Hereditary Platelet Function Hereditary Platelet Function DefectsDefects

Rob McFarlane, MDRob McFarlane, MD

January 20, 2006January 20, 2006

ObjectivesObjectives

Review platelet morphology and its role in Review platelet morphology and its role in primary hemostasisprimary hemostasis

Understand the pathophysiology of the major Understand the pathophysiology of the major inherited platelet defects, including: Bernard-inherited platelet defects, including: Bernard-Soulier syndrome, Glanzmann’s thrombasthenia, Soulier syndrome, Glanzmann’s thrombasthenia, and the storage pool defects and the storage pool defects

Understand the laboratory methods used to Understand the laboratory methods used to diagnose and classify the hereditary platelet diagnose and classify the hereditary platelet function defectsfunction defects

Primary Hemostasis: The PlateletPrimary Hemostasis: The Platelet

Anuclear discoid cell (3-5 microns) arising from Anuclear discoid cell (3-5 microns) arising from megakaryocytes in bone marrowmegakaryocytes in bone marrow

4-5 day maturation, 9-10 day life span4-5 day maturation, 9-10 day life span Bilamellar membrane contains multiple Bilamellar membrane contains multiple

invaginations with an open canalicular system:invaginations with an open canalicular system: Attached to intracellular dense tubular system, Attached to intracellular dense tubular system,

forming an interconnecting network (membrane forming an interconnecting network (membrane complex) throughout the cellcomplex) throughout the cell

Facilitates secretion of granulesFacilitates secretion of granules

Platelet organellesPlatelet organelles

Mitochondria, golgi, ribosomes, Mitochondria, golgi, ribosomes, peroxisomes, lysosomesperoxisomes, lysosomes

Two platelet-specific storage granules:Two platelet-specific storage granules:Alpha granules: Platelet Factor 4 (heparin Alpha granules: Platelet Factor 4 (heparin

binding chemokine), PDGF, fibrinogen, binding chemokine), PDGF, fibrinogen, fibronectin, plasminogen activator inhibitor I fibronectin, plasminogen activator inhibitor I (PAI I), Factors V, VIII,and vWF(PAI I), Factors V, VIII,and vWF

Dense bodies: histamine, epinephrine, Dense bodies: histamine, epinephrine, serotonin, ADP, calciumserotonin, ADP, calcium

Platelet cytoskeletonPlatelet cytoskeleton

Composed of cross-linked actin filaments Composed of cross-linked actin filaments coating the inner surface of the lipid coating the inner surface of the lipid bilayerbilayer

Regulates the shape of the resting plateletRegulates the shape of the resting platelet Interacts with transmembrane receptorsInteracts with transmembrane receptorsPlatelet activation, intracellular protein Platelet activation, intracellular protein

phosphorylation cascade and subsequent phosphorylation cascade and subsequent contraction leads to extrusion of platelet contraction leads to extrusion of platelet organellesorganelles

Platelet morphologyPlatelet morphology

Numerous G-protein receptors or adhesion Numerous G-protein receptors or adhesion receptors (integrins) are present on the cell receptors (integrins) are present on the cell surfacesurface transmembrane heterodimers composed of alpha and transmembrane heterodimers composed of alpha and

beta subunits, responsible for adhesion and signal beta subunits, responsible for adhesion and signal transductiontransduction

Glycoproteins are designated I (large) to IX (small); a Glycoproteins are designated I (large) to IX (small); a and b were added when electrophoretic techniques and b were added when electrophoretic techniques allowed for resolution of single bands to separate allowed for resolution of single bands to separate bandsbands

Glycoprotein receptorsGlycoprotein receptors

GP Ib-V-IX; complex of four gene GP Ib-V-IX; complex of four gene products, serves as a receptor for vWF; products, serves as a receptor for vWF; adhesion; Bernard-Soulieradhesion; Bernard-Soulier

GP IIb-IIIa; most abundant, recognizes GP IIb-IIIa; most abundant, recognizes four adhesive receptors: fibrinogen, four adhesive receptors: fibrinogen, fibronectin, vitronectin, and vWF; fibronectin, vitronectin, and vWF; aggregation; Glanzmann’s aggregation; Glanzmann’s

Others: Others: GP Ia, IIa; GP VI: collagen receptorsGP Ia, IIa; GP VI: collagen receptors

Primary hemostasisPrimary hemostasis

Extremely dynamic, complicated, and Extremely dynamic, complicated, and continuous interaction between vessel, continuous interaction between vessel, platelet, and plasma componentsplatelet, and plasma components

