Lecture 5, fall 2014 pdf

Lecture 5

Disorders of Primary Hemostasis Qualita6ve Platelet Disorders

Von Willebrand Disease

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Func6onal disorders of platelets

•  Congenital Disorders –  Disorders of Adhesion

•  Bernard-‐Soulier Syndrome •  Platelet-‐type von Willebrand Disease

–  Disorders of Aggrega6on •  Glanzmann Thrombasthenia

–  Disorders of Secre6on •  Storage Pool Disorders (SPD)

–  Dense Granule disorders –  Alpha Granule disorders

•  Signal Transduc6on defects/Platelet Release defects –  Defec6ve TXA2 Pathway –  Receptor Signaling Defects

•  Acquired Disorders

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Nature Reviews

Inherited Disorder of Platelet Dysfunc6on

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Phospholipase A2 Phospholipase C

PL PIP2

TXA2

PGG2 PGH2

Arachidonic Acid IP3 DAG

CA2+

Protein Kinase C

Protein Phosphorylation

CA2+

Procoagulant Activity

Shape Change

GpIIb/IIIa Receptor

a-granules

Dense Bodies

ATP ADP 5HT Ca2+

PF4 B-TG PAI-1 vWF Fibrinogen Factor V PDGF

TXA2

Fibrinogen

Adrenaline

ADP Thrombin

TxA2 *

Arachidonic Acid DAG Lipase

COX-1

Thromboxane Synthetase

Platelet Activation Pathways

TXA2 *

TXB2

Low Collagen High Collagen

Aspirin Inhibition

Created by Krystal McGarvey, Applications Specialist, Chrono-log Corp.

Reference: Platelets in Thrombotic and Non-thrombotic Disorders. 2002. Pages: 119, 127-129, 222-223, 238-239, 339, 361, 371, 471-472

Laboratory Inves6ga6on of Primary Hemostasis

•  Includes tests for platelet number and func6on –  Platelet Count –  Peripheral Blood Smear Evalua6on –  Platelet Func6on Analyzer (PFA)

•  Bleeding Time –  VerifyNow (Accumetrics) –  Platelet Aggrega6on

Bleeding Time •  Bleeding 6me—overall test of hemosta6c func6on •  Measures

1.  Vessel integrity 2.  Platelet integrity 3.  Protein/Platelet interac6on

–  Methods –  Duke (1912)—ear lobe –  Ivy (1941)– volar surface of forearm with blood

pressure cuff inflated to 40 mm of mercury –  Meckel (1969)—standardized template device –  Reference range = 2-‐9 minutes

–  Prolonged in: •  Thrombocytopenia •  Platelet disorders •  vWD •  Low or abnormal fibrinogen •  Vascular disorders

–  Disadvantages 1.  Lack of consistency with the results 2.  No correla6on with pre-‐surgical bleeding 3.  No evidence to suggest that it will predict a

post-‐surgical bleed –  Procedure

�  Standardized cut made in forearm �  1 mm deep and 5 mm long

�  40 mm Hg pressure (blood pressure cuff) used to provide constant hemosta6c stress

–  Reference range: generally 2 -‐ 9 minutes 8

PFA-‐100: Platelet Func6on Screen

•  Test cartridges containing: 1.  Collagen/Epinephrine 2.  Collagen/ADP

•  Monitors platelet adhesion and aggrega6on •  Results reported as a “Closure 6me” in seconds (CT) •  Correla6on to Bleeding Time

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The PFA-‐100® System Simulates In Vivo Condi6ons

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PFA-‐100® Test Cartridge Injured Blood Vessel

Collagen

Agonist

Flow

Platelet Plug

VerifyNow •  Cartridge-‐based system that uses fibrinogen-‐coated beads

•  Method 1.  Citrated blood is drawn into each of two sample channels in a

disposable cartridge 2. Mixed with platelet agonist FLLRN and Fibrinogen coated polystyrene

beads by a steel ball 3.  Light is transmiied through the sample 4.  Agglu6na6on occurs between ac6vated platelets and the fibrinogen-‐

coated beads such that they fall out of suspension -‐à increased light transmission

5.  Reported as PAU (platelet aggrega6on units)

OPTICAL PLATELET AGGREGOMETRY: BORN PRINCIPLE

PPP BLANK, NO MAGNET PRP with

MAGNET PRP with MAGNET PRP with

MAGNET PRP with MAGNET

AGONIST ADDED TIME MINUTES

0%

100%

LIGHT IN LIGHT OUT

L I G H T

T R A N S M I S S I O N

Monophasic Curve

Platelet Aggregation

Platelet Aggrega6on §  Primary wave

§  Reversible §  Measures ability of platelets

to respond to an external agonist and to start to aggregate

§  Without enough s6mulus or without an intact prostaglandin pathway à TXA2 – platelets disaggregate

§  Secondary wave §  Irreversible §  Results in complete release of

dense granules contents, most importantly ADP

Graphic accessed URL hip://evolvels.elsevier.com/sec6on/default.asp?id=1138_ccalvo7_0001, 2008.

