Qualitative Platelet Disorders Shejal patel Do

Platelet Production Average platelet life span is 7-10 days. Platelets are lost through senescence, activation, or other processes. New platelets arise from bone marrow megakaryocytes at a rate of 1 x 1011 to 3 x 1011 platelets per day. This production can increase 10 fold during times of high demand. Platelet production is mostly regulated by hormone thrombopoietin(TPO) and its cellular receptor, c-Mpl. TPO binds to megakaryocytes and hematopoietic stem cells via c-Mpl which activates several signaling pathways in megakaryocytesmegakaryocytes proliferation and differentiation--> platelet production.

Platelet Production TPO produced in liver cells. In addition to TPO, stem cell factor, interleukin 3, interleukin 6, and interleukin 11 are all required for optimal megakaryocyte development. Megakaryocyte growth and differentiation involve endomitosis and polyploidization, where nucleus divides and cell does not. Next there is cytoplasmic maturation and eventually projection of multiple filamentous pseudopodial structures called proplatelets. Fragmentation of the proplatelets leads to release of new platelets. Platelets are removed from circulation by monocyte macrophage system.

Main molecular mechanisms affected in malignant megakaryopoiesis and platelet function defects. Main molecular mechanisms affected in malignant megakaryopoiesis and platelet function defects. Schematic representation of (A) TPO/MPL signaling pathway together with (B) the most relevant transcriptional regulators of megakaryocyte commitment, differentiation, and maturation. GP2B (CD41) is a component of the GPIIb-IIIa glycoprotein complex. GP9 (CD42a), GP1BA (CD42b), and GP1BB (CD42c) are components of the GPIb-IX-V glycoprotein complex. HSC, hematopoietic stem cell; MEP, megakaryocyte-erythroid progenitor; MK, megakaryocyte. Elisa Bianchi et al. Blood 2016;127:1249-1259 ©2016 by American Society of Hematology

Platelet Stucture

When vascular endothelium is injured, platelets adhere to exposed intimal surface primarily though vWF. Platelet adhesion results in generation of intracellular signals that lead to activation of the platelet glycoprotein(GP) IIb/IIIa receptor and resultant platelet aggregation. Activated platelets release granule contents, including nucleotides, adhesive proteins, growth factors, and procoagulants that serve to promote platelet aggregation and blood clot formation, and influence the environment of the forming clot. During platelet aggregation, additional platelets are recruited to the site of injury, leading to formation of an occlusive platelet thrombus. Platelet plug is stabilized by the fibrin mesh that develops simultaneously as the product of the coagulation cascade. Figure 5: After an injury in the vessel wall, activation of platelets begins to start. It involves its adhesion to the subendothelium surface. Interaction between receptors like GPIb-V-IX, GPIa-IIa, and subendothelial compounds like vWf and collagen triggers the release of platelet granule contents accelerating aggregate formation.

Qualitative Platelet Disorders Disorders of platelet adhesion Disorders of platelet aggregation Disorders of platelet secretion and signal transduction Disorders of platelet procoagulant activities Other abnormalities Acquired disorders of platelet function

Disorders of platelet adhesion

Case 1 20 yo female who history of chronic refractory ITP comes to see you in clinic. She was diagnosed at age 1. Her platelets are around 60k. She bled after a splenectomy and has heavy periods requiring frequent iron infusions. Her bleeding with the splenectomy got under control with platelet transfusions. She has not had significant response with ITP directed treatments: steroids, IVIG, win Rho, rituxan and splenectomy. Her coags are normal. Her cbc shows wbc of 5 with normal diff, hgb 12 and platelets 60. You suspect she does not have ITP and look at her peripheral smear.

Case 1 Platelet aggregation study is done which shows normal response to ADP, epinephrine, thrombin and collagen while a decreased response to ristocetin.

Bernard-Soulier Syndrome autosomal recessive. Found in populations with consanguinity. Gene is on chromosome 17 prolonged bleeding time(>20mins), very large platelets, and thrombocytopenia. Have bleeding because their platelets are unable to adhere to von willebrand factor in subendothelial matrix bc of deficiency of GPIb-IX complex.

Bernard-Soulier Syndrome Diagnosis Platelet aggregation response to ADP, epinephrine, thrombin and collagen are normal and response to ristocetin is decreased or absent (a feature shared with severe vWD). * vWF and f8 are normal in BSS unlike vWD. Addition of normal vWf does not restore ristocetin induced agglutination of platelets because of GPIb deficiency while it does in vWD. Confirm diagnosis with flow cytometry showing decreased platelet surface GPIb.

