Hemostasis (PDF) 16/05/2022

16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone MOD - VASCULAR PATHOLOGY – Bonecchi- HEMOSTASIS The term hemostasis is normally used to define a mechanism that takes place when there are problems in vessels, but also a mechanism that maintains the blood in a fluid state (1st meaning...

16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone MOD - VASCULAR PATHOLOGY – Bonecchi- HEMOSTASIS The term hemostasis is normally used to define a mechanism that takes place when there are problems in vessels, but also a mechanism that maintains the blood in a fluid state (1st meaning). In order to maintain the blood fluid, there are many regulators. Besides maintaining homeostasis, hemostasis guarantees the rapid formation of clots to prevent blood loss when there is an injury (2nd meaning). On the contrary, thrombosis is the pathological counterpart of hemostasis. While the latter is always happening, even if vessels are damaged, thrombosis, indeed, happens in pathological conditions. There are many differences, but the most important is the location where the blood clot is formed: • in hemostasis, homeostatic clot is formed in vessel wall, to repair the vessel at the site of injury; • in thrombosis, thrombus is formed inside the lumen of intact vessels, which is a dangerous situation. Both hemostasis and thrombosis, are dictated by the same processes that involve three main components: 1. Endothelial cells and other cells of the vascular wall 2. Platelets 3. Factors of the coagulation cascade Under normal conditions, there is a very delicate balance between pro and anti-thrombotic activities of blood, allowing the maintenance of the blood fluidity. If this balance is destroyed, there will be excessive bleeding or, at the contrary, thrombosis. When vessel wall is ruptured: 1- Vasoconstriction 2- Activation of platelets, that will provide the surface for the coagulation cascade to start 3- Coagulation cascade, that will stabilize the clot with a net of fibrin In the image on the right, there is the explanation of two concepts: • primary hemostasis, the process until platelets adhesion and activation (platelet plug formation); • secondary hemostasis, up to the activation of coagulation cascade (fibrin clot formation) 1 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone Primary includes vessels and platelets, secondary, the coagulation cascade, the purpose of these processes is to stop the bleeding as an injury occurs. Hemostatic process can be divided in steps: a. Vasoconstriction, b. 1ary hemostasis, c. 2ary hemostasis, d. Clot dissolution and fibrinolytic system, since, in order to repair the damage, it is necessary to dissolve platelet plug and fibrin clot beforehand. A- VASOCONSTRICTION At the beginning, when there is an issue, like an injury in the vessel, the very first step is vasoconstriction because endothelial cells release an inflammatory mediator, endothelin, that will bind to receptors, present in smooth muscle cells that by contracting induce vasoconstriction. This step is fast, it is a reflex vasoconstriction due to the release of this mediator. The purpose of this step is to reduce blood loss. B- PRIMARY HEMOSTASIS Then things get more complex in terms of mediators, molecules, receptors. First, during primary hemostasis, platelets start to adhere to the matrix on the rupture. They are able to bind the sub-endothelium thanks to the recognition of vWF, Von Willebrand Factor (represented as the small red dots on the rupture). First vWF binds to collagen, in turn platelets will bind to the factor. Once platelets adhere, they change shape (from roundish, they become flat with dendritic protrusions), release granule content/ undergo degranulation, meaning they get activated (there is the release of inflammatory mediators that recruit other platelets at the plug site) and finally aggregate (with each other, not only to the endothelium). This mechanism is complex, dangerous when/where not needed, becoming pathological. For example, in COVID, a very important issue is thrombosis, where patients start to have disseminated intravascular coagulation. So, this process is potentially lethal if not controlled, that is why there are so many regulators. 2 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone Platelets In the image on the left, they are the purple small dots (red ones are RBCs). Platelets are anucleated, with a short life-span. Their main function is to perform hemostasis, where they perform a double action: first they form the primary hemostatic plug, then they provide a surface for coagulation cascade. Even if they’re anucleated, they have all normal cell functions. Additionally, they present granules (alpha and delta), important in hemostasis. The molecules contained in the granules are for example, in alpha ones: P-selectin, fibrinogen, fibronectin, chemokines, cytokines (also pro/anti-inflammatory ones), coagulation factors. The delta granules are instead dense, since they contain a lot of calcium precipitate, crucial for the activation of the coagulation system, but also ADP/ATP, histamine, serotonin and epinephrine. Platelets derive from the same progenitor of the hematopoietic stem cells. In the bone marrow there is the megakaryocyte, the father of the platelets, which form following fragmentation (platelets formation), after which they will end up in blood. The cytokines stimulating differentiation from stem cells to megakaryocyte are now known, so it is possible to promote differentiation, giving patients those specific factors. During primary hemostasis, platelets start to adhere to sub-endothelium, and after adhesion they also change shape, presenting protrusions. They will subsequently degranulate and, after secretion, aggregate with other platelets. There are specific receptors for adhesion, as well as for aggregation. 