Biphasic Curve

ANATOMY of a BIPHASIC AGGREGATION CURVE

ATP

ATP

Resting disk-shaped

cells

Activation: shape change Irreversible Aggregation

2

3

4

ADP

time

aggr

egat

ion

(%)

PRIMARY WAVE

REVERSIBLE

AGGREGATION

1

SECONDARY WAVE

MAXIMUM AAGREGATION

DIS-AGGREGATION

PLT Aggrega6on: WB

•  Parallel electrodes (DC) immersed in saline-‐diluted whole blood

•  Add agonist

•  PLTs aggregate on electrodes, reducing current

•  Change is current directly propor6onal to level of PLT aggrega6on

Graphic accessed URLhip://evolvels.elsevier.com/sec6on/default.asp?id=1138_ccalvo7_0001, 2008.

The aggrega6ng platelets form a layer on the electrodes, and current is impeded by the platelet layer. Resistance (Ω) is propor6onal to aggrega6on, providing a tracing that resembles op6cal aggregometry.

Agonists •  Collagen

–  Membrane defects –  General ability of platelets to aggregate –  SPD, RD, NSAID

•  Epinephrine –  Membrane defects –  COX –  NSAID

•  Arachidonic Acid –  Most useful in detec6ng aspirin-‐like deficiencies –  Aspirin, NSAID

•  ADP –  Membrane defects –  COX, SPD –  NSAID

•  Ristoce6n –  Membrane defects –  Measures agglu7na7on –  Differen6ate between BS vs. vWD, vWD 2B vs. Platelet-‐type vWD

Aggrega6on

Agglu6na6on

ADP

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Collagen and Arachidonic Acid

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Epinephrine

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Ristoce6n

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ADP and Arachidonic Acid

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Collagen (low dose and high dose)

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Ristoce6n (low dose and high dose)

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Bernard-‐Soulier Syndrome (BSS) •  Laboratory findings

–  Thrombocytopenia –  Giant platelets

•  5-‐8 um vs 20-‐30 um diameter

–  Prolonged PFA/BT –  Abnormal aggrega6on with ristoce6n –  Decreased to absent expression of

GPIb and or GPIX (CD42b, CD42a) –  CD61 = GPIIIa

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Green = control, Red = patient FS = size

Absence of: •  CD42a, 42b, and 42c •  (components of the Ib/IX

complex)

Bernard-‐Soulier Syndrome •  Laboratory Findings

–  Mild to moderate thrombocytopenia common •  30 – 200 x109/L

–  Giant platelets on peripheral blood smear –  Platelet aggrega6on studies

•  Absent aggrega6on with ristoce6n •  Normal aggrega6on with all other agonists

Why is platelet agglu6na6on with ristoce6n s6ll abnormal when vWF is added?

In vitro aggrega6on does not first require adhesion

Why is platelet aggrega6on normal with other agonists?

Missing GPIb/IX receptor

Glanzmann Thrombasthenia (GT)

•  First described in 1918 – Switzerland –Dr. Glanzmann

–  Described in children from a 6ny village – Le Valais in Swiss Alps – where intermarriage was common

•  Autosomal recessive disorder involving one of two genes coding for either GPIIb or GPIIIa – both found in chromosome 17q

•  GPIIb/IIIa func6on in platelet aggrega6on – binding to fibrinogen

•  Bleeding appears during the 1st year of life –  Epistaxis, gingival bleeding, purpura, heavy

menorrhagia

•  Three clinical presenta6ons –  Type I – severe deficiency with less than 5%

IIb/IIIa receptors present –  Type II – mild-‐moderated deficiency with

5-‐20% IIb/IIIa receptors present –  Type III – normal to almost normal amount

of IIb/IIIa receptors present but defec6ve func6on

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Glanzmann Thrombasthenia (GT)

•  Laboratory findings –  Prolonged PFA/BT –  Normal platelet count –  Normal platelet retrac6on –  Abnormal platelet aggrega6on response to ADP, Arachidonic Acid, Collagen, Epinephrine –  Normal platelet aggrega6on response to ristoce6n –  Decrease expression by flow cytometry – confirmatory diagnosis

•  GPIIb (CD41) or •  GPIIIa (CD61)

•  Why is aggrega6on with ristoce6n normal?

