# Presentation: 682431

## Slide 3

**Notes:**
Hemostasis is a complex sequence of interrelated events involving the vessel wall, platelets, and the coagulation system.  Following injury to the blood vessel, platelets adhere to exposed subendothelium by a process (adhesion) that involves the interaction of a plasma protein, von Willebrand factor (vWF), and a specific protein on the platelet surface, glycoprotein Ib (GPIb).  Adhesion is followed by recruitment of additional platelets that form clumps, a process called aggregation (cohesion).  This platelet-platelet interaction involves binding of fibrinogen to specific platelet surface receptors - a complex comprising glycoproteins IIb-IIIa (GPIIb-IIIa).  In the resting state, platelets do not bind fibrinogen; platelet activation induces a conformational change in the GPIIb-IIIa complex, leading to fibrinogen binding and aggregation. 

## Slide 4

**Notes:**
Activated platelets release contents of their granules (secretion or release reaction), such as adenosine diphosphate (ADP) and serotonin from the dense granules, which causes recruitment of additional platelets.  Platelets also play a major role in hemostasis by contributing to coagulation mechanisms; several key enzymatic reactions in blood coagulation occur on the platelet membrane lipoprotein surface. Together, the fibrin generated, the platelets, and red cells lead to clot formation and restoration of hemostasis.

## Slide 5

**Notes:**
Under the electron microscope a number of organelles can be discerned, including mitochondria (Mt) and three types of granules:  the dense granules (DG), the alpha granules, and vesicles containing acid hydrolases.  Platelets have an active machinery to produce and use ATP.  They have very limited capacity to synthesize new proteins.  Thus, when aspirin irreversibly inactivates cyclooxygenase, platelets cannot replenish the enzyme with new synthesis.

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**Notes:**
The various granule constituents are shown.  The contents of these granules are secreted on platelet activation.

## Slide 7

**Notes:**
The platelet surface has glycoproteins that are associated with specific interactions or function. The GPIIb-IIIa complex binds fibrinogen and mediates aggregation.  GPIb binds vWF and mediates platelet adhesion to subendothelium.

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**Notes:**
On platelet activation a number of responses can be shown to occur, including a change in shape from disk to sphere, platelet aggregation, granule release or secretion, and thromboxane (TxA2) production.  Some of these products induce further platelet activation as a positive feedback.  These include secreted ADP and TxA2.

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**Notes:**
AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; CO, cyclooxygenase; DG, diacylglycerol; IP3, inositol 1,4,5 trisphosphate; MLC, myosin light-chain; PAF, platelet activating factor; PIP2, phosphatidylinositol bisphosphate; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; R, receptor; vWD, von Willebrand Factor

A number of physiologic agonists interact with specific receptors on the platelet surface to induce a series of responses. The agonists include ADP, epinephrine, TxA2, thrombin, PAF, collagen, vasopressin, and serotonin.  Platelet activation initiates the production or release of several intracellular messenger molecules, including Ca2+ ions, products of phosphoinositide (PI) hydrolysis, DG, IP3, TxA2, and cyclic nucleotides [cAMP].  These modulate the various discernable platelet responses of Ca2+ mobilization, protein phosphorylation, aggregation, secretion, and liberation of arachidonic acid.  The interaction between the agonist receptors on the platelet surface and the key intracellular effector enzymes (e.g. PLA2,PLC, and AC) are mediated by a group of GTP-binding proteins that are modulated by GTP. On platelet stimulation, phosphoinositides are hydrolyzed by PLC to DG and various inositol phosphates, including IP3. IP3 functions as a messenger to mobilize Ca2+ from an  intracellular source.  DG activates PKC and this results in the phosphorylation of a 47-kD protein, pleckstrin.  On activation, platelets release arachidonic acid from phospholipids, which is mediated by PLA2.  The free arachidonic acid is converted by COX to prostaglandins G2 and H2, and subsequently by thromboxane synthetase to TxA2. Together, these events lead to aggregation and secretion.

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**Notes:**
Most platelet agonists activate platelets via G protein-coupled receptors on the platelet surface. Thrombin activates human platelets through the protease activated receptors (PAR), PAR-1 and PAR-4, which are coupled to Gq-, G12-, and Gi-mediated effector pathways. Cleavage of PAR-1 by thrombin between arginine 41 and serine 42 exposes a new N-terminus that serves as a tethered ligand.

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**Notes:**
Platelets contribute to the blood coagulation mechanisms by providing the surface on which a number of the enzymatic reactions occur, including prothrombin and FX activation.

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**Notes:**
Disorders of platelet function are characterized by mucocutaneous bleeding manifestations that are highly variable among patients with different underlying defects; excessive hemorrhage may follow surgical procedures or trauma.  Platelet counts and morphology are normal in most patients with platelet dysfunction.  While the majority of these patients have a prolonged bleeding time, it is not always the case.  Platelet aggregation and secretion studies provide evidence for the defect; however, these parameters are not universally predictive of the severity of clinical manifestations. 

