What

's

new

in

using

platelet

research

To

unravel

thrombopathies

and

other

human

disorders

This

review

on

platelet

research

focuses

on

defects

of

adhesion

cytoskeletal

organisation

signal

transduction

and

secretion

Platelet

defects

can

be

studied

by

different

laboratory

platelet

functional

assays

and

morphological

studies

Easy

bruising

or

a

suspected

platelet-based

bleeding

disorder

is

of

course

the

most

obvious

reason

to

test

the

platelet

function

in

a

patient

However

nowadays

platelet

research

also

contributes

to

our

understanding

of

human

pathology

in

other

disciplines

such

as

neurology

nephrology

endocrinology

and

metabolic

diseases

Apart

from

a

discussion

on

classical

thrombopathies

this

review

will

also

deal

with

the

less

commonly

known

relation

between

platelet

research

and

disorders

with

a

broader

clinical

phenotype

Classical

thrombopathies

involve

disorders

of

platelet

adhesion

such

as

Glanzmann

thrombastenia

and

Bernard-Soulier

syndrome

defective

G

protein

signalling

diseases

with

impaired

phospholipase

C

activation

and

abnormal

platelet

granule

secretion

disorders

such

as

gray

platelet

disorder

and

delta-storage

pool

disease

Other

clinical

symptoms

besides

a

bleeding

tendency

have

been

described

in

disorders

and

Duchenne

muscular

dystrophy

due

to

adhesion

defects

and

also

in

disorders

of

impaired

Gs

signalling

in

Hermansky

Pudlack

disease

and

Chediak

Higashi

disease

with

abnormal

secretion

Finally

platelet

research

can

also

be

used

to

unravel

novel

mechanisms

involved

in

many

neurological

disorders

such

as

depression

and

autism

with

only

a

subclinical

platelet

defect

Introduction

Normal

hemostasis

prevents

spontaneous

bleeding

and

traumatic

hemorrhage

by

a

coordinated

sequence

of

cellular

and

biochemical

reactions

to

the

ultimate

formation

of

a

stable

platelet-fibrin

aggregate

-LSB-

-RSB-

Platelets

under

normal

circumstances

circulate

in

close

contact

to

the

endothelial

cell

lining

of

the

vessel

wall

and

respond

to

vascular

damage

by

adhering

to

subendothelial

structures

Platelet

adhesion

is

the

first

step

in

the

hemostatic

plug

formation

-LSB-

-RSB-

The

major

platelet

receptors

involved

in

this

process

are

the

von

Willebrand

factor

-LRB-

vWF

-RRB-

receptor

GP

-LRB-

glycoprotein

-RRB-

Ib/IX/V

the

collagen

integrin

receptor

α2β1

and

the

fibrinogen

integrin

receptor

-LRB-

Fig.

-RRB-

Subsequent

platelet

spreading

is

conducted

by

cytoskeleton

proteins

including

the

structural

subunit

of

the

microtubules

the

αβ-tubulin

heterodimer

filamin

and

actin

-LRB-

Fig.

-RRB-

The

cytoskeleton

is

responsible

for

the

shape

of

the

resting

platelet

and

carries

out

contractile

events

such

as

the

secretion

of

granules

and

retraction

of

clots

by

activated

cells

Fig.

Schematic

model

of

the

main

components

involved

in

platelet

adhesion

and

the

cytoskeleton

proteins

Platelet

adhesion

and

its

subsequent

activation

by

calcium

release

is

mainly

regulated

by

the

platelet

receptor

after

binding

to

fibrinogen

or

the

RGD

domain

of

vWF

the

main

vWF

receptor

GPIb/IX/V

and

the

collagen

receptor

α2β1

Microtubules

together

with

the

cytoplasmic

actin-rich

cytoskeleton

are

responsible

for

the

platelet

structure

Different

actin

binding

proteins

have

been

identified

in

platelets

such

as

filamin

A

myosin

and

dystrophin

b

Schematic

model

of

G

protein

signal

transduction

in

platelets

regulated

by

Gq

for

platelet

activation

by

the

ultimate

step

of

calcium

release

Gi

and

Gs

further

influence

the

platelet

activation

by

respectively

inhibiting

and

stimulating

the

intracellular

cAMP

formation

c

Schematic

model

of

platelet

secretion

The

second

amplification

step

in

platelet

activation

is

the

release

of

alpha

and

dense

granules

in

platelets

guarantying

irreversible

platelet

activation

Platelet

adhesion

also

initiates

multiple

intracellular

G

protein-coupled

signalling

pathways

-LRB-

Fig.

-RRB-

Stimulation

of

Gq

by

different

ligands

such

as

adenosine

diphosphate

-LRB-

ADP

-RRB-

and

thromboxane

-LRB-

-RRB-

results

in

platelet

activation

by

stimulating

phospholipase

C

and

a

release

of

calcium

from

the

intracellular

stores

This

platelet

activation

process

is

enhanced

when

Gi

is

activated

and

inhibited

when

Gs

is

activated

both

by

modulating

the

intracellular

cAMP

level

Platelet

adhesion

and

activation

eventually

results

in

secretion

from

platelet

organelles

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

Resting

platelets

circulate

as

discoid

anuclear

cells

and

consist

of

a

lipid

bilayer

and

an

internal

dense

tubular

system

where

calcium

is

sequestered

The

platelet

cytoplasm

contains

mitochondria

glycogen

particles

lysosomes

and

the

platelet-specific

storage

granules

the

α-granules

and

dense

granules

-LRB-

Fig.