Adhesion, Activation (Secretion), Adhesion, Activation (Secretion), AggregationAggregation

AdhesionAdhesion Vascular injury exposes the pro-coagulant Vascular injury exposes the pro-coagulant

components of the sub-endothelial extracellular components of the sub-endothelial extracellular matrix: collagen, proteoglycans, and fibronectinmatrix: collagen, proteoglycans, and fibronectin

Platelets are exposed to these components in a Platelets are exposed to these components in a rolling fashionrolling fashion

vWF acts as an adhesion bridge between the vWF acts as an adhesion bridge between the platelet GP Ib-V-IX complex and exposed platelet GP Ib-V-IX complex and exposed collagen; platelets also adhere to fibronectincollagen; platelets also adhere to fibronectin

However, vWF-GPIb bridge is the only However, vWF-GPIb bridge is the only association strong enough to overcome blood association strong enough to overcome blood flow shearing forceflow shearing force

SecretionSecretion

Shape change via cytoskeletal activation: Shape change via cytoskeletal activation: spherical with extending pseudopodsspherical with extending pseudopods

Platelet granules are released thru canalicular Platelet granules are released thru canalicular systemsystem

Cytoplasmic activation of eicosanoid pathway Cytoplasmic activation of eicosanoid pathway (TXA2), decreased cAMP, and mobilization of (TXA2), decreased cAMP, and mobilization of Ca++Ca++

Phospholipids are translocated to cell surface Phospholipids are translocated to cell surface membrane (phosphatidylserine)membrane (phosphatidylserine)

Binding surface for factor Va and Xa (along with Ca++) forms Binding surface for factor Va and Xa (along with Ca++) forms prothrombinase complex; secondary hemostasisprothrombinase complex; secondary hemostasis

AggregationAggregation

Promoted by ADP and TXA2 release Promoted by ADP and TXA2 release ADP induces a conformational change of ADP induces a conformational change of

the IIbIIIa receptor, allowing fibrinogen the IIbIIIa receptor, allowing fibrinogen bindingbinding

Platelets aggregate via fibrinogen bound Platelets aggregate via fibrinogen bound to IIbIIIa receptorsto IIbIIIa receptors

Auto-catalytic reaction activating other Auto-catalytic reaction activating other plateletsplatelets

Formation of primary hemostatic plugFormation of primary hemostatic plug

Glanzmann’s ThrombastheniaGlanzmann’s Thrombasthenia

Eduard Glanzmann (1887-1959), Swiss Eduard Glanzmann (1887-1959), Swiss pediatricianpediatrician

Reported a case of a bleeding disorder Reported a case of a bleeding disorder starting immediately after birthstarting immediately after birth

W. E. Glanzmann:W. E. Glanzmann:Hereditäre Hereditäre hämorrhägische Thrombasthenie. Ein hämorrhägische Thrombasthenie. Ein Beitrag zur Pathologie der Blutplättchen.Beitrag zur Pathologie der Blutplättchen.Jahrbuch für Kinderheilkunde, 1918; 88: 1-Jahrbuch für Kinderheilkunde, 1918; 88: 1-42, 113-141. 42, 113-141.

Glanzmann’sGlanzmann’s IIbIIIa most abundant platelet surface receptor IIbIIIa most abundant platelet surface receptor

(80,000 per platelet)(80,000 per platelet) IIbIIIa complex is a Ca++ dependent IIbIIIa complex is a Ca++ dependent

heterodimerheterodimer Genes for both subunits are found on Genes for both subunits are found on

Chromosome 17Chromosome 17 Disease is caused by mutations (substitution, Disease is caused by mutations (substitution,

insertion, deletion, splicing abnormalities) in insertion, deletion, splicing abnormalities) in genes encoding for IIb or IIIa resulting in genes encoding for IIb or IIIa resulting in qualitative or quantitative abnormalities of the qualitative or quantitative abnormalities of the proteinsproteins

Fundamental defect of thrombasthenic Fundamental defect of thrombasthenic patients is the inability of the platelets to patients is the inability of the platelets to aggregateaggregate

Other problems: platelets do not spread Other problems: platelets do not spread normally on the subendothelial matrix (due normally on the subendothelial matrix (due to lack of IIbIIIa – vWF/fibronectin to lack of IIbIIIa – vWF/fibronectin interaction)interaction)

