06 - platelet disorders and fibrinolysis

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6 Platelets Disorders and Fibrinolysis

Key Points Platelets are highly complex cells that participate in

critical reactions central to hemostasis and thrombosis, including adhesion to subendothelium, secretion of granule contents, aggregation, and provision of membrane surface for activation of coagulation factors.

Abnormalities of either platelet number or platelet function can play an important role in the balance of hemostasis and thrombosis.

Almost unique to laboratory medicine and pathology, assessment of platelet pathology may be determined in real time upon living cells obtained from the patient.

Von Willebrand factor is a multimeric protein synthesized by endothelial cells and megakaryocytes that plays a central role in platelet adhesive interactions.

Platelet counts and platelet function are affected by auto-immune processes, a wide variety of drugs, and a number of acquired disorders.

Platelet Structure Glycocalyx = outer membrane Submembranous microtubules Contractile myofilaments Open canalicular system Dense tubular system with calcium sequestration

pump Alpha granules = platelet fibrinogen, platelet-derived

growth factor (PDGF), von Willebrand factor, Factor V binding protein multimerin, P-selectin, B-thromboglobulin, heparin-neutralizing factor (PF) 4.

Dense core granules = nonmetabolic pools of ADP, ATP, 5-hydroxytryptamine (5-HT) and calcium.

Platelet membrane glycoproteins (receptor for adhesive ligands)

GP IIb/IIIa = most abundant; integrin; ligand for fibrinogen, von Willebrand, fibrinectin, vitronectin

GP Ib/Ix = second most abundant; leucine-rich glycoprotein; ligand for vWF, thrombin

Phospholipids (phosphatidylserine) = procoagulant surface

Adhesion

Aggregation

Secretion

Pathophysiology of primary hemostasis Thrombin and collagen (activators) conformational

change of platelets release of alpha and dense granules change in the conformation of GPIIb/IIIa (binds with fibrinogen leading to aggregation); GP Ib/Ix (binds with collagen of veins); P-selectin (binds with P-selectin of monocytes, neutrophils, damaged endothelial cells tissue factor expression)

Platelet activities in hemostasis and their laboratory measurements

1. Platelet count and mean platelet volume 2. Reticulated platelet count 3. Screening studies of platelet function

a. Template bleeding time = global test; prolonged in platelet


b. Use of PFA-100 (Platelet Function Analyzer) Principle: Anticoagulated blood under high sheer pressure goes through a narrow hole coated with collagen, epinephrine, ADP; closure time is measured. Prolonged closure time: Profound impairment of platelet function, vWD, thrombocytopenia, anemia. This test has low sensitivity and specificity.

c. Use of platelet aggregometer Principle: Measures aggregation in the first channel and secretion in the second channel. First channel = change of transmittance Second channel = release of ATP measured through luciferin-luciferase chemoluminescence

d. Direct measurement of serotonin to monitor platelet secretion

e. Clot retraction Principle: Contractile activities of activated platelets; delayed in thrombocytopenia and Glanzmann thrombasthenia

f. Flow cytometry quantitative measurement of glycoprotein receptors using monoclonal antibodies

Quantitative Platelet Disorders (thrombocytopenia)

Congenital Thrombocytopenia

Due to mutation of MYH9 gene, WAS gene Big platelets = May Hegglin anomaly (MYH9 gene

mutation) Deficiency/abnormality of GP Ib/IX = Bernard -Soulier

syndrome (big platelets) Congenital amegakaryocytic thrombocytopenia =

mutation of thrombopoietin receptor MPL

Bernard-Soulier Syndrome

Increased Platelet Destruction Immune-mediated destruction

ITP o Acute o Chronic

Alloimmune Drug-induced

Non-immune destruction/consumption DIC Thrombotic Thrombocytopenic Purpura (TTP) Hemolytic Uremic Sydrome (HUS) Dilution or Distribution Disorders

Thrombocytopenia results when production and replacement of PLTS cant keep up with rate of destruction.

Causes

Impaired or Decreased Production

Megakaryocyte aplasia

BM Replacement

Ineffective poiesis

Distribution/ Dilution Disorders

Increased Destruction

Immune

Non-Immune


Immune (Idiopathic) Thrombocytopenic Purpura = ITP Cause - antibodies against viral epitopes or antibodies

against platelet membrane glycoproteins IIb-IIIa or Ib-IX, and are of the IgG type

RES system clears sensitized PLTs Acute children

Viral Infection


Non-Immune PLT Destruction Causes

PLT exposure to non-endothelial surfaces Mechanical heart valve

Activation of coagulation DIC

PLT consumption by endovascular injury TTP HUS

Thrombotic Thrombocytopenic Purpura (TTP)

TTP Mechanism ULVWF multimers are normally digested by von Willebrand cleaving protease, ADAMTS13. In TTP, the absence of ADAMTS13 allows release of ULVWF, triggering platelet activation. TTP, thrombotic thrombocytopenic purpura; ULVW, unusually large von Willebrand factor. A Disintegrin-like And Metallprotease with ThromboSpondin