3 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone Receptors on platelet membrane As other leukocytes, platelets have on their surface receptors: - selectins, in alpha granules. For example, when platelets degranulate, P-selectin comes to the surface - GPCRs, like ADP receptor, thrombin receptor (PAR), TXA2 (thromboxane A2) receptor. - integrins, expressed normally by leukocytes, not by endothelial cells. The most important are: o glycoprotein IIb/IIIa: binds to fibrinogen, for platelet aggregation o glycoprotein Ib (GpIb): binds vWF, for adhesion to sub-endothelium of collagen Platelet adhesion to ECM The endothelium is ruptured, sub-endothelium binds vWF, that will bind the GpIb, on platelets surface, necessary to overcome the high shear forces of flowing blood. Platelets also express another integrin, GpIIb-IIIa, that can bind a molecule of fibrinogen, and one molecule of fibrinogen can bind two GpIIb-IIIa, on two different platelets, promoting their aggregation. To recap: 1- injury of the endothelium, collagen fibers are exposed and bind vWF. 2- vWF binds to GpIb, bound to platelets. (Remember that the receptor doesn’t bind collagen directly but it binds the vWF) 3- GpIIb-IIIa binds fibrinogen (indeed, one fibrinogen can bind two GpIIb-IIIa), which results in platelet aggregation A deficiency of these factors (vWFs, Gps) will result in thrombosis or bleeding, since platelets are not correctly bound to sub-endothelium or to each other’s. von Willebrand Factor Von Willebrand factor (vWF) is a multimeric glycoprotein, mediating the adhesion and aggregation of the platelets. Platelets aggregation is obtained by GpIIb-IIIa binding vWF. Endothelial cells themselves produce the factor, which is present in granules (Weibel-Palade bodies) that are released at damaging. It is also present in platelets’ alpha granules. 4 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone vWF is not only in endothelial cells but also in plasma, meaning there is a detectable concentration in the blood (7-10 mg/mL), that is important in diagnostic. Platelet activation The last thing that platelets do after they bind to endothelium is to activate. This will be important for the change in shape and degranulation. The activation is mediated by GPCRs: - thromboxane A2 receptor (NB. Thromboxane is produced by both COX I and COX II pathways. Indeed, when taking aspirin, it blocks these pathways, and consequently thromboxane A2, which is the reason why it has an anticoagulant function. In fact, thromboxane A2 is a strong inducer of platelet activation and aggregation); - ADP receptor. - thrombin receptor. Other activating factors are: thrombospondin, epinephrine, platelet activating factor. Platelet activation results in the release of calcium from the open canalicular system = secretion of granules. Platelet activation and secretion Secretion of granules by platelets is important to release fibrinogen found in the alpha granules, important for platelet aggregation. Alpha granules also contain: fibrinogen, vWF, factor VIII, platelet-derived growth factor (PDGF) which promotes healing, platelet factor IV, which prevents formation of active thrombin inhibitor from heparin and anti-thrombin III. Dense core granules instead release ADP, also activating platelets, inflammatory mediators (serotonin), calcium (very important, to activate coagulation cascade). When platelets change shape, they also rearrange membrane lipids: phosphatidyl serine which is usually on the inner membrane of the platelet, flips out to the outer membrane where it plays a role in binding prothrombin, important for the activation of the coagulation cascade. Flipping out guarantees a negative starting surface where the coagulation cascade can start. This change gives a binding site for many coagulation factors. 5 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone Platelets adhesion to the vessel wall Platelets’ adhesion process isn’t much different than leukocytes’ behavior during extravasation. Platelets start rolling on the endothelium, then adhere and aggregate on each other. So, all the molecules expressed by leukocytes, are also expressed by platelets even if they’re used slightly differently. Adhesion molecules are used to bind to the sub-endothelium. Platelet aggregation This is a very important step, because platelets need to form a very strong clot to stop the bleeding. The process is mediated by GpIIb-IIIa (integrin) that binds fibrinogen. Fibrinogen is present both in alpha granules of platelets and in blood (soluble) produced by the liver. This soluble fibrinogen is very important, because once it is necessary, it can bind to