Also, alpha granule fibrinogen is Also, alpha granule fibrinogen is decreased to absentdecreased to absent

AR inheritanceAR inheritancePatients present with wide spectrum of Patients present with wide spectrum of

diseasediseaseLike thrombocytopenic bleeding: skin, Like thrombocytopenic bleeding: skin,

mucous membrane (petichiae, mucous membrane (petichiae, echymoses), recurrent epistaxis, GI echymoses), recurrent epistaxis, GI hemorrhage, menorrhagia, and immediate hemorrhage, menorrhagia, and immediate bleeding after trauma/surgerybleeding after trauma/surgery

ICH, joint, muscle bleeding uncommonICH, joint, muscle bleeding uncommon

Glanzmann’s patients are stratified into Glanzmann’s patients are stratified into three groups based on complex three groups based on complex expression:expression:Type I less than 5 percent GPIIbIIIa, absent Type I less than 5 percent GPIIbIIIa, absent

alpha granule fibrinogenalpha granule fibrinogenUsually as a result of IIb gene mutationUsually as a result of IIb gene mutation

Type II <20 percent, fibrinogen presentType II <20 percent, fibrinogen presentType III >50 percent; “variant” Type III >50 percent; “variant”

thrombasthenia; qualitative disorderthrombasthenia; qualitative disorder

DiagnosisDiagnosis

Platelet count and morphology are normalPlatelet count and morphology are normalBleeding time prolongedBleeding time prolongedThe hallmark of the disease is severely The hallmark of the disease is severely

reduced or absent platelet aggregation in reduced or absent platelet aggregation in response to multiple agonists ie ADP, response to multiple agonists ie ADP, thrombin, or collagen (except Ristocetin)thrombin, or collagen (except Ristocetin)

Flow cytometry: decreased mAb Flow cytometry: decreased mAb expression of CD41 (GPIIb) and CD61 expression of CD41 (GPIIb) and CD61 (GPIIIa)(GPIIIa)

Platelet Aggregation StudiesPlatelet Aggregation Studies

Platelet-rich plasma (PRP) is prepared from Platelet-rich plasma (PRP) is prepared from citrated whole blood by centrifugationcitrated whole blood by centrifugation

Inactive platelets impart a characteristic turbidity Inactive platelets impart a characteristic turbidity to PRPto PRP

When platelets aggregate after injection of an When platelets aggregate after injection of an agonist, the turbidity falls, and light transmission agonist, the turbidity falls, and light transmission through the sample increases proportionallythrough the sample increases proportionally

The change in light transmission can be The change in light transmission can be recorded on an aggregometerrecorded on an aggregometer

AgonistsAgonists

Different concentrations of each agonist Different concentrations of each agonist are usedare used

ADP: biphasic pattern:ADP: biphasic pattern:First wave: low concentration, reversible First wave: low concentration, reversible Second wave: high concentration, irreversibleSecond wave: high concentration, irreversible

Other agonistsOther agonists

Epinephrine: triphasic (resting platelets, Epinephrine: triphasic (resting platelets, primary aggregation, secondary primary aggregation, secondary aggregation)aggregation)

Other agonistsOther agonists

Collagen, arachidonic acid, Calcium Collagen, arachidonic acid, Calcium ionophore, PAF are potent agonists and ionophore, PAF are potent agonists and induce a single wave of irreversible induce a single wave of irreversible aggregationaggregation

Ristocetin (antibiotic): aggregation can be Ristocetin (antibiotic): aggregation can be reproduced with metabolically inert, reproduced with metabolically inert, formalin-fixed plateletsformalin-fixed platelets

Defective risto-induced aggregation is Defective risto-induced aggregation is characteristic of Bernard-Souliercharacteristic of Bernard-Soulier

Problems with platelet aggregation Problems with platelet aggregation studiesstudies

Numerous variables affect aggregation:Numerous variables affect aggregation:Anticoagulant (sodium citrate best)Anticoagulant (sodium citrate best)Plt count in PRPPlt count in PRPPlt size distributionPlt size distributionTime of dayTime of dayTemporal relation to meals and physical activityTemporal relation to meals and physical activity

Bernard-Soulier SyndromeBernard-Soulier Syndrome

First described in 1948 by Jean Bernard and First described in 1948 by Jean Bernard and Jean-Pierre Soulier; French hematologistsJean-Pierre Soulier; French hematologists

