Haemostasis and Thrombosis Pre-Reading PDF

MED2100/MED2200 Haematology Active Learning Workshop PRE-READING Haemostasis and thrombosis Objectives After participating successfully in this session and related activities, you should have learnt to: understand the way that the haemostatic system is deranged in disease understand th...

MED2100/MED2200 Haematology Active Learning Workshop PRE-READING Haemostasis and thrombosis Objectives After participating successfully in this session and related activities, you should have learnt to: understand the way that the haemostatic system is deranged in disease understand the role of the coagulation pathway in normal haemostasis understand the effects of clotting factor deficiency on routine diagnostic coagulation tests interpret basic coagulation diagnostic tests in a clinical context understand the way that acquired coagulation defects can be distinguished from inherited problems on history introduction to basic concepts of anticoagulant treatment Instructions to Students The material should be read prior to the session. Part I Background Information: Haemostasis In order to understand the way that the haemostatic system is deranged in disease it is first necessary to appreciate how it functions normally. Blood vessels are lined on their luminal side with endothelial cells, which suppress the haemostatic mechanism. When an injury occurs to the blood vessel the endothelial cells are damaged exposing subendothelial matrix. The matrix contains elements such as collagen and tissue factor, which stimulate the haemostatic response. The haemostatic response includes the following: 1. platelet aggregation at the site of injury 2. activation of the coagulation pathway 3. vasoconstriction caused by release of local inflammatory mediators and cytokines Platelet aggregation and coagulation are complementary processes with interacting components. Activated coagulation factors, in particular thrombin, activate platelets. In response, activated platelets provide a negatively charged phospholipid surface, which is required for assembly of coagulation factors. 1  MED2100/MED2200 Formation of a primary haemostatic plug by platelets Platelets - Platelets arearetiny tiny (2-3µm) cells without anucleate cells, which a nucleus, measuring aboutare 2–3 produced micrometersininbone marrow size. They are from their - precursor produced in cell thethe megakaryocyte. bone Thousands marrow from a precursor of platelets cell called bud off fromThousands the megakaryocyte. the surface of of a single megakaryocyte. Platelets circulate in the blood for a few days before being destroyed in the platelets bud off from the surface of a single megakaryocyte. After entering the bloodstream, platelets circulate forsystem reticuloendothelial a few days before being (principally broken the down Normal spleen). in the reticuloendothelial blood contains system, from 150-400 x mainly 9 -1 in the spleen. Normal blood contains about 150 to 400 billion platelets per liter (150–400 × 10 L 10⁹ L⁻¹).. => Platelets normally Under normal show platelets conditions, little or no tendency do not to adhere stick to the to blood cells lining normal endothelial vessels, known cells. as Upon injury toendothelial the bloodcells. vessel wall the However, subendothelial when a blood vesselmatrix is exposed. is injured, Thislayer the underlying matrix has called thea high affinity for the large protein subendothelial matrix isvon Willebrand exposed. Factor This matrix has (vWF) a strong which is for attraction produced by endothelial a large protein called cells. VWF bound to von Willebrand matrix Factor (VWF),is which capable of binding is produced platelets,cells. by endothelial especially under conditions of high flow rate. After the initial tethering of platelets by vWF a series of other binding reactions occur When VWF binds to the exposed matrix, it can also bind platelets, especially when blood flow leading to stable adhesion of platelets to the site of injury. Platelet-platelet binding then leads is rapid. This initial tethering of platelets by VWF is followed by other binding reactions that tofirmly formation of the haemostatic plug. anchor the platelets to the injury site. Subsequently, platelets bind to each other, leading to the formation of the haemostatic plug. ⑦ The secondary activation of platelets leading to their irreversible binding at the site of injury involve the release by platelets of further activators (platelet agonists) such as the prostaglandin derivative thromboxane A2 and adenosine diphosphate (ADP). This secondary activation can be blocked therapeutically using agents which inhibit the enzyme cyclo- oxygenase (eg Aspirin) which is responsible for thromboxane A2 production. Alternatively, drugs which block the ADP receptor (clopidogrel, ticlopidine) are also used to block platelet activation. Von Willebrand Factor vWF is a