-RRB-

The

α-granules

contain

proteins

such

as

platelet

factor

β-thromboglobulin

platelet

derived

growth

factor

fibrinogen

fibronectin

thrombospondin

plasminogen

activator

inhibitor

I

and

vWF

Dense

bodies

are

rich

in

serotonin

ATP

ADP

and

calcium

Fig.

microscopy

-LRB-

original

magnification

-RRB-

of

platelets

showing

the

dense

tubular

system

-LRB-

DTS

-RRB-

microtubules

-LRB-

MT

-RRB-

open

canalicular

system

-LRB-

OCS

-RRB-

alpha

granules

-LRB-

G

-RRB-

glycogen

-LRB-

Gly

-RRB-

and

the

dense

bodies

-LRB-

DB

-RRB-

The

study

of

platelet

adhesion

G

protein

signalling

and

secretion

is

particularly

useful

for

our

understanding

of

several

clinical

disorders

-LRB-

Table

-RRB-

The

most

obvious

reason

is

of

course

the

study

of

platelet

defects

in

patients

with

an

isolated

platelet

disorder

leading

to

bleeding

or

thrombosis

in

order

to

gain

additional

information

on

the

different

pathways

involved

in

platelet

function

The

analysis

of

human

disorders

in

which

the

defective

platelet

phenotype

is

just

one

part

of

its

clinical

spectrum

to

unravel

novel

biological

and

genetic

mechanisms

involved

in

the

disease

Also

using

platelet

functional

and

morphological

studies

can

be

used

as

a

tool

to

find

novel

pathways

involved

in

more

complex

disorders

usually

caused

by

more

than

one

gene

defect

Recently

it

became

obvious

that

the

molecular

pathways

involved

in

more

complex

human

disorders

such

as

diabetes

type

or

neurological

disorders

such

as

schizophrenia

migraine

bipolar

disorder

and

depression

can

also

be

better

understood

by

studying

platelet

signalling

and

secretion

Due

to

space

limitations

it

is

impossible

to

give

a

full

overview

of

all

human

disorders

studied

today

by

means

of

platelet

research

This

review

describes

some

well-known

as

well

as

some

less

common

disorders

to

illustrate

how

platelet

research

contributes

to

the

understanding

of

thrombopathies

-LSB-

-RSB-

as

well

as

the

broader

future

of

this

research

outside

its

classical

field

of

thrombosis

and

hemostasis

-LRB-

Table

-RRB-

Table

and

non-syndromic

platelet

defects

and

the

implicated

genes

according

to

the

defective

platelet

pathwayType

of

platelet

defectIsolated

platelet

disordersDisorders

including

a

platelet

defectDisorders

studied

by

functional

platelet

assaysAdhesion

and

cytoskeletal

defectsGlanzmann

thrombasteniaMay-Hegglin

anomaly

Fechtner

syndrome

Epstein

syndrome

and

Sebastian

syndromeNeurological

disorders

as

bipolar

disorder

schizophrenia

depression

autismProlonged

bleeding

timeAll

macrothrombocytopenia

prolonged

bleeding

timeNo

other

clinical

problemsAll

leucocyte

inclusions

Epstein

Fechtner

nephritis

deafness

or

SyndromeDuchenne

Muscular

DystrophyMacrothrombocytopenia

prolonged

bleeding

timeProlonged

bleeding

after

surgeryNo

other

clinical

problemsMuscle

degenerationGPIbα

GPIbβ

or

GPIXDMDG

protein

signalling

defectsADP

receptorInducible

Gsα

hyperfunction

syndromeSubclinical

platelet

defectProlonged

bleeding

timeProlonged

bleeding

time

after

traumaNo

other

clinical

problemsBrachydactyly

increased

alkaline

phosphatase

and

neurological

or

growth

receptorPACAP

overexpressionProlonged

bleeding

timeProlonged

bleeding

timeNo

other

clinical

problemsMental

retardation

and

defectsGray

platelet

disorderHermansky

Pudlack

diseaseNeurological

defect?Prolonged

bleeding

timeProlonged

bleeding

timeNo

other

clinical

problemsAlbinism

lysosomal

defect?HSP

storage

Pool

diseaseChediak

Higashi

diseaseProlonged

bleeding

timeProlonged

bleeding

timeNo

other

clinical

problemsAlbinism

immunological

lethal

defect?LYSTThe

following

implicated

genes

are

indicated

in

integrin

integrin

GPIbα

