Platelet Disorders Kanwar Kahlon M.D.

Division of Hematology & Oncology

PLT ANATOMY / FUNCTION

PLT Anatomy

Alpha granules

Dense granules PLTs = a fundamental unit of

hemostasis

External PLT Anatomy

PLT

GPIb-V-IX receptor (ristocetin-vWD interaction)

P2Y12 receptor (ADP receptor)

GPIIb-IIIa receptor (fibrinogen bridging)

PAR-1 receptor (thrombin)

GPVI (collagen receptor)

P2Y1 receptor (ADP receptor)

Internal PLT Anatomy

Mito-chondria

Microtubules

Glycocalyx

TXA2

DENSE GRANULES (ADP, ATP, serotonin, Ca2+)

ALPHA GRANULES (PF4, fI, fV, fVIII/vWF, Ig, RANTES, BTG)

Surface connecting system

fXIII COX

PLT Activation • PLTs become activated by two primary mechanisms:

• Shear-induced activation • Bypass of exposed endothelium induces shape changes and

activation • Chemical activation

• Typically arises from local environment and other activated PLTs

• Both of the pathways are at work in the bleeding individual

PLT Shear & Chemical Release

Shear induces PLT shape change, adhesion to endothelium, and activation This enhances granule secretion, PLT recruitment, and clot propagation

PLT shear induces adhesion (via vWF and GPIb-V-IX) and

activation (shape change & granule release)

Aggregation occurs via fibrinogen-GPIIb-IIIa interaction;

clot propagation by additional PLT activation

PLT plug formation: putting it together

PTT, or APTT, starts here, to make fibrin:

PT goes from here to fibrin:

(also activates other factors)

(also activates IX)

Stages of Hemostasis

• Primary Hemostasis • von Willebrand’s factor and platelets

• Secondary Hemostasis • Coagulation cascade

• Tertiary Hemostasis • Cross-linking of fibrin strands • Clot maturation and wound healing

Thrombocytopenia

Platelet dysfunction may show up as petechiae, but something this prominent is usually thrombocytopenic (unless its vasculitic)

PLT related bleeding

Epistaxis Petechial rash

Cerebral hemorrhage

Anatomic distribution of bleeding diathesis Plt dysfunction or

vWD Coagulation

factor deficiency

Petechiae Body cavity bleeding

Anatomic sites Skin

Mucosal surfaces (oral, nasal, GI, GU)

Deep tissues Muscles Joints

Petechiae Common Uncommon

Ecchymoses Superficial, small Deep, extensive (hematomas)

Bleeding after minor cuts Yes No

Bleeding after surgery Immediate Delayed

Common thrombocytopenias

• Splenomegaly/hypersplenism • Chronic Hepatitis C • Cirrhosis • Sepsis • DIC • Drug induced • Immune thrombocytopenia (ITP, HIV) • Pseudothrombocytopenia

Pseudothrombocytopenia

• Platelet clumping in EDTA (lavender top tubes), with decreased automated Plt quantitation

• Redraw CBC in tubes with citrate or heparin anticoagulant

Case 1

• 66 yo man presents with petechiae of the extremities, and ecchymoses, within several weeks of an URI. Plts are 3,000. He is given IVIg (1 gm/kg), and Dexamethasone 40 mg daily for 4 days is started. His Plts increase quickly, and he is discharged the following day. CBC within 1 wk reveals a complete response. Plts remain normal several months later.

Case 2

• 54 yo woman with “cutaneous lupus”, taking hydroxychloroquine, presents with petechiae of the extremities, oral purpura, and diffuse ecchymoses. Plts are 5,000. Steroids are started, and IVIg 2 gm/kg is given over 2 days, without a response. Despite treatment for ~10 days in the hospital, Plts remain <10,000. Weekly Romiplostim is initiated, as is a 4 wk course of Rituxan. Plts are >100,000 2 wks later. Steroids are tapered slowly over several months. However, 2 wks after steroids are stopped, she presents again with ecchymoses, purpura, and petechiae, and Plts are <1,000.

