1. Outline of the lecture Histamine and H1, H2 – antihistamines
Haemostasis: a short overview
Drugs used in the diseases of haemostasis

2. Haemostasis
Hemostasis stops bleeding from an injured vessel. It is the first stage of wound healing.
Thrombosis is the pathological formation of a haemostatic plug resulting from inappropriate activation of haemostatic mechanisms (without the damaged vascular wall). It can lead to local ischemia/ necrosis or distant thromboembolism.
Bleeding (= excessive bleeding).

3. Haemostasis
When endothelial injury occurs, the endothelial cells stop secretion of inhibitors of platelet aggregation and coagulation. Instead, they secrete von Willebrand factor and other factors which promote adhesion and activation of platelets.
Hemostasis has three major steps:
1) vasoconstriction
2) primary hemostasis = platelet phase: temporary formation of a loose platelet plug
3) secondary haemostasis involving blood coagulation and formation of a firm clot from a cross-linked fibrin

4. Haemostasis

5. Haemostasis
Endothelial cells of intact vessels prevent blood clotting and thrombus formation with a heparin-like molecules (heparans) and thrombomodulin and prevent platelet aggregation with nitric oxide and prostacyclin (= prostaglandin I2).

6. Primary haemostasis Vascular phase:
vasoconstriction limits blood flow (blood loss) and makes platelets adhesion easier
vasoactive molecules are adrenaline, tromboxane A2, serotonine and others

7. Primary haemostasis: platelet phase

8. The role of platelets (adhesion)
Circulating platelets bind collagen with surface collagen-specific glycoprotein Ia/IIa/IIb receptors.
The adhesion is strengthened further by the large circulating protein, von Willebrand factor (vWF), which forms links between the platelets glycoprotein Ib and the collagen fibrils. This adhesion activates the platelets.

9. Platelet activation
Activated platelets release the contents of stored granules into the plasma.
The granules include ADP, serotonin, platelet-activating factor (PAF), vWF, platelet factor 4, and thromboxane A2 (TXA2), which, in turn, activate additional platelets.
Platelet`s integrin membrane glycoprotein IIb/IIIa increases its affinity to bind fibrinogen.
Prostacyclin (PGI2) is released by endothelium. It causes vasodilation and inhibits the release of granules that would lead to activation of additional platelets and the coagulation cascade (vasodilation, inhibition of aggregation).

10. Platelet activation
Activated platelets change its shape from spherical to stellate, release the contents of stored granules into the plasma (more than 200 compounds), expose membrane receptors and negatively charged phospholipids
The granules release molecules which activate additional platelets
adenosine diphosphate (ADP)
serotonin
thromboxane A2 (TXA2)
platelet-activating factor (PAF)
vWF, platelet factor 4, …

11. Platelet aggregation
Platelet`s integrin membrane glycoprotein IIb/IIIa increases its affinity to bind fibrinogen
and the fibrinogen cross-links with glycoprotein IIb/IIIa supporting aggregation of adjacent platelets.

12. Blood coagulation (secondary haemostasis)
The coagulation pathways are a series of reactions, in which a zymogen (inactive enzyme precursor, proenzyme) of a serine protease and its glycoprotein cofactor are activated to become an active enzymatic complex that then catalyzes the next reaction in the cascade (cleave the zymogen to activate another protease), ultimately resulting in cross-linked fibrin.

13. Blood coagulation
The coagulation cascade is classically divided into three pathways.
The tissue factor (formerly known as the extrinsic pathway), and
contact activation pathways (formerly known as the intrinsic pathway),
both activate the "final common pathway" of factor X, thrombin and fibrin.
It was previously thought that the coagulation cascade consisted of two pathways of equal importance joined to a common pathway. It is now known that the primary pathway for the initiation of blood coagulation is the tissue factor pathway.

14. The formation of a clot. The coagulation cascade features a simple amplification unit that is repeated several times: A cofactor protein (green) binds to an enzyme (pink) on a phospholipid surface. This triumvirate complex is recognized by a circulating zymogen precursor (yellow). After a brief interaction, the complexed enzyme cleaves the zymogen and activates it. This new enzyme is incorporated into the next amplification unit for activation of the next precursor molecule.
The tissue factor–Factor VIIa–membrane complex begins in vivo coagulation when tissue factor is exposed to blood components after injury.
Factors XII and XI begin the contact phase–activation (intrinsic) pathway.
Platelets provide the majority of phospholipid surfaces to support coagulation.
Prothrombin (Factor II) is the precursor to thrombin, the final enzyme of the cascade, which releases Fragment 1.2 and thrombin during activation.
Thrombin removes fibrinopeptides from fibrinogen, forming fibrin.
When fibrin oligomerizes and is cross-linked, it provides surfaces for the fibrinolytic pathway, which comprises another set of activators and enzymes that cleave the clot and release fragments [fibrin degradation products (FDPs), D-dimers] into the circulation.
Thrombin also acts in a proinflammatory manner by activating protease-activated receptors (PARs) on platelets and endothelial cells.

