8131MED - Y1 Coagulopathy Lecture 04&08 March 2024.pdf

Inherited & Acquired Coagulopathy 8131Med Y1 4th March 2024 & 8th March 2024 Indu Singh [email protected] Learning Objectives  Overview & mechanisms of coagulation  Overview & mechanisms of fibrinolysis  Discuss the factors/regulators that limit clot formation and how alterations in this pr...

Inherited & Acquired Coagulopathy 8131Med Y1 4th March 2024 & 8th March 2024 Indu Singh [email protected] Learning Objectives  Overview & mechanisms of coagulation  Overview & mechanisms of fibrinolysis  Discuss the factors/regulators that limit clot formation and how alterations in this process can lead to pathology.  Diagnosis and treatment of venous thrombosis.  Explain the mechanism of action of the main drugs that modify haemostasis and thrombolysis.  Discuss the genetic and environmental factors that predispose to thrombosis. HAEMOSTASIS COAGULATION & FIBRINOLYSIS 4th March 2024 What is Haemostasis? Process by which bleeding is spontaneously arrested within the body Formation of fibrin clot or thrombus Breakdown & removal Main aim  reduce blood loss & maintain blood in fluid state within the vessels Involves four major systems Primary Haemostasis Vascular system (blood vessels) Platelets Secondary Haemostasis Coagulation system (procoagulant & anticoagulant) Fibrinolytic system Haemostasis Reactions internal or external damange collagen released, activate platelets and release serotinin also from platelet wall serotinin triggers vasoconstriction fibrin for stable clot Overall Picture Role of Platelets in Haemostasis Adhesion  Platelets bind to vascular sub-endothelium at sites of injury Activation  Upon adhering to injured vessel wall, platelet surface receptors mediate intracellular events resulting in internal phosphorylation of proteins and calcium mobilisation  Platelets undergo shape change with protrusion of membrane-bound cytoplasmic extensions Aggregation  Begins after activation induced cytoskeletal reorganisation is complete  Availability of ADP results in expression of the GpIIb-IIIa  Binding of fibrinogen allows for platelet-platelet interconnection Coagulant activities of platelets  Plasma membrane of activated platelets provides a phospholipid surface for assembly of tenase & prothrombinase complexes bridge between blood vessel wall and platelets release contents 2b3a to upper layer need to be sealed as blood oozes activated platelets can bind to other activated platelets platelets exposed to collagen narrowing of blood vessel bridge to other platelets initiate coagulation pathway reducing space increases blood pressure thrombolysis to clear clot binds to vWF Classical Pathways of Haemostasis  Intrinsic Pathway » Exposure to negatively charged surfaces, e.g. phospholipids, collagen, subendothelial surfaces » Contact initiation → IXa+VIIIa → Xa+V → IIa → fibrin clot changed - starts activation activated, then lead to factor X activation thrombin fibrinogen-fibrin clotting cascade only after activation  Extrinsic Pathway » Tissue damage & exposure to tissue factor (TF) » TF complexes with VIIa & initiates pathway » TF+VIIa →Xa+V →IIa → fibrin clot when wall breaks activates factor 7  Common Pathway » Intrinsic or Extrinsic activation → Xa+V → IIa → fibrin clot then common pathway Coagulation Cascade & Regulation Intrinsic Pathway Extrinsic Pathway “Contact green = intrinsic IX Activation” YELLOW - REGULATORS “TF Pathway” TFPI XI by negative charge activated XIIa Prekallikrein HMW Kininogen PL Ca2+ Common Pathway Prothrombin XIa activates factor IX Antithrombin X TF:VIIa ACT ON FACTOR X AND THROMBIN phospholipid IXa in blood PL, Ca2+(Tenase) VIIIa activate factor X Xa factor X activated - prothombinase to thrombin PL, Ca2+ (Prothrombinase) PROTHROMBIN - THROMBIN Va XIII Thrombin Activated Protein C, Protein S PREVENT