors (PARs) found on the intact vascular endothelial cells adjacent to the area of vascular injury. So these cells are stimulated to release some specific factors important for the formation of the clot, which are important because we want the clot only in the area of damage.

Among PARs receptors PAR1 is also expressed on platelets and is also the most sensitive receptor for thrombin: That means that thrombin can also act on first hemostasis stimulating the aggregation of platelets.

So thrombin activates the release from these cells of prostacyclin PGI2, which interestingly has an opposite effect to that of thrombin. Prostacyclin PGI2, has a specific receptor bound to a Gs protein. Since this receptor has an opposite action to Gq protein, it is stimulates the inhibition of platelet aggregation and stimulates vasodilation. A similar effect is caused by nitric oxide, which is also released by healthy tissue and whose release is also caused by thrombin.

These cells are also stimulated by thrombin to release t-PA modulator whose role is to produce plasmin which in turn is able to degrade fibrin, so thrombin is important for forming the clot but also for the activation of the processes that block coagulation, meaning that there is a very fine regulation of the clot formation. T-PA is the most active when it is bound to a fibrin meshwork. T-PA can be inhibit by another molecule called plasminogen activator inhibitor 1 (there is also 2 but as a lesser effect) and it is present when we have a lot of thrombin and high inflammatory, so we have a lot of cytokines, meaning that the damage has just started. Moreover thrombin stimulates the release, always from the same cells, of the profibrinolytic protein which is an activator of t-PA.

We also have alpha2-antiplasmin which neutralizes free plasmin in the circulation preventing systemic degradation of plasma fibrinogen.

Other factors to keep in mind which play an important role in the limitation of the hemostatic process and are always produced by healthy tissue, are:

• antithrombin III which is a protein able to inhibit thrombin, whose main effect, as we said, is the activation the coagulation cascade, so blocking it means inhibiting the formation of a stable clot. This protein also inhibit other components of the coagulation cascade: factor IX, X and XI. These interactions are enhanced by heparin-like molecules expressed at the surface of intact endothelial cells, which activate antithrombin III. So antithrombin III gives the message to not coagulate because it is not needed.
• protein C and S are vitamin K dependent protein and are able to slow the coagulation by inactivating factor 5 and 8. They are part of a negative feedback pathway, Thrombin infact, can stimulate the production of these proteins.
• tissue factor pathway inhibitor TFPI is also produced. **We saw that tissue factor is very important so when we limit it, we are preventing prevents the activation of factor IX and X.

So the procoagulant factor are already in the plasma and need to be activated, this cascade is activated by the damaged tissue through signals such as phosphatidyl serin and tissue factor. They are membrane bound and localized in the site of injury. On the other hand the anticoagulant factors are produced by the healthy endothelium and are released by cells, these molecules are soluble and can travel in the blood so that we have a balance between these two categories of molecules. Hemostasis is finally regulated in order to achieve 2 main goals:

• The clot must be restricted in the area of damage
• The size of the clot must be kept under control, because if it grows too much it will block the blood flow.

Question 4

What are the causes of pathological thrombi? what are they?

Answer 4

Why do we need to control the formation of thrombi? because we have pathologically activated thrombi, a thrombus is a clot that is blocking the blood flow. So they pose the risk of vessels obstruction and reduction or even stop of the blood flow. Thrombosis is the name of a pathology in which we have an excess of thrombi in an area where there is healthy tissue. The size of these not needed clots can even block the lumen.

Three different situations can lead to Thrombosis, this condition can also cause each other and are closely related one to the other:

1. Injury to the endothelium which is no more healthy and is dysregulated, when this happen the formation of the thrombi mostly happens in heart and arterial circulation. the Injury might be caused by hypertension or turbulent flow, hyperlipidemia, elevated blood glucose in diabetes mellitus, traumatic vascular injury and also some infections. The injury will promote the platelet adhesion to the injured site and we will have the initiation of the coagulation cascade through tissue factor
2. abnormal blood flow, for example when the blood flow is no more laminar, so there is a state of turbulence or stasis. In this case atherosclerotic plaques and bifurcations of blood vessels can create areas of turbulent flow and this can lead to endothelial injury. More precisely local stasis can result from formation of an aneurysm, from myocardial infarction and cardiac arrhythmias, it can also lead to venous thrombi, which typically occur in the deep veins of the leg. The absence of laminar blood flow allows platelets to come into close proximity to the vessel wall. Stasis also inhibits the flow of fresh blood into the vascular bed, so that activated coagulation factors in the region are not removed or diluted. Abnormal blood flow promotes endothelial cell activation, which leads to a prothrombotic state
3. There are disorders such as hypercoagulability causing an heightened coagulation response to vascular injury, resulting from, for example, primary (genetic) disorders .Very often it is caused by a factor V mutation which is controlled by protein c, so protein c causes an excess of factor 5. Then we have non genetic secondary causes, for example, we find hypercoagulability in the antiphospholipid syndrome. Whit this illness we start producing antibodies against lipids, blocking the start of platelets aggregation. Oral contraceptives can also cause the synthesis of coagulation factors, or even in the postpartum period we can have venous stasis. Moreover during surgery we can have venus stasis. So in general, with this disease the formation of clots happen more frequently than what is necessary. Hypercoagulability is normally less frequent then the other causes.

Question 5

What are the three classes of drugs that can act on thrombosis? speak about the three classes of antiplatelet agents

Answer 5

What can we use to cure thrombosis? There are three main classes of drugs: antiplatelet agents, anticoagulants and thrombolytic agents.