large multimeric (made of multiple repeated units) protein, which is synthesised by endothelial cells, which line the luminal surface of blood vessels. Some of the protein is secreted directly into the blood or sub-endothelial matrix while the remainder is stored in cytoplasmic granules available for release at times of stress. vWF has multiple binding sites for collagen. When bound to collagen vWF becomes activated and binds platelet surface receptors avidly leading to the adhesion and activation of platelets at sites of vessel injury. vWF has another important role, which is to act as a carrier for the clotting Factor VIII. Without vWF, Factor VIII has a very short survival time in blood. & WF acts as a carrier for Factor F~ 2 * vWF activates for collagen  Proteins ↓ MED2100/MED2200 cleaved ↓ Clotting factors activated The coagulation reaction Normal blood contains an array of proteins, which take part in the coagulation reaction. Many of these proteins are enzymes which circulate in inactive forms (zymogens). Cleavage at specific sites within the protein activates the clotting factors allowing them to take part in the reaction. Other components of the coagulation system are co-factors which are not catalytically active but which accelerate the catalytic activity of other factors. Most clotting factors are made in the liver. It is also important to remember that the coagulation reaction can only work efficiently when the clotting factors are assembled on a charged lipid membrane such as the surface of an activated platelet. This in turn can only occur in the presence of calcium. * Coagulation can only occur The coagulation reaction can be thought of as occurring in several steps: in the presence of calcium. Initiation lining · The main goal of the initiation phase of clotting is to Endothelial The principal task of the initiation phase of produce small amounts of an enzyme called coagulation is the production of small quantities of damaged thrombin. the enzyme thrombin. ↓ When the lining of a blood vessel (the endothelium) Tissue factor At the outset, breach of the endothelium exposes is damaged, it exposes tissue factor, a protein exposed tissue factor, which is expressed by a wide variety ↓ found in cells outside blood vessels. Tissue factor of extravascular cells. Tissue factor is the key Binds to Factor 7 is crucial for starting the clotting process. It binds protein responsible for initiating the coagulation ↓ with Factor VII, a protein that is normally present in TF + Factor 7 activa reaction. Tissue factor binds and activates Factor tes Factor Y small amounts in the blood. When Factor VII - # VII which is present in trace quantities in blood. ↓ attaches to tissue factor, it becomes active and Activated Factor VII in complex with tissue factor then activates other proteins, like Factor X. Factor Factor X changes then activates downstream factors (Factor X) X can then help change prothrombin into thrombin, prothrombin thrombine to which movingin theturn areprocess clotting capable of activating prothrombin forward. to thrombin. Amplification Tiny quantities of thrombin are capable of activating other clotting factors and cofactors in a positive feedback reaction. If this positive feedback system is allowed to proceed then larger quantities of thrombin are produced. Clot formation If large enough quantities of thrombin are produced then cleavage of fibrinogen to fibrin will occur. Fibrin then spontaneously polymerises to form a clot. 3  MED2100/MED2200 · Regulation Thrombin When thrombingeneration also is produced, it also activates triggers Factors Sa < by & negativefeedback negative balance. cofactors down feedback proteolysing Va clotting important mechanisms, mechanisms These mechanisms and VIIIa. The helpers, to keepwhich clottingact thework procoagulant by breaking positive cofactors and Va and in and ta negative VIIIa. regulatory Both positive mechanisms (clot-promoting) operate and negative simultaneously (clot-limiting) processesand,happen depending at the sameon the strength of the initial stimulus and other time, and depending on how strong the original local factors, clotting signal is,the & coagulation the process either self terminate or progress to clot own or continue to form a clot. cascade will either stop on will its formation. Negative Negative regulators regulators of clotting of the include activated coagulation protein C, which,reaction include along with its activated partner Protein S, protein C which, together with its breaks down cofactors Va and VIIIa to reduce cofactor ProteinAnother clotting. S, degrades key negativethe procoagulant regulator is Protein C + Proteins cofactors Va and VIIIa. # Negative regulators : antithrombin III, which directly blocks thrombin, ↓ Factor Sa and a8 Factor Another Xa, major and other enzymes regulator negative involved in is antithrombin III which is a direct breaks