MEET THE PLATELET

K. Krishnan MD Department of Internal

Medicine

Acknowledgements My teachers at PGI,

Chandigarh, Hammersmith Hospitals, UK and U of Michigan, Ann Arbor

American Society of Hematology for images

LEARNING OBJECTIVES

Understand platelet development and function

Understand the classification of platelet disorders

Understand the clinical manifestations of platelet disorders

Understand the methods available to diagnose platelet disorders

Understand the pharmacological agents used to treat platelet disorders

PLATELET HEMATOLOGY

Platelet development and kinetics Platelet tests Clinical aspects of platelet disorders Qualitative platelet disorders

Platelet function disorders Congenital Acquired

Quantitative platelet disorders Thrombocytopenia Thrombocytosis

Platelet therapeutics

PLATELET DEVELOPMENT Small anucleate fragments formed from the

megakaryocyte cytoplasm Characteristic discoid shape Hematopoeitic stem cells are converted into MGKs by

exposure to the specific growth factor, thrombopoietin Tpo initiates a maturation program

Amplifies the megakaryocyte DNA Synthesis of platelet-specific proteins Cytosketal elements, membrane systems and receptor proteins

are bulk produced Platelet production begins when microtubules aggregate in the

cell cortex, elaborate pseudopodia These pseudopodia develop into proplatelets Platelets are assembled at the end of proplatelets Microtubules deliver intracellular organelles into these

proplatelets Platelets are released from the ends of proplatelets

PLATELET KINETICS

Platelets are produced in bone marrow by megakaryocytes

MGKs produce platelets by cytoplasmic shedding into bone marrow sinusoids

1000-5000 platelets per MGK 35k to 50k platelets per microl of whole

blood per day Platelet life span 8-10 days Removed from circulation by monocyte-

macrophage system

Determinants of megakaryocytopoiesis and thrombopoiesis.

Battinelli E et al. PNAS 2001;98:14458-14463

©2001 by National Academy of Sciences

EARLY MEGAKARYOCYTE

INTERMEDIATE STAGE MEGAKARYOCYTE

MATURE MEGAKARYOCYTE

PLATELET FUNCTIONS

Adhere to sites of vascular injury Generate biological mediators Secrete granule contents Form multicellular aggregates Serve as a nidus for plasma coagulation

reactions

PLATELET FUNCTIONS

For these platelet functions, Structural rearrangements Utilize multiple membrane receptors

Bind small molecule mediators Bind adhesive glycoproteins and constituents of

vascular endothelium Activate a network of complex signaling pathways

HOW TO ASSESS PLATELETS

Automatic/Manual Platelet count Peripheral smear Bone marrow examination and specialised

tests Platelet function testing

PFA test/screening test Specific tests using platelet aggregometry (many

methods/instruments) Thrombin, Collagen, ADP, Arachidonic acid, Ristocetin

Antibody assays

CLINICAL FEATURES IN PLATELET DISORDERS

Splenomegaly/Chronic liver disease Petechiae or dry purpura

Begins in the dependent portions of the body due to venous pressure-ankles and feet in an ambulatory patient

Occurs when platelet count decreases; not seen in disorders of platelet function

Differentiate dry non-palpable purpura from palpable purpura seen in vasculitis eg. Henoch-Schonlein purpura

Wet purpura- look in mouth, oral mucosa Sign of severe thrombocytopenia Denotes risk for significant hemorrhage

Excessive bruising Seen in disorders of platelet function and number

CLINICAL FEATURES OF PLATELET DISORDERSHIGH PLATELET COUNT

Thrombocytosis Symptoms due to high platelet count

Easy bruising Bleeding due to platelet dysfunction Thrombotic tendencies TIAs Erythromelalgia Mild splenomegaly

BRUISING

PU

RP

UR

A

PURPURA

PU

RP

UR

A

Seen in dependent areas of the body

Palpable purpura: Henoch-Schonlein Purpura

Date of download: 6/10/2012Copyright © 2012 American Medical Association.

All rights reserved.