Bernard J, Soulier JP: Sur une nouvelle variete de dystrophie Bernard J, Soulier JP: Sur une nouvelle variete de dystrophie thrombocytaire hemarroagipare congenitale. Sem Hop Paris thrombocytaire hemarroagipare congenitale. Sem Hop Paris 24:3217, 1948 24:3217, 1948

AR; characterized by moderate to severe AR; characterized by moderate to severe thrombocytopenia, giant platelets, and thrombocytopenia, giant platelets, and perfuse/spontaneous bleedingperfuse/spontaneous bleeding

Basis for the disease is deficiency or dysfunction Basis for the disease is deficiency or dysfunction of the GP Ib-V-IX complex of the GP Ib-V-IX complex

Bernard-Soulier SyndromeBernard-Soulier Syndrome Decreased GP Ib-V-IX leads to decreased platelet Decreased GP Ib-V-IX leads to decreased platelet

adhesion to the subendothelium via decreased binding of adhesion to the subendothelium via decreased binding of vWFvWF

Approximately 20,000 copies of GP Ib-V-IX per plateletApproximately 20,000 copies of GP Ib-V-IX per platelet GP 1b: heterodimer with an alpha and beta subunitGP 1b: heterodimer with an alpha and beta subunit The gene for GP Ib alpha is located on chromosome 17; The gene for GP Ib alpha is located on chromosome 17;

GP Ib beta: chromosome 22; GPIX and V: chromosome GP Ib beta: chromosome 22; GPIX and V: chromosome 33

Most mutations are missense or frameshifts resulting in Most mutations are missense or frameshifts resulting in premature stop codonspremature stop codons

Most mutations involve GP Ib expression (rare GP IX Most mutations involve GP Ib expression (rare GP IX mutations have been described; no mutations in GP V)mutations have been described; no mutations in GP V)

DiagnosisDiagnosis

Prolonged bleeding time, Prolonged bleeding time, thrombocytopenia (plt<20 K), peripheral thrombocytopenia (plt<20 K), peripheral smear shows large platelets (mean smear shows large platelets (mean diameter >3.5 microns)diameter >3.5 microns)

DiagnosisDiagnosis

Platelet aggregation studies show normal Platelet aggregation studies show normal aggregation in response to all agonists aggregation in response to all agonists except Ristocetin (opposite pattern than except Ristocetin (opposite pattern than thrombasthenia)thrombasthenia)

Flow cytometry: decreased expression of Flow cytometry: decreased expression of mAbs to CD 42b (GPIb), CD42a(GPIX), mAbs to CD 42b (GPIb), CD42a(GPIX), CD42d(GPV)CD42d(GPV)

May-Hegglin anomaly: AD; giant platelets, May-Hegglin anomaly: AD; giant platelets, thrombocytopenia, Dohle-like inclusions thrombocytopenia, Dohle-like inclusions (larger, more angular)(larger, more angular)

Neutrophils are functional; only 40% of Neutrophils are functional; only 40% of patients may have bleeding diathesispatients may have bleeding diathesis

Storage Pool DefectsStorage Pool Defects

Classified by type of granular deficiency or Classified by type of granular deficiency or secretion defect (ASA)secretion defect (ASA)

Dense body deficiency, alpha granule Dense body deficiency, alpha granule deficiency (gray platelet syndrome), mixed deficiency (gray platelet syndrome), mixed deficiency, Factor V Quebecdeficiency, Factor V Quebec

Dense body deficiencyDense body deficiency

decreased dense decreased dense bodies (ADP, ATP, bodies (ADP, ATP, calcium, calcium, pyrophosphate, 5HT)pyrophosphate, 5HT)

Normal platelet Normal platelet contains 3-6, 300 contains 3-6, 300 micron dense bodiesmicron dense bodies

Described in inherited disorders ie Described in inherited disorders ie Hermansky-Pudlak syndrome, Wiskott-Hermansky-Pudlak syndrome, Wiskott-Aldrich syndrome, Chediak-Higashi Aldrich syndrome, Chediak-Higashi syndrome, and Thrombocytopenia with syndrome, and Thrombocytopenia with absent radius (TAR) syndromeabsent radius (TAR) syndrome

Wiskott-AldrichWiskott-Aldrich

X-linked, genetic defect in WASp (protein X-linked, genetic defect in WASp (protein responsible for actin cytoskeleton responsible for actin cytoskeleton formation in hematopoetic cells)formation in hematopoetic cells)

characterized by thrombocytopenia (with characterized by thrombocytopenia (with platelet storage pool defect), eczema,and platelet storage pool defect), eczema,and recurrent infectionsrecurrent infections