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Storage Pool Disease

•  Affect the secre6on phase of platelet func6on •  Autosomal dominant or autosomal recessive mode of inheritance

–  Dense Granules Deficiency •  Decrease or absence of dense granules on EM •  Morphologically normal appearing platelets on peripheral blood smear

•  Prolonged PFA/BT •  Abnormal aggregaAon due to lack of ADP in Dense Granules •  Abnormal aggrega6on with ADP, Epinephrine à normal primary wave BUT blunted secondary wave

•  Low levels of collagen – collagen requires endogenous ADP and this is lacking

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•  Alpha Granules Deficiency

–  Absence of the alpha granules causes the platelets to appear agranular on peripheral blood smear (EM)

–  Mgk synthesis of the alpha granules is normal BUT there are defects involving targe6ng endogenously synthesized proteins to developing alpha-‐granules

–  Platelet aggrega6ons studies are normal /decreaased in alpha granule deficiency

–  AKA gray platelet syndrome

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•  Gray Platelet Syndrome –  Congenital platelet disorder –  Marked decreased or absence of

platelet alpha-‐granules –  Large platelets with few granules

àgiving the “gray” appearance –  Bleeding is usually mild to moderate

but can be exacerbated by aspirin –  Clinical: easy bruising, menorrhagia,

and excessive postpartum or postopera6ve bleeding

–  Typical Lab Findings •  Usually normal platelet count with

variable morphology •  Platelet aggrega6on shows normal

primary wave but absence of secondary wave when s6mulated with ADP, epinephrine, arachidonic acid –

•  Ristoce6n agglu6na6on is normal

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Gray Platelet Syndrome

•  Quebec Platelet Defect –  Deficiency of α-‐granule

mul6merin – a protein that binds FV within the α-‐granule à decreased content of platelet FV

–  Abnormal proteolysis of alpha-‐granule proteins due to increased levels of platelet urinary-‐type plasminogen ac6vator

–  Platelets are morphologically normal by light microscopy

–  Slight thrombocytopenia

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Platelet and neutrophil images of GPS patients in comparison with controls.

Gunay-Aygun M et al. Blood 2010;116:4990-5001

©2010 by American Society of Hematology


•  ScoD Syndrome –  Due to a defect in a platelet

mechanism required for blood coagula6on

–  Defec6ve procoagulant ac6vity of platelets

–  During normal platelet ac6va6on – PS on the inner leaflet is transported to the outer membrane surface – provides a binding site form the tenase and prothrombinase complexes

–  In Scoi Syndrome the mechanism for transloca6ng PS is defec6ve à impaired thrombin genera6on

Nature.com 35


•  SPD versus RD – need EM to differen6ate between the two –  SPD may have decreased number of dense bodies –  RD will have normal number of dense bodies

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Hermansky -‐ Pudlak Syndrome •  Due to a decreased number of dense granules •  Described in 1959 by Hermansky and Pudlak

–  Described a 55-‐year old man with oculocutaneous albinism and history of frequent bruising following minimal trauma

•  Autosomal recessive disorder à muta6on in the HPS1 gene on chromosome 10q23 –  HSP1 responsible for produc6on and control of melanosomes, dense granules, and lysosomes

•  Most commonly found in Swiss Alps and Puerto Rico

•  Triad phenotype 1.  Albinism—blond hair pale skin 2.  Prolonged bleeding due to storage pool granular deficiency

•  Platelet func6on requires dense granules filled with proaggrega6on chemical reagent 3.  Accumula6on of ceroid pigment in lysosomal organelles

•  Ceroid à wax-‐like substance made by certain cells •  Ceroid accumula6on may cause organ dysfunc6on [intes6nes, lungs, kindeys]

•  Lab findings –  Normal PT/PTT, BT variably normal to prolonged –  Platelet aggrega6on shows blunted response in biphasic curves –  Diagnosis made by EM à absence of dense granules