SCURVY

Arch Dermatol. 2010;146(8):938-938. doi:10.1001/archdermatol.2010.162

PLATELET FUNCTION DISORDERS Defects of platelet-vessel wall interaction (disorders of

adhesion) Von Willebrand disease Bernard Soulier syndrome

Defects in platelet- platelet interaction (disorders of aggregation) Congenital afibrinogenemia Glanzman’s thrombasthenia

Disorders of platelet secretion and abnormalities of granules Storage pool deficiency Quebec platelet disorders

Disorders of platelet secretion and signal transduction Defects in platelet- agonist interaction (TXA2, COX, Collagen, ADP)

Defects in cytoskeletal regulation Wiskott- Aldrich syndrome

Disorders of platelet coagulant-protein interaction (membrane phospholipid defects) Scott syndrome

INHERITED PLATELET DISORDERS

Rare, heterogenous group Not often seen in clinical practice Yet fascinating abnormalities that provide

insight into normal platelet biochemistry and physiology

INHERITED PLATELET DISORDERS

Disorders of Platelet membrane Platelet granule packaging Hereditary macrothrombocytopenias Platelet signaling disorders Platelet coagulant function disorders

PLATELET MEMBRANE DISORDERSGLANZMAN’S THROMBASTHENIA

“Weak platelets” Platelets carry out most of the functions Platelet count is normal Platelet morphology is normal Platelets adhere normally to vascular

endothelium Platelets secrete granules and perform

normal signalling functions Platelets DO NOT AGGREGATE due to loss

of GpIIb/IIIa receptor Normally this complex binds fibrinogen linked

into multicellular aggregates

PLATELET MEMBRANE DISORDERSGLANZMAN’S THROMBASTHENIA

Inherited Most are compound heterozygotes Life long mucosal bleeding Life long platelet transfusions Recombinant Factor VII

Acquired Rare, autoantibodies that bind to GpIIb/IIIa

epitopes Seen in ITP and in patients with normal counts Steroids may not work Immunotherapy/Rituxan may work

BERNARD SOULIER SYNDROME

Autosomal recessive Gp1b deficiency or defect Gp1b is the principal receptor for vWF No functioning Vwf receptor Platelets cannot adhere to vascular endothelium Giant platelets and thrombocytopenia

Large size due to lack of interaction between actin binding proteins in platelet cytoskeleton and cytoplasmic domain of gp1b

Lack of gp1b bound sialic acid residues causes shortening of platelet survival leading to thrombocytopenia

Platelet transfusions, DDAVP and fibrinolytic inhibitors like EACA

WHAT IS THIS?

ACQUIRED QUALITATIVE PLATELET DISORDERS

Drugs Aspirin

Treat with platelet transfusions for severe bleeding NSAIDs Glycoprotein inhibitors like Abciximab ADP receptor antagonists like Clopidrogel

Uremia Toxic effects of uremia plasma, impaired platelet-

vessel wall adhesion and increased production of NO Platelet transfusions ineffective Treat with dialysis, DDAVP, conjugated estrogens

Myeloproliferative disorders Myelodysplastic disorders

WH

AT IS

TH

IS?

How does it happen?

PS

EU

DO

-TH

RO

MB

OC

YTO

PEN

IA

Pseudothrombocytopenia secondary to platelet satellitism is illustrated in this image. Platelets are shown to adhere to the cytoplasmic membrane of two of the PMNs present on this peripheral blood smear. This phenomenon is an in vitro artifact that occurs with EDTA anticoagulant. Collection of the blood specimen in either sodium citrate or heparin corrects the abnormality.

CLASSIFICATION OF THROMBOCYTOPENIA Impaired or decreased production

Congenital May –Hegglin anomaly Bernard- Soulier syndrome Wiskott- Aldrich syndrome TAR Congenital amegakaryocytic thrombocytopenia

Neonatal Infective/viral Drug induced Acquired

Increased platelet destruction Immune

ITP Drug induced HIT

Non-immune Thrombocytopenia in pregnancy and pre-eclampsia HIV TTP DIC HUS Drugs

Disorders related to distribution or dilution Splenic sequestration Kasabach-Merritt syndrome Hypothermia Loss of platelets- massive blood transfusion, extracorporeal circulation

