Hereditary platelet function defects
Platelet physiology § Megakaryopoiesis is regulated by TPO (synthesised by liver) § TPO binds receptor, c-mpl on circulating platelets and BM megas and is then internalised and degraded § Total mass of cells thus determines free TPO levels and regulates megakaryopoiesis and platelet production § Mean life span of 10 days § Upon vessel wall damage § Adhesion § vWF facilitates initial adhesion via GP1b/IX/V complex § slows down platelets and allows binding via GPIIb/IIIa (via vWF) and GPIa/IIa (via collagen) § Reduced GP1b/X/V = Bernard Soulier § Reduced GPIIb/IIIa = Glanzmann’s thrombasthenia § Aggregation and secretion § Activated by a range of agonists § ADP, thrombin, thromboxane § Receptor stimulation results in G protein interactions that trigger intracellular metabolic pathways § Shape change via cytoskeletal changes § Release of alpha and dense granule contents § Stimulation of phospholipase A2, liberation of thromboxane A2 and activation of GP IIb/IIIa receptors
Suggestive history § ISTH § 2 or more of the following without blood transfusion or § 1 or more of the following with blood transfusion or § 1 symptom recurring on 3 distinct occasions § Nose bleeding >10 minutes in absence of trauma § Cutaneous bleeding with no or minimal trauma § Prolonged bleeding from trivial wounds >15 minutes § Oral cavity, including tooth extraction, post tonsillectomy etc. bleeding requiring medical attention § Spontaneous GI bleeding § Menorrhagia or bleeding from other mucosal sites § Positive family history § 2 or more distinct bleeding sites § A single bleed so severe as to require blood transfusion § Need to stop certain drugs before testing (aspirin, clopidogrel, NSAIDs, antihistamines, beta-blockers, penicillin / cephalosporins. Aminophylline and some foods e.g. garlic)
Investigation FBC § +/- citrated sample
Blood film § Platelet size
Coagulation screen § VWD 2N
Platelet adhesion testing PFA-100 § Aspirates whole blood under constant vacuum through a capillary and a microscopic aperture within a membrane § Membrane is coated with collagen and either ADP or epinephrine § Platelet plug forms at the aperture and eventually occludes = closure time § Sensitive for Glanzman’s and Bernard Soulier § Limited sensitivity for storage pool disorders § Not sensitive to vascular-collagen disorders or fibrinogen levels/ fibrin generation § Neither bleeding time or PFA are a perfect screening test § Coll/ epi = primary screening test § Coll/ ADP = normal in aspirin § Affected by § Platelet count § Haematocrit § Diet § Aspirin § vWF levels – need to measure vWF levels if abnormal result § Abnormal tests should be repeated to exclude transient defect
[also Retention in glass column and Baumgartner’s technique]
Platelet aggregation § Assay based on measuring the decrease in light absorbance that occurs in platelet rich plasma when platelets aggregate. § Aggregometer is set with platelet poor plasma to demonstrate 100% light transmittance, and platelet rich plasma is used to set the baseline at 0%. § Different agonists are then added to separate test aliquots, and as platelets aggregate light transmittance increases and results are plotted on moving graph paper. § Agonists include ADP, adrenaline, collagen and ristocetin, (arachidonic acid U46619 (thromboxane receptor agonist), TRAP (thrombin receptor activating peptide), heparin (HIT)) § Recommended that full dose response curves are obtained with each agonist § In thrombocytopenic samples (<120) it is best to adjust the control sample to the same platelet count or perform studies on washed platelets in which the count can be adjusted (neither technique is perfect) § Limited sensitivity for storage pool disorders (25% will have normal platelet aggregation) § ADP release § Low concentrations of ADP cause primary or reversible aggregation. § ADP initially binds its receptor and releases intracellular Ca which causes a shape change (reflected by a small initial change in absorbance) § Fibrinogen