Platelet secretion defects. Komal Arora, MD

Transcription

1 Platelet secretion defects Komal Arora, MD

2 COMPONENTS OF NORMAL HAEMOSTASIS Blood vessels Platelets Plasma coagulation factors Their inhibitors The fibrinolytic system

3 Small fragments of cytoplasm derived from megakaryocytes μm in diameter No nucleus Lipid bilayer membrane Beneath the outer membrane lies the marginal band of microtubules Cytoplasm has three types of platelet granules: the δ granules, α granules, and lysosomal granules.

4 Platelet membrane site of interaction with the plasma environment and with the damaged vessel wall consists of phospholipids cholesterol glycolipids; glycoproteins, named GpI GpIX. Sphingomyelin and Phosphatidylcholine predominate in the outer leaflet phosphatidyl- ethanolamine, - inositol and - serine in the inner leaflet. After platelet activation the membrane also expresses binding sites for several coagulation proteins such as factor XI and factor VIII. Contractile system dense microtubular system circumferential microfilaments (maintain the disc shape) Actin (main constituent), myosin and calmodulin (regulatory calcium- binding protein)

5 Dense (δ) granules α Granules Lysosomal vesicles ATP PF4 Galactosidases ADP β-thromboglobulin Fucosidases Calcium Fibrinogen Hexosaminidase Serotonin Factor V Glucuronidase Pyrophosphate Thrombospondin Cathepsin P selectin (CD62) Fibronectin Glycohydrolases Transforming growth factor-beta (1) Catecholamines (noradrenaline/adrenaline) GDP/GTP PDGF PAI-1 Histidine-rich glycoprotein α 2 Macroglobulin Plasmin inhibitor P selectin (CD62) + others

6 Adhesion and aggregation forming the primary haemostatic plug Release of platelet activating and procoagulant molecules Provision of a procoagulant surface for the reactions of the coagulation system

7 When the vessel wall is damaged, the subendothelial structures (basement membrane, collagen, and microfibrils) are exposed. Platelets interact with exposed extracellular matrix components, consisting of a large number of adhesive macromolecules including collagen, von Willebrand factor (vwf), fibronectin, laminin, fibulin and thrombospondin. Surface- bound vwf binds to GpIb on circulating platelets, resulting in an initial monolayer of adhering platelets.

8 Binding via GpIb initiates activation of the platelet via a G- protein mechanism. Once activated, platelets immediately change shape from a disc to a tiny sphere with numerous projecting pseudopods.

9 After adhesion of a single layer of platelets to the exposed subendothelium, platelets stick to one another to form aggregates. Fibrinogen, fibronectin, and the glycoprotein Ib IX and IIbIIIa complexes are essential at this stage Certain substances (agonists) react with specific platelet membrane receptors to promote platelet aggregation and further activation. exposed collagen fibres ADP Thrombin Epinephrine Serotonin Arachidonic acid metabolites including TXA 2. At the site of an injury, locally damaged red cells release ADP, which further activates platelets.

10 GpIIb/IIIa ADP (P2Y12) ADP Thrombin Thromboxane PAF DAG PKC PLCγ Fibrinogen G q PLCβ PIP2 Pleckstrin Src FcRγ IP3 TXA2 α PI3K ADP Ca ++ PGG2/PGH2 GpIb/IX/V Arachidonic acid P2XI Ca ++ PLA2 PL Secretion

11 Defects in platelet vessel wall adhesion von Willebrand disease (deficiency or defect in plasma VWF) Bernard Soulier syndrome (deficiency or defect in GPIb) Defects in platelet platelet aggregation Congenital afibrinogenemia Glanzmann's thrombasthenia (deficiency or defect in GPIIb IIIa) Disorders of platelet secretion and signal transduction mechanisms Abnormalities of granules Storage pool deficiency Quebec platelet disorder Signal transduction defects (primary secretion defects) Abnormalities in arachidonic acid pathways and thromboxane A2 synthesis Impaired liberation of arachidonic acid Cyclooxygenase deficiency Thromboxane synthase deficiency Defects in cytoskeletal regulation Wiskott Aldrich syndrome Disorders of platelet coagulant protein interaction Scott syndrome