inhibitor down of the enzymes clotting. Antithrombin is further activated by thrombin and Factor Xa as well as other coagulation serine proteases. OR The activity heparin) of heparin, a substance found on the surface of Antithrombin 111 (3) (activated by antithrombin is stimulated by heparin present on endothelial ↓cells or administered blood vessel cells or used as a medication to therapeutically. and Factor X prevent excessive clotting. blocks thrombin THE COAGULATION LABORATORY · There are several laboratory tests that are commonly used to examine the coagulation system in vitro. It is however essential to remember that the interpretation of a laboratory tests has to take into Several consideration lab tests the history are used to check how welland the physical examination of patients. Many patients have m abnormal in vitro blood’s clotting coagulation system studies works. However, and do not have a bleeding problem, and the converse is it's important also true. Isthat to remember imperative test resultsthat a don’t alone bleeding give thedisorder full is strongly supported by the patient’s history, family picture.history Doctorsandalso physical need to lookexamination. at the patient's history, family history, and physical examination, as In order to prepare samples for testing in the some people might have abnormal test results without laboratory blood is collected from the patient any actual bleeding problems, and vice versa. and immediately mixed with sodium citrate, which binds To prepare calcium a blood sample and prevents for testing, blood activation is collected from the patient and mixed of the coagulation reaction. Further calcium with sodium citrate. can beSodium added citrate binds to calcium, subsequently towhich allow stops the the clotting process for testing. Later, coagulation reactions to proceed. Citrated more calcium is added to let the clotting reactions blood is centrifuged in the laboratory to start again. separate The blood is the red cells, then centrifuged (spunleukocytes in a machine) andto platelets separate the components: red cells, white cells the from the plasma, which contains clotting (leukocytes), factors. and plateletsTheare separated clear plasmafrom the is separated from the blood cells plasma. The plasma, which contains the clotting and used in most coagulation factors, is usually used laboratory tests. If required for most coagulation tests. If the platelets needed, can platelets can bealso collected separately be separated and used from for the redtests specific cells that and checkleukocytes and used in how well the platelets assays function.of platelet function. 4  MED2100/MED2200 summ The prothrombin time This The is a simple Prothrombin Timecoagulation test where (PT) test measures citrated how quickly bloodplasma clots. In is thispre-warmed and a(plasma test, citrated plasma mixturewith of thromboplastin (tissue factor) and calcium added. A clot forms after approximately 12-18 added sodium citrate to prevent clotting) is warmed, and a mix of thromboplastin (also known as tissue factor) secs. Thromboplastin (tissue factor) is a lipoprotein which combines with factor VII to form a and calcium is added. A clot usually forms within 12–18 seconds. complex in which factor VII is activated. This results in the activation of factor X, the formation of the prothrombinase Thromboplastin is a proteincomplex and with that combines the Factor generation of thrombin. VII to activate it. ThisAactivated normal Factor prothrombin VII then time is triggers therefore Factor dependent X, which helps formonthe the presence of complex. prothrombinase normal concentrations of factors This complex generates VII, anaintact thrombin, tenase key enzyme in complex (factors X, V, II) and fibrinogen. Deficiency in any of these factors or the presence of blood clotting. an inhibitor against any of these factors will result in prolongation of the prothrombin time. A normal PT depends on the right levels of Factors VII, X, V, and II and fibrinogen. If any of these factors are In the orlaboratory, missing reduced, or ifthe thromboplastin something is blockingisthem, derived it willfrom rabbitfor take longer brain a clotor bovine to form, lung. in resulting Different a prolonged commercial preparations are available which when used according to the manufacturer's PT. instruction result in variable coagulation time. In an attempt to minimise inter-laboratory In labs, thromboplastin used in the test can come from animal sources, such as rabbit brain or cow lung. Since variability world-wide an international thromboplastin standard has been introduced. This different types of thromboplastin can give different clotting times, an international thromboplastin standard was allows the laboratory to standardise the thromboplastin they use. The standardised result created to make test results more consistent worldwide. obtained