glycoprotein

Ibalpha

GPIbß

glycoprotein

Ibbeta

GPIX

glycoprotein

IX

purinergic

receptor

TXASR

thromboxane

receptor

nonmuscle

myosin

heavy

chain

DMD

Dystrophin

XLαs

extra-large

stimulatory

G

protein

alpha

subunit

PACAP

pituitary

adenylate

cylase-activating

peptide

HSP

Hermansky

Pudlack

genes

through

LYST

lysosomal

trafficking

regulator

When

considering

type

of

platelet

tests

A

clinical

platelet-based

bleeding

problem

is

of

course

the

main

reason

to

investigate

platelet

function

and

morphology

The

diagnostic

approach

to

easy

bruising

or

a

suspected

platelet-based

bleeding

disorder

includes

a

careful

history

and

physical

examination

of

the

patient

as

well

as

different

laboratory

investigations

such

as

the

Ivy

bleeding

time

platelet

aggregation

tests

ATP

secretion

platelet

adhesion

by

the

platelet

function

analyzer

-LRB-

-RRB-

and

platelet

morphology

by

electron

microscopy

-LSB-

-RSB-

Reviewing

the

medical

history

can

already

establish

whether

the

disorder

is

hereditary

or

acquired

The

specific

clinical

findings

useful

in

the

differential

diagnosis

of

coagulation

versus

platelet-based

disorders

are

summarized

in

Table

Frequently

mucocutaneous

bleedings

characterize

abnormalities

of

platelet

function

In

contrast

hemorrhage

into

synovial

joints

and

deep

muscular

hemorrhage

are

signs

of

severe

hereditary

coagulation

disorders

and

very

rare

events

in

disorders

of

platelets

vessels

or

acquired

coagulation

disorders

Inherited

disorders

of

platelet

function

are

further

subdivided

based

on

the

functions

or

responses

that

are

abnormal

and

therefore

can

belong

to

different

subgroups

including

abnormal

platelet

adhesion

signalling

and

secretion

-LSB-

-RSB-

Platelet-based

bleeding

disorders

are

usually

classified

according

to

abnormalities

of

platelet

function

platelet

number

-LRB-

thrombocytopenia

-RRB-

or

both

-LSB-

-RSB-

Table

presentation

of

coagulation

and

platelet-based

bleeding

disordersClinical

symptomsDisorders

of

coagulationDisorders

of

plateletsPetechiae

epistaxisRareCharacteristicSuperficial

ecchymosesCommon

large

and

solitaryCharacteristic

small

and

multipleBleeding

from

superficial

cuts

and

bruisesMinimalPersistant

often

profuseDelayed

bleedingCommonRareDeep

dissecting

hematomasCharacteristicRareHemarthrosisCharacteristicRare

Functional

and

morphological

platelet

studies

in

patients

with

mainly

a

neurological

metabolic

or

another

clinical

problem

but

no

obvious

bleeding

problem

are

usually

not

performed

for

diagnostic

purposes

but

rather

for

research

aims

In

such

patients

novel

insights

are

expected

to

result

from

the

platelet

research

studies

which

are

still

preliminary

today

but

in

the

future

hopefully

will

help

to

better

define

when

to

ask

for

what

type

of

platelet

tests

in

a

given

patient

Thrombopathies

Glanzmann

thrombasthenia

and

the

Bernard-Soulier

syndrome

-LRB-

BSS

-RRB-

are

two

rare

inherited

disorders

of

platelet

adhesion

Glanzmann

thrombasthenia

-LRB-

MIM

-RRB-

is

an

autosomal

recessive

disorder

characterized

by

prolonged

bleeding

time

and

abnormal

clot

retraction

-LSB-

-RSB-

The

hallmark

of

this

disease

is

severely

reduced

or

absent

platelet

aggregation

in

response

to

various

physiological

platelet

agonists

such

as

ADP

thrombin

and

collagen

The

defect

is

caused

by

mutations

in

one

of

the

integrin

genes

or

encoding

the

receptor

complex

Lack

of

expression

or

qualitative

defects

in

results

in

a

disturbed

interaction

between

activated

platelets

and

adhesive

glycoproteins

-LRB-

fibrinogen

at

low

shear

and

vWF

at

high

shear

-RRB-

that

bridge

adjacent

platelets

during

platelet

aggregation

-LRB-

Fig.

