Lecture 8, 9, 10 PDF

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Lecture 8, 9 and 10 Haemostasis Content Table         General points Vascular integrity Platelets Coagulation factors Coagulation inhibitors Fibrinolysis Laboratory tests and drugs Disorders of haemostasis General points     The role of haemostasis is to maintain a balance between normal...

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Lecture 8, 9 and 10

Haemostasis Content Table        

General points Vascular integrity Platelets Coagulation factors Coagulation inhibitors Fibrinolysis Laboratory tests and drugs Disorders of haemostasis

General points   

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The role of haemostasis is to maintain a balance between normal blood function and minimal blood loss following an injury This is achieved by keeping clotting and bleeding at a good balance 5 main components work together for haemostasis o Vascular integrity o Platelets o Coagulation factors o Coagulation inhibitors o Fibrinolysis

Endothelium o Maintains vascular structure for haemostasis o Produce anti haemostatic influences  Prostacyclin and nitric oxide (NO)  Vasodilatory properties  Good endothelium prevents adhesion in platelets o Endothelial cells produce  Von willeband factor (vWF) which binds to factor VIII

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 Tissue plasminogen activator (TPA) Ratio of blood components Erythrocytes : Platelets : Leukocytes is 700:40:1

Vascular integrity   

Response is vasoconstriction Effect is immediate Effect lasts for a few minutes before other mechanisms take over

Platelets        

Formed in the bone marrow Formed from fragmentation of megakaryocyte cytoplasm Each megakaryocyte can create 4000 platelets Size: 2-4μm Lifespan: 9-10 days Normal platelet count: 150 – 400 x109 /L o Transfusion only required at >50x109/L Destroyed in spleen and liver by specialized macrophages (Kupffer cells) Platelets do not have a nuclear and can therefore not repair themselves

Platelet structure, components, and their function 

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Glycocalyx o Structure made from glycoproteins  e.g. GPIa, GPIb, GPIIb o Allows the adhesion of platelets to surfaces (e.g. collagen) that are expressed in damaged vessels Glycogen o Energy store Dense tubular system o Site of prostaglandin and thromboxane A2 synthesis Submembraneous filaments (platelet contractile protein) o Makes platelet aggregation irreversible Granules o Electron dense granules (δ granules)  Contains: Ca2+ (e.g. useful in haematopoeisis), ADP, ATP, serotonin (chemical vasoconstrictor) o α granules  Contains: platelet derived GH, fibrinogen, vWF, factors V and VIII Open canalicular system o Allows easy and fluid granular release

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Plasma membrane

Platelet production and regulation 

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Production pathway: Endomitotic synchronous nuclear replication o Cytoplasmic volume increases as number of nuclear lobes increases in multiples of 2 (division+growth) o Cytoplasm becomes granular o Granular content released as platelets o From stem cell to platelet it takes 10 days Regulation o Thrombopoeitin (TPO) stimulates platelet production  TPO is degraded and internalized by platelets (negative feedback) o IL-3 and GM-CSF stimulate myeloid precursor cells  Stem cell Common myeloid progenitor  [Only GM-CSF]* Myeloblast ([+variable]* basophil, neutrophil, eosinophil, monocytes)  [+EPO]* Erythrocyte  [+TPO] Megakaryocyte (thrombocytes/platelets) *Not in curriculum for this lecture. Just know that TPO causes the conversion of the common precursor into megakaryocyte instead of other things

Platelet clot formation      

GPIa will directly adhere to the collagen surface GPIb adheres to vWF molecules assosciated with collagen from broken endothelial cells GPIIb associates to GPIIIa on the plasma membrane which adheres to vWF Platelets secrete ADP and thromboxane A2 to increase aggregation Platelets swell due to ADP (facilitates adhesion) Positive feedback causes further ADP and TXA2 release until a platelet plug is produced

Coagulation factors 

Coagulation factors: Co-factors, serine proteases, coagulation related proteins Coagulation factor

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Description name

Active form

I

Fibrinogen

Fibrin

II*

Prothrombin

Serine protease

III

Tissue factor

Co-factor

V

Labile factor

Co-factor

VII*

Proconvertin (stable factor)