12

13

14 Dense granule SPDs (δ- SPDs) mild to moderate bleeding diathesis Platelet counts and morphology are usually can be seen as a singular clinical entity or as part of other hereditary disorders, such as Chediak- Higashi Hermansky- Pudlak syndrome Thrombocytopenia- absent radius syndrome Griscelli syndrome Lab Assay Decreased aggregation response to ADP, epinephrine, and collagen with normal aggregation to arachidonic acid and ristocetin Decreased adenosine triphosphate release by lumiaggregometry Decreased mepacrine uptake or release by flow cytometry are observed. EM: show decreased dense granules.

15 Alpha granule SPDs (α- SPD) Gray platelet syndrome has decreased alpha granules Macrothrombocytopenia ARC Syndrome (Arthrogryposis renal dysfunction- cholestasis) Normal platelet count Enlarged platelets Absence of granules Quebec platelet disorder AD (2 families in Quebec) Mild thrombocytopenia Defective epinephrine- induced platelet aggregation The α- granules appear morphologically normal The amount of the a- granule proteins factor V, multimerin, fibrinogen, vwf, fibronectin, osteonectin, and TSP is decreased, and many are substantially degrade due to the enhanced expression of urokinase- type plasminogen activator (u- PA) in α granules Rare α/δ- SPD has features of both disorders

16 Drouin A et al. Blood 2001;98:

17 Defects in platelet- agonist interaction Receptor defects: thromboxane A2, collagen, ADP, epinephrine Defects in G- protein activation Gq deficiency Defects in phosphatidylinositol metabolism Phospholipase C- β2 deficiency Defects in calcium mobilization Defects in protein phosphorylation (pleckstrin)

18 Collagen receptor defects (Intracellular pathway: collagen binds initially to either the GPIa GPIIa (α2β1) or GPVI, leading to subsequent binding to the other receptor which reinforces adhesion and generation of intracellular signals including Ca 2+ mobilization and protein phosphorylation) mucocutaneous bleeding manifestations Platelets deficient in GpIa or GpVI selective impairment in platelet collagen interaction collagen- induced platelet aggregation is markedly reduced platelet aggregation responses to other agonists preserved Selective impairment in platelet responsiveness to epinephrine Congenital deficiencies of the α2- adrenergic receptor associated with a decreased platelet response to adrenaline have been reported

19 Thromboxane A2 receptor defect TXA 2 is formed upon platelet activation as the consequence of the action of phospholipase A2 that releases arachidonic acid from membrane phospholipids. The formed TXA 2 is released and can bind to its Gq- coupled TXA 2 R, producing a feed- back amplification loop Several homozygous and heterozygous patients, suffering from lifelong mucosal bleeding and easy bruising reported Arg60Leu substitution in the TXA 2 R Impaired platelet aggregation responses to TxA2 analogs as well as collagen

20 ADP receptor defect ADP interaction with platelets is mediated by multiple receptors (P2Y1, P2Y12, P2X1) P2Y1 mediates Ca2+ - mobilisation, shape change and starts aggregation P2Y12 promotes the formation of large and stable platelet aggregates

21 Rare patients with an autosomal recessive hereditary disease and mild bleeding Decreased and reversible platelet aggregation in response to ADP (despite a normal shape change and Ca2+- mobilisation) Platelets show identical functional changes to normal platelets treated with the anti- platelet drugs clopidogrel for which P2Y12 is the pharmacologic target

22 GTP- binding proteins are a group of heterotrimeric (consisting of α, β and γ subunits) proteins Constitute the link between surface receptors and intracellular enzymes Defect of Gαq expression One patient with lifelong mucocutaneous bleeding and decreased platelet aggregation, calcium mobilisation and secretion to multiple agonists including ADP, thrombin and TxA2 (P2Y1, TxA2R and the PAR receptors are linked into Gαq)

23 Decreased expression of Gα1 Gαi inhibits agonist- stimulated adenylyl cyclase activity in platelets One case with chronic bleeding with a severely decreased aggregation with ADP Congenital Gsα hypo- or hyperfunction syndromes Gsα activation increases platelet camp levels and inhibits agonist- stimulated platelet responses. Two siblings and an individual have been described with inducible Gas hyper- activity that was manifest as a bleeding diathesis, markedly prolonged bleeding times, variable mental retardation, and skeletal malformations. Platelet aggregation was reportedly normal, but the platelets had increased sensitivity to inhibition by agents that elevate camp levels.