Bernard-Soulier Syndrome Treatment Significant bleeding or surgical procedure: platelet transfusions (use leukodepleted platelets and try to keep at minimal because of isoimmunization. Bleeding despite platelets would try Novo7. Menses-hormonal suppression, amicar Minor mucosal bleeding or as prophylaxis: amicar, DDAVP

Disorders of Platelet Aggregation

Case 2 18 yo male comes to hematology clinic for evaluation of bleeding disorder after significant bleeding after a tonsillectomy. He has no chronic medical problems and not had any other surgeries other than tonsillectomy. He is on no medications and doesn’t take any over the counter meds. No family history of bleeding. He has nose bleeds about 5 times a year and they last for about 40 minutes. He also notes that he easily bruises. Labs show a normal cbc with diff, normal PT/PTT and peripheral smear not remarkable. What makes you think this could be a platelet function disorder?

Platelet function disorders Clinical Manifestations Bleeding after invasive procedures Epistaxis that lasts longer than 30 mins or causes anemia or hospital admission Extensive bruising or soft tissue hematoma Gingival bleeding Mennorhagia Bleeding with child birth Spontaneous hemarthrosis and deep hematomas are unusual

Case 2(back to…) You check von willebrand ag and assay and it is normal. You check a platelet aggregation and it shows normal response to ristocetin induced platelet aggregation and absent platelet aggregation in response to ADP, epinephrine, and collagen. You suspect this is Glanzmann Thrombasthenia so you order an additonal test…

Glanzmann Thrombasthenia Autosomal recessive, gene on chromosome 17. Found in populations where consanguinity is common. Primary abnormality is a quanitative or qualititative defect in GPIIb-IIIa complex (can send flow cytometry to confirm diagnosis). Poor aggregation with ADP, collagen, epi. Normal ristocetin induced aggregation. Morphologically normal platelets and normal count Absent clot retraction Prolonged bleeding time Coags normal, this can help distinguish from congenital afibrinogenemia (prolonged PT, PTT, TT and absent fibrinogen) which also has absence of platelet aggregation.

Glanzmann Thrombasthenia Type Level between 20 to 50% is needed to prevent bleeding. 50% of normal number of gpiib-iiia complexes per platelet is sufficient for platelet aggregation. risk of bleeding doesn’t always correlate with extent of gpiib/iiia defieciency

Glansmann Thrombasthenia Treatment Localized bleeding can use topical thrombin or antifibrinolytic agents Invasive procedures use prophylactic platelet transfusion If platelets don’t work in a patient bleeding despite platelets because of alloantibodies then can give recombinant factor VIIa. Menses-hormonal suppression, amicar

Disorders of Platelet Secretion and Signal Transduction

Case 3 18 yo male comes to clinic for evaluation of chronic easy bruising and recurrent epistaxis. Epistaxis lasts 50 minutes at times and he has had to go to the ER previously. He has had no surgeries. On physicial exam he has scattered bruises on his extremities. His labs show a wbc of 6.2 with normal diff, hgb 13,plts 190, nml coags, factor 8, vWF ag and activity. You next order a platelet aggregation which shows no secondary wave in response to ADP and epinephrine and decreased aggregation with collagen. Response to ristocetin is normal. Your diagnose the patient with a platelet secretion defect.

Storage Pool Disease Deficiency of granules or their constituents that results in a defect in ADP release from activated platelets and abnormal secretion dependent platelet aggregation. The extension phase of the clot formation and platelet activation is mediated in large part by the release of stored compounds from platelet granules. Platelet aggregation shows absence of second wave of aggregation upon stimulation of platelet rich plasma with ADP or epinephrine; responses to collagen, thromboxane and arachidonic acid also may be impaired. Prolonged bleeding time

Types of Granules in Platelet Cytoplasma ⍺ (alpha) granules contain a variety of proteins some derived from plasma others synthesized by megakaryocytes 𝛿 (delta) granules or dense body contain ADP, adenosine triphosphate, calcium, serotonin, and pyrophosphate.

⍺ Storage Pool Disease Gray Platelet syndrome-thrombocytopenia(avg 60-100K) and prolonged bleeding time. Peripheral smear shows platelets with a gray appearance. Inheritance pattern is variable-autosomal recessive, autosomal dominant, and sex-linked patterns have been described. Platelet aggregation are variable with no classical response pattern to ADP, epinephrine, thrombin or collagen. Primarily diagnosis made with electron microscopy which shows a near complete absence of ⍺ granule in platelets Treatment-platelet transfusion.

ASH Image Bank Gray platelet

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012593/ (A) EM of thin sections of a platelet from patient 1(V-20) showing absence of α-granules and abundant channels of the open canalicular system (labeled as OCS) in comparison with EM of a control platelet (B) showing normal alpha granules (labeled as AG). DTS: Dense tubule system. (C) Light microscopy of the peripheral smear of GPS patient 1(V-20) showing a large pale gray platelet (arrow) without the cytoplasmic color of α-granules, in comparison with a normal smear (D)

⍺ Storage Pool Disease Quebec platelet disorder-autosomal dominant. Platelet aggregation deficiency is most striking with epinephrine. Normal to reduced platelet counts According to Hoffman bleeding unresponsive to platelet transfusion Some studies favor fibrinolytic inhibitors to control bleeding.