platelets and induce aggregation. Aggregation is also promoted by vWF, ADP, TxA2, and thrombin, and all other molecules that bind GPCRs on platelets, leading to the activation of GpIIb-IIIa NB. Integrins on surface are not activated, the signaling of GPCRs is able to activate them and once activated they can bind fibrinogen, inducing aggregation. C- SECONDARY HEMOSTASIS: the clotting mechanism The primary plug is not so stable (because of blood flowing, which is characterized by a strong force, so a strong plug is needed to stop the bleeding). So, over the plug of platelets there is the activation of the coagulation cascade. Tissue factor will be released by endothelial cells, important to induce the cascade. It’s important to notice that all these processes are orchestrated by endothelial cells, thanks to the production of factors that allow the binding of platelets to the broken endothelium, the activation of the cascade and so on. Then, phospholipid complex expression allows the binding of coagulation factors. The most important is the final step, activation of thrombin. Thrombin will promote fibrinogen into fibrin monomers which successively assemble fibrin network that will stabilize the primary plug. So, this will be called secondary hemostatic plug. Main event in coagulation cascade is the conversion in prothrombin in thrombin. 6 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone Coagulation cascade Coagulation cascade is a series of enzymatic reactions mediated by factors indicated in roman numbers. Each reaction in the pathway results from the assembly of a complex composed of: - enzyme (activated coagulation factor) - substrate (proenzyme form of the coagulation factor) - cofactor (reaction accelerator) In the picture on the right, it’s possible to see different factors: pink ones are inactivated, the green ones, with suffix “a” are activated. The same activated molecule will become an enzyme that will convert and activate another factor (e.g., IX to IXa). There are two pathways, one is the intrinsic, the other is the extrinsic. The tissue factor from endothelial cells induces the extrinsic pathway. So, in this case, tissue injury leads to the release of tissue factor, which will be then activated. Both pathways are converging into a common one. The central event is conversion of factor X in Xa, which in turn converts factor II/prothrombin into factor IIa/Thrombin (Notice the double nomenclature in roman numbers and names). Thrombin will convert fibrinogen in fibrin (soluble), while factor XIIIa, activated by thrombin, will cross-link the fibrin fibers (so it becomes insoluble). Ca2+ presence is fundamental for the common pathway, especially for: - factor X activation, - conversion of pro-thrombin in thrombin, - cross-link of fibrin fibers. All these factors are assembled and activated on the surface of activated platelets and held together by calcium ions. 7 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone Thrombin Thrombin is a very crucial and powerful (it can cleave and activate hundreds of receptors) enzyme in the cascade. It is not only involved in the cleavage of fibrinogen, but it also activates other factors: XI, VIII, V. So, it is a sort of amplifier of the coagulation cascade: activating other factors will lead to the generation of more thrombin (positive feedback cascade). It also activates factor XIII, involved in the last step when there is cross-linking of fibrin. An additional function of thrombin is activation of a family of GPCRs, called Proteases Activated Receptors (PARs). Thrombin is able to cleave the N terminal of the PAR, the extracellular portion, so the new end terminal becomes the new agonist of the receptor. They are very particular receptors, because they carry their own ligand in their sequence, so thrombin is not the agonist of this receptor, but the activator. PARs are expressed on endothelium, monocytes, dendritic cells, T lymphocytes. This means that thrombin also activates inflammatory cells. This capability is linked to the fact that inflammatory reactions and hemostasis happen simultaneously. Besides fighting the infection, in inflammation, we also need regeneration, healing. Fibrinogen - fibrin Fibrinogen is a very important component in our blood (3 g/l), produced by the liver and it is indeed soluble. It is converted into fibrin monomers by thrombin. These monomers are then cross-linked by factor XIIIa. D- FIBRINOLYTIC CASCADE. Once the final clot is formed, with the possibility to have some leukocytes entrapped, there is the need to remove it slowly, since it is necessary to repair the endothelium first. The clot is removed through digestion by plasmin, generated by a circulating precursor, called plasminogen. Plasmin is a general protease with wide variety of substrates, but has two major functions: Fibrinolysis Inhibition of fibrin polymerization It is activated by Plasminogen Activators (PAs) which are several: 1. tissue-PA: synthesized principally by endothelium and is most active when bound to fibrin, so at clot presence. Main way to activate plasminogen. 2. Urokinase-like PA (u-PA): present in plasma and in various tissues; it can activate plasmin in the fluid phase. 