• Secondary ITP 20% of total ITP cases

Misc systemic infection 2%

SLE 5%

APS 2%

CVID 1%

CLL 2% Evan’s 2% ALPS, post-tx 1% HIV 1% HCV 2%

H pylori 1% Postvaccine 1%

Primary 80%

Cines, Blood, 2009

Immune thrombocytopenia

ASH-SAP, 5th ed.

Harrington’s Classic Experiment

Harrington, J Lab Clin Med, 1951

Epitope Spreading Leads to Diversity of Platelet Targets

Cines, N Eng J Med, 2002

Glycoprotein IIb/IIIa

autoantibody

Glycoprotein Ib/IX

Anti- glycoprotein

Ib/IX

Anti glycoprotein

IIb/IIIa

Glycoprotein Ib/IX B-cell clone 1 T-cell clone 1 Glycoprotein

IIb/IIIa

Glycoprotein Ib/IX

Activated macrophage

Glycoprotein IIb/IIIa

Antibody-coated platelet

Fcγ

CD40 CD154

CD40

TCR TCR

B-cell clone 2 T-cell clone 2

Molecular Mimicry in ITP

Aster, Blood, 2009

Clearance of Antibody-Coated Platelets by Phagocyte Fcγ Receptors

1. Karpatkin, Lancet, 1997 2. Psaila, J Clin Invest, 2008

Antibody-Coated Platelet[1] Fcγ Family of Receptors[2]

Low to medium

Low to medium

Low to medium

Low to medium

Low to medium

High Affinity for Fc Fragment

Cell membrane

Production and Survival of Platelets in ITP

1. Harker, Br J Haematol, 1970 2. Branehög, Br J Haematol, 1974 3. Stoll, Blood, 1985 4. Ballem, J Clin Invest, 1987

Platelet Survival, Days

Study Platelets Normal Subjects ITP Pts Turnover

(x Normal) Harker 1970[1] Allogeneic* 9.9 0.34 4.9 Branehög 1974[2] Allogeneic† 6.9 0.67 2.3 Stoll 1985[3] Autologous 8.0 2.9 0.9 Ballem 1987[4] Autologous 9.6 2.8 0.6

*Subjects with platelet counts > 25 x 109/L received autologous platelets. †Subjects with platelet counts > 20 x 109/L received autologous platelets.

Smears

ASH Image Bank

Blood

Marrow aspirate

Bleeding Manifestations in ITP

Purpura

Ecchymoses, petechiae

CNS hemorrhage (rare)

Goals of ITP Therapy in Adults • Maintain a safe platelet count with minimal toxicity • May need to individualize treatment strategy based on

Plt count (<30,000), assessment of bleeding risk, and patient preference

Neunert, Blood, 2011

Recommended ‘Safe’ Platelet Ranges

Br J Haematol, 2003

Clinical Situation Platelets

General dentistry Extractions Regional dental block

≥ 10 x 109/L ≥ 30 x 109/L ≥ 30 x 109/L

Surgery Minor Major

≥ 50 x 109/L ≥ 80 x 109/L

Pregnancy Vaginal delivery Caesarean section Spinal/epidural anesthesia

> 50 x 109/L > 80 x 109/L > 80 x 109/L

*British Committee for Standards in Hematology General Hematology task force

Therapy Options

Provan, Blood, 2010

Clinical Situation Therapy Options

First line (initial treatment)

Corticosteroids: prednisone, dexamethasone, methylprednisolone

IVIg Anti-D

Second line

Azathioprine Cyclosporine A

Cyclophosphamide Danazol Dapsone

Mycophenolate mofetil Rituximab

Splenectomy TPO receptor agonists (romiplostim and eltrombopag)

Vinca alkaloids Treatment for patients failing first- and second-line therapies

Category A*: TPO receptor agonists Category B†: campath-1 H, combination of first- and second-

line therapies, combination chemotherapy, HSCT *Sufficient data to support recommendation. †Minimal data to support recommendation; potential for considerable toxicity.