24 Mild bleeding diathesis Abnomalities in aggregation and secretion in response to weaker agonists (ADP, epinephine, PAF); but not to stronger agonists such as AA and high concentrations of collagen.

25 Platelet activation induced conformational change in the GPIIb IIIa complex and fibrinogen- binding to platelets is an inside- out signal transduction- dependent process patient with markedly abnormal platelet aggregation and receptor activated pleckstrin phosphorylation who had decreased activation of the platelet GPIIb IIIa complexes despite the presence of normal number of these platelet receptors with intact ligand (fibrinogen) binding capacity Such a defect in GPIIb IIIa activation secondary to abnormalities in upstream signaling events may be a more common mechanism for blunted aggregation (particularly primary wave)

26 Defects in the liberation of arachidonic acid from phospholipids mild to moderate bleeding manifestations abnormal aggregation and secretion associated with impaired liberation of arachidonic acid during platelet stimulation Deficiencies of cyclooxygenase and thromboxane synthase aspirin- like platelet defect have dysfunctional arachidonic acid (AA) metabolism; mostly with autosomal dominant inheritance and a mild bleeding phenotype

27 RUNX1 haplodeficiency thrombocytopenia and predisposition to leukemia abnormal aggregation secretion responses, and protein phosphorylation dense granule and alpha- granule deficiency deficiencies of platelet PKC- q, PF4, myosin light chain, and 12- lipoxygenase. GATA- 1 mutations impaired responses to collagen and ristocetin macrothrombocytopenia

28 Platelet transfusions and 1- desamino- 8- d- arginine vasopressin (DDAVP) Because of the wide disparity in bleeding manifestations, therapeutic approaches need to be individualized Allogeneic BMT has been successfully performed with complete correction in patients with thrombasthenia and the Wiskott Aldrich syndrome Rarely required in patients with congenital platelet function disorders

29 Uremia Myeloproliferative neoplasms Essential thrombocythemia Polycythemia vera Chronic myelogenous leukemia Primary Myelofibrosis Acute leukemias and Myelodysplastic syndromes Dysproteinemias Cardiopulmonary bypass Acquired von Willebrand Disease Acquired Storage pool deficiency Antiplatelet antibodies Liver disease Drugs and other agents

30

31 Patients with a familial bleeding diathesis and abnormalities in agonist- mediated platelet aggregation and secretion are common Yet in the vast majority of these patients the underlying molecular mechanisms leading to the platelet dysfunction are unknown The well- characterized entities such as thrombasthenia, the Bernard Soulier syndrome, and storage pool deficiencies are rare or Evidence is now available for the existence of patients with specific abnormalities in the signaling events The larger group of currently poorly characterized patients lumped into such categories as primary secretion or activation defects

32 Rao AK. Congenital disorders of platelet secretion and signal transduction. In: Colman, RW, Hirsh, J, Marder, VJ, Clowes, P, George, JN, eds. Hemostasis and Thrombosis. Basic Principles and Clinical Practice, 6th edn. Philadelphia, PA: Lippincott, Williams and Wilkins 2001: Rao AK. Inherited defects in platelet signaling mechanisms. Sem Thromb Hemost 2004;30: Hirata T, Kakizuka A, Ushikubi F, et al. Arg60 to Leu mutation of the human thromboxane A2 receptor in a dominantly inherited bleeding disorder. J Clin Invest 1994;94: Nurden P, Savi P, Heilmann E, et al. An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb- IIIa complex function. J Clin Invest 1995;95: