INHERITED PLATELET DISORDERS · Disorders of platelet function Inherited Platetel Function...

INHERITED PLATELET DISORDERS

Federica MELAZZINI, MD PhD federica.melazzini@gmail.com

Department of Internal Medicine

University of Pavia & IRCCS Policlinico San Matteo Foundation Pavia, Italy

Platelets

• Blood elements with discoid shape which originate from the fragmentation of the cytoplasm of megakaryocytes

• Average life: 7 days

• Average diameter: 2-3 mm

• Mean platelet volume: 8-11 fL

• Normal platelet count: 150 to 400 x 109/L

1. Initiation

2. Propagation

3. Maintenance of a blood clot

Hemostasis is organized in three stages

1. Initiation

disorders of primary hemostasis

2. Propagation

disorders of secondary hemostasis

3. Maintenance of a blood clot

disorders of stabilization (improper processing and

cross-linking of fibrin strands)

inappropriate fibrinolysis

Disorders of Hemostasis

Primary hemostasis (1): constriction of the vessel

1. reduce blood flow

2. increase the interaction of platelets with the components of the vessel wall

Nat. Rev. Cardiol. doi:10.1038/nrcardio.2012.91

Primary hemostasis (2): formation of the platelet plug

1. Platelet adhesion: vWF/GPIb-IX-V interaction, collagen/GPIa-IIa/GPVI interaction

2. Platelet activation: influx of calcium, extrusion of platelet storage granule contents, further activation of platelets, irreversible platelet-platelet aggregation (fibrinogen/GPIIb-IIIa

interaction), formation of the platelet plug and transformation of platelet surface into a phospholipid platform appropriate to support the enzymatic reaction of secondary hemostasis

Nat Rev Urol 11, 51–58 (2014) modified from Nat Rev Immunol 13, 34–45 (2013)

Secondary hemostasis: formation of fibrin clot

1. exposure of tissue factor (TF) usually hidden in the intact vessel wall 2. sequential activation of blood-based serine proteases and their cofactors (blood clotting factors)

initiated by TF interacting with FVII 3. formation of fibrin strand and of a hemostatic clot

Formation of a stable clot

Thrombin activates FXIII which covalently cross-links fibrin strands to form a stable clot which will be retained long enough for vessel repair to occur

Major category of most common hemostatic disorders

Pathway Major component Salient clinical

features Classic disorders

Primary hemostasis Blood vessel

Platelets vWF

Bruising Ecchymoses Oozing from

mucosa Teleangectasia

HTT, vWD Trombocytopenia

Dysfunctional platelets

Secondary hemostasis

Clotting proteins Fibrinogen

Delayed hematomas

Hemophilia A and B Vitamin K deficiency

Liver disease

Fibrinolysis tPA

a2-plasmin inhibitor PAI-1

Delayed bleeding Poor wound healing

Dysfibrinogenemia a2-plasmin inhibitor

Liver disease

Disorders of platelet function

Inherited Platetel Function Disorders

1. Defect of PLT adhesion (VWF receptor GP1b/IX/V, collagen receptors GPVI, α2β1); 2. Defect of PLT activation (via G protein-coupled receptors, e.g., the ADP

receptors,P2Y1, P2Y12); 3. Defect of signal transduction pathways and secretion (α granules, dense granules); 4. Defect of aggregation for platelet thrombus formation (fibrinogen and WVF receptor

αIIbβ3); 5. Defect of presentation of the negatively-charged phospholipid procoagulant surface

for coagulation factor interaction on activated platelets.

Acquired disorders 1. Drugs 2. Liver disease 3. Kidney disease 4. Cancer 5. Autoimmune disorders

Disorders of platelet function

Inherited Platetel Function Disorders

1. Defect of PLT adhesion (VWF receptor GP1b/IX/V, collagen receptors GPVI, α2β1); 2. Defect of PLT activation (via G protein-coupled receptors, e.g., the ADP

receptors,P2Y1, P2Y12); 3. Defect of signal transduction pathways and secretion (α granules, dense granules); 4. Defect of aggregation for platelet thrombus formation (fibrinogen and WVF receptor

αIIbβ3); 5. Defect of presentation of the negatively-charged phospholipid procoagulant surface

for coagulation factor interaction on activated platelets.

Acquired disorders 1. Drugs 2. Liver disease 3. Kidney disease 4. Cancer 5. Autoimmune disorders