**Notes:**
Various laboratory tests used to assess and diagnose platelet function disorders are shown. In general, the correlation between the bleeding symptoms and the prolongation in the bleeding time or the platelet responses in the platelet aggregation studies is weak. The findings on the aggregation studies do not predict the severity of bleeding symptoms. 

## Slide 15

**Notes:**
In the studies performed using platelet-rich plasma (PRP), as is the practice in most hospital clinical laboratories, platelet aggregation in response to increasing concentrations of agonists such as ADP and epinephrine consists of two phases - the initial wave of aggregation (primary aggregation) resulting from the interaction of the agonist with the platelet receptors, and the secondary irreversible aggregation related, at least in part, to the release of dense granule contents and TxA2 synthesis.  Thus, patients with impaired release of dense granule contents or TxA2 production generally demonstrate a primary wave but not the second wave of aggregation.  Patients with thrombasthenia or congenital afibrinogenemia have a markedly decreased or absent primary wave of aggregation as well.

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**Notes:**
The table provides a classification of congenital disorders of platelet function based on the platelet functions or responses that are abnormal. 

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**Notes:**
This slide shows the major mechanisms leading to platelet adhesion, involving GPIb and plasma vWF. In patients with defects in platelet-vessel wall interactions, adhesion of platelets to subendothelium is abnormal.  The two disorders in this group are von Willebrand disease (vWD), due to a deficiency or abnormality in plasma vWF, and Bernard-Soulier syndrome, where platelets are deficient in GPIb (and GPV and IX) and the binding of vWF to platelets is abnormal. 

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**Notes:**
Salient features in patients with the Bernard-Soulier syndrome are shown.

## Slide 19

**Notes:**
In patients (red tracings) with vWD (type I) or Bernard-Soulier syndrome (BSS), platelet aggregation studies using platelet-rich plasma show diminished aggregation response to ristocetin with normal responses to ADP and epinephrine.  The response to thrombin (low concentrations) may be impaired in BSS.

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## Slide 21

**Notes:**
DG, diacylglycerol; PIP2, phosphatidylinositol bisphosphate; IP3, inositol 1,4,5 trisphosphate; R, receptor

Disorders characterized by abnormal platelet-platelet interactions (aggregation) arise because of a severe deficiency of plasma fibrinogen (congenital afibrinogenemia) or because of a quantitative or qualitative abnormality  of the platelet membrane GPIIb-IIIa complex (Glanzmann thrombasthenia).  The activated GPIIb-IIIa complexes constitute the platelet binding sites for fibrinogen; this binding is a prerequisite for platelet aggregation. 

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**Notes:**
Clinical features of Glanzmann thrombasthenia are shown.

## Slide 23

**Notes:**
Platelets from patients with thrombasthenia or afibrinogenemia fail to aggregate in response to all physiologic platelet agonists, and even the primary wave of platelet aggregation is absent. 

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## Slide 25

**Notes:**
AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; CO, cyclooxygenase; DG, diacylglycerol; IP3, inositol 1,4,5 trisphosphate; MLC, myosin light-chain; PAF, platelet activating factor; PIP2, phosphatidylinositol bisphosphate; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; R, receptor; vWD, von Willebrand Factor

Patients with defects in platelet secretion and signal transduction are a heterogeneous population grouped together for convenience of classification rather than on the basis of an understanding of the specific underlying abnormality.  The major common characteristic in these patients is an inability to release intracellular granule contents upon activation of PRP with agonists such as ADP, epinephrine, and collagen.  In aggregation studies the second wave of aggregation is blunted or absent.  A small proportion of these patients have a deficiency of dense granule stores (storage pool deficiency).  Recent studies show that in some of the other patients, the impaired secretion results from aberrations in the signal transduction events that govern secretion and aggregation. 

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**Notes:**
Disorders of granules, secretion, and signal transduction arise due to abnormalities in the granules per se (storage pool deficiency [SPD], the Quebec platelet disorder) or in mechanisms that regulate release of granule contents (secretion).

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**Notes:**
Deficiency of Granule Stores:  The term storage pool deficiency (SPD) refers to patients with deficiencies in platelet content of dense granules (-SPD), -granules (-SPD) or both groups of granules (-SPD).  In electron micrographs, -SPD platelets have a marked decrease in dense granules, and the total platelet and granule ATP and ADP contents are decreased.  Other dense granule substances, including serotonin, calcium, and pyrophosphate, are also decreased. -SPD has been reported in association with other inherited systemic disorders, such as the Hermansky-Pudlak syndrome (associated with oculocutaneous albinism), the Chediak-Higashi syndrome, and the Wiskott-Aldrich syndrome.  Abnormal granule morphogenesis is considered as a mechanism for -SPD.  In patients with isolated deficiency of platelet -granule contents (gray platelet syndrome) the megakaryocytes and platelets are selectively deficient in -granule proteins, such as platelet factor 4, -thromboglobulin, fibrinogen, and the platelet-derived growth factor.  The platelets appear gray on the peripheral smear and impaired platelet aggregation and secretion have been reported consistently in response to thrombin but not other agonists.  Gray platelet syndrome appears to result from a defect in the packaging of -granule proteins into the granules in megakaryocytes. 