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Chediak -‐ Highashi Syndrome •  Autosomal recessive disorder resul6ng in recurrent infec6ons with ocular, neurological, and skin

manifesta6ons •  Described in 1943 by a Cuban pediatrician à Chediak and Higashi gavedetailed, published descrip6on in

1954 •  Caused by a muta7on in the LYST gene

–  Lysosomal trafficking regulator gene on chromosome 1 –  Abnormal membrane fluidity with uncontrolled granule membrane fusion –  Giant cytoplasmic granules in all granule-‐containing cells (leukocytes, melanocytes and platelets) –  Platelets have deficient or reduced storage pools of ADP, ATP, and serotonin à loose platelet

aggrega6on forma6on

•  Clinical manifesta7on –  Decreased pigmenta6on of the hair and eyes –  Photophobia, Nystagmus –  Large eosinophilic, peroxidase-‐posi6ve inclusion –  Pa6ents are suscep6ble to bacterial infec6ons

•  Laboratory findings –  Normal platelet counts, prolonged bleeding 6me –  Normal PT/aPTT –  Leukocytes with darkly stained giant granula6on –  Platelet aggrega6on decreased with collagen and ADP

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ASH Image Bank

Acquired Platelet Defects

•  Cardiopulmonary Bypass •  Chronic Renal Failure

–  Seen in uremic pa6ents related to the accumula6on of waste products in the blood –  Prolonged PFA/BT –  Decreased aggrega6on with collagen –  Secondary aggrega6on with ADP and epinephrine is decreased à abnormal secretory response –  Platelet procoagulant ac6vity is defec6ve

•  Myeloprolifera6ve Disease and Acute Leukemia

•  Drugs –  Aspirin –  Alcohol –  An6bio6cs –  Cardiopulmonary Bypass Surgery

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Cardiopulmonary Bypass •  Causes a deple6on of α-‐granules •  Func6onal defect results from increased platelet ac6va6on and fragmenta6on in the

bypass mechanical process

•  Causes of defects and granule deple6on a.  Aggrega6on of platelets by fibrinogen absorbed onto the surfaces of the bypass

circuit material b.  Hypothermia c.  Complement ac6va6on d.  Mechanical trauma and shear stresses e.  Bypass pump-‐priming solu6ons

•  Lab findings –  Increases the BT by >30 minutes –  Platelet fragments –

Ø Typically platelet func6on returns to normal ~ 1-‐3 hours awer surgery Ø Platelet count returns to normal several days later

–  Thrombocytopenia can be amplified by hemodilu6on as blood passes through the bypass mechanism

–  Significant post-‐surgical bleeding is seen in 3% of pa6ents 40

Uremia •  Related to accumula6on of waste products in the blood including

inhibitory and dialyzable molecules 1.  BT correlates with severity of disease 2.  Procoagulant ac6vity may be impaired 3.  Nitric Oxide may inhibit platelet func6on 4.  Thought to be due to impaired platelet-‐vessel interac6on 5.  Hemosta6c abnormality partly corrected by RBC transfusion or EPO

•  Failue of HGB to quench excess NO synthesis may be partly responsible for platelet dysfunc6on

Lab Tests in Disorders of Primary Hemostasis

vWD—Disorder of Primary Hemostasis

}  Most common of the congenital bleeding disorders }  1-‐2 % of the general popula6on }  Symptoma6c in only about 1/10,000

}  1926 – Erik von Willebrand à 5 y-‐o-‐f and her family who lived on the Åland Islands – Hereditär pseudohemofili, 1926

}  Ini6ally described as “pseudohemophilia”

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vWD—Disorder of Primary Hemostasis

} Clinical manifesta6ons } Mucocutaneous bleeding of varying severity in males and females 1. Ecchymoses 2. Epistaxis 3. Gastrointes6nal bleeding 4. Menorrhagia

} Defec6ve platelet adhesion } Reduced FVIII levels

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vWF •  Large mul6meric protein – ranges from 600 kD to >20 million kD

–  Synthesized by endothelial cells and megakaryocytes •  Endothelial cells source of plasma vWF