THROMBOCYTOPENIA

Impaired or decreased platelet production Megakaryocyte hypoplasia

Usually congenital and include Fanconi anemia, thrombocytopenia with absent radii

(TAR syndrome), Wiskott- Aldrich syndrome, Bernard- Soulier syndrome, May Heglin anomaly, congenital amegakaryocytic thromobocytopenia

Ineffective thrombopoeisis Megaloblastic anemia

Miscellaneous Viral Marrow infiltration by malignancy, myelofibrosis

MAY-HEGGLIN ANOMALY

MAY-H

EG

GLIN

AN

OM

ALY

A macrothrombocyte is present in this view. The PMNs have blue cytoplasmic inclusions bordering the cell surface membrane. These inclusions contain precipitated non-muscle myosin heavy chains characteristic of this group of congenital quantitative platelet disorders. Neutrophil function in this disorder is normal.

CONGENITAL AMEGAKARYOCYTIC THROMBOCYTOPENIA

AR disorder causing bone marrow failure Seen in infancy Platelet count <20 Petechiae and physical anomalies Develop aplastic anemia, MDS and leukemia Stem cell transplantation is curative Mutations in the c-mpl gene leading to loss of

the thrombopoietin receptor function Loss of TPO receptor function causes

reduction in MGK progenitors and high TPO levels

ACQUIRED HYPOPLASIA

Drugs Chemotherapy drugs Zidovudine Ethanol Interferon therapy Anticonvulsants Antibacterial agents like chloramphenicol

INFECTION INDUCED THROMBOCYTOPENIA

Many viral and bacterial infections without DIC

Infections affect platelet survival and production; immune mechanisms can also be at work (Infectious mononucleosis, early HIV)

At times, bone marrow exam may be required for occult infections

THROMBOCYTOPENIAINCREASED PLATELET DESTRUCTION

Immune thrombocytopenic purpura Acute

Disorder of children Abrupt onset Follows an infection usually nonspecific respiratory or

GI virus Diagnosis is clinical Most patients recover without treatment within 3

weeks Severe cases can be treated with IVIG, platelet

transfusions and splenectomy Occasionally seen in adults

THROMBOCYTOPENIAINCREASED PLATELET DESTRUCTION

Chronic ITP 20-50 yrs of age Females:males 2:1 Mucocutaneous bleeding, menorrhagia, recurrent

epistaxis or easy bruising Immune mediated destruction of platelets Autoantibodies against platelet glycoproteins

CLINICAL PICTURE OF ACUTE AND CHRONIC ITP

Characteristics Acute Chronic

Age at onset 2-6 yrs 20-50 yrs

Sex predilection None Female over male 3:1

Prior infection Common Unusual

Onset of bleeding Sudden Gradual

Platelet count <20 30-80

Duration 2-6 wk Months to years

Spontaneous remission

90% Uncommon

Seasonal pattern High in winter/spring

None

Therapy 70% steroid responsive

30% steroid responsive

Splenectomy rare Splenectomy<45 yr 90% response >45 yr 40% response

BONE MARROW IN ITPMEGAKARYOCYTIC HYPERPLASIA

PhlWHWWegmasia Cerulea Dolens

Bawrham K, Shah T. N Engl J Med 2007;356:e3.WHA

HEPARIN-INDUCED THROMBOCYTOPENIA (HIT)

Differs from other drug induced thrombocytopenias Thrombocytopenia never severe ie <20k Not associated with bleeding but with thrombosis

Antibody to a complex of platelet specific PF4 and heparin (anti-PF4/heparin)

Antibody activates platelets through the FcYR II a receptor; also activates endothelial cells

Many patients exposed to heparin develop this antibody though not all develop HIT and even less develop HITT

HIT

Both standard heparin and LMWH can cause HIT-former more common

Heparin exposure 5-10 days Rarely HIT can develop several days after

heparin discontinued called delayed onset HIT

Diagnostic algorithm 4Ts Thrombocytopenia Timing of platelet drop Thrombosis oTher cause of thrombocytopenia not evident