## Slide 28

**Notes:**
A representative tracing from a patient with dense granule SPD or a secretion defect is shown.  The primary wave of aggregation with ADP or epinephrine is generally present with a loss of the secondary wave.  The response to ristocetin is generally normal.

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**Notes:**
The Quebec platelet disorder is an autosomal dominant disorder associated with abnormal proteolysis of -granule proteins, deficiency of platelet -granule multimerin, and markedly impaired aggregation with epinephrine as a striking feature.  Multimerin, a multimeric factor V-binding protein, is decreased in the -granule. On immunoblot analyses, platelet factor V is degraded along with several other -granule proteins, including fibrinogen, vWF, thrombospondin, osteonectin, fibronectin, and P-selectin. 

## Slide 30

**Notes:**
AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; CO, cyclooxygenase; DG, diacylglycerol; IP3, inositol 1,4,5 trisphosphate; MLC, myosin light-chain; PAF, platelet activating factor; PIP2, phosphatidylinositol bisphosphate; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; R, receptor; vWD, von Willebrand Factor

Defects in Platelet Signal Transduction:  Signal transduction events reflect processes resulting from the initial interaction of agonists with specific receptors on platelets and leading to events such as G-protein activation and activation of effectors such as PLC and PLA2.  If the key components in platelet signal transduction mechanisms are the surface receptors, the G-proteins that modulate their link with intracellular effectors, and the effectors, evidence now exists for specific platelet abnormalities at each of these levels. 

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**Notes:**
Signal Transduction Defects: Defects in platelet-agonist interaction (receptor defects) are exemplified by patients with impaired responses because of an abnormality at the level of platelet surface receptors for a specific agonist.  Such receptor defects have been documented for collagen, ADP, and TxA2 and epinephrine.  Several patients have been identified who have a relatively mild bleeding diathesis and impaired dense granule secretion, although their platelets have normal granule stores.  In some patients abnormalities in signal transduction events including Ca2+ mobilization, pleckstrin phosphorylation, G-protein activation, and PLC activation have been documented.  Recently, platelet dysfunction has been associated with specific deficiencies in Gq, Gi1, PLC-2 isozyme, and protein kinase C-. 

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**Notes:**
Defects in Thromboxane A2 Production: A major platelet response during activation is liberation of arachidonic acid from phospholipids and its subsequent enzymatic oxygenation to TxA2.  Patients have been described with defects in the liberation of arachidonic acid as well as the level of cyclooxygenase and thromboxane synthase.

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**Notes:**
Defects in Platelet Procoagulant Activities:  These patients have an abnormality in interactions of platelets with proteins of the coagulation system, so that the platelet contribution to the coagulation cascade (platelet coagulant activity) is impaired.  The best described is Scott syndrome, where the platelets have impaired binding of factor Va-Xa and factor VIIIa-IXa, resulting in impaired factor X and prothrombin activation,  and an  abnormality in platelet factor III activity.  These patients have an abnormality in the translocation of phosphatidylserine (PS) from the inner platelet membrane location to the platelet surface on platelet activation.  

**Notes:**
Recently, a number of disorders have been described with abnormalities in platelet structural or cytoskeletal proteins or genes.  These include the group of disorders associated with macrothrombocytopenia and mutations in the MYH-9 gene.  Other examples are the Wiskott-Aldrich syndrome and platelet spherocytosis.

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**Notes:**
Platelet transfusions have been the major therapeutic modality to manage bleeding in patients with intrinsic platelet defects and this approach needs to be individualized, because of the wide variability in bleeding diathesis.  Platelet transfusions are indicated to manage bleeding events and in relation to surgical procedures to prevent excessive bleeding.  Platelet transfusions are associated with the risk of viral transmission and allergic reactions.  Of importance, platelet transfusions may lead to production of antibodies against specific glycoproteins, particularly in patients with thrombasthenia and Bernard-Soulier syndrome. 

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**Notes:**
Other options for the management of patients with congenital platelet defects include DDAVP and recombinant FVIIa.
A viable alternative is intravenous administration of DDAVP which shortens the BT in a substantial number of these patients,  particularly those with normal dense granule stores.  This response appears to be dependent on the underlying platelet mechanism leading to the platelet dysfunction.  Most patients with thrombasthenia appear not to respond to DDAVP infusion with a shortening of the BT.  More recently, evidence is becoming available that administration of recombinant Factor VIIa is beneficial in some patients, particularly with thrombasthenia. However, as of this time, neither DDAVP nor rFVIIa is approved in the United States for use in patients with platelet function defects.