Let’s start with antiplatelet agents. They target the formation of a localized platelet plug in response to endothelial injury. These are very useful for prophylactic and therapeutic strategies against **infarction caused by thrombosis in coronary and cerebral arteries.

Which are the targets that can be used to inhibit platelets aggregation? we have cyclooxygenase COX inhibitors like cardioaspirin, or phosphodiesterase PDE inhibitors and ADP receptor pathway inhibitors, which are foundamental for the formation of the bridge for platelet aggregation.

cyclooxygenase are produced by many cells including platelets, they work on a substrate called arachidonate which originates form phospholipids. These compounds can transform arachidonate into prostaglandins, prostacyclins and thromboxanes. So with cardioaspirin or other cyclooxygenase inhibitors we block the synthesis of prostaglandins, thereby inhibiting the platelet granule release reaction and interfering the with normal platelet aggregation. And since platelets have no nucleus, when we have the blockage of cyclooxygenases the effect lasts for the entire life of the platelets. On the other hand COX inhibitors also block the formation of prostacyclins which have anti-platelets effect So we must pay attention to not administer too much of this drug. The antiplatelet activity of COX inhibitors is already present in 80 mg of compound, so 80mg is the dosage needed to inhibit cyclooxygenases in a constant way in the platelets without affecting too much the ability of endothelial cell to produce prostacyclins. This happens since COX are permanently inhibited in the platelets but not in the endothelium, where prostacyclins can still be synthetised since there is a nucleus.

Other useful compounds are phosphodiesterase PDE inhibitors. We know that PDE can degrade cAMP to obtain AMP, so by blocking PDE we have an increase of cAMP which is important because it blocks the aggregation of platelets. A famous PDE inhibitor is dipyridamole. These drugs have a low effect which can be increased when used combination with other drugs like cardioaspirin or warfarin.

We also have ADP receptors inhibitors but maybe instead of inhibitors a better term would be antagonists. Most of these drugs are irreversible antagonists, so they block this pathway by blocking the action of ATP. We have different generations of these drugs:

• the first generation is represented by Ticlopidine. This ****is a prodrug, meaning that it needs to be converted after the administration into the actual active drug. ticlopidine is used when patients cannot stand aspirin which is usually the first choice drug and the safest since we know it the best.
• the second generation compound that has replaced ticlopidine is clopidogrel which has a quicker action even though it is still a prodrug. This drug has less side effects, especially in the bone marrow. Another compound is prasugrel which is similar to clopidogrel but has a quicker action, that means we will have a larger amount of active drug in our body, which, at a too high dosage might cause bleeding.
• the third generation compound is ticagrelor, a competitive antagonist which is reversible, so a lot safer. Another point in favour of this drug is that it is not a prodrug so it does not need to be elaborated and acts immediately.

A part from these 3 famous types of antiplatelet agents, we also have:

• antagonists of GPIIb-IIIa: if we block GPIIb- IIa, we also block the formation of the bridge among platelets, so fibrinogen does not form the bridge. This compounds are very powerful for platelet aggregation in vitro. Even in this case there are 2 classes: eptifibatide and tirofiban but also a monoclonal antibody called abciximab. The main side effect is bleeding. the monoclonal antibody is the only one which is irreversible, with the other drugs we are inhibiting in a permanent way the formation of the fibrinogen bridge with other platelets.
• Thrombin receptor antoagonists or PAR-1 antagonists: for example vorapaxar blcks PAR-1 receptor and prevents the platelet aggregation. What is interesting is that this antagonist is mainly affecting platelet aggregation but not the coagulation platelets.

Question 6

Speak about anticoagulants

Answer 6

These compounds are used to prevent the thrombotic disease or treat emergencies when a thrombus is blocking the blood flow needing an immediate intervention. These drugs target the coagulation cascade but have bleeding as a side effect, so the concentration of the dose needs to be carefully controlled.

Warfarin is a synthetic compound which tries to mimic a natural compound called dicumarol, dicumarol was discovered around 100 years ago in Canada and North Dakota were farmers changed the food for the cattle, and instead of using corn they introduced sweet clover, but at a certain point they used a spoiled harvest that led the cattles to suffer from bleeding. In this way it was found that dicumarol is present in spoiled sweet clover. Since warfarin is similar, we need to pay attention to the bleeding effect. Also, Warfarin has a small therapeutic window.

This drug affects vitamin K which influences some important coagulant factors like II, VII, IX and X, but vitamin K is also required for the synthesis of these factors. So, these proteins are biologically inactive as unmodified polypeptides but gain activity by post-translational carboxylation which is mediated by reduced vitamin K. This allows the binding of Ca2+ ions to the proteins, which are needed to efficiently binding to phospholipid surfaces. Knowing this we can understand why the regeneration of reduced vitamin K is essential for the sustained synthesis of biologically functional clotting factors II, VII, IX, and X, all of which are critical components of the coagulation cascade.

Warfarin does not block vitamin K directly but blocks the reductase enzyme called epoxide reductase or VKROC1 that catalyses the generation of reduced vitamin K.

A single dose of warfarin has no effect for approximately 18–24 hours because the shorter half life of the coagulations factors is factor VII’s, so we have to wait a bit.

Also, we have to consider that there is also a small population of patients is genetically resistant to warfarin because of mutations in their epoxide reductase gene.

Warfarin, beside having a narrow therapeutic window,, is a complex drug, because it is usally highly bound to albumin (99% of this drug will be bound to albumin), so if we administer it with other compounds that bind to albumin there might be an interaction causing side effect. So people under warfarin treatment need to have bloodwork done continuously to prevent the side effect of bleeding.