CLINICAL TEACHING POINTS ABOUT HIT

Early recognition; HIT remains a clinical diagnosis

Thrombosis can be arterial and/or venous When HIT suspected, doppler legs Anticoagulate when HIT suspected even in the

absence of thrombosis because of higher rate of thrombosis (alternate AC followed by 3-6 months of warfarin)

Risk of thrombosis persists for about 1 month after diagnosis of HIT

Do not introduce warfarin alone in setting of HIT or HITT as it may precipitate thrombosis especially venous gangrene. Start after several days of alternate anticoagulation

ALTERNATE ANTICOAGULANTS IN HIT/HITT

Direct thrombin inhibitors Argatroban Lepirudin

Both approved in the US Bivalirudin

Effective but not FDA approved

Antithrombin-binding polysaccharide Fondaparinux Effective but not FDA approved in the US

Anti-Xa Danaproid

No longer available in the US

PREGNANCY AND THROMBOCYTOPENIA

You are asked to see a pregnant patient with thrombocytopenia.

What is the differential diagnosis?

Differential diagnosis of thrombocytopenia in pregnancy

MAHAThrombocytopenia

Coagulopathy

HTNLiver disease

Renal disease

CNS Time of onset

ITP ------ Mild to severe ------- -------- --------- --------- ---------Anytime common in first tri

Gestational -------- Mild ------- --------- --------- --------- --------- 2nd-3rd tri

Preeclampsia

MildMild to moderate

Absent to mild

Mod- to severe

------- Protein Seizures 3rd trim

HELLPModerate to severe

Mod to severe MildAbsent to severe

Mod to severeAbsent to moderate

Absent to moderate

3rd trim

HUS Mod to severe Mod to severe AbsentAbsent to mild

Absent Mod to severeAbsent to mild

Post partum

TTP Mod to severe Severe Absent Absent AbsentAbsent to moderate

Absent to severe

2nd- 3rd

trim

AFLP Mild Mild to mod SevereAbsent to mild

SevereAbsent to mild

Absent to mild

3rd tri

NON-IMMUNE MECHANISMS OF PLATELET DESTRUCTION

Thrombocytopenia in pregnancy and preeclampsia Gestational thrombocytopenia

Commonest cause Usually mild Healthy with no prior history of thrombocytopenia Mechanism unknown Return to normal a few weeks after delivery

NON IMMUNE CAUSES OF PLATELET DESTRUCTION

Thrombocytopenia in preeclampsia and hypertensive states in pregnancy Thrombocytopenia occurs in about 15- 20% of

preeclampsia Some have microangiopathic hemolysis,

elevated liver enzymes, and low platelet count-HELLP syndrome

Thrombocytopenia is due to platelet destruction Perhaps an underlying low grade DIC or ?

Immune process Delivery is the treatment for this condition-

thrombocytopenia will resolve in a few days post delivery

MICROANGIOPATHIC HEMOLYTIC ANEMIA (MAHA)

NON IMMUNE CAUSES OF PLATELET DESTRUCTION

Thrombotic thrombocytopenic purpura Triad of microangiopathic hemolytic anemia,

thrombocytopenia, neurological abnormalities Sometimes the pentad- fever + renal dysfunction Four types

Single acute episode Recurrent episodes Drug induced Chronic relapsing-rare form, starts in infancy

TTP

Hyaline thrombi in end arterioles and capillaries

Hyaline thrombi are composed of platelets and von Willebrand factor with little or no fibrin or fibrinogen

Deposition of these platelet-vWf thrombi leads to thrombocytopenia

Degree of thrombocytopenia is related to extent of microvascular platelet aggregation

RBCs flowing under arterial pressure fragment when they have to manouever these thrombi in the microvessels

TTP

Thrombotic lesions give rise to other manifestations Organ ischemia

Neurological Visual Abdominal-pain due to mesenteric ischemia, bleeding

due to thrombocytopenia Renal

Overwhelming renal damage is not usual; if so, consider HUS

TTP

Hemolysis can be severe Smear shows marked decrease in platelets,

RBC polychromasia and RBC fragmentation (microspherocytes, shistocytes) called MICROANGIOPATHIC HEMOLYTIC ANEMIA

Coagulation tests remain normal

TTP

Accumulation of unusually large von Willebrand factor (ULVWF)

In the plasma, ULVWF is rapidly cleaved by a VWF cleaving metalloprotease also called “ a disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs” (ADAMTS 13)

SO WHAT HAPPENS IN TTP?