𝛿 Storage Pool Disease Likely autosomal dominant On peripheral smear platelets appear normal and platelet number is adequate. Platelet aggregation show impaired second wave of aggregation when stimulated by ADP, epinephrine or thrombin. Could measure platelet ADP and ATP to help with diagnosis. Primarily diagnosis made by electron microscopy which would show lack of dense granules. Treatment platelet transfusion

Normal platelet Whole mount EM. Arrow is dense granule C absent dense granules

𝛿 Storage Pool Disease associated disorders Hermansky-Pudlak syndrome(HPS)-autosomal recessive disorder with oculocutaneous albinism, lysosomal granule defects and platelet dense granule deficiency. Chediak-Higashi syndrome(CHS)-autosomal recessive disorder with oculocutaneous albinism, dense granule deficiency, immunodeficiency and progressive neurological deterioration. In accelerated phase of disease can develop thrombocytopenia. This disease arises from mutations in lysomal trafficking regulator (LYST) gene on chromosome 1.

Defect in platelet signal transduction and platelet activation Disorders of platelet membrane receptors can affect platelet extension and aggregation. Platelet membrane receptors that can be affected are P2Y12 ADP receptor (Plavix targets this receptor), TxA2 receptor, collagen receptors(GPIa-IIa and GPVI) and epinephrine receptor. G proteins are a link between surface receptors and intracellular effector enzymes. Defects in G proteins cause impairment of platelet signal transduction.

Disorders of platelet procoagulant activities

Disorders of Platelet Procoagulant activities Scott Syndrome-extremely rare inherited disorder that occurs due to lack of exposure of procoagulant phosphatidylserine (PS) to external leaflet of the plasma membrane of activated platelets and other hematologic lineages. This compromises thrombin generation and thus clot formation. Only 3 cases of Scott Syndrome reported

Other abnormalites

Other abnormalities Wiskott-Aldrich syndrome-WAS-an x linked inherited disorder affecting T lymphocytes and causes thrombocytopenia, eczema and immunodeficiency. Bleeding manifestations are variable. In WAS there have been several platelet abnormalities including dense granule deficiency and deficiencies of GPIb, GPIIb-IIIa, and GPIa reported. RUNX1-patients with RUNX1 mutations have hereditary thrombocytopenia, platelet dysfunction and a predisposition to acute leukemia.

Acquired disorders of platelet function

Case 4 27 yo female comes to see you for increased bruising. She has some anxiety and is on zoloft. She denies heavy menses, epistaxis or any bleeding issues with surgeries or procedures. Her labs are unremarkable. Is your next step going to be to order a platelet aggregation study? No, question whether zoloft could be cause by chronology.

Acquired disorders of platelet function Drugs and other agents Antiplatelet antibodies Acquired von willebrand disease Acquired storage pool deficiency Cardiopulmonary bypass Dysproteinemias Acute leukemia and MDS Myeloproliferative disorders Uremia

Schematic cartoon representing the proteins mutated in inherited platelet function disorders. Schematic cartoon representing the proteins mutated in inherited platelet function disorders. Light blue ovoids represent α-granules, dark blue spheres represent δ-granules. LAD-III, leukocyte adhesion deficiency-III syndrome; VWD, von Willebrand disease. Elisa Bianchi et al. Blood 2016;127:1249-1259 ©2016 by American Society of Hematology

References Simon et al. “Platelet function defects”. Hemophilia (2008), 14, 1240-1249. Cuker et al. “Disorders of platelet number and function”. ASH-SAP 6th edition, p 249-276. Nurden. “Qualitative disorders of platelets and megakaryocytes”. Journal of Thrombosis and Haemostasis, 3: 1773-1782. Sharathkumar et al. “Platelet Function Disorders” 2nd edition. http://exodontia.info/files/Treatment_of_H_mophilia_2008._Platelet_Functio n_Disorders.pdf Bolton-Maggs et al. “ A review of inherited platelet disorders with guidelines for their management on behalf of the UKHCDO” BJH, 135, 603- 633.

References Diz-Kucukkaya. “Inherited platelet disorders including Glanzmann thrombasthenia and Bernard-soulier syndrome”. American Society of Hematology Education Program. December 6, 2013. p 268-275. Bianchi et al. ”Genomic landscape of megakaryopoiesis and platelet function disorder”. Blood 2016 127: 1249-1259. Ghoshal et al. “Overview of Platelet Physiology: Its Hemostatic and Nonhemostatic Role in Disease Pathogenesis”. The Scientific World Journal vol 2014, article ID 781857, p1-16 Fritsma. “Platelet structure and function”. Clin Lab Sci 2015; 28(2): 125. Dan Longo. Harrison’s Hematology and Oncology 2010 p224-234.

References Hoffmann et al. Hematology Basic Principles and practice 3rd edition. Page 2154- 2172