-RRB-

The

Bernard-Soulier

syndrome

-LRB-

MIM

-RRB-

is

caused

by

abnormalities

in

the

GP

Ib/IX/V

receptor

complex

due

to

mutations

in

the

genes

for

GPIbα

GPIbβ

or

GPIX

-LRB-

but

there

are

no

reports

of

BSS

affecting

the

GPV

gene

-RRB-

-LSB-

-RSB-

It

is

an

autosomal

recessive

disorder

with

moderate

to

severe

macrothrombocytopenia

decreased

platelet

survival

and

often

a

spontaneous

bleeding

tendency

The

bleeding

events

can

be

severe

but

are

usually

controlled

by

platelet

transfusion

Most

heterozygotes

with

a

few

exceptions

do

not

have

a

bleeding

diathesis

BBS

platelets

aggregate

normally

in

response

to

physiological

agonists

-LRB-

ADP

and

collagen

-RRB-

have

a

weak

response

towards

low

concentrations

of

thrombin

and

do

not

agglutinate

when

platelet

rich

plasma

is

stirred

with

ristocetin

or

botrocetin

-LSB-

-RSB-

Defects

in

G

protein

signaling

resulting

in

an

isolated

platelet

defect

-LSB-

-RSB-

are

expected

to

be

caused

by

a

mutant

G

protein-coupled

receptor

-LRB-

GPCR

-RRB-

since

these

can

be

cell-specific

while

the

G

proteins

and

their

downstream

effectors

in

this

pathway

are

ubiquitously

expressed

A

dominantly

inherited

mutation

-LRB-

-RRB-

in

the

Gq-coupled

receptor

was

described

in

patients

with

a

mild

bleeding

disorder

characterized

by

defective

platelet

aggregation

responses

to

and

its

analogues

-LRB-

MIM

-RRB-

-LSB-

-RSB-

In

cultured

cells

the

mutant

was

shown

to

impair

phospholipase

C

-LRB-

PLC

-RRB-

activation

Patients

can

be

heterozygous

-LRB-

with

some

PLC

activation

left

-RRB-

or

homozygous

-LRB-

without

PLC

activation

-RRB-

for

this

mutation

but

all

have

a

life-long

history

of

mucosal

bleeding

and

easy

bruising

but

no

episodes

of

major

bleeding

such

as

hematuria

gastrointestinal

bleeding

or

hemarthrosis

-LSB-

-RSB-

The

Gi-coupled

ADP

receptor

-LRB-

Fig.

-RRB-

is

responsible

for

the

sustained

full

aggregation

response

to

ADP

deficiency

-LRB-

MIM

-RRB-

is

an

autosomal

recessive

bleeding

disorder

characterized

by

excessive

bleeding

prolonged

bleeding

time

and

abnormalities

that

are

very

similar

to

those

observed

in

patients

with

secretion

defects

-LRB-

reversible

aggregation

in

response

to

weak

agonists

and

impaired

aggregation

towards

low

concentrations

of

collagen

and

thrombin

-RRB-

except

for

the

severely

impaired

response

to

ADP

-LSB-

-RSB-

Study

of

the

heterozygous

defect

revealed

platelets

that

undergo

a

normal

first

wave

of

ADP

induced

aggregation

but

abnormal

ATP

secretion

with

different

agonists

-LSB-

-RSB-

A

defective

platelet

secretion

is

described

for

patients

with

absent

alpha

granules

-LRB-

gray

platelet

syndrome

-RRB-

or

abnormal

dense

granules

-LRB-

delta-storage

pool

disease

δ-SPD

-RRB-

-LSB-

-RSB-

Gray

platelet

syndrome

or

α-SPD

-LRB-

MIM

-RRB-

owes

its

name

to

the

fact

that

the

typically

enlarged

platelets

devoid

of

α-granule

staining

present

with

a

gray

color

in

a

Wright-stained

blood

smear

-LSB-

-RSB-

Most

cases

are

sporadic

though

some

family

studies

suggest

an

autosomal

dominant

inheritance

Affected

members

have

a

life-long

history

of

mucocutaneous

bleeding

which

may

vary

from

mild

to

moderate

in

severity

prolonged

bleeding

time

mild

thrombocytopenia

abnormally

large

platelets

and

an

isolated

reduction

of

the

platelet

α-granule

content

The

molecular

defect

-LRB-

s

-RRB-

in

α-SPD

have

not

yet

been

defined

and

further

insights

into

the

molecular

mechanisms

responsible

for

platelet

exocytosis

-LRB-

as

the

SNARE

proteins

-RRB-

will

help

in

the

search

for

causes

of

human

platelets

secretory

disorders

δ-SPD

-LRB-

MIM

-RRB-

may

present

as

an

isolated

platelet

function

defect

or

can

be

associated

with

a

variety

of

other

congenital

defects

-LRB-

see

further

-RRB-

δ-SPD

is

characterized

by

a

bleeding

diathesis

of

variable

degree

mildly

to

moderately

prolonged

skin

bleeding

time

-LRB-

fully

related

to

the

amount

of

ADP

or

serotonin

contained

in

the

granule

-RRB-

abnormal

platelet

secretion

induced

by

several

agonists

and

a

reduced

platelet

aggregation

The

δ-SPD

platelets

have

decreased

levels

of

the

dense

granule

contents

ATP

and

ADP

serotonin

calcium

and

pyrophosphate

-LRB-

Fig.