then adds to the cell to cell contact and reversible aggregation occurs. At low concentrations of ADP the platelets may dissociate after this phase. § Higher concentrations of ADP cause an irreversible secondary wave aggregation which is associated with the release of dense and alpha-granules as a result of activation of the arachidonic acid pathway. § The primary wave is obscured by the secondary wave and defects will be missed if only high concentrations are used. § Collagen § This results in a single wave of aggregation after a lag phase, which results from activation of the arachidonic acid pathway § Ristocetin § This reacts with vWF and the membrane receptor to induce platelets to clump together (agglutination) and does not activate any of the aggregation pathways. § Arachidonic acid § AA induces TXA2 generation and granule release even if there is a defect of agonist binding to the surface membrane or the phospholiase induced release of endogenous arachidonate. § Aggregation only impaired if further steps in the pathway are impaired such as inhibition of cyclooxygenae (aspirin effect). § Pitfalls § Centrifugation – red cell contamination may cause apparent incomplete aggregation § Time – for 30 minutes after PRP preparation, platelets are unresponsive to agonists § Platelet count – low counts may cause slow/ weak aggregation § pH - <7.7 inhibits aggregation, >8.0 enhances aggregation § mixing speed - <800 or >1200rpm slows aggregation § haematocrit § temperature § dirty cuvette/ air bubbles in cuvette
Platelet granular content and release § ATP/ADP content and release (bioluminescence) § Platelet factor V release (Quebec platelet syndrome) § Electron microscopy § Platelet granule defects § Changes in platelet ultrastructure eg MHY-9 defects (previously classified as May-Heggelin, Flechtner, Sebastian, Epstein syndromes)
Flow cytometry § Uses much smaller volume of bloods § Glycoprotein surface expression used to diagnose § Glanzman thombasthenia (GPIIb or IIIa/ CD41 or 61) and Bernard Soulier (GPIb/ CD42b) § Scott syndrome (annexin V binding) § Fibrinogen binding § Annexin binding (to phosphatidylcholine) § P-selectin surface expression
Genetic analysis § Wiskott Aldrich § Offered to some families with severe platelet function disorders to allow prenatal diagnosis
Repsonse patterns
Adhesion Pseudo or platelet VWD § AD § Mutations in platelet GP1b resulting in increased sensitivity to ristocetin § Reduced HMW VWF multimers § Important to distinguish from type IIb VWD as treated with platelets rather than VWF concentrates § Distinguised from type IIb by performing RIPA with donor platelets
Bernard Soulier syndrome § Deficieny of platelet GP Ib/V/IX complex § Decreased binding of vWF – reduced adherence to endothelium esp. where high shear stress § Giant platelets § Thrombocytopenia – due to reduced platelet survival § Normal aggregation except reduced in ristocetin § Same pattern of results as vWD, but not corrected by addition of normal plasma § Increased platelet nucleotides – suggestive of large platelets (normal in vWD) § Flow for accurate platelet count and for GP1b § Carriers mostly asymptomatic
Aggregation Glanzmann’s thombasthenia § Fail to form aggregates because of reduced GPIIb/IIIa (CD41/ CD61) § Type 1 and type 2 § Type 1: <10% GP IIb/IIIa complexes and absent platelet fibrinogen § Type 2: up to 30% GP IIb/IIIa with reduced platelet fibrinogen § Prolonged PFA100 § Abnormal aggregation in all except ristocetin § In ristocetin, may have a primary response only § Confirm diagnosis by flow for GP IIb/IIIa (absent CD41 and 61 on flow) § Treat with platelet transfusions § Allo-immunisation and Abs to GP IIb/IIIa may develop § rVIIa can be used to arrest bleeding § Acquired form due to auto-Abs to GPIIb/IIIa (rare)