Hermansky-PudlakHermansky-Pudlak

Described in 1959 by Hermansky and PudlakDescribed in 1959 by Hermansky and Pudlak AR, tyrosinase-positive oculocutaneous AR, tyrosinase-positive oculocutaneous

albinism, ceroid-like deposition in lysosomes of albinism, ceroid-like deposition in lysosomes of the RES and marrowthe RES and marrow

Highest prevalence in Puerto RicoHighest prevalence in Puerto Rico May be associated with pulmonary fibrosis, IBD, May be associated with pulmonary fibrosis, IBD,

and recurrent infectionsand recurrent infections quantitative deficiency of dense granules leading quantitative deficiency of dense granules leading

to mild-moderate bleeding diathesisto mild-moderate bleeding diathesis

Chediak-HigashiChediak-Higashi

described by Beguez Cesar in 1943, Steinbrinck described by Beguez Cesar in 1943, Steinbrinck in 1948, Chédiak in 1952, and Higashi in 1954 in 1948, Chédiak in 1952, and Higashi in 1954

AR; abnormal microtubule formation and giant AR; abnormal microtubule formation and giant lysozomal granules are present in phagocytes lysozomal granules are present in phagocytes and melanocytesand melanocytes

No degranulation/chemotaxis = recurrent No degranulation/chemotaxis = recurrent bacterial infectionsbacterial infections

Partial oculocutaneous albinismPartial oculocutaneous albinism Dense-body granules decreased/absentDense-body granules decreased/absent

Thrombocytopenia with absent Thrombocytopenia with absent radius (TAR)radius (TAR)

First described in 1951First described in 1951AR, characterized by absent radii, AR, characterized by absent radii,

thrombocytopenia (with storage pool thrombocytopenia (with storage pool defect), and other abnormalities of the defect), and other abnormalities of the skeletal, GI, cardiovascular systemskeletal, GI, cardiovascular system

Etiology unclearEtiology unclearHemorrhage is the major cause of Hemorrhage is the major cause of

mortalitymortalityPX is good if survive the two yearsPX is good if survive the two years

DiagnosisDiagnosis

Platelet aggregation studies may show Platelet aggregation studies may show diminished response to low concentration diminished response to low concentration collagencollagen

ADP and epinephrine show diminished ADP and epinephrine show diminished second wave responsesecond wave response

Ristocetin shows normal aggregationRistocetin shows normal aggregationEM: lack of dense bodiesEM: lack of dense bodies Increased ATP:ADP ratio within plateletsIncreased ATP:ADP ratio within platelets

Alpha granule deficiencyAlpha granule deficiency

Alpha storage pool deficiency, Gray Platelet Alpha storage pool deficiency, Gray Platelet SyndromeSyndrome

First described by Raccuglia in 1971First described by Raccuglia in 1971 Normal platelets contain approximately 50 Normal platelets contain approximately 50

granules (PF4, beta-thromboglobulin, PDGF, granules (PF4, beta-thromboglobulin, PDGF, fibrinogen, vWF, Factor V, fibronectin)fibrinogen, vWF, Factor V, fibronectin)

Patients lack granules, present with lifelong, mild Patients lack granules, present with lifelong, mild to moderate mucocutaneous bleedingto moderate mucocutaneous bleeding

DiagnosisDiagnosis

Prolonged bleeding time, mild Prolonged bleeding time, mild thrombocytopeniathrombocytopenia

Agranular, large “gray” platelets on Agranular, large “gray” platelets on peripheral smearperipheral smear

Aggregation studies: decreased to absent Aggregation studies: decreased to absent response to collagenresponse to collagen

SummarySummary

Morphology and role of the platelet in Morphology and role of the platelet in primary hemostasisprimary hemostasis

Adhesion: GP1b-V-IX; Bernard-Soulier; Adhesion: GP1b-V-IX; Bernard-Soulier; aggregates with everything but Ristocetinaggregates with everything but Ristocetin

Activation (Secretion): dense body Activation (Secretion): dense body deficiency (associated syndromes), alpha deficiency (associated syndromes), alpha granule deficiencygranule deficiency

Aggregation: GPIIb-IIIa; Glanzmann’s; no Aggregation: GPIIb-IIIa; Glanzmann’s; no aggregation except for Ristocetinaggregation except for Ristocetin

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