FACTOR V AND VIII ACTIVATION need negative activators not normally in blood Fibrinogen requires thrombin Fibrin XIIIa Monomer XLFibrin Polymer Prothrombinase & Tenase complex Factor X and cofactor V  The prothrombinase complex consists of the serine protein, Factor Xa & the protein cofactor, Factor Va » The complex assembles on negatively charged phospholipid membranes in the presence of calcium ions » The prothrombinase complex catalyzes the conversion of prothrombin (Factor II) to thrombin (Factor IIa)  Extrinsic tenase complex: FVIIa binds to exposed TF forming the TF/factor VIIa complex activate factor X  Intrinsic tenase: FVIIIa/FIXa complex Regulation of Haemostasis  Fine balance between systems that control the formation & breakdown of clots » Coagulation system → proteins that lead to clot formation » Fibrinolytic System → proteins that lead to break down & removal of clot » Anticoagulation system → proteins that regulate coagulation & fibrinolytic system  This balance can be disrupted by » ￪ or ￬ levels of proteins » Abnormal protein structure/function » Abnormal cell (platelet or endothelial) structure/function » Anticoagulants, antiplatelet & thrombolytic drug therapies activated with trigger keep blood flowing normally and naturally coagulation proteins Factors that limit clot formation  Natural inhibitors of clotting factors: » Tissue Factor Pathway Inhibitor (TFPI) » Antithrombin III » Protein S regulators » Protein C » When activated, these proteins inactivate specific clotting factors » These regulatory mechanism serve to control the coagulation response and limit the extension of the clot Coagulation Cascade Regulation  Antithrombin » Serine protease inhibitor (SERPIN) & acts as pseudosubstrate » Inhibits IIa (thrombin), IXa, Xa, XIa, XIIa, kallikrien & plasmin » Heparin can ↑ AT inhibition ~1000x » Half life 60-70 hours  Protein C Vitamin K dependent, if low Not want to be too low » Vitamin K dependent glycoprotein » Thrombin:thrombomodulin complex cleaves  activated protein C (APC) thrombin receptor on endothelial lining thrombin attaches to form complex - breaks down and cleaves protein C » APC works with phospholipid, calcium, protein S to inactivate  Va + VIIIa  leads to ↓ thrombin generation inactivates co-factor V and VIII PROTEIN C ACTS HERE » tPA  ↑ fibrinolysis » Half life 6-7 hours REGULATES FIBRINOLYSIS SHORT HALF LIFE Coagulation Cascade Regulation  Protein S » Vitamin K dependent glycoprotein » Nonenzymatic cofactor for APC mediated inactivation of Va & VIIIa » APC independent inactivation by direct binding to VIIIa (tenase complex) & Va + Xa (prothrombinase complex) » Half life 96 hours Much longer half life Dosage and timing of medication taken into account  Protein Z & Z dependent protease inhibitor (ZPI) » Vitamin K dependent plasma protein » Z + ZPI = Z:ZPI catalyses inhibition of Xa (↑ ZPI ~1000x) requires protein C » Levels ↑ from birth & stabilise in adults Coagulation Cascade Regulation Further research needed for these proteins in re: thrombophilia  Heparin Cofactor II » Serine protease inhibitor (SERPIN) » ONLY inhibits thrombin depending on condition and blood tests - sometimes better as avoiding thrombus to bleeding or bleeding to thrombus » Levels ↑ by heparin, heparan sulfate or demratan sulfate  Tissue factor pathway inhibitor (TFPI) » Protease inhibition of Xa & TF:VIIa complex » Associated with lipoproteins (70-80%) & ↑ with age (? hormonal involvement) unknown, but hormonal involvement Fibrinolytic pathway released at same time when blood vessel injured rate of release and action is much slower than factor X and thrombin another protein if progressing slowly start breaking down fibrin when activated Clot = damage to blood vessels, to reduce bleeding THROMBOSIS clot from intact blood vessels, then released from clot - embolus