-RRB-

It

was

estimated

that

of

patients

with

a

congenital

abnormality

of

the

platelet

function

have

δ-SPD

-LSB-

-RSB-

The

inheritance

pattern

is

autosomal

recessive

in

some

families

while

autosomal

dominant

in

others

but

the

molecular

players

responsible

for

δ-SPD

are

still

unknown

Human

disorders

comprising

a

platelet

defect

Defects

in

platelet

adhesion

and

subsequent

platelet

activation

can

also

be

due

to

an

alteration

in

the

platelet

cytoskeletal

organization

which

consists

of

the

microtubules

and

F-actin

coupled

to

myosin

filamin

and

dystrophin

-LSB-

-RSB-

Mutations

in

these

widely

expressed

proteins

result

in

a

broader

clinical

phenotype

May-Hegglin

anomaly

-LRB-

MIM

-RRB-

Fechtner

syndrome

-LRB-

MIM

-RRB-

Epstein

syndrome

-LRB-

MIM

-RRB-

and

Sebastian

syndrome

-LRB-

MIM

-RRB-

are

characterized

by

macrothrombocytopenia

with

or

without

different

types

of

leukocyte

inclusions

which

can

only

be

differentiated

by

an

accurate

ultrastructural

examination

-LSB-

-RSB-

In

addition

patients

with

Epstein

or

Fechtner

syndrome

also

suffer

from

nephritis

deafness

and

congenital

cataracts

Recently

it

became

obvious

that

Sebastian

platelet

syndrome

May-Hegglin

anomaly

Fechtner

and

Epstein

syndrome

have

mutations

in

the

same

gene

encoding

the

nonmuscle

myosin

heavy

chain

polypeptide

-LSB-

-RSB-

This

gene

is

expressed

in

platelets

monocytes

granulocytes

the

kidney

the

auditory

system

but

also

in

a

lot

of

other

tissues

deficiency

results

in

an

alteration

of

the

composition

and

agonist-induced

reorganization

of

the

platelet

cytoskeleton

-LSB-

-RSB-

The

cytoskeletal

defect

could

also

explain

the

abnormal

platelet

formation

from

megakaryocytes

resulting

in

thrombocytopenia

and

giant

platelets

in

deficiency

Why

patients

with

May-Hegglin

anomaly

Fechtner

syndrome

Epstein

syndrome

and

Sebastian

platelet

syndrome

have

different

signs

and

symptoms

in

other

tissues

than

their

common

defect

in

platelets

still

remains

to

be

elucidated

Duchenne

muscular

dystrophy

-LRB-

DMD

-RRB-

is

an

X-linked

recessive

disease

-LRB-

MIM

-RRB-

characterized

by

progressive

degeneration

of

muscle

resulting

in

early

death

from

respiratory

or

cardiac

failure

DMD

is

caused

by

mutations

in

the

dystrophin

gene

a

membrane-associated

cytoskeletal

protein

Evidence

for

a

role

of

dystrophin

in

platelets

started

with

the

observation

that

DMD

patients

tend

to

bleed

more

during

spinal

surgery

for

scoliosis

than

do

patients

during

the

same

surgery

with

other

underlying

conditions

-LSB-

-RSB-

Other

C-terminal

isoforms

of

dystrophin

due

to

differential

promoter

usage

and/or

alternative

splicing

at

the

end

of

the

gene

have

been

identified

in

platelets

-LRB-

-RRB-

the

retina

-LRB-

-RRB-

and

in

the

peripheral

-LRB-

-RRB-

and

central

nervous

systems

-LRB-

-RRB-

It

is

well

established

that

platelets

contain

a

complex

membrane

cytoskeleton

that

resembles

at

least

in

part

the

cytoskeleton

found

in

muscle

but

a

role

for

dystrophin

during

platelet

activation

still

remains

to

be

clarified

-LSB-

-RSB-

Recent

studies

showed

a

role

for

dystrophin

in

normal

controls

during

platelet

spreading

and

adhesion

by

regulating

the

α2β1

receptor

but

this

was

not

studied

in

DMD

patients

-LSB-

-RSB-

Another

study

describes

a

normal

platelet

function

in

DMD

patients

and

blames

the

selective

defect

of

primary

hemostasis

in

DMD

to

impaired

vessel

reactivity

-LSB-

-RSB-

Patients

with

an

abnormal

signal

transduction

are

a

heterogeneous

group

combined

of

defects

in

platelet

G

protein-coupled

receptors

-LRB-

GPCR

-RRB-

the

G

proteins

and

their

effectors

Due

to

an

extreme

complex

regulation

between

those

key

components

-LRB-

Fig.

-RRB-

the

incidence

of

this

class

of

defects

is

definitely

underestimated

and

the

underlying

molecular

defects

for

the

signaling

problems

are

still

largely

unknown

Platelet

Gs

activity

is

easily

determined

using

the

platelet

aggregation-inhibition

test

which

gives

a

value

for

the

Gs

activity

based

on

the

inhibition

of

platelet

aggregation

by

the

rapid

generation

of

cAMP

after

incubation

with

different

Gs

agonists

such

as

prostacyclin

or

prostaglandin

-LRB-

Fig.