Differential Diagnosis

• Disseminated intravascular coagulation (Consumptive coagulopathy) • Thrombotic thrombocytopenic purpura/ Hemolytic uremic syndrome

DIC (Consumptive Coagulopathy)

• Uncontrolled activation of the coagulation cascade: unregulated thrombin generation mediated by activation and injury of the endothelium or by mechanical injury to tissues leading to excessive exposure of tissue factor and/or lipid surfaces

• Infection • Malignancy • Obstetric complications • Tissue Injury

The Coagulation Cascade

Fibrinogen

VIIIa VIIa

Phospho- lipid

Phospho- lipid

Phospho- lipid

Xa

Va

IXa TF

II IIa

Fibrin

XIa

IX

X

V

VIII

Clinical Manifestations of DIC

• Asymptomatic • Bleeding

• The primary association with DIC when symptomatic

• Thrombosis • Rare (seems to be associated with infectious causes of DIC)

Laboratory Diagnosis

• Microangiopathic hemolysis • Thrombocytopenia • Prolonged PT and/or PTT • Elevated thrombin time (TT) • Decreased fibrinogen • Increased D-dimer

Caveats to Laboratory Diagnosis

• Though numerous coagulation parameters may be abnormal in DIC (elevated PT/PTT/D-dimer, decreased platelets and fibrinogen) not every one needs to be present to make the diagnosis

• Relative change may be important (dropping fibrinogen or platelets)

Management of DIC • Treat the underlying cause • Early and aggressive blood product support in

symptomatic cases • Platelets • Plasma • Cryoprecipitate • Red cells

Case 3

A 41 year-old woman with no significant past medical history presents to her primary care provider with shortness of breath and bruising. She is taking no medications. There is no family history of any bleeding disorder. She denies fever, chills, nausea, vomiting, diarrhea. Physical examination reveals scattered petechiae and ecchymoses, a I/VI systolic ejection murmur, and a normal mental status.

Laboratory Data

WBC 9,600 Hct 18% Platelets 16,000 PT 12.1 s PTT 24.6 s Creatinine 0.9 LDH 775

Thrombotic Thrombocytopenic Purpura/Hemolytic Uremic Syndrome

• Congenital • Idiopathic • Drug-related • Toxin-related

Diagnosing thrombotic microangiopathy (TMA)

• Hemolytic Anemia • Mechanical – schistocytes • Response – reticulocytosis • Increased LDH • Hyperbilirubinemia • Suppressed haptoglobin (undetectable)

• Thrombocytopenia • Often in the range of 20-60,000/mcL

Basic pathophysiology

• Endothelial attack upon circulating red cells and platelets; RBCs fragmented ↑LDH

• Platelets: microthrombi (& decreased Plts) organ dysfunction ↑LDH

• ADAMTS-13 testing is informative for TTP • Conditions of Intense Endothelial Activation

• Hypertensive emergency • Scleroderma Renal Crisis • Disseminated Cancer/Chemotherapy

• Hemolytic Uremic Syndrome (HUS) • Atypical HUS – complement mediated TMA

Moake JL, 2002. New England Journal of Medicine, 2002;347(8):589-600

Clinical Pearl

• Think twice about making the diagnosis of TTP when the PT and/or PTT is abnormal - DIC is much more common

Drugs Associated with TTP

• Clopidogrel (Plavix) • Ticlopidine (Ticlid) • Cyclosporin • Mitomycin C • Gemcitabine

Infections Associated with TTP or HUS

• E. coli 0157:H7 • Shigella (verotoxin producing) • HIV

Etiology of TTP

• Deficiency of von Willebrand cleaving protease • Congenital ADAMTS13 deficiency • Acquired through development of an inhibitor (antibody)