is referred to as the International standardized time. Most laboratories report their prothrombin Most labs reporttime PT asas a ratio a ratio, of the the comparing standardised patient’s PT patients prothrombin to a control time over PT, which is called the control the International value. This Normalized ratio Ratio is known (INR). The INRas allows the international PT results tonormalized be comparedratio (INR).between accurately In theory a prothrombin different labs. time reported as an INR should be reproducible anywhere in the world. This to a large extent is true The activated partial thromboplastin time =>> This examines the complete coagulation pathway with the exception of factor VII. Pre- warmed The APTT citrated test checks plasma the entire with is incubated bloodaclotting process and phospholipid except for Factor activator of VII. theFirst, citrated contact plasma factors. is The Immu warmed, then activator mixedawith is usually a phospholipid negatively charged and an activator. surface, whichThis activator serves is usually to activate the atwo negatively charged main contact coagulation factors (factors surface that activates FactorsXII, andXI XII and (known XI). Afteras ancontact appropriate incubation factors). After someperiod time,to allow is calcium foradded, the Factors and 12. formation of factor XIa, calcium is added and the time for clot formation monitored. With the and the time taken for a clot to form is recorded. addition of calcium, the XIa formed in the first incubation can now activate factor IX generating - The The IXa. Factor XIa produced latter in concertinwiththe first stepFVIII factor now and phospholipid form the Tenase complex which Factor 9a cleaves activatesfactor FactorX IX, generating Xa. Factor IXa. turning it into With help from Factor VIII and phospholipids, With the formation of factor Xa, the assembly ofFactor IXa forms the Tenase complex, which the next major coagulation complex, the prothrombinase X, activates Factor turning takes it intoThis place. Factorserves Xa. to activate Prothrombin producing Once Factor Xa is present, the next main thrombin Prothrombinasewhich actsprothrombinase, complex, to convert fibrinogen to fibrin and forms. This resulting in clot formation. complex activates prothrombin to produce Athrombin, prolonged APTT which thenisconverts the result of deficiency fibrinogen into offibrin, anyresulting of the above in clot named coagulation formation. factors. An inhibitor directed against these A prolonged factors APTT will also means result there in the could be a of the prolongation deficiency in one of these clotting factors, or APTT. there might be an inhibitor blocking them. The thrombin time This is a one step assay where thrombin is added to citrated plasma. The test examines the ability of exogenous thrombin to clot plasma fibrinogen. added thrombin Plasma fibrinogen Fibrin 5  - MED2100/MED2200 The thrombin time is prolonged in: a) Fibrinogen deficiency b) In the presence of thrombin inhibitors (e.g. heparin) c) In the presence of inhibitors of fibrin polymerisation. Differentiation between a deficiency in a coagulation factor and the presence of an inhibitor 2 - In Whenall the tests clotting indicated time aboveit could is prolonged, prolongation of the mean either clotting a lack time isfactor of a clotting a reflection of either (deficiency) or the a deficiency presence ofstate or an indication something that blocksofthe theclotting presence of an(an process inhibitor. To differentiate inhibitor). between To figure out which onethese it is, a two possibilities, mixing test is used. the mixing test is used. In this assay, varying mixtures of the patient and normal plasma are prepared and the coagulation tests performed on the mixed plasma sample. In a mixing test, the patient’s plasma is mixed with normal plasma, often in a 50:50 ratio. Then the Commonly clotting test ais50:50 mixture is used. mixed In case of a deficiency state one expects that this mixture ⑳ repeated on this sample. will yield a completely normal clotting time. In the presence of an inhibitor the time will remain If there is a deficiency in a clotting factor, the normal plasma provides the missing factor, and clotting prolonged. time should return to normal. Example: A patient’s APTT is 86 secs with a control of 36 secs. The 1:1 mix is 84 secs. This If there is an inhibitor, even with the normal plasma added, the clotting time will still be prolonged. result is consistent with an inhibitor responsible for the prolongation of the APTT. If on the other Example: -If a patient's APTT is 86 seconds (compared to a normal 36 seconds) and the 1:1 mix gives hand the APTT 84 seconds, this fully corrected suggests on mixing an inhibitor. But iftothe 36-38 secs, APTT then in the thissample mixed would have been corrects consistent to around 36–38 with a deficiency state causing the prolonged