15. The coagulation cascade
Tenase complex
Prothrombinase
complex
Green-Activation
Red-Inhibition

16. Tissue factor pathway (extrinsic)
The main role of the tissue factor pathway is to generate a "thrombin burst,“
Thrombin is the most important constituent of the coagulation.
Following damage to the blood vessel, FVII leaves the circulation and comes into contact with tissue factor (TF) expressed on stromal fibroblasts and leukocytes, forming an activated complex (TF-FVIIa).
TF-FVIIa activates FIX and FX.
FVII is itself activated by thrombin, FXIa, plasmin, FXII and FXa.
The formation of FXa by TF-FVIIa is almost immediately inhibited by tissue factor pathway inhibitor (TFPI).
FXa and its co-factor FVa form the prothrombinase complex, which activates prothrombin to thrombin.
Thrombin then activates other components of the coagulation cascade, including FV and FVIII (which activates FXI, which, in turn, activates FIX), and activates and releases FVIII from being bound to vWF.
FVIIIa is the co-factor of FIXa, and together they form the tenase complex, which activates FX; and so the cycle continues.

17. Contact activation pathway (intrinsic)
The contact activation pathway begins with formation of the primary complex on collagen by high-molecular-weight kininogen (HMWK), prekallikrein, and FXII (Hageman factor).
Prekallikrein is converted to kallikrein and FXII becomes FXIIa. FXIIa converts FXI into FXIa.
Factor XIa activates FIX, which with its co-factor FVIIIa form the tenase complex, which activates FX to FXa.
The minor role that the contact activation pathway has in initiating clot formation can be illustrated by the fact that patients with severe deficiencies of FXII, HMWK, and prekallikrein do not have a bleeding disorder.

18. Final common pathway Thrombin has a large array of functions:
Its primary role is the conversion of fibrinogen to fibrin by removal of the two small peptides (fibrinopeptides A and B) from fibrinogen, thereby creating the fibrin monomers that oligomerize.
In addition, it activates Factors VIII and V and their inhibitor protein C (in the presence of thrombomodulin), and
It activates Factor XIII, which forms covalent bonds that crosslink the fibrin polymers (transamidation) that form from activated monomers.
It activates platelets.
It activates the inhibitor of fibrinolysis TAFI
The actions of thrombin are remarkably diverse, with influence on inflammation, tumor metastasis, cell proliferation, and angiogenesis, among others.

19. Final common pathway
Following activation by the contact factor or tissue factor pathways, the coagulation cascade is maintained in a prothrombotic state by the continued activation of FVIII and FIX to form the tenase complex, until it is down-regulated by the anticoagulant pathways.

20. Important cofactors in the coagulation cascade
Calcium and phospholipid (a platelet membrane constituent) are required for the tenase and prothrombinase complexes to function.
Calcium mediates the binding of the enzyme complexes via the terminal gamma-carboxy residues to the phospholipid surfaces expressed by platelets, as well as procoagulant microparticles or microvesicles shed from them. Calcium is also required at other points in the coagulation cascade.
Vitamin K is an essential cofactor to a hepatic gamma-glutamyl carboxylase, the enzyme that adds a carboxyl group to glutamic acid residues on factors II, VII, IX and X (Vitamin K-dependent factors), as well as anticoagulant Protein S and Protein C.

21. Vitamin K cycle
Warfarin
Vitamin K
active
inactive

22. Regulation of the coagulation cascade
Both coagulation and fibrin formation must be confined to a smaller possible area to achieve local hemostasis in response to bleeding from injury without causing disseminated coagulation or impaired blood flow
Two systems work to reach it:
Endogenous anticoagulants: Plasma contains protease inhibitors that rapidly inactivate the coagulation proteins as they escape from the site of vessel injury: antithrombin, proteins C and S
Fibrinolysis: The central process is conversion of inactive plasminogen to the proteolytic enzyme – plasmin. Plasmin limits the extension of thrombus by proteolytic digestion of fibrin.

23. Important regulators (inhibitors) in the coagulation cascade
Protein C is a major physiological anticoagulant.
It is a vitamin K-dependent serine protease enzyme that is activated by thrombin into activated protein C (APC).
Protein C is activated in a sequence that starts with Protein C and thrombin binding to a cell surface protein thrombomodulin.
The activated Protein C, along with protein S and a phospholipid as cofactors, degrades the two important cofactors in enzyme complexes, FVa and FVIIIa. This slows coagulation by an order of magnitude. Quantitative or qualitative deficiency of proteins C or S may lead to thrombophilia.
Impaired action of Protein C (activated Protein C resistance), for example by having the "Leiden" variant of Factor V or high levels of FVIII also may lead to a thrombotic tendency.