DIAGNOSIS & TREATMENT Thrombosis  Most Common cause of death in developed world: CVD & Stroke  Formation of thrombus in blood vessel » Thrombus = clot » Embolus  moving throughout body  embolism into lungs - pulmonary embolism » Thrombophilia  susceptibility/predisposition increased platelets at risk of clot thrombus formation DVT - go into deep veins  Most people with thrombophilia do not develop thrombosis  Underlying cause is an imbalance between » Procoagulant + anticoagulant systems in blood stream » Profibrinolytic + fibrinolytic systems » Inherited (primary) or Acquired (secondary) at risk of thrombosis Alterations that lead to pathology  Thrombophilia also called hypercoagulable state increased platelets  The most common causes: need to be careful of risk, but not significant but higher if mutations » Factor V Leiden (a mutation in the F5 gene at position 1691) highest risk of thrombosis rule out inherited genetic predisposition » Prothrombin G20210A, a mutation in prothrombin (at position 20210 in the 3' untranslated region of the gene) » The rare forms of congenital thrombophilia are typically caused by a deficiency of natural anticoagulants {More details later} Inherited Thrombophilia Strong Indicators  Antithrombin deficiency       Protein C deficiency Protein S deficiency Activated protein C resistance Factor V Leiden Prothrombin 20210 Homocystinuria Supportive Indicators  ↑ I, II, VIII, IX & XI  Hyperhomocysteinaemia  Dysfibrinogenaemia  Hypofibrinolysis Supportive Indicators  Hypoplasminogenaemia & dysplasminogenaemia  ↓ protein Z & Z dependent protease inhibitor (ZPI)  ↓ TFPI  Sickle cell disease  tPA deficiency  ↑ PAI-1  Methylene tetra hydrofolate reductase (MTHFR) polymorphisms  XIII polymorphisms  ↑ thrombin-activatable fibrinolysis inhibitor (TAFI) Acquired Thrombophilia Strong Indicators  Malignancy blood vessles cut, then clot  Chemotherapy » Antiangiogenic therapies  Chronic myeloproliferative neoplasm (CMPN)  HIT coagulation tests not normal heparin can induce thrombocytopaenia  Nephrotic Syndrome  Intravascular coagulation & DIC  TTP  Oral contraceptives & oestrogen  Antiphospholipid syndrome  PNH  Wegner granulamatosis Supportive Indicators  Inflammatory bowel disease  Varicose veins  Systemic lupus erythematosus  Venous vascular anomalies  Progesterone therapy  Infertility ‘therapy’  Hyperhomocysteinaemia  HIV infection  Dehydration  EPO therapy Arterial Thrombosis white thrombus (venous = red thrombus)  Risk factors lifestyle factors » Smoking » Hypertension » ↓ HDL & ↑ LDL » Diabetes » Peripheral arterial disease » Family history of CVD  Treatment » ↑ homocysteine  folate » Antiplatelet therapies break down thrombus high molecular weight heparin » HMWH/LWMH » Surgery if in critical part of body or drugs cannot break down Arterial Thrombosis Atheroma Endothelial damage lead to platelets sticking to the wall Activation of coagulation Thrombin Fibrin Exposure of blood to subendothelial tissue collagen, tissue factor, ADP & vWf released Thromboxane A2 Platelet adhesion Platelet aggregation Plateletfibrin mass Arterial thrombosis Diet & Thrombosis Research Group, Department of Medical Laboratory Science, RMIT University Arterial Thrombosis increases pressure and blood pushed back to heart bumps where thrombi formed Source: http://www.familydoctor.co.uk/htdocs/CORONARY/CORON02_8.jpg (Accessed 13.08.06) Source: http://www.trauma.org/vascular/images/vein02.jpg (Accessed 13.08.06) Source: http://heme-coag.uthscsa.edu/wwwthromb97/30artthrom.gif (Accessed 13.08.06) Risk Factors for VTE (Venous thromboembolism) clot breaks off  Factor V Leiden  20-40% » APC + Va + IIa (thrombin) » ↑ when combined with other risk factors  Homocysteine  10% » Enzyme defect  endothelial cell dysfunction ↔ homocysteine ↑  Prothrombin 20210  6% » ↑ prothrombin generation  Protein C & S 