Serine protease

VIII

Antihaemophilic factor

Co-factor

IX*

Christmas factor

Serine protease

X*

Stuart-Prower factor

Serine protease

XI

Plasma thromboplastin antecedent

Serine protease

XII

Hageman factor

Serine protease

XIII

Fibrin stabilizing factor

Serine protease

*Vitamin K dependent Coagulation cascade o [Initiation 1 – extrinsic (on the membrane/exterior)] o Initiated by tissue factor (TF) o TF is not normally in contact with the blood o It becomes exposed to the blood upon injury/damage o TF binds to FVIIa and Ca2+ o FX associates to [TF+FVIIa+Ca2+]complex and becomes converted to FXa o FXa down regulates [TF+FVIIa] activity by activating Tissue factor pathway inhibitor T(FPI) o (negative feedback)

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FXa assosciates with FII and Ca2+ FII is converted to FIIa [Amplification 2 – intrinsic (on the interior of the cell)]

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[Propagation 3 - intrinsic] Formation of intrinsic tenase complex

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Formation of prothrombinase complex

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Burst of thrombin generation

Coagulation inhibitors 

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Tissue factor pathway inhibitor (TFPI) o Inhibits VIIa and Xa o 1st inhibitor to act o Found in plasma and platelets o Accumulation is due to platelet activation Antithrombin III o Inhibits serine proteases. Mainly factor Xa and thrombin o Synthesised in the liver Protein S (PS) and Protein C (PC) o Vit K dependent o Synthesised in the liver o protein S  PS inactivates Va and VIIIa o Protein C (when activated)  Enhances PS action  Helps with fibrinolysis (clot breakdown) by inactivating tPA* inhibitor  Inactivates factors Va and VIIIa *tPA = tissue plasminogen activator

Fibrinolysis

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Plasminogen --[tPA][XIIa][Streptokinase]*--> Plasmin Fibrin –[Plasmin] fibrin degradation products *Powerfully enhances the reaction and can be administered Plasminogen --[Tissue plasminogen activator inhibitor-1 (tPAI-1)]--> α2-antiplasmin

Laboratory tests and drugs 

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Bleeding time o Normal bleeding time using “Ivy template” is 3-8 minutes o Abnormal bleeding time is caused by:  Abnormal platelet function  Low platelet count Prothrombin time (PT) o Measurements of factor VII, X, V, prothrombin and fibrinogen o Normal PT is 10-14 (time for blood to clot) o Prolonged PT can be due to liver disease and during anticoagulant treatment (OAT) (e.g. warfarin) o PT is standardised as international normalised ratio (INR) – A measure that monitors OAT  This means that the sensitivity of the tissue used (see below) is standardised internationally o Method  Recombinant tissue factor/thromboplastin (used to be extracted from animals) is mixed with human plasma  Interactions between the two causes the blood to clot Activated partial thromboplastin time (APTT) o Measurements of factors VIII, IX, XI, and XII but also X, V, prothrombin and fibrinogen (see above) o Normal APTT is 3-40 seconds o Not internationally standardised o Prolonged APTT is caused by haemophilia, and heparin treatment Warfarin o Initially a rat poison o Vit K antagonist. Decreases Vit K dependant factors at varying degrees o Treatment is monitored using INR every 6 weeks (or more if unstable) o INR therapeutic range is 2-4, 2-3 for most conditions and 2.5-3.5/4 (depending on the country) with mechanical heart valves o Various foods and drugs affect INR (e.g. antibiotics) o Action is not immediate o Does cross the placenta o Prescribed for e.g. atrial fibrillation, deep vein thrombosis, heart valve replacement o Conclusion: Hard to control and administer on a wide range of patients Heparin o Stimulates antithrombin III action on thrombin and factor Xa o Immediate action o Low molecular weight:  Administered via subcutaneous injections  Highly bioavailable (95%) o Used to prevent clots during surgery o Does not cross the placenta New antithrombotic drug targets (some still in development) o Theoretically any clotting factor in the enzyme cascade could be targeted o Only thrombin and FXa are common to both pathways meaning they are possibly more efficient drug targets for anticoagulation  In model systems, FXa was shown to activate clotting over a wider concentration

