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INHERITED PLATELET DISORDERS
Federica MELAZZINI, MD PhD [email protected]
Department of Internal Medicine
University of Pavia & IRCCS Policlinico San Matteo Foundation Pavia, Italy
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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
INHERITED PLATELET DISORDERS
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1. Initiation
2. Propagation
3. Maintenance of a blood clot
Hemostasis is organized in three stages
INHERITED PLATELET DISORDERS
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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
INHERITED PLATELET DISORDERS
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Primary hemostasis (1): constriction of the vessel
1. reduce blood flow
2. increase the interaction of platelets with the components of the vessel wall
INHERITED PLATELET DISORDERS
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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
INHERITED PLATELET DISORDERS
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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
INHERITED PLATELET DISORDERS
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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
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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
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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
INHERITED PLATELET DISORDERS
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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
INHERITED PLATELET DISORDERS
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Nurden AT & Nurden P, 2015. Jurnal of Thrombosis and Haemostasis; 13(S1): S2-S9
PATHOLOGIES OF GENES AFFECTING SURFACE CONTITUENTS
INHERITED PLATELET DISORDERS
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PATHOLOGIES OF GENES AFFECTING SURFACE INTRACELLULAR PROTEINS
Nurden AT & Nurden P, 2015. Jurnal of Thrombosis and Haemostasis; 13(S1): S2-S9
INHERITED PLATELET DISORDERS
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Different types of bleeding
INHERITED PLATELET DISORDERS
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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
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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
INHERITED PLATELET DISORDERS
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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
INHERITED PLATELET DISORDERS
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INHERITED PLATELET DISORDERS
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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
GPS
INHERITED PLATELET DISORDERS
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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
GPS
INHERITED PLATELET DISORDERS
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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
GPS
INHERITED PLATELET DISORDERS
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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
GPS
INHERITED PLATELET DISORDERS
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Light transmission aggregometry (LTA)
INHERITED PLATELET DISORDERS
The Chrono-log Model 700whole blood/optical 2-channel lumiaggregometer (Chrono-log, Havertown, PA)
FIRST-STEP LABORATORY TEST: AGGREGOMETRY
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Light transmission aggregometry (LTA)
INHERITED PLATELET DISORDERS
Multiplate multiple platelet function 5-channel impedance analyzer. (Roche Diagnostics, Burgess Hill, West Sussex, UK)
FIRST-STEP LABORATORY TEST: AGGREGOMETRY
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INHERITED PLATELET DISORDERS
Platelet agonist: collagen, ADP, epinephrine, thrombin, Arachidonic Acid
Ligh
t
tran
smis
sio
n
0%
100%
minutes
“In vitro” platelet aggregation by densitometric method of Born
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ADP
TRAP
Arachidonic acid
Collagen + Convulxin Collagen
Ristocetin
INHERITED PLATELET DISORDERS
Light transmission aggregometry (LTA)
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Shape change
Baseline level
Primary wave
Secondary wave
Maximal
aggregation
Inflection point
Light transmission aggregometry (LTA): NORMAL platelet aggregometry response
INHERITED PLATELET DISORDERS
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ADP 4 mcM ADP 2,5 mcM
ADP 2 mcM
ADP 1,25 mcM
INHERITED PLATELET DISORDERS
Light transmission aggregometry (LTA): Patterns of aggregation at different ADP concentrations
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Aggregometer
Ristocetin 1,5 mg/ml
Ristocetin 1 mg/ml
Ristocetin 0,5 mg/ml
INHERITED PLATELET DISORDERS
Light transmission aggregometry (LTA): Patterns of aggregation at different Ristocetin concentrations
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INHERITED PLATELET DISORDERS
SUMMARY
PATHOLOGIES OF GENES AFFECTING SURFACE CONTITUENTS
Glanzmann’s Thromboasthenia
Bernard Soulier Syndrome
PATHOLOGIES OF GENES AFFECTING SURFACE INTRACELLULAR PROTEINS
Dense granule deficiency
Hermansky – Pudlak Sydrome (HPS)
Chediak-Higashi Syndrome (CHS)
Alfa granule deficiency
Gray Platelet Syndrome (GPS)
Arthrogryposis, Renal Disfunction and Cholestasis Syndrome
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INHERITED PLATELET DISORDERS
SUMMARY
PATHOLOGIES OF GENES AFFECTING SURFACE CONTITUENTS
Glanzmann’s Thromboasthenia
Bernard Soulier Syndrome
PATHOLOGIES OF GENES AFFECTING SURFACE INTRACELLULAR PROTEINS
Dense granule deficiency
Hermansky – Pudlak Sydrome (HPS)
Chediak-Higashi Syndrome (CHS)
Alfa granule deficiency
Gray Platelet Syndrome (GPS)
Arthrogryposis, Renal Disfunction and Cholestasis Syndrome
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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)
Glanzmann’s Thromboasthenia
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• Autosomal recessive
• Platelet surface receptor disorder of GPIIb/IIIa, qualitative or quantitative
• Severe bleeding tendency