APTT. seconds, it would indicate a deficiency as the cause of the prolonged APTT. Prolongation of the APTT with normal prothrombin and thrombin times I Consider deficiency of factor XII, XI, IX, VIII X - X 7 Prolongation of the PT with normal APTT and thrombin times - ~ X 10 , 5 , 2 Heparin therapy Consider factor VII deficiency. (7) · -v ~ · Multiple deficiencies Prolongation of the PT and the APTT with normal thrombin time (10 , 5, 2) Here the defect must lie in the common pathway including deficiencies of factors X, V, or II. In practice the most common scenario for this include patients receiving Warfarin or patients with liver disease. In both situations several coagulation factors will be deficient. Prolongation of the APTT, PT and thrombin time The two most likely causes are Heparin therapy or the presence of a complex deficiency state (deficiency in several coagulation proteins including fibrinogen). A common scenario for this would be disseminated intravascular coagulation. Other possible causes include abnormal fibrinogen or fibrinogen deficiency. = The information derived from the simple screening test coupled with the clinical picture will The information determine what from a basiccoagulation additional screening test, combined testing with the is required. patient’s Thus symptoms, in patients with ahelps prolonged determine APTT, if more INR a normal clotting andtests are needed. a significant For example, bleeding history ifone a patient should hassuspect a prolonged APTT, a of a deficiency either factor normal INR, VIII, and IX, o of a history r XI. The mode bleeding, of inheritance a deficiency in Factor helps 8, 9, or in 11 isdirecting the clinician likely. Knowing how in deciding these conditions are inherited can also help the doctor decide which factor to test first. If a APTT which factor to assay first. Patients who are asymptomatic yet have prolonged with a normal patient shows noINRsymptoms should bebutsuspected as having has a prolonged APTTFactor and XII deficiency. a normal INR, it may suggest a Factor 12 deficiency. 6  MED2100/MED2200 Haemophilia · - Haemophilia results from a deficiency of one of the clotting factors, most often Factor VIII (Haemophilia Hemophilia is causedA)byor Factor IX (Haemophilia a deficiency B). The in a clotting factor, genesFactor usually for Factors VIII and IXA)are 8(Haemophilia or on the X chromosome and therefore only one copy of the gene is present in males. Factor 9 (Haemophilia B). The genes for Factors 8 and 9 are located on the X chromosome, so Female carriers of the abnormal gene are usually asymptomatic. Genetic tests including males only have restriction one copy fragment of each length gene. This isSouthern polymorphisms, why males with the blotting andabnormal gene show direct sequencing can usually identify symptoms, abnormalities while female carriersin the Factor typically VIIIhave do not or Factor IX gene symptoms. of carriers. Genetic tests,Alike significant restriction proportion fragment lengthof polymorphisms, haemophilia cases result from Southern new mutations blotting, and directand therefore can sequencing, the family history identify gene is normal. abnormalities in carriers. Some cases of haemophilia occur due to new mutations, so there The might beclinical no familypattern ofofbleeding history in severe haemophilia consists of bleeding into joints the disorder. (haemarthrosis), muscle (haematomas), bleeding from cuts or scratches which is typically delayed In severe or bleeding after haemophilia, surgery.include symptoms Joint and muscle into bleeding bleeding jointscan occur spontaneously (haemarthrosis), or after muscles minor trauma.bleeding (haematomas), The delay in bleeding from from small cuts orcuts occurs (often scratches because primary or delayed), haemostasis is mainly bleeding after mediated by platelet adhesion and vasoconstriction at the site of blood vessel injury. However, surgery. Joint and muscle the consolidation bleeding can of the haemostatic happenisspontaneously response dependent on theor after minor of generation injury. The by thrombin delay theincoagulation bleeding from cutsincluding proteins occurs because initial Factors VIII andclotting IX. is handled by platelet adhesion and vessel Onlynarrowing, but stable small amounts clotting of Factors VIIIneeds and IXthrombin production, are required which for normal requires Factors haemostasis 8 and and severe 9. haemophilia occurs when 120kg, < 50 kg or > 120kg, less < 50 kg or > 120kg, less less than 25% change than 25% change in than 25% change in in plasma drug plasma drug plasma drug concentration, no concentration, no dose concentration, no dose dose adjustment adjustment adjustment Age AUC for plasma drug AUC for plasma drug AUC 32% higher after concentration up to concentration 1.5 fold 65 years, no dose 2.0 fold higher after higher after 65 years, no adjustment though 