-RRB-

A

congenital

Gs

hyperfunction

syndrome

was

described

in

three

patients

of

two

unrelated

families

due

to

a

paternally

inherited

functional

polymorphism

in

the

extra-large

stimulatory

G-protein

gene

-LRB-

XLαs

-RRB-

and

its

overlapping

cofactor

ALEX

-LSB-

-RSB-

This

XLαs

variant

is

associated

with

Gs

hyperfunction

in

platelets

leading

to

an

increased

trauma-related

bleeding

tendency

but

is

also

accompanied

by

neurological

problems

and

brachydactyly

-LRB-

MIM

-RRB-

A

subsequent

study

revealed

eight

additional

patients

who

paternally

inherited

the

same

XLαs

polymorphism

presenting

with

platelet

Gs

hyperfunction

brachydactyly

increased

alkaline

phosphatase

and

neurological

problems

or

growth

deficiency

-LSB-

-RSB-

Megakaryocytes

and

platelets

express

the

Gs-coupled

receptor

for

which

both

the

pituitary

adenylyl

cyclase-activating

polypeptide

-LRB-

PACAP

-RRB-

and

the

vasointestinal

peptide

-LRB-

VIP

-RRB-

are

specific

agonists

Studies

in

two

related

patients

with

a

partial

trisomy

revealed

three

copies

of

the

PACAP

gene

and

elevated

PACAP

concentrations

in

plasma

The

patients

suffer

from

multiple

neurological

-LRB-

epilepsy

hypotonia

convulsions

mental

retardation

tremor

psychotic

hyperactive

behavior

-RRB-

gastro-intestinal

-LRB-

diarrhea

vomiting

-RRB-

and

endocrinological

-LRB-

hypoplasia

of

the

pituitary

gland

hypogonadotropic

hypogonadism

-RRB-

problems

and

have

a

pronounced

bleeding

tendency

-LRB-

MIM

-RRB-

-LSB-

-RSB-

The

basal

cAMP

level

in

the

patients

platelets

was

strongly

elevated

providing

a

basis

for

the

strongly

reduced

platelet

aggregation

The

signalling

pathway

also

mediates

megakaryocyte

maturation

and

platelet

formation

-LRB-

unpublished

results

-RRB-

Patients

with

PACAP

overexpression

have

a

mild

thrombocytopenia

a

normal

platelet

survival

relatively

small

platelets

and

their

bone

marrow

examination

reveals

almost

no

mature

megakaryocytes

There

exist

two

rare

syndromic

forms

of

the

δ-SPD

the

Hermansky-Pudlak

syndrome

-LRB-

HPS

-RRB-

and

the

Chediak-Hygashi

syndrome

-LRB-

CHS

-RRB-

HPS

-LRB-

MIM

-RRB-

consists

of

several

genetically

different

autosomal

recessive

disorders

which

share

the

clinical

manifestations

of

oculocutaneous

albinism

bleeding

and

lysosomal

ceroid

storage

resulting

from

defects

of

multiple

cytoplasmic

organelles

melanosomes

platelet-dense

granules

and

lysosomes

hypopigmentation

-LSB-

-RSB-

HPS

can

arise

from

mutations

in

at

least

eight

different

genes

known

to

date

-LRB-

to

-RRB-

all

coding

for

proteins

involved

in

the

formation

trafficking

or

fusion

of

intracellular

vesicles

of

the

lysosomal

lineage

-LSB-

-RSB-

CHS

-LRB-

MIM

-RRB-

is

also

an

autosomal

recessive

disorder

characterized

by

variable

degrees

of

oculocutaneous

albinism

large

peroxidase-positive

cytoplasmic

granules

in

hematopoietic

and

non-hematopoietic

cells

δ-SPD

recurrent

infections

neutropenia

and

an

accelerated

chronic

lymphohistiocytic

infiltration

phase

The

only

known

CHS-causing

gene

LYST

codes

for

a

large

protein

of

unknown

function

but

it

seems

that

CHS

is

a

disease

of

vesicle

trafficking

-LSB-

-RSB-

Most

CHS

patients

present

in

early

childhood

and

die

before

the

age

of

years

unless

treated

by

bone

marrow

transplantation

-LSB-

-RSB-

About

of

patients

exhibit

a

much

milder

clinical

phenotype

and

survive

to

adulthood

but

develop

progressive

and

often

fatal

neurological

dysfunction

Human

disorders

examined

by

functional

and

morphological

platelet

assays

Some

mainly

polygenetic

disorders

can

also

be

studied

by

using

platelets

although

the

patients

only

present

with

a

subtle

subclinical

platelet

phenotype

It

is

not

easy

to

define

disorders

such

as

diabetes

type

or

some

neurological

diseases

according

to

a

defective

adhesion

G

protein

signalling

and

secretion

since

the

platelet

defect

is

not

yet

well

studied

and

usually

overlaps

different

pathways

This

part

of

the

review

will

only

briefly

focus

on

the

use

of

platelet

research

in

our

understanding

of

neurological

disorders

It

has

been

known

for

years

that

certain

cellular

functions

are

very

similar

in

platelets

and

in

neurosecretory

cells

-LSB-

-RSB-

but

the

link

between

functional

platelet

studies

and

neurological

defects

is

novel

A

platelet

and

a

neuron

contain

both

mitochondria

and

dense

core

vesicles

in

which

transmitters

-LRB-

such

as

serotonin

-RRB-

are

stored

Platelets

release

serotonin

upon

activation

-LRB-

Fig.

-RRB-

and

the

neuronal

membrane

is

facilitated

by

a

calcium

dependent

excitation-excretion

coupling

mechanism

In

addition

both

platelets

and

neurons

contain

functional

neurotransmitter

and

neuromodulator

receptor

sites

on

their

outer

membrane

such

as

adrenoceptors

serotonin

receptors

and

serotonin

transporters

More

recently

it

was

shown

that

platelets

also

express

GABA

and

glutamate

receptors

-LSB-

-RSB-

Serotonin

uptake

and

release

by

platelets

and

serotonin

plasma

levels

have

been

quantified

and

found

to

be

altered

in

patients

with

bipolar

disorder

schizophrenia

depression

aggression

autism

migraine

etc.