5-8% » Anticoagulant role not fulfilled & is a ↑↑ risk factor  Dysfibrinogenaemia  3% » Accelerated clot formation  Antithrombin  2-4% » Anticoagulant role not fulfilled & is a ↑↑ risk factor  Dysplasminogenaemia  target but ≤ 5.0 & patient should be at 1.0 or below not bleeding Nil 1-2 days & ↓ dose reduce dose If INR is just above target, then ↓ dose INR 5.0 – 9.0 & bleeding absent Cease warfarin  identify cause of ↑ INR If at risk of bleeding  give vitamin K Check INR 24 hours later & monitor as soon as return to 5.0 - start warfarin again INR > 9.0 & bleeding absent Cease warfarin  identify cause of ↑ INR If at risk of bleeding  give vitamin K or Prothrombinex & FFP if indicated Check INR 24 hours later & monitor Resume warfarin when INR ~5.0 Any clinically significant bleeding Cease warfarin  identify cause of ↑ INR with warfarin implicated as If at risk of bleeding  give vitamin K or contributing factor Prothrombinex & FFP if indicated Check INR 24 hours later & monitor Resume warfarin when INR near target range Modified from MJA Volume 181:9; 492 Nov 2004 Therapeutic Ranges for Warfarin Clinical Indication Deep vein thrombosis (DVT) Pulmonary embolus (PE) Atrial fibrillation (AF) Cardioversion Post surgery – TKR/THR + others Recurrent venous thromboembolism AVR/MVR Thromboembolism related to antiphospholipid syndrome + others Target INR Range don't restart warfarin until back to 2-3 antibodies against coagulation factors high APTT and PT 2.0 – 3.0 for these conditions 3.0 – 4.0 Mechanism of Action of Direct Thrombin Inhibitor bind at calaytic site also to Argatroban binds directly to and inhibits the active site of free and fibrin/clotbound thrombin. Antithrombin is not involved in this reaction. Bivalirudin & dabigatran directly inhibit thrombin by interacting at the same site but with their own specific binding characteristics. Mechanism of Action of LMWH - Anti-Factor Xa Blocks factor X unfractionated blocks directly prevents activation of factor lowmolecular weight blocks clot formation Antithrombin (AT) molecules bind a pentasaccharide region on heparin polysaccharide chains, producing an allosteric change that activates AT, facilitating FXa binding. endothelial wall repaired - clot removed only when endothelial wall break down Need to break down plasminogen stop unnecessary conversion in blood, start acting on plasminogen if clot in vessel convert plasminogen to plasmin blocks plasminogen breaks down fibrin in clot needs to see degradation of factor V and VII plasminogen works slowly can stop breaking down fibrin plsmin acts faster - fibrin fibrinogen degradation double bond for single fibrins into complex factor XIII - strengthens separation - release D-dimer into blood Abnormal Fibrinolysis  Fibrinolysis removes fibrin from intra & extravascular sites  Defective fibrinolysis can have catastrophic clinical consequences in terms of both bleeding & thrombotic events plasminogen acts faster - keeps bleeding and clotting as clot not stabilised  Excessive liberation of plasminogen activators » Ca prostrate » Extensive surgery too much cutting of blood vessels  Impaired fibrinolytic inhibition Or too much antiplasmin - plasmin will not break down fibrin  Streptokinase or urokinase therapy from kidney cells  Some snake venoms anticoagulant effect also fibrinolytic effect  Associated with DIC (secondary) if therapy - increases Abnormal Fibrinolysis  Plasmin (serine protease)  digests a wide variety of proteins breaks down proteins » Fibrin » Fibrinogen » Factor V » Factor VIII  Plasminogen activator released by vessel wall or tissues after trauma damaged blood vessel might have atherosclerosis in blood vessel or fatty streak - damage » Exercise suddenly a lot of exercise when sedentary or too high rates » Emotional stress can also affect flow Thrombolytic Therapy  Treatment