• Faulty platelet aggregation
• Diminished clot retraction
Glanzmann’s Thromboasthenia
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Platelet aggregation in healthy
vWF
GPIb
GPIIb-IIIa
Glanzmann’s Thromboasthenia
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vWF
GPIb
GPIIb-IIIa
GT
vWF
Platelet aggregation in Glanzmann Thrombasthenia (GT)
Glanzmann’s Thromboasthenia
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Platelet aggregation in Glanzmann Thrombasthenia (GT)
Glanzmann’s Thromboasthenia
100
1 2 3 4 5
0
20
40
60
80
Minutes
ADP 20 mcM
Collagen 20 mcg/ml
ristocetin 1.5 mg/ml
Agg
rega
tio
n %
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Flow cytometry: GP expression on platelets surface
Glanzmann’s Thromboasthenia
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)
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Glanzmann’s variant (ITGA2B/ITGB3- related thrombocytopenia)
Isotype
patient
Healthy control
Glanzmann’s Thromboasthenia
• Autosomal dominant
• Macrothrombocytopenia
• Gain of function mutation in ITGA2B/ITGB3
• Moderate to severe bleeding diathesis
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• 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
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Von Willebrand factor
Thrombin GPIIIa
myosin IIA
actin filaments
signalling to
cytoskeleton
GPIbα
GPIbβ
GPIX
GP
V
cell membrane
GPIIb
GPIb-IX-V complex
AR
P2
/3
WASP (Wiskot-AldrichSyndromeProtein)
Bernard-Soulier syndrome (BSS)
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Platelet aggregation in healthy
vWF
GPIb
GPIIb-IIIa
Biallelic Bernard-Soulier syndrome
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BSS
vWF
vWF
GPIb
GPIIb-IIIa
Platelet aggregation in Bernard-Soulier syndrome (BSS)
Biallelic Bernard-Soulier syndrome
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“In vitro” platelet aggregation on platelet rich plasma: biallelic Bernard-Soulier syndrome
Biallelic Bernard-Soulier syndrome
100
1 2 3 4 5
0
20
40
60
80
Minutes
ristocetin 3 mg/ml
ristocetin 1,5 mg/ml
collagen 20 mcg/ml
ADP 5 mcM
Agg
rega
tio
n %
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• 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
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Expression of glycoproteins on the surface platelet membrane in BSS Bolzano patients
Monoallelic Bernard-Soulier syndrome
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INHERITED PLATELET DISORDERS
Summarizing laboratory test….
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INHERITED PLATELET DISORDERS
Summarizing laboratory test….
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INHERITED PLATELET DISORDERS
Summarizing laboratory test….
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INHERITED PLATELET DISORDERS
SUMMARY
PATHOLOGIES OF GENES AFFECTING SURFACE CONTITUENTS
Glanzmann’s Thromboasthenia
Bernard Soulier Syndrome
PATHOLOGIES OF GENES AFFECTING SURFACE INTRACELLULAR PROTEINS
Dense granule deficiency
Hermansky – Pudlak Sydrome (HPS)
Chediak-Higashi Syndrome (CHS)
Alfa granule deficiency
Gray Platelet Syndrome (GPS)
Arthrogryposis, Renal Disfunction and Cholestasis Syndrome
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INHERITED DEFECT OF PLATELET GRANULES
INHERITED PLATELET DISORDERS
- δ storage pool deficiency: most common, bleeding diathesis - α storage pool deficiency: macrothrombocytopenia, mild to
moderate bleeding
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Dense granule deficiency
Hermansky – Pudlak Sydrome (HPS)
• 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
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Dense granule deficiency
Chediak-Higashi Syndrome (CHS)
• Autosomal Recessive
• Mutation in LYST
• Bleeding Diathesis mild to moderate
• Immunodeficiency
• Albinism
• Neurological disorders
• Patients frequently die from lymphoproliferative disorders
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Alfa Granule Deficiencies
Gray Platelet Syndrome (GPS)
• Autosomal Recessive
• Mutation in NBEAL2
• Macrothrombocytopenia
• Bleeding diathesis mild to moderate
• Splenomegaly
• Bone marrow fibrosis
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Alfa Granule Deficiencies
Gray Platelet Syndrome (GPS) - variant
• Autosomal Dominant
• Mutation in GFI1B
• Macrothrombocytopenia
• Bleeding diathesis mild
to moderate
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Alfa Granule Deficiencies
Arthrogryposis, Renal Disfunction and Cholestasis Syndrome
• Autosomal Recessive
• 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
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INHERITED PLATELET DISORDERS
Summarizing laboratory test….
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INHERITED PLATELET DISORDERS
Summarizing laboratory test….
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INHERITED PLATELET DISORDERS
Summarizing laboratory test….
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INHERITED PLATELET DISORDERS
Summarizing laboratory test….
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INHERITED PLATELET DISORDERS
Summarizing laboratory test….
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DIAGNOSTIC ALGORITHM FLOWCHART FOR IPD ACCORDING TO SSC AND ISTH
P. Gresele 2015, for the subcommittee on platelet physiology
INHERITED PLATELET DISORDERS
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INHERITED PLATELET DISORDERS
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
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INHERITED PLATELET DISORDERS
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
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INHERITED PLATELET DISORDERS
Platelet Transfusion
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INHERITED PLATELET DISORDERS
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
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INHERITED PLATELET DISORDERS
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..)
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INHERITED PLATELET DISORDERS
Federica MELAZZINI Università di Pavia - Fondazione IRCCS Policlinico San Matteo