3. Bacterial enzyme streptokinase, which is a PA, avoiding coagulation, degrade the clot and to better enter the host. 8 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone It’s important to mention also the regulation systems. The first step of regulation is that tissue-PA is mostly active when bound to fibrin that needs to be degraded. Additional steps of regulation are plasmid inhibitors: we do not want to eliminate the plug fast in order to repair the tissue. There are at least 2 systems of regulation of plasmin: 1. α2-plasmin inhibitor: binds free plasmin. 2. plasminogen activator inhibitor (PAI): binds to tissue plasminogen activator (T-pa). Production of the two is increased by thrombin and inflammatory cytokines, to avoid removal of clot before tissue repair. T-pa is given to patient with a thrombus, in order to dissolve the clot. To recap, the image below represents the whole picture of hemostasis. remember that: - in platelet granules there are chemoattractants to attract more platelets to the plug. - Fibrinogen is already present for platelet aggregation and will then be cleaved to turn not fibrin and for the clot. Endothelium Endothelial cells have both anti-thrombotic and prothrombotic activity. But in the picture (which represents the homeostatic condition) it is possible to see that the balance is shifted towards anti-thrombotic function, because they produce more anti-thrombotic molecules in comparison to thrombotic ones, since it is more important to avoid thrombi on a daily basis and keep the blood fluid. Still, the endothelium is ready to produce thrombotic factors if necessary, in order to induce all the steps of hemostasis. 9 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone In the scheme on the right, anti-thrombotic and thrombotic molecules can be classified in 3 different categories: - Inhibition /promotion of platelet aggregation - Inhibition/promotion of coagulation - Inhibition/promotion of fibrinolysis. Antithrombotic properties They are three: 1- Antiplatelet effects: - Prostacyclin (PGI2), derived by arachidonic acid cascade, from COX pathway, produced by endothelial cells. Its effect is very potent. It inhibits platelet adhesion and aggregation, as well as it is a vasodilator. - Nitric oxide: vasodilator and inhibitor of platelet adhesion and aggregation to endothelial cells; - Expression of Adenosine diphosphatase: enzyme degrading ADP. ADP is a mechanism of activation of platelets. So, by producing this enzyme there is inhibition platelet aggregation. Production of these three elements allows endothelial cells to have an antithrombotic property through an antiplatelet effect. 2- Anticoagulant effects: In normal condition endothelial cells tend to block coagulation. These effects are achieved thanks to: - Membrane-associated heparin-like molecules on surface of endothelial cells. They act trough antithrombin molecule, so the complex heparin-anti thrombin will first inhibit thrombin and then inactivate other coagulation factors. So, these heparin-like molecules don’t directly work on thrombin, but they do so thanks to the formation of the complex. 10 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone Thrombomodulin, binds thrombin inhibiting its activity. Then the complex thrombomodulin-thrombin inhibits protein C, a mechanism of regulation of the coagulation system, normally active and which degrades factor Va and VIIIa. Tissue factor pathway inhibitor produced by endothelial cells. Antithrombin III is the heparin cofactor, and its function is: - Inhibition of the activity of thrombin and others (factors IXa, Xa, XIa and XIIa) when in the complex heparinantithrombin (endothelium, liver and megakaryocytes) Antithrombin III is activated by binding to heparin-like molecules on endothelial cells The last antithrombotic property is the expression of tissue plasminogen activator. Endothelial cells by producing it will have a fibrinolytic effect, and consequently an anti-thrombotic property. Lastly, the prothrombotic activity of endothelium is: - production of vWF, necessary for binding of platelets to sub-endothelial collagen, - Procoagulant effects, thanks to tissue factor production, to start the coagulation cascade - Antifibrinolytic effects, thanks to production of inhibitors of plasminogen activator (PAIs), blocking fibrinolytic effect. So, by producing all these mediators, it is possible to maintain hemostasis, an homeostatic state, where the blood is kept fluid, but at the same time produce the formation of a plug fast if necessary. 11 16/05/2022 Sbobinator: Claudia Daidone Reviewer: Marta Rainone Some further clarifications: Heparin is blocking thrombin function (important to remember when giving to patients), thanks to the formation of a complex with antithrombin III which it activates. However, if a thrombus is detected in a patient, administering heparin wouldn’t really help: the thrombus is already formed, so it would be useless to stop the activity. In case the thrombus has occluded a vessel and there is an ischemic situation, if the patient is rapidly given a fibrinolytic or anticoagulant agent in high doses, it will cause a reperfusion injury. Especially in the brain, it’s important to remember not to give high doses of fibrinolytics, like t-PA, too fast, in order to prevent this injury. 12