65 years; use 110mg dose adjustment though twice daily if ≥ 75 years of age Concomitant antiplatelet Aspirin alone – Aspirin alone – caution Aspirin alone – caution agents caution Dual agents – increased Dual agents – increased Dual agents – bleeding risk bleeding risk increased bleeding risk Australian Stroke prevention in TGA registered TGA registered TGA registered Approval by non-valvular AF with ≥ 1 PBS authority PBS authority PBS authority indication risk factor (SPAF) Prevention VTE after PBS authority PBS authority PBS authority elective total hip or knee replacement Treatment of acute DVT TGA registered PBS authority PBS authority and prevention of recurrent VTE (Adapted from: Tran H, Joseph J, Young L, McRae S, Curnow J, Nandurkar HH, Wood P, McLintock C. New oral anticoagulants – a practical guide on prescription, laboratory testing and peri- procedural/bleeding management. Internal Medicine Journal 2014, 44(6):525-36) 13  MED2100/MED2200 Platelet Disorders INVESTIGATION OF THE PATIENT WITH A SUSPECTED PLATELET DISORDER Platelet count This is part of the full blood count. The presence of thrombocytopenia (low platelet count) should be confirmed by examination of a blood film which serves to confirm the presence of true thrombocytopenia. Once a low platelet count is identified it is important to work out the cause of the reduction in the count. To this end, the examination of other haematological parameters is essential. There are numerous causes of thrombocytopenia and the patient work-up will be primarily reliant on the history, physical examination and results of the full blood count. In general the classification of thrombocytopenia is based on the mechanism of reduction in platelet count: a) Excess platelet destruction, such as immune mediated, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura etc. b) Decreased platelet production, such as acute leukemia, aplastic anaemia etc. c) Platelet pooling, such as in patients with splenomegaly. d) Ineffective platelet production (thrombopoiesis) due to B12 and folate deficiency, myelodysplasia, etc. An increase in the platelet count (thrombocytosis or thrombocythaemia) is either primary or secondary. Reactive (secondary) thrombocytosis follows acute or chronic inflammation, infections, malignancy, splenectomy, and acute or chronic bleeding. Differentiation between reactive and primary thrombocytosis can at times be difficult. A thorough history and physical examination are essential. The presence of splenomegaly should point in the direction of a primary event. In the laboratory, assessment of platelet function may be of some help. Platelets from patients with primary thrombocythemia can have defective in vitro function, which helps identify that the cells are abnormal. Platelet aggregation This is examined in an aggregometer. The ability of platelets suspended in plasma (platelet rich plasma) to respond to a variety of agonists is examined. Commonly used agonists include ADP, adrenalin, thrombin, collagen, arachidonic acid and the antibiotic Ristocetin. The most common cause of abnormal platelet aggregation is the use of Aspirin or anti- inflammatory medications. These drugs modify platelet function in a reversible or, irreversible manner. The most com m on irreversible modifier of platelet function is A spirin. The use of a dose as low as 100 mg of this drug will inhibit platelet aggregation for up to one week (the life time of a platelet). It is therefore essential that patients are taken off Aspirin and all anti-inflammatory medications before the performance of platelet function studies. Platelet aggregation is dependent on: a) An intact platelet membrane structure b) Fibrinogen c) An intact prostaglandin pathway Abnormalities in any of the above will result in an abnormal platelet response. 14  MED2100/MED2200 The prostaglandin pathway This starts with the liberation of arachidonic acid from the platelet membrane. This essential fatty acid is oxidised by the enzyme cyclooxygenase resulting in the elaboration of several different forms of prostaglandin endoperoxides. These are subsequently converted to thromboxane A2 by the enzyme thromboxane synthetase. Thromboxane A2 is a very potent vasoconstrictor and mediator of platelet aggregation. It is a labile product and is rapidly broken down to thromboxane B2. Aspirin and other anti-inflammatory medications inhibit the enzyme cyclooxygenase. Aspirin irreversibly acetylates the enzyme and the defect lasts for the lifetime of the platelet. Other anti-inflammatory medications are reversible in their effects on the enzyme. The effect of aspirin on platelet function is characterised by failure of the platelet to demonstrate a secondary wave of aggregation in response to agonists such as adrenalin, ADP, and collagen in low concentrations. Arachidonic acid mediated platelet aggregation is completely inhibited by aspirin. 15