Hemolytic Uremic Syndrome

Self-limiting Bacterial toxins in blood stream damage renal

capillary endothelia Release ULVWF Adult form associated

w/immunosuppressive agents or chemotherapeutics

Dialysis usually required

Thrombocytopenia Distribution/Dilution Disorders Spleen sequestration Extracorporeal circulatory devices Massive transfusions

Qualitative Defects Definition: Platelets do not function normally May be either

Congenital or acquired Intrinsic or extrinsic defect

Normal Response to Various Agonists Tracings

Acquired Qualitative Defects Extrinsic - platelets are normal; environment is

abnormal and impacts platelet function Lots of possible etiologies

Uremia, high concentration monoclonal protein, high concentration FDP/fdp, some drugs, etc.

Platelet transfusion of no benefit Intrinsic - platelets are abnormal; environment is

normal Drugs - big one

Aspirin - irreversible acetylation of active center of cyclo-oxygenase

Ibuprofen - reversible inhibition of cyclooxygenase

Bleeding time approximately doubles within 2 hours of taking one baby aspirin tablet

Remember other agonists, primarily thrombin, will also cause secretion of platelet dense granules and so aggregation is simply delayed

Congenital Qualitative Defects Intrinsic

Most are inherited as autosomal recessive defect (few exceptions)

Platelet itself is defective All do not lead to bleeding disorder Platelet transfusion may be helpful if

bleeding becomes a problem Characterized Disorders

Adhesion Aggregation Secretion

Abnormal granule content Inability to secrete granule

contents

Microangiopathic hemolytic anemia

Thrombocytopenia

Neurologic Abnormalities

Renal Dysfunction

Children

E.coli O157

Renal failure

Hemolytic anemia Thrombocytopenia


Bernard-Soulier Syndrome Laboratory Findings

Mild to moderate thrombocytopenia common Platelets appear large on blood films Platelet aggregation studies

Why is platelet agglutination with ristocetin still abnormal when vWF is added? Missing GPIb/IX receptor

Why is platelet aggregation normal with other agonists? In vitro aggregation does not first require adhesion .

Contrast Platelet Aggregation VWD vs Bernard Soulier

VWD

Addition of vWF corrects VWD agglutination with ristocetin - receptor okay.

Glanzmann Thrombasthenia Platelet aggregation defective Inherited in autosomal recessive pattern

Consanguinity often noted in affected families Missing or defective GPIIb/IIIa complex What is the significance of a deficiency of this

receptor? Platelets are unable to aggregate. 3 subtypes of Glanzmanns depending upon the

number and quality of GPIIB/IIIa Signals induced by platelet stimulation (via adhesion

or platelet agonist) result in a conformational change in the receptor that leads to exposure of its ligand binding site

ADP Collagen Epinephrine Ristocetin

Why is agglutination with Ristocetin normal? GPIb/IX is intact.

Storage Pool Disorders Defects in secondary aggregation Deficiency of contents of one of granules Inheritance is variable (heterogeneous group) Bleeding is usually mild to moderate but can be

exacerbated by aspirin Clinical: easy bruising, menorrhagia, and excessive

postpartum or postoperative bleeding

Gray Platelet Syndrome Alpha granule deficiency Clinical: mild bleeding Laboratory:

Thrombocytopenia: 60 - 100 x 103/mL Platelets are large and appear gray on

Wrights stained smear (hence the name) Marked deficiency of alpha granules Dense granules are normal Platelet aggregation studies are fairly

normal since 2o aggregation wave due primarily to dense granule release

Secretion Disorders Inability to Release Dense Granule Contents

Platelet ADP content is normal Rare disorders Abnormalities may be in the platelet stimulus-

response coupling Involves the metabolic pathways leading

from receptor occupation to platelet aggregation and secretion

Includes defects in the arachidonic acid pathway Deficiency of cyclo-oxygenase Deficiency of thromboxane synthetase

Often called aspirin-like defects Granules are normal by electron microscopy Bleeding with mild, just as with aspirin, supporting

fact that there are other pathways to activate platelets (e.g., thrombin) that do not require this pathway

Thrombocytosis Platelet counts greater than upper reference range

Reactive process (Counts usually dont exceed > 750 x109/L)

Rare hemorrhage or thrombotic episodes

PLT morphology normal BM megakaryocytes may be

increased Primary T or Myeloproliferative disorders

(Counts often exceed 1000 x109/L Common hemorrhage or

thrombotic episodes Abnormal PLT morphology

Large in size Dysplastic appearance

BM megakaryocytes increased

Von Willebrand Disease Type 1 Partial quantitative deficiency Type 2 Qualitative deficiency Type 3 complete deficiency Acquired von Willebrand Disease

Acquired Disorders of Platelet Function Myeloproliferative disorders Acute leukemias and myelodysplasias Dysproteinemia Uremia Acquired storage pool disease Antiplatelet antibodies Drugs

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06 - platelet disorders and fibrinolysis

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