Storage pool defects § Either deficiency of granules or failure of normal secretion Dense granule disorders (d SPD) Dense granules containATP, ADP, calcium pyrophosphate and serotonin § Hermansky-Pudlak syndrome § AR § Oculocutaneous albinism § Total absence of dense bodies § Commonest genetic disorder in Puerto Rico § Can treat with DDAVP § Chediak-Higashi syndrome § AR § Oculocutaneous albinism, infections, LPD and neutrophil peroxidase positive inclusions § Classical finding is very large peroxidase-positive cytoplasmic granules in neutrophils § Death in first decade § Reduced or irregular dense bodies § Idiopathic deficiency/ δ–storage pool disease
α-granule disorders (CD62P = P-selectin) § Alpha-granules contain PDGF and PF4 § Grey platelet syndrome § Large and agranular, thrombocytopenia § Contents of alpha granules are absent eg. PF4, PDGF, VWF, fibrinogen § Platelet nucleotides are normal § EM demonstrates reduced or absent granules § Aggregation – reduced response to collagen and ADP § Associated with myelofibrosis § P selectin retained and flow will be positive for this if platelets activated first § Platelets appear grey on blood film § Treate with DDAVP +/- platelets § Paris-Trousseasu or Jacobson syndrome § AD, thrombocytopenia § Giant abnormal alpha granules – can’t release their contents normally § Associated mental retardation, cardiac abnormalities, cranio-facial abnormalities § Quebec platelet syndrome or factor V quebec § AD – very rare § Low factor V within platelet α-granules, but normal plasma levels § Defective procoagulant activity due to failure in assembly of prothrombinase complex § Urokinase plasminogen is released in large quantities upon plt activation, therefore § Unresponsive to platelet transfusion, treat with anti-fibrinolytics § No characteristic aggregometry pattern § Arthrogryposis-renal dysfunction-cholestasis
Combined alpha and dense granule disorders § Rare - gene defect currently unknown
Platelet receptors and signal transduction pathways § Normal platelet count and morphology § Decreased primary aggregation to one or more agonists, accompanied by the absence of secondary aggregation to some or all § Nucleotide content and release will help to distinguish between SPDs Thrombaxane A2 receptor defects § Impaired aggregation to arachidonic acid with preserved ristocetin response § Defects in COX or thromboxane synthase produce a similar pattern, but have preserved aggregation to prostaglandin or synthetic thromboxane U46619 ADP receptor defects § Aggregation to ADP is reduced and rapidly reversed § Due to mutations in ADP receptor P2Y12 which is needed for large aggregates to form (P2Y1 receptor is involved initially) § Clopidogrel produces the same phenotype (P2Y12 is the clopidogrel target) Collagen receptor defects § Defects in GPVI or GPIa/IIa § 10 cases described Adrenoreceptor defects § Absence of aggregation to adrenaline Signalling § Not well characterised § Post receptor signalling pathways § Defects in G-protein subunits and phospholipase C enzymes § Many of these enzymes are expressed in multiple tissues but seem to be relatively deficient in the platelet
Phospholipid surface Scott syndrome § AR § Primary haemostatic function is intact but there is failure of scramblase activity once platelet activated § Allows phosphatidylserine and phosphatidylenthanolamine to move from the inner to the outer membrane § Platelets have a reduced ability to promote factor X and prothrombin activation § Due to decreased surface exposure of phosphatidylserine plus a reduced shedding of microvesicles § Aggregation is normal § Flow to demonstrate absent PS on surface of activated platelets § Prothrombin consumption index test
Hereditary macrothrombocytopenias – MYH9 mutations § AD § MYH9 mutations are the most common (encodes non-muscle myosin II-A heavy chain – involved in cytoskeleton in megas, platelets and other tissues) § Previously classified as: § May-Heggelin anomaly § Fechtner syndrome § Sebastian syndrome § Epstein syndrome § Thrombocytopenia (20-130) with very large platelets and varying platelet dysfunction (usually mild) § Often associated with Granulocyte inclusions (Dohle-like bodies) § Phenotype very variable – may be associated with other congenital abnormalities including: § Glomerulonephritis (urinalysis should be part of the workup) § Sensorineural deafness § Cataracts § Electron microscopy § Definitive diagnosis requires demonstration of a genetic defect in MYH9 § Management – variable as phenotype varies greatly – depends on personal and family history § As for other mild platelet disorders