-LSB-

-RSB-

Many

epidemiological

studies

try

to

link

these

changes

in

activity

of

the

serotonin

transporter

or

changes

in

the

density

and

responsiveness

of

the

serotonin

receptor

and

the

adrenoceptor

on

the

platelet

membrane

of

these

patients

with

genetic

polymorphism

in

the

corresponding

genes

As

for

other

epidemiological

studies

solid

evidence

for

any

linkage

is

not

obvious

but

it

is

irrefutable

that

platelet

studies

have

been

invaluable

in

enabling

an

insight

to

be

gained

into

the

role

of

serotonin

in

a

number

of

psychiatric

and

neurological

disease

-LSB-

-RSB-

Major

advances

are

expected

of

platelet

research

for

this

field

in

the

near

future

since

it

is

now

obvious

that

besides

the

serotonin

pathway

which

was

only

the

tip

of

the

iceberg

many

other

pathways

are

chaired

between

platelets

and

neurons

as

well

as

the

many

gene

products

responsible

for

the

regulation

of

granule

formation

transport

secretion

and

endocytosis

Conclusions

Platelet

research

is

an

expanding

field

originally

studying

isolated

thrombopathies

caused

by

the

imbalance

between

thrombosis

and

hemostasis

but

more

recently

being

able

to

bring

novel

insights

in

our

understanding

of

human

pathology

in

other

clinical

disciplines

such

as

neurology

endocrinology

and

metabolic

diseases

Platelets

are

easily

accessible

cells

and

different

techniques

are

possible

to

study

platelet

function

and

morphology

under

basal

and

activated

conditions

Defects

in

platelet

adhesion

G

protein

signalling

and

secretion

can

result

from

mutations

in

platelet-specific

genes

leading

to

isolated

thrombopathies

or

from

mutations

in

widely

expressed

genes

leading

to

broader

clinical

phenotype

including

a

platelet

defect

In

addition

to

using

platelet

research

for

diagnostic

purposes

these

platelet

functional

and

morphological

studies

can

also

be

used

for

research

aims

From

the

close

collaboration

between

clinicians

of

different

disciplines

geneticists

and

the

functional

platelet

research

unit

novel

insights

in

the

pathogenesis

of

different

human

disorders

are

to

be

expected

in

the

near

future

by

using

this

strategy

O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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Children with cerebral palsy exhibit greater and more regular postural sway than typically developing children Following recent advances in the analysis of centre-of-pressure -LRB- COP -RRB- recordings , we examined the structure of COP trajectories in ten children -LRB- nine in the analyses -RRB- with cerebral palsy -LRB- CP -RRB- and nine typically developing -LRB- TD -RRB- children while standing quietly with eyes open -LRB- EO -RRB- and eyes closed -LRB- EC -RRB- and with concurrent visual COP feedback -LRB- FB -RRB- . In particular , we quantified COP trajectories in terms of both the amount and regularity of sway . We hypothesised that : -LRB- 1 -RRB- compared to TD children , CP children exhibit a greater amount of sway and more regular sway and -LRB- 2 -RRB- concurrent visual feedback -LRB- creating an external functional context for postural control , inducing a more external focus of attention -RRB- decreases both the amount of sway and sway regularity in TD and CP children alike , while closing the eyes has opposite effects . The data were largely in agreement with both hypotheses . Compared to TD children , the amount of sway tended to be larger in CP children , while sway was more regular . Furthermore , the presence of concurrent visual feedback resulted in less regular sway compared to the EO and EC conditions . This effect was less pronounced in the CP group where posturograms were most regular in the EO condition rather than in the EC condition , as in the control group . Nonetheless , we concluded that CP children might benefit from therapies involving postural tasks with an external functional context for postural control . Introduction Poor postural control has been suggested to underlie the delays and deviations in motor skill acquisition and development observed in children with cerebral palsy -LRB- CP -RRB- -LRB- e.g. , Berger et al. 1984 ; Liao et al. 1997 ; Liao and Hwang 2003 -RRB- . In particular , children with CP encounter problems during static upright standing in altered sensory environments -LRB- Cherng et al. 1999 ; Rose et al. 2002 -RRB- and when rapid weight shifts during standing are required , either in gait initiation -LRB- Stackhouse et al. 2007 -RRB- or in reaction to external perturbations -LRB- Nashner et al. 1983 ; Woollacott and Burtner 1996 -RRB- . Postural control is often assessed by means of posturography , that is , the quantitative analysis of centre-of-pressure -LRB- COP -RRB- trajectories as measured with a force platform . Several descriptive statistics of the COP time series , which are often derived by averaging out the assumed noisy or random character of postural sway , have been shown to change with various motor , sensory and cognitive processes involved in the control of standing -LRB- e.g. , Horak 2006 ; Woollacott and Shumway-Cook 2002 -RRB- . In the past two decades , however , new concepts and methods for studying postural control have been introduced based on the assumption that the act of maintaining an erect posture may be viewed as a stochastic process -LRB- Collins and De Luca 1993 ; Newell et al. 1997 ; Riley et al. 1999 ; Frank et al. 2001 -RRB- . Based on these insights , new measures have been developed for examining the time-varying or dynamical characteristics of COP trajectories . These dynamical measures may be more informative with regard to changes in postural control with task constraints , aging or pathology than the more global summary statistics that have been typically employed for this purpose , or at least provide important additional information -LRB- see Baratto et al. 2002 ; Raymakers et al. 2005 ; Roerdink et al. 2006 -RRB- . Recent research has suggested that the regularity of COP trajectories is a particularly revealing and theoretically important dynamical measure . As a case in point , Roerdink et al. -LRB- 2006 -RRB- found that COP trajectories were more regular -LRB- as indexed by reduced sample entropy -RRB- in stroke