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Thrombin is the final effect of blood coagulation and the most potent platelet agonist (platelet activator) Rivaroxaban (Xarelto)  Gaining NICE approval for a growing number of conditions  10x more expensive that warfarin  No monitoring required  Initially the drug lacked an antidote = risk of overdose  Acts on FXa Direct thrombin inhibitors  Dabigatran etexilate (Pradaxa)  Only oral administration  Ximelagatran (Exanta)  1st oral direct thrombin inhibitor  Investigated for oral use with no monitoring  Taken off the market due to liver damage reports Antiplatelet agents  Aspirin  Irreversible COX inhibitor  Inhibits thromboxane A2 production  Used long term/low dose to prevent heart attacks, strokes, and blood clots in high risk individuals  Used short term/high dose after a heart attack to prevent another heart attack or the death of cardiac tissue  ADP receptor inhibitors  Ticlopidine o Used in patients that:  Cannot tolerate aspirine  Have a high risk of stroke o Side effects include risk of neutropenia  Clopidogrel (Plavix, BMS, Sanofi-Aventis) o Action may be related to ADP receptor on platelet membrane o Indications  Prevention of vascular ischemic events in patients with symptomatic atherosclerosis  Myocardial infarction  Used in combination with aspirin to prevent thrombosis after intracoronary stent o Clopidogrel is the drug of choice presently. (2nd top selling drug*)  May be used with anticoagulants

Disorders of haemostasis Abnormalities of the vessel wall 

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Characterized by vascular bleeding o Easy bruising and purpura o Little bleeding from small blood vessels and in the skin and mucous membrane Bleeding time test and other haemostasis tests are usually normal/healthy Type of vascular disorders o Hereditary (Hereditary haemorrhagic telangiectasia)  Affects 1.2m people worldwide (uncommon)  Affects men as equally as women

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Autosomal dominant (not sex linked) Defects in at least 3 genes. But only one gene causes the disorder in any single family Abnormally thin capillary walls Symptoms (depending on which gene was inherited the severity can be from mild to severe)  Dilated microvascular swelling that can easily rupture (telangiectasia) o Nose tongue and lips o Nosebleeds and gastrointestinal blood loos o Chronic iron deficiency is frequent o 90-95% of cases the patient has a few telangiectasia on the skin of the faceand/or hands by middle age o 90-95% nosebleeds by adulthood (daily=severe / infrequent=minor) o 20-25% bleeding in the stomach or intestines but rarely before 50yo  Treatment:  Embolization (inducing a block in the blood flow)  Laser treatment (e.g. nosebleeds)  Tranexamic acid (e.g. dental treatment)  Other hereditary vascular disorders:  Connective tissue disorders  Giant cavernous haemangioma Aquired  Causes: infections, drugs reactions, trauma, old age, steroid use  Categories  Purpura simplex – Common benign disorder in women of child bearing age  Senile purpura – Caused by a loss of skin elasticity and vascular collagen atrophy after age. Purpura usually in forearms and hands  Infection associated purpura – Bacterial or viral infections o e.g. Measles pathogens can cause vascular damage

Platelet disorders  

Small (but larger than vessel wall abnormalities) bleeding into the skin and mucous membrane Thrombocytopenia (opposite of thrombocytothemia) o Deficiency in platelet number (>150x109/L) o Caused by  Failure of platelet production  Most common cause of thrombocytopenia  Usually caused by bone marrow failure due to o Aplastic anaemia or leukaemia o Drug/viral induced toxicity  Diagnosis: o Clinical history o Peripheral blood count o Blood film o Bone marrow examination  Increased destruction of platelets  Mainly caused by autoantibodies (antibodies produced by the body) attaching to the platelet surface  Two types of autoimmune (idiopathic) thrombocytopenia purpura (ITP) o Chronic ITP  Relatively common  Mainly in 15-50yo women  Asymptomatic or insidious onset of bleeding