Nurden AT & Nurden P, 2015. Jurnal of Thrombosis and Haemostasis; 13(S1): S2-S9

PATHOLOGIES OF GENES AFFECTING SURFACE CONTITUENTS

PATHOLOGIES OF GENES AFFECTING SURFACE INTRACELLULAR PROTEINS

Nurden AT & Nurden P, 2015. Jurnal of Thrombosis and Haemostasis; 13(S1): S2-S9

Different types of bleeding

Evaluation of a patient with bruising/bleeding

Bleeding history Amount, type, location, timing, clinical setting

Medical history Liver disease, kidney disease, medications, previous surgery

Family history X-linked disease, autosomal disease, consanguinity, ethnicity

Physical examination Type and location of bruising and hematomas, presence of

splenomegaly

Screening labs CBC with differential, peripheral blood smear,

complete chemistry panel, PT/PTT

Screening labs CBC with differential, peripheral blood smear,

complete chemistry panel, PT/PTT

Advanced testing

Primary hemostasis Secondary hemostasis

Clot stability and

fibrinolysis

Evaluation of a patient with bruising/bleeding

Advanced testing

Primary hemostasis Secondary hemostasis

Clot stability and

fibrinolysis

Thrombocytopenia Citrate tube to exclude pseudo-thrombocytopenia Antiplatelet antibodies vWD vWF:Ricof vWF:Ag FVIII:C Platelet dysfunction Platelet aggregation Vessel disorders Bleeding time Exclude collagen disorders

Factor deficiency Individual factor levels Factor inhibitors Mixing studies Factor level Bethesda titer LAC, ACA Fibrinogen disorder Fibrinogen D-dimer Thrombin clot time Reptilase time

Factor XIII deficiency Factor XIII level Hyperfibrinolysis Fibrinogen D-dimer PAI-1 a2-antiplasmin inhibitor deficiency

FIRST-STEP LABORATORY TEST: BLOOD SMEAR

Platelet Size Small WAS

Normal All the remainind disorders

Large BSS GPS

GT- variant PT-vWD

VCS MYH9-RD

Platelet Morphology "grey" or "pale" platelet GPS

Other cells morphology Abnormal giant granules in CHS

eosinophils, basophils

& monocytes

Dyseritropoiesis GATA1

Large BSS GPS

GT- variant PT-vWD

VCS MYH9-RD

& monocytes

Large BSS GPS

GT- variant PT-vWD

VCS MYH9-RD

& monocytes

Large BSS GPS

GT- variant PT-vWD

VCS MYH9-RD

& monocytes

Light transmission aggregometry (LTA)

The Chrono-log Model 700whole blood/optical 2-channel lumiaggregometer (Chrono-log, Havertown, PA)

FIRST-STEP LABORATORY TEST: AGGREGOMETRY

Multiplate multiple platelet function 5-channel impedance analyzer. (Roche Diagnostics, Burgess Hill, West Sussex, UK)

FIRST-STEP LABORATORY TEST: AGGREGOMETRY

Platelet agonist: collagen, ADP, epinephrine, thrombin, Arachidonic Acid

minutes

“In vitro” platelet aggregation by densitometric method of Born

Arachidonic acid

Collagen + Convulxin Collagen

Ristocetin

Shape change

Baseline level

Primary wave

Secondary wave

Maximal

aggregation

Inflection point

Light transmission aggregometry (LTA): NORMAL platelet aggregometry response

ADP 4 mcM ADP 2,5 mcM

ADP 2 mcM

ADP 1,25 mcM

Light transmission aggregometry (LTA): Patterns of aggregation at different ADP concentrations

Aggregometer

Ristocetin 1,5 mg/ml

Ristocetin 1 mg/ml

Ristocetin 0,5 mg/ml

Light transmission aggregometry (LTA): Patterns of aggregation at different Ristocetin concentrations

SUMMARY

Glanzmann’s Thromboasthenia

Bernard Soulier Syndrome

Dense granule deficiency

Hermansky – Pudlak Sydrome (HPS)

Chediak-Higashi Syndrome (CHS)

Alfa granule deficiency

Gray Platelet Syndrome (GPS)

Arthrogryposis, Renal Disfunction and Cholestasis Syndrome

SUMMARY

Eduard Glanzmann was a Swiss pediatrician who first discovered the condition of thrombasthenia in 1918. Formerly known as “hereditary hemorrhagic thrombasthenia”, Glanzmann recognized a disorder that was not attributed to an abnormal number of platelets, but rather a faulty clot retraction […]. […] Glanzmann’s encounter with a symptomatic 7-year-old girl led him to study the disease within families. Noting a familial pattern and symptoms manifesting in children, he considered a possible hereditary component”.

Eduard Glanzmann (Luzern 1887 - Bern 1959)

• Autosomal recessive

• Platelet surface receptor disorder of GPIIb/IIIa, qualitative or quantitative

• Severe bleeding tendency

• Faulty platelet aggregation

• Diminished clot retraction

Platelet aggregation in healthy

GPIIb-IIIa

Platelet aggregation in Glanzmann Thrombasthenia (GT)

1 2 3 4 5

Minutes

ADP 20 mcM

Collagen 20 mcg/ml

ristocetin 1.5 mg/ml

Flow cytometry: GP expression on platelets surface

PAC1 binding to control and GT platelets before (grey) and after (red) platelet stimulation with ADP 10 µM

GPIIb/IIIa expression using different mAb against GPIIb (clones SZ22, P2, A2A9/6) or GPIIIa (clones SZ21, SAP)

Glanzmann’s variant (ITGA2B/ITGB3- related thrombocytopenia)