ADAMTS 13 and TTP

Management of TTP

• Daily plasmapheresis with plasma exchange until platelet count >150,000, then taper slowly

• Monitor CBC, retics, LDH, creatinine

Course of TTP

• About 90% of cases of TTP improve with plasmapheresis • About 30% of these patients relapse, yet a fair number of these

respond to another course of plasmapheresis • About 10% of patients have refractory disease after a prolonged

course of pheresis

Second Line Therapy for TTP

• Immunosuppresants • Steroids • Cyclophosphamide • Rituximab

Keir LS. Hemolytic Uremic Syndrome. Hematol Oncol Clin N Am. 2015;29:525-539.

Shiga Toxin (STx) producing E. Coli (STEC)

STx binds to Gb3 on colonic epithelium, endothelial

cells, renal mesangial cells,

Cell death with organ specific dysfunction

HUS

vWF and thrombosis in HUS

Etiology of atypical HUS • Appears to result from abnormalities in complement

regulatory proteins (factor H, factor I, others), complement activation, and the development of endothelial injury

Nester CM, Barbour T, Rodriguez de Cordoba S, Dragon-Durey MA, Fremeaux-Bacchi V, Goodship THJ, et al. Atypical HUS: State of the Art. Molecular Immunology, 2015;67:31-42) George JN, Nester CM. Syndromes of Thrombotic Microangiopathy. New England Journal of Medicine, 2014;371:654-666.

79% of episodes occur postpartum

“Thickened arteriolar and capillary walls, prominent endothelial swelling and detachment, subendothelial swelling and detachment, subendothelial accumulation of cell debris, and fibrin and platelet-rich thrombi obstructing vessel lumina is characteristic of all TMAs.” – Nester, et al.

C3 C3b C3a

C3bBbC3b

Factor H Factor I

MCP (CD46)

Factor H

C5

C5b MAC

Eculizumab

C5a

Factor B

Fact

or D

C3b(H2O)Bb Fluid Phase C3 Convertase

C3bBb AP C3 Convertase

• Altered binding sites reduce cell surface binding/protection • Deletions predispose to FH autoantibodies • FHR dimers may competitively antagonize Factor H

Factor B

Fact

or D

• Implicated mutations in MCP – a surface bound cofactor for Factor I – primarily reduce copy number

• Other mutations in Factor I reduce C3b inactivation capacity

• Decreased binding to MCP • Increased affinity for Factor B • Decreased susceptibility of C3

convertase to normal decay

Nester CM, Barbour T, Rodriguez de Cordoba S, Dragon-Durey MA, Fremeaux-Bacchi V, Goodship THJ, et al. Atypical aHUS: State of the Art. Molecular Immunology, 2015 (67:31-42.

Heparin-mediated destruction • Heparin-mediated destruction is a common form of drug-induced

thrombocytopenia • Also called heparin-induced thrombocytopenia (HIT)

• In this case, heparin + PLT factor 4 (PF4) form a neoantigen on the PLT surface

• This neoantigen induces an antibody response resulting in PLT clearance

• Testing revolves around detecting the heparin-dependent antibody • Stopping heparin should cause PLT count to normalize within a few

days

HIT

• HIT is particularly dangerous because it decreases PLT counts, but may also induce a hypercoagulable state in some patients

• When the antibodies interact with the PLTs, they can cause their activation

• This can induce clotting despite the decreased counts

• Patients with HIT and Thrombosis (called HITT) require anticoagulation

Heparin induced thrombocytopenia (HIT)

4T score for HIT

• ≤ 3: low prob (1.6%) • 4-5: int prob (28.6%) • 6-8: high prob (100%)

Diagnosis of HIT

• Screen for Ab to Heparin/PF4 (ELISA)

• Serotonin release assay • confirmatory test

Management of suspected HIT

• STOP Heparin • Use non-heparin agent: direct thrombin inhibitor (eg, argatroban), or

fondaparinux • Await confirmatory testing

Questions?