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Mechanism:  Autoantibodies present in plasma and on platelet surface  Autoantibodies usually target glycoprotein IIb/IIIa or Ib expressed on platelets  When sensitised/marked it gets destroyed in the spleen and liver by macrophages  Platelet lifespan is reduced to as little as a few hours o Acute ITP  Affects kids under 10  Majority of onset happens following vaccination or a viral episode (chicken pox, measles)  Post viral cases occur when a viral antigen is absorbed on the platelet surface causing IgG antibodies to attack the platelet reducing platelet count to >20x109/L  Spontaneous remission is usual  Minority of cases develop chronic ITP  Sequestration (abnormal distribution) of platelets  In a normal state the spleen contains ~30% of all platelets  Up to 90% of platelets may be sequestered in the spleen o Causes splenomegaly (enlargement of the spleen) Thrombocytopathy o Consided when clinical signs of thrombocytopenia are present but platelet count is normal o Hereditary  Rare but can interfere with each phase of the platelet reaction (activation, adhesion, secretion, aggregation) o Acquired  Much more common  Aspirin induced thrombocytopathy  Irreversibly inactivates COX enzyme  Thromboxane A2 –[COX]Arachidonic acid  This results in platelet aggregation being inhibited  Longer bleeding time  Leads to haemorrhaging in patients with thrombocytopenia  Haematological malignancy  E.g. acute myeloid leukaemia  E.g. any myeloproliferative disorders and myeloma Diagnosis of platelet disorders o Blood count and blood film examination  If normal blood time then further diagnostics are done for thrombocytopathy  If prolonged blood time bleeding time then the defect is usually acquired (not hereditary) o Bone marrow biopsy to ascertain failure of platelet production in thrombocytopenic patients o Patients with hereditary defects require further testing to define specific abnormality Research o The mechanism for bone marrow cell over-stimulation was identified (increase platelet production) o TPO is responsible for signalling bone marrow cells to produce platelets o Exact cells that responded to this signal are unknown o Thrombopoeitin receptor (Mpl) studies on blood cells in the bone marrow allowed the identification of the exact cells involved in making platelets o Study reveals that TPO did not directly stimulate megakaryocytes but several generation old precursors (stem cells and progenitors)

Defective blood coagulation

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Bleeding is often into joints (haemarthrosis) or soft tissues o Haemarthrosis causes a restriction in movement and significant pain Hereditary o Haemophilia A  Most common hereditary clotting factor deficiency  Deficiency in FVIII  Caused by a mutation that leads to o Under-production due to mutation o Clinical syndrome of haemophilia  Prevalence is 30-100 per million  X-linked recessive disorder (sex linked)  All men with defective gene will have haemophilia  All sons of haemophiliac men are normal  All daughters are carriers  Can be a spontaneous mutation despite no family history  Clinical features vary depending on degree of deficiency in FVIII  Severe haemophilia: o Bleeding into muscles (less freq.) and joints (more freq.) o Knees elbows and ankles most commonly affected o Majority of bleeds require treatment o Pain in affected areas o Intracranial haemorrhaging (main cause of death from disease)

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Treatment  FVII replacement therapy o Risk: e.g. HIV-contaminated donations (old)* o Usually recombinant FVIII is used  For milder cases: 1-amino-8-D-arginine vasopressin (DDAVP) is used to mobilize FVIII from endothelial cells  Treatment can include a combination of both  Gene therapy – Phase 1 trials carried out  Diagnosis  Prolonged APTT o Identifies a deficiency in clotting factors but cannot specifically detect FVIII deficiency  Confirmed by FVIII clotting assay  DNA technology can be used for carrier detection and antenatal diagnosis  Chorionic biopsies at 8-10 weeks of gestation provide DNA for analysis Haemophilia B (christmas disease)

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X-linked recessive (see above) Mutation leads to FIX deficiency Prevalence: 15-20 per million Clinically undistinguishable from haemophilia A Diagnosis:  Prolonged APTT  Confirmed after FIX clotting assay  Treatment: FIX replacement therapy von Willerbrand’s disease  Prevalence:  1 per 100 – no symptoms  1 pet 10,000 – clinically significant  Usually autosomal dominant  Mutation in the von Willerbrand factor (vWF)  Most patients are heterozygous for VWF gene  vWF is a large protein found in the plasma  Promotes platelet adhesion to damaged endothelium and other platelets  Carrier for FVIII  Classification:  Type 1 and 3: Partial reduction or nearly complete absence of VWF molecules (number)  Type 2 : Abnormal form of the protein (function)  Diagnosis  Prolongued APTT  Reduced FVIII clotting activity  Reduced plasma VWF  Impaired platelet aggregation...