24. Important regulators (inhibitors) in the coagulation cascade
Antithrombin is a serine protease inhibitor (serpin) that degrades the serine proteases: thrombin, FIXa, FXa, FXIa, and FXIIa.
It is constantly active, but its adhesion to these factors is greatly increased by the presence of heparan sulfate (a glycosaminoglycan on the enothelial cells) or the administration of heparins (different heparinoids increase affinity to Fxa and thrombin).
Quantitative or qualitative deficiency of antithrombin (inborn or acquired, e.g., in proteinuria) leads to thrombophilia.
Tissue factor pathway inhibitor (TFPI) limits the action of tissue factor (TF). It also inhibits excessive TF-mediated activation of FIX and FX.

25. Fibrinolysis
At any stage of blood clotting, the amount of clot depends on the balance between the activity of coagulation and fibrinolysis
Thrombosis occurs when the coagulation system is innaproprietly initiated and activated.
The fibrinolytic system counterbalance thrombosis.
Plasmin (serine protease) is generated by proteolytic cleavage of plasminogen, an inactive plasma protein synthesized in the liver.This cleavage is catalyzed by tissue plasminogen activator (t-PA), which is synthesized and secreted by endothelium.
Plasmin proteolytically cleaves fibrin into fibrin degradation products.

26. Fibrinolysis

27. Fibrinolysis

28. Fibrinolysis
inhibition of fibrinolysis
activation of fibrinolysis

29. The prothrombin time (PT) is the number of seconds required to make a clot starting from the tissue factor (TF)–Factor VIIa–phospholipid unit.
The activated partial thromboplastin time (APTT) is the number of seconds required to make a clot starting from the contact activation pathway. It is a partial clotting time because TF is not present.
Both pathways merge at Factor Xa, which forms an amplification unit with Factor Va and lipid.
Inhibitors of Factor Xa block the amplification unit and prevent thrombin formation.
Tests are available to measure the presence of prothrombin Fragment 1.2 generated during thrombin production, the inactive thrombin-antithrombin complex (TAT), fibrinopeptide A (FPA) released during formation of fibrin, and fragments of cross-linked fibrin released during fibrinolysis of the clots [fibrin degradation products (FDPs), D-dimers].

30. Laboratory monitoring of coagulation

31. aPTT

32. Prothrombin time (Quick assay)

33. Coagulation factors Number and/or name Function I (fibrinogen)
Forms clot (fibrin)
II (prothrombin)
Its active form (IIa) activates I, V, VII, VIII, XI, XIII, protein C, platelets
Tissue factor
Co-factor of VIIa (formerly known as factor III)
Calcium
Required for coagulation factors to bind to phospholipid (formerly known as factor IV)
V (proaccelerin, labile factor)
Co-factor of X with which it forms the prothrombinase complex VI
Unassigned – old name of Factor Va
VII (stable factor)
Name: Pro Convertin - Activates IX, X
VIII (Anti Hemophilic factor A)
Co-factor of IX with which it forms the tenase complex
IX (Anti Hemophilic Factor B or Christmas factor)
Activates X: forms tenase complex with factor VIII
X (Stuart-Prower factor)
Activates II: forms prothrombinase complex with factor V
XI (plasma thromboplastin antecedent)
Activates IX
XII (Hageman factor)
Activates factor XI and prekallikrein
XIII (fibrin-stabilizing factor)
Crosslinks fibrin

34. Related molecules Number and/or name Function von Willebrand factor
Binds to VIII, mediates platelet adhesion
prekallikrein
Activates XII and prekallikrein; cleaves HMWK
high-molecular-weight kininogen (HMWK)
Supports reciprocal activation of XII, XI, and prekallikrein
fibronectin
Mediates cell adhesion
antithrombin III
Inhibits IIa, Xa, and other proteases;
heparin cofactor II
Inhibits IIa, cofactor for heparin and dermatan sulfate ("minor antithrombin")
protein C
Inactivates Va and VIIIa
protein S
Cofactor for activated protein C (APC, inactive when bound to C4b-binding protein)
protein Z
Mediates thrombin adhesion to phospholipids and stimulates degradation of factor X by ZPI
Protein Z-related protease inhibitor (ZPI)
Degrades factors X (in presence of protein Z) and XI (independently)
plasminogen
Converts to plasmin, lyses fibrin and other proteins
alpha 2-antiplasmin
Inhibits plasmin
tissue plasminogen activator (tPA)
Activates plasminogen
urokinase
plasminogen activator inhibitor-1 (PAI1)
Inactivates tPA & urokinase (endothelial PAI)
plasminogen activator inhibitor-2 (PAI2)
Inactivates tPA & urokinase (placental PAI)
cancer procoagulant
Pathological factor X activator linked to thrombosis in cancer

35. Coagulation cascade

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48. Coagulation cascade