that helps break down formed thrombus  All work by activating fibrinolytic system » Plasminogen  plasmin  Used to prevent post thrombotic syndrome & post myocardial infarction (heart attack)  Includes the following » Streptokinase  Derived from beta haemolytic streptococci » rt-PA  recombinant tissue plasminogen activator  Affinity for fibrin  Expensive  Aspirin may be used in combination  Needs to be closely monitored clinically  NO laboratory control for short term treatment acts like t-PA easy to control and adjust needs to be clinically monitored Mechanism of action thrombolytic drugs  Fibrinolysis - breakdown fibrin in the clot (natural process)  Thrombolysis – dissolution of the thrombus (therapeutic) fibrin v thrombus  Convert thrombus bound plasminogen to plasmin thus lysing the clot  Most fibrinolytic agents work by cleaving the Arg/Val bond in inactive proenzyme - plasminogen to form active enzyme – plasmin  Most used agents that act as substitute for naturally occurring t-PA are: » Alteplase (genetically engineered copy of the naturally occurring t-PA) » Reteplase (recombinant modified form of t-PA with longer half life than t-PA) no evidence to show one drug better than other » Streptokinase (a polypeptide derived from beta-hemolytic streptococci bacteria) » Urokinase (an enzyme produced by kidneys and extracted from human urine) Thrombolytic Therapy alpha 2 antiplasmin not in body, given as therapy Source: http://www.ovc.uoguelph.ca/BioMed/Courses/Public/Pharmacology/pharmsite/98409/Blood/hem_thromb.html Environmental & Genetic Risk Factors for VTE Crous-Bou M, Harrington LB, Kabrhel C. Environmental and Genetic Risk Factors Associated with Venous Thromboembolism. Semin Thromb Hemost. 2016;42(8):808-820 Genetic factors that predispose to thrombosis  Factor V Leiden mutation  Prothrombin G20210A mutation  Protein C deficiency std genetic conditions  Protein S deficiency  Antithrombin deficiency  Antiphospholipid syndrome/antibodies (anticardiolipin antibodies, anti-beta 2-glycoprotein antibodies, or lupus anticoagulant) Factor V Plasma glycoprotein: Mol wt 330kDa & 2224 amino acids Coded by a complex 25 Exons - 80kb gene on chromosome 1 Activated factor V facilitates conversion of prothrombin to thrombin Circulates as a single-chain protein in a precursor in active form Converted to an active 2-chain form by thrombin or factor Xa Thrombin cleaves it at 3 separate sites to activate factor V Following cleavage, the 2 chains are linked via a divalent metal ion bridge also requires protein C and S to activate- regulators Factor V Leiden - impact on coagulation Factor V Binds to phospholipid surface via the light chain It is inactivated by activated protein C & its cofactor protein S Predominantly synthesized in the liver (plasma factor V) and megakaryocytes (platelet factor V stored in alpha granules) Factor V has a binding protein in platelets (multimerin) which is analogous to vWF for factor VIII Plasma concentration of factor V is about 7-10ug/ml with a half life of approximately 12 hours Factor V Leiden mutation leads to abnormal Factor V activity & APC resistance activated Protein C resistance Inactivation of mutant 506 FVa:Q FV Leiden Mutation (V Arg 506 Gln) Eliminates cleavage site 506 Prevalent in about 2–13% of population Accounts for about 90% - APC Resistance Accounts for about 20–60% of VTE cases FV Leiden (single point mutation) APC cleavage sites 306 506 Heterozygotes have 2 to 5-fold increased thrombotic risk 679 Homozygotes have 80 to100-fold increased thrombotic risk greater risk FVa heavy chain 2+ Ca FVa light chain Mutation results in a 10-fold lower inactivation rate of FVa Prothrombin G20210A mutation Mutation in factor V & prothrombin gene are associated with thrombophilia (DVT) G20210A variant of prothrombin gene & G1691A polymorphism in Factor