•  Gene for vWF is located on chromosome 12p •  178 kB, 52 exons

45 Hoffman: Adapted from Ginsburg D, Bowie EJW: Molecular geneAcs of von Willebrand disease. Blood 79:2507, 1992.)

Synthesis of vWF

}  vWF synthesized in endothelial cells and megakaryocytes 1.  Stored in Weibel-‐Palade bodies of

endothelial cells 2.  Stored in α-‐granules of platelets

Steps in synthesis of vWF 1.  First synthesized as a pre-‐

pro-‐vWF monomer 2.  DimerizaAon occurs in ER 3.  Pre-‐pro-‐vWF monomers

linked together at the carboxyl terminal end

4.  Dimeric molecules pass to the Golgi apparatus

5.  Dimers mulAmerize 6.  Propep6de is cleaved off

à mature subunit 46

N-Terminal Multimerization

C-Terminal Dimerization

High Molecular

Weight Multimer

ER Golgi

vWF Release

Valentijn K M et al. Blood 2011;117:5033-5043

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Func6on of vWF }  vWF serves two important biologic func6ons

1.  Serves as a carrier protein for plasma FVIII a.  VWF protects Factor VIII in circula6on b.  VWF co-‐localizes FVIII at sites of vascular injury

2.  Serves as a ligand that binds to the gpIb receptor on platelets to ini6ate platelet adhesion to the damaged endothelium a.  VWF binds to extravascular collagen b.  Platelets adhere to the bound vWF c.  Adherent platelets become ac6vated

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Platelets

Clotting factors Vessel wall

VWF

Func6on of vWF

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Elsevier

50 Elsevier

Classifica6on of vWD •  vWD – extremely heterogenous, complex disorder with > 20 dis6nct

subtypes

•  Types of vWD

1.  Quan6ta6ve Defects

•  Type 1 –  Par6al quan6ta6ve deficiency –  Autosomal dominant

•  Type 3 –  Complete absence/severely decreased –  Autosomal recessive

2.  Qualita6ve Defects

•  Type 2 –  2A –  2B –  2M –  2N –  Autosomal recessive

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Subgroups

Type I vWD

}  Most common type of vWD }  80% of pa6ents with vWD fall into this category

}  Caused by heterozygous muta6on leading to a par6al quan6ta6ve deficiency of vWF }  Gene6c abnormality in ONE of the vWF alleles }  Accounts for a 50% reduc6on in vWF }  Mild secondary deficiency in FVIII

}  Endothelial cells and platelets contain normal, but reduced levels of vWF }  DDAVP can induce the release the stored vWF

}  Bleeding symptoms range from asymptoma6c to mild

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Type I vWD }  Lab findings }  Normal to decreased

1.  FVIII (aPTT) 2.  vWF:Ac6vity (Ristoce6n Cofactor) 3.  vWF:An6gen

4.  Prolonged BT •  (PFA-‐100—Col/EPI, Col/ADP)

5.  Propor6onal decrease of ALL vWF mul6mers

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Type 3 vWD

}  Most severe form of the disease

}  Results from the homozygous muta6on leading to a deficiency of vWF with absent or profound deficiency in levels of plasma vWF

}  Autosomal recessive

}  vWF levels are <5% }  FVIII is markedly cleared from the plasma with levels below 5-‐10% }  FVIII is not as severely depressed as in severe Hemophilia A }  Spontaneous bleeding }  Severe mucocutaneous bleeding }  Sow 6ssue/musculoskeletal bleeding

}  1-‐5% of case }  Prevalence increases in regions of consanguineous marriages

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Type 2A vWD

•  Muta6ons commonly occur in the A2 region

•  Presence of only the smaller vWF mul6mers in plasma à reduced binding to platelets •  LOSS platelet-‐dependent funcAon

•  Two proposed mechanisms: ▫  Abnormal assembly and secre6on of

large vWF mul6mers ▫  Increased suscep6bility of vWF to

proteolysis in circula6on

•  Pa6ents exhibit moderate to severe mucocutaneous bleeding

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Type 2B •  Muta6on in the A1 domain of the vWF

gene •  Absence of the high-‐molecular-‐weight

mul6mers –  Caused by “gain of func7on” muta6on in

vWF à increased affinity to bind to the gpIb platelet receptor

–  Spontaneous binding of vWF to platelets –  Large mul6mers are synthesized but

rapidly cleared due to increased binding to platelets

–  Thrombocytopenia

•  DDAVP contraindicated à would cause increased thrombocytopenia as platelets would be hyper-‐reacAve to the released vWF

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Type 2M vWD

•  Muta6ons in Exon 28 in A1 domain

•  Defect leads to decreased or absent binding of vWF to platelet gpIb receptor

•  Decreased platelet dependent func6on

•  Normal mul6mer profile

•  Plasma binding to FVIII is normal

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FVIII GPIb collagen RGDSGPIIb/IIIacollagen