Familial chronic relapsing TTP Deficiency or absence of the Vwf cleaving

protease Sporadic

Autoantibody against the protease causing deficiency or loss of function

Measurement of the vWF protease enzyme (not rapid enough for clinical use)

THROMBOCYTOPENIA IN THE ICU Sepsis is commonest Often multifactorial, exact cause may be difficult to

pinpoint Infection, sepsis, shock Heparin Other drugs DIC Massive blood transfusion Post transfusion purpura CPR Cardiopulmonary bypass ARDS Pulmonary emboli Intravascular catheters

DRUG INDUCED THROMBOCYTOPENIA

Drug dependent antibodies specific for the drug structure and bind tightly to the platelets by the Fab region in the presence of the drug

Platelets seem to be the favorite target of these drug dependent antibodies

When should DIT be suspected? Unexpected occurrence of thrombocytopenia Recurrent episodes of thrombocytopenia with quick

recovery Misdiagnosis of ITP Beware of quinine containing agents like tonic water,

bittter lemon; foods such as tahini containing sesame seeds, herbal remedies like Jui herbal tea

List of drugs from www.ouhsc.edu/platelets

ANTITHROMBOTIC AGENTS AND THROMBOCYTOPENIA

Presents as acute ITP 0.1% - 2% of patients have severe

thrombocytopenia within several hours of exposure to Abiciximab, Tirobifan or Eptifibatide

About 12% can become acutely thrombocytopenic after second exposure to Abiciximab

Immediate reactions are due to presence of naturally occurring antibodies against structural elements of abiciximab or due to structural changes to GpIIb/IIIa induced by binding of Tirobifan and Eptifitabide.

Immune-Mediated Thrombocytopenia.

Warkentin TE. N Engl J Med 2007;356:891-893.

THROMBOCYTOPENIA

Dysplastic megakaryocytes Myelodysplastic syndromes Chemotherapy effects

Failure of function of megakaryocytes due to defects in DNA synthesis

B12 deficiency Folate deficiency

DYSPLASTIC MEGAKARYOCYTE

DYSPLASTIC MEGAKARYOCYTE

APPROACH TO THROMBOCYTOPENIA

Plt <150

Hb and WBC count

Normal

Smear

Fragmented red cells

DIC/TTP

Normal RBC, platele

ts norma

l

Consider Drug

s, Infection, ITP, Congenital

Abnormal

Bone marrow exam

THROMBOCYTOSIS

Reactive thrombocytosis Associated with blood loss and surgery Post splenectomy Iron deficiency anemia Inflammation and disease Stress or exercise

Clonal thrombocytosis Polycythemia vera CML Myelofibrosis Primary or Essential thrombocythemia MDS associated

THROMBOCYTOSIS IN CML

MICROMEGAKARYOCYTES IN PERIPHERAL BLOOD

PLATELET THERAPEUTICS

Platelet transfusions Platelet pheresis Manipulation of the immune system

IVIG, Steroids, Rituxan, Splenectomy, immunosuppressives Prevention of complications Reduction of platelet number

Hydrea Suppression of megakaryocyte platelet production

Anagrelide Stimulation of megakaryocyte production

Thrombopoeitin mimetics or TPO mimetics Romiplostim Eltromobag

Inhibitors of platelet aggregation Aspirin, Clopidrogel, NSAIDs Gp IIb/IIIa inhibitors Dipyridamole

THROMBOPOEITIN MIMETICS Romiplostim Trade name is Nplate TPO receptor agonist Route: subcutaneous Mechanism: Like

endogenous TPO- increases platelet production by binding and activating TPO receptor

Indications: Chronic ITP Dose titration based on

platelet count

Eltromobag Trade name is

Promacta TPO receptor agonist Route: oral Mechanism: similar

to Nplate Indications: Chronic

ITP Dose titration based

on platelet count