Isotype

patient

Healthy control

• Autosomal dominant

• Macrothrombocytopenia

• Gain of function mutation in ITGA2B/ITGB3

• Moderate to severe bleeding diathesis

• Autosomal recessive • Frequency: 1 in 1 million • Severe thrombocytopenia with giant platelets • Severe bleeding tendency • Severe defect of the GPIb/IX/V on platelet surface • Defective GPIba-vWF interaction

Biallelic Bernard-Soulier syndrome

Von Willebrand factor

Thrombin GPIIIa

myosin IIA

actin filaments

signalling to

cytoskeleton

GPIbα

GPIbβ

cell membrane

GPIb-IX-V complex

WASP (Wiskot-AldrichSyndromeProtein)

Bernard-Soulier syndrome (BSS)

Platelet aggregation in healthy

GPIIb-IIIa

Platelet aggregation in Bernard-Soulier syndrome (BSS)

“In vitro” platelet aggregation on platelet rich plasma: biallelic Bernard-Soulier syndrome

1 2 3 4 5

Minutes

ristocetin 3 mg/ml

ristocetin 1,5 mg/ml

collagen 20 mcg/ml

ADP 5 mcM

• Autosomal dominant • Frequency: 1/500 • Variable thrombocytopenia with enlarged platelets • Variable bleeding tendency • Mild defect of the GPIb/IX/V on platelet surface

and mild defect of GPIba-vWF interaction

Monoallelic Bernard-Soulier syndrome

Expression of glycoproteins on the surface platelet membrane in BSS Bolzano patients

Monoallelic Bernard-Soulier syndrome

Summarizing laboratory test….

SUMMARY

INHERITED DEFECT OF PLATELET GRANULES

- δ storage pool deficiency: most common, bleeding diathesis - α storage pool deficiency: macrothrombocytopenia, mild to

moderate bleeding

• Autosomal Recessive

• 9 different HPS mutation/variant

HPS encodes for complexes require for the

formation of lysosome-related organelles

• Variable mucocutaneous bleeding

• Significant post-surgical bleeding

• Oculocutaneous albinism (impaired vision)

• IBD

• Progressive Pulmonary Fibrosis

• Mutation in LYST

• Bleeding Diathesis mild to moderate

• Immunodeficiency

• Albinism

• Neurological disorders

• Patients frequently die from lymphoproliferative disorders

Alfa Granule Deficiencies

• Mutation in NBEAL2

• Bleeding diathesis mild to moderate

• Splenomegaly

• Bone marrow fibrosis

Gray Platelet Syndrome (GPS) - variant

• Autosomal Dominant

• Mutation in GFI1B

• Bleeding diathesis mild

to moderate

• Mutation in VPS33B protein

• Pale platelets, Aggregation after ADP and

AA pathological

• Mild bleeding diathesis

• Arthrpgryphosis multiplex congenita

• Kidney disfunction

• Cholestasis

• Failure to thrive

• Usually die during 1st year of life

DIAGNOSTIC ALGORITHM FLOWCHART FOR IPD ACCORDING TO SSC AND ISTH

P. Gresele 2015, for the subcommittee on platelet physiology

Need for multidisciplinary comprehensive care - Education to patients - Appropriate dental hygiene - pre-conception genetic counselling Conservative management of minor bleeds - local pressure, packing with gauze or gel foams (e.g. for epistaxis),

cauterization, sutures - antifibrinolytics (tranexamic acid), for bleeding involving mucosal sites Systemic hemostatic agents - Desmopressin acetate (DDAVP): ↑release VWF and FVIII from the endothelial, ↑ platelet adhesiveness and aggregation

Need for multidisciplinary comprehensive care - Education to patients - Appropriate dental hygiene - pre-conception genetic counselling Conservative management of minor bleeds - local pressure, packing with gauze or gel foams (e.g. for epistaxis),

cauterization, sutures - antifibrinolytics (tranexamic acid), for bleeding involving mucosal sites Systemic hemostatic agents - Desmopressin acetate (DDAVP): ↑release VWF and FVIII from the endothelial, ↑ platelet adhesiveness and aggregation

Platelet Transfusion

Recombinant human activated factor VII (rFVIIa) recommended for patients with IPFD with a history of platelet antibodies and platelet refractoriness In patients with GT, it is prudent to use rFVIIa for the treatment or prophylaxis of bleeding in those with severe mutations and absent platelet surface αIIbβ3 expression Use of rFVIIa in platelet function disorders is safe

Currative treatment HSCT GT pts with severe bleeding phenotype, persistent and recurrent life-threatening bleeding, refractory to available effective and safe hemostatic treatment, extremely poor quality of life Gene therapy Successful transduction of CD34+ cells was demonstrated in vitro from two patients with GT using murine leukemia retrovirus vectors with expression of αIIbβ3 on transduced megakaryocytes. This resulted in correction of GT. (Wilcox et al..)

Federica MELAZZINI Università di Pavia - Fondazione IRCCS Policlinico San Matteo

federica.melazzini@gmail.com

INHERITED PLATELET DISORDERS · Disorders of platelet function Inherited Platetel Function...

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