D1 D2 D‘D3 A1 A2 A3 D4 B1B2 B3 C1 C2N C

Type 2N vWD

•  Also referred to as “autosomal hemophilia” or the Normandy variant •  Caused by muta6ons in the FVIII binding region of vWF

•  Markedly decreased affinity for binding to FVIII –  Rapid turnover of the unbound FVIII à reduced levels •  Lab findings

1.  Decreased FVIII 2.  Normal vWF an6gen and ac6vity 3.  Normal bleeding Ames (PFA-‐100) 4.  Platelet binding to vWF is normal 5.  Similar to “mild” hemophilia

•  GeneAc counseling and treatment is different from hemophilia

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FVIII GPIb collagen RGDSGPIIb/IIIacollagen

D1 D2 D‘D3 A1 A2 A3 D4 B1B2 B3 C1 C2N C

Pseudo-‐von Willebrand Disease – (Platelet type vWD)

}  NO gene6c defect of the vWF molecule – vWF molecule is NORMAL

}  “Gain in func6on” muta6on in the platelet gpIb receptor }  Increased affinity of platelets for vWF }  Enhanced clearance of vWF and platelets from circula6on

}  Defect is in the platelet à standard approaches to trea6ng vWD are not helpful

}  Lab findings 1.  Loss of high molecular weight mul6mers 2.  Platelet count is low** 3.  Platelet aggrega6on with low dose ristoce6n (RIPA)

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Acquired vWD

•  Qualita6ve, structural, or func6onal disorder of vWF not inherited and is associated with an increased risk of bleeding

•  Associated with –  Autoimmune clearance – lymphoprolifera6ve, MGUS, SLE, hypothyroidism

•  Autoan6bodies à increased clearance of vWF from plasma

–  Fluid shear stress-‐induced proteolysis – aor6c stenosis, LVAD

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Aspect Acquired vWD Congenital vWD Personal History Late onset bleeding Early onset bleeding

Family History Nega6ve Posi6ve

AVWS associated disorder

Posi6ve Nega6ve

Laboratory associated disorder

Inhibitor to vWF Gene6c muta6on

Treatment •  Remission awer IVIg •  Short lived response

awer vWF-‐containing product

vWF-‐containing product

Assays for vWD

•  Platelet Func6on Screen (BT) •  vWF an6gen assay •  vWF ac6vity assay •  FVIII:C

•  Mul6mer Analysis

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Assays for vWD

•  vWF:An6gen –  Immunoassay that measures the concentra6on of vWF protein in plasma

•  Actual protein responsible for binding to FVIII and gp Ib/IX/V complex –  Detects all forms of vWF (func6onal and nonfunc6onal forms) –  Cannot discriminate between mul6mer size

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Patient vWF å

Testing well

Reagent beads coated with anti-vWF

å

åå

å

å

å

å

åå

å

å

Incubate

LIA based tes7ng

Instrument reading—changes in optical density secondary to aggregates

Assays for vWD

•  vWF:Ac6vity –  Ristoce6n cofactor assay (gold standard)

•  Measures the ability of vWF (pa6ent) to induce agglu6na6on of normal fixed platelets in the presence of Ristoce6n

•  Mix paAent’s plasma + normal donor platelets + ristoceAn à platelet aggluAnaAon reacAon occurs on platelet aggregometer

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Assays for vWD

•  vWF:Ac6vity –  Latex par6cle enhanced immunoturbidimetric assay

•  Specific anA-‐vWF monoclonal anAbody adsorbed onto latex reagent directed against the platelet binding site of vWF (gp Ib receptor)

•  Reacts with vWF in the pa6ent’s plasma •  Degree of agglu6na6on is directly propor6onal to ac6vity of vWF in

pa6ent's plasma –  Mix paAent’s plasma + latex beads coated with an anA-‐vWF

monoclonal anAbody è aggluAnaAon of parAcles

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Assays for vWD

•  FVIII ▫  Circula6ng level of FVIII ▫  Clot-‐based assay that measures the ability of plasma FVIII to shorten

the clo}ng 6me in FVIII-‐deficient plasma

•  Mul6mer Analysis ▫  QualitaAve assay (electrophoresis) to depict the variable

concentraAons of different-‐sized vWF mulAmer

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Science

Lecture 5, fall 2014 pdf