Congenital amegakaryocytic thrombocytopenia § AR § Diagnosis § Severe congenital thrombocytopenia with virtually absent amegakaryocytes in marrow is highly suggestive § Mutations in MPL gene required for definitive confirmation (mutations in MPL result in abnormal function or expression of the thrombopoietin receptor) § Management § Platelet transfusion § Stem cell transplant – progresses to aplastic anaemia Amegakaryocytic thrombocytopenia with radioulnar syntosis § AD – only described in a small number of families § Due to mutations in HOX A11 § Management – platelet support + HSCT
Thrombocytopenia with absent radii § AR (or AD) § Molecular basis unknown § Severe thrombocytopenia § Bilateral absent radii § Cow’s milk intolerance, renal and cardiac abnormalities, facial capillary haemangiomas § Platelet count improves with age § Some develop AML in later life § Diagnosis § Distinctive clinical features § Reduced megakaryocytes in the bone marrow with abnormal maturation § Management § Platelet transfuions in infancy – usually not needed beyond this §
Wiskott-Aldrich Syndrome § X-linked recessive disease, WAS gene § Microthrombocytopenia, eczema and immunodeficiency (T and B cell) § AI disorders esp haemolytic anaemia and vasculitis § Malignancy (usually lymphoreticular) § Treat with HLA matched, irradiated and CMV neg platelets § Definitive treatment = BMT
Management § Registered with haemophilia centre § Green card etc § Life style advice / avoid NSAIDs etc § Tranexamic acid § Including gauze soaked in § DDAVP § Usually works for storage pool defects § Not usually for Glanzmann’s, Scott, occasional severe form of BSS § Platelets § Allo-immunisation or Abs to their missing GP § HLA matched § rFVIIa § licensed for GT where platelet refractoriness has been demonstrated § Babies of women with severe bleeding disorders eg. BSS, GT may be at risk of alloimmune thrombocytopenia if mum has platelet antibodies § Monitored for HLA and anti GPIIb/IIIa and GPIb antibodies § Plasma exchange has been used during pregnancy to reduce antibody titre § Transplantation may be appropriate § WAS
Acquired platelet dysfunction § Drugs § Aspirin – irreversible acetylation of platelet COX-1 and resulting inhibition of thromboxane A2 synthesis § Effect lasts as long as the platelet § NSAIDs – reversible inhibition of COX-1 (normal platelet function restored rapidly § Clopidogrel – bind irreversibly to the platelet purinergic receptor P2Y12 and inhibit platelet responses to ADP § American College of cardiology recommends cessation of aspirin 7-10days and clopidogrel 5 days prior to surgery § Aciximab – GPIIb/IIIa antagonist, inhibit platelet aggregation. § Bleeding in about 10%, but ICH and death are rare § Abciximab disassociates more slowly than others (eg. Tirofibran) § β-lactam antibiotics – most severe with low albumin § more unbound drug to interact with platelet surface § contribute to bleeding when there is a co-existing haemostatic defect § Uraemia § Increased NO impairs platelet-vessel wall interactions § Accumulation of guandinosuccinic acid which is the donor for NO § Improved with dialysis because of removal of GSA § Anaemia, because reduced axial flow means less platelet contact with vessel wall § Cirrhosis § Myeloma § PPs get adsorbed to platelet surface and cause dysfunction § Can also get acquired VWDS § Plasmapheresis may be helpful § Myeloproliferative disorders § Aquired VWD due to loss of HMWM § Increased platelet numbers deplete VWF § DDAVP doesn’t correct, cytoreduction is most appropriate treatment § Also associated with aortic stenosis § Cardiopulmonary by-pass § Blood bank platelets |
Coagulation >