V gene (APCR) present in about 1/5th population with family history of venous thrombosis Prevalence in normal population approximately 3% G → A translation at nucleotide 20210 in prothrombin gene Leads to abnormal RNA metabolism increasing Factor II (prothrombin) levels and clot formation – however, this is unreliable as a screening test relative increase in prothrombin Most common lab tests to detect this mutation are PCR coupled with restriction-endonuclease digestion, gel electrophoresis & RT-PCR Protein S Protein S (PS) is one of many natural anticoagulants that keep your clotting mechanism in check It is the cofactor stimulating activity of Protein C, APC and TFPI 60% is bound to C4B-binding protein (inactive form) PS Deficiency can lead to venous thrombosis Acquired deficiency due to liver or kidney or inflammatory bowel diseases also seen in pregnant women Single-chain vitamin K dependent protein is synthesized by hepatocytes, megakaryocytes and endothelial cells Gene for protein S (PROS1) is found on chromosome 3q11.1 Nearly 300 mutations in this gene result in autosomal dominant hereditary thrombophilia need to be monitored Protein C Factors Va & VIIIa are powerful cofactors in coagulationenhancing activity of serine proteases (Factors VII, IX, X, XI & XII) acts on Vitamin K dependent proteins Both factors Va & VIIIa specifically inactivated by components of Protein C pathway & it’s cofactor Protein S Protein C is the key inactivating enzyme Single-chain vitamin K dependent protein (other vit K dependents- prothrombin, factors VII, IX & X) Synthesized by Liver Activated protein C resistance if mutated with Factor V Leiden Protein C Gene for protein C found on chromosome 2 is 11kb of DNA with 9 exons & 8 introns Thrombin binds to thrombomodulin on the surface of vascular endothelial cells. Thrombin/Tm complex activates protein C Protein C is activated by cleavage at Arg 169-Leu170 Activated protein C (APC) is inhibited by the specific inhibitor, Protein C inhibitor (PCI) added risk Protein C Deficiency Normal Type I Type II PC PC PC PC PC PC quantitiative normal PC PC PC PC Normal molecules Absent molecules Variant molecules Normal activity & antigen Reduced antigen & reduced activity Normal antigen but reduced activity Functional & antigenic assays will detect Functional assay will detect One mutated copy of gene Antigenic assay will NOT detect APC Resistance Common in the general caucasian population (~5%) with Autosomal dominant inheritance Most common cause of hereditary thrombophilia (~30%) A G-to-A base substitution at nucleotide 1691 in exon 10 of the Factor V gene linked to APCR Can be hereditary or acquired APC is first step of inactivating the activated FV protein & downstream decreasing affinity for FX APCR prolongs inactivation of this molecule (can not cleave FVa & FVIIIa) APC Resistance alone is not a significant risk factor, but combined with other risk factors, greatly increases risk of thrombosis combined - increases thrombosis Take Home Message  VTE General Risk Factors: Age, gender, family history, obesity, cancer, oral contraceptives, HRT, pregnancy, immobilization, major surgery & increased coagulation factors (particularly FVIII, FIX & FXI)  VTE Genetic Risk Factors: FVL, Prothrombin 20210A, non “O” blood group » Documented evidence suggest about 19682 SNP’s identified in 10887 genes associated with DVT by Genome Wide Association Study (GWAS) in 2009  Genetic risks have been added to clinical prediction to show better discriminating ability » However, cost effectiveness of using genetics in risk factor prediction should be considered relative to clinical benefit.  In high-risk population, prediction model may be helpful in improving prophylaxis Factir V Leiden higher risk Thank you Questions???? 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