patients than in healthy elderly while COP trajectories became progressively less regular with recovery . These findings may be viewed in line with the notion of pathological regularity versus healthy complexity -LRB- e.g. , Goldberger 1996 , 1997 -RRB- . Moreover , when performing a secondary cognitive task COP trajectories became less regular -LRB- Roerdink et al. 2006 -RRB- . The latter finding was replicated in young healthy adults , albeit only in the more attention-demanding eyes closed condition -LRB- Donker et al. 2007 -RRB- . Based on these results , a direct relation was proposed between the regularity of COP time series and the amount of attention invested in postural control -LRB- or , inversely , between COP regularity and the degree of automatism of postural control ; see Donker et al. 2007 ; Roerdink et al. 2006 -RRB- . In particular , the introduction of a secondary cognitive task withdraws attention from the regulation of posture -LRB- i.e. , towards the performance of the attention-demanding secondary task -RRB- , resulting in less regular COP trajectories . This interpretation is in line with the claim and corresponding findings of Wulf and colleagues that adopting an external focus of attention is beneficial for the performance and learning of motor skills , whereas an internal focus of attention -LRB- e.g. , directing attention to one 's own body and bodily movements -RRB- can be detrimental as it may disrupt the ` automatisms ' with which well-learned skills are normally performed -LRB- for a review see Wulf and Prinz 2001 ; see also Wulf et al. 2001 ; McNevin and Wulf 2002 ; McNevin et al. 2003 -RRB- . When applied to CP children this insight may imply that their postural control may be improved by means of instructions or task conditions that direct their attention away from the regulation of posture itself to its consequences in the world . Providing CP children with concurrent visual feedback about postural sway may help create a more external focus of attention -LRB- i.e. , the external consequences of postural sway become readily apparent via visual cursor movements -RRB- . Visual feedback of postural sway provides a functional context for postural control -LRB- e.g. , minimise cursor movements -RRB- , which is likely to enhance the performance of the primary task -LRB- cf. , Lin et al. 1998 ; Van der Weel et al 1991 ; Volman et al. 2002 -RRB- . For example , Volman et al. -LRB- 2002 -RRB- showed that the quality of reaching movements of the affected arm in children with spastic hemiparesis could be enhanced when reaching to press a light switch to turn on a light -LRB- task with functional context -RRB- compared to reaching to a marker -LRB- non-functional task -RRB- . We therefore expected that postural control could benefit from postural visual feedback as it constitutes a task with a functional external context for postural control , inducing a more external focus of attention . Although postural visual feedback is sometimes used by physical therapists to train postural control and steadiness -LRB- i.e. , the ability to maintain a given posture with minimal sway , Nichols 1997 -RRB- , evidence for this practice is limited -LRB- e.g. , Rougier 2003 ; Ledebt et al. 2005 -RRB- . The recent conceptual and methodological developments in the analysis of COP trajectories alluded to in the preceding provide an alternative approach for studying the consequences of concurrent visual feedback for postural control and thus of its potential for possible therapeutic applications . Motivated by this combination of theoretical and practical considerations , the aim of the present study was -LRB- 1 -RRB- to compare postural sway fluctuations of children with CP with that of typically developing -LRB- TD -RRB- children and -LRB- 2 -RRB- to investigate the effect of visual information on postural sway in CP children and in TD children . For this purpose , the availability of visual information was manipulated relative to standing with eyes open -LRB- EO condition -RRB- by means of visual deprivation -LRB- standing with eyes closed , EC condition -RRB- and external provision of COP feedback -LRB- FB condition -RRB- . Two kinds of measures were used to analyse the COP trajectories recorded during those conditions , the one scale-dependent and the other scale-independent . The conventional , scale-dependent measures pertained to the ` amount of sway ' , whereas the more recent scale-independent measures pertained to the ` regularity of sway ' -LRB- see Methods section for details -RRB- . We hypothesised that -LRB- 1 -RRB- compared to TD children , CP children exhibit a greater amount of sway and more regular sway and -LRB- 2 -RRB- concurrent visual feedback -LRB- providing a functional external context for postural control , creating a more external focus of attention -RRB- decreases both the amount of sway and sway regularity in TD and CP children alike , while closing the eyes -LRB- increasing task difficulty through deprivation of visual information , promoting a less automatic mode of postural control -RRB- has opposite effects . Methods Participants Ten children with congenital hemiplegia or cerebral palsy -LRB- CP children ; six boys , four girls ; mean age = 7 years , range = 5 -- 11 years -RRB- , reduced to nine in the analyses -LRB- see below -RRB- and nine typically developing children -LRB- TD children ; five boys , four girls ; mean age = 8 years , range = 5 -- 11 years -RRB- without known motor impairments or movement-related disorders volunteered to participate in the experiment . All children and their parents gave their informed consent prior to participation . The experiment was carried out in compliance with the Helsinki Declaration and was endorsed by the VU University Medical Centre Amsterdam . Procedure and apparatus Children stood upright on a 1 m × 1 m custom-made strain gauge force plate1 with their arms hanging relaxed alongside the body . Their feet were placed parallel to the anterior -- posterior axis of the force plate and the distance between the feet corresponded to the width of the pelvis . Children wore their own shoes , including ankle-foot ortheses and/or insoles to correct for leg-length discrepancies . Children participated in three conditions that were presented in random order : standing upright with eyes open -LRB- EO condition -RRB- , standing upright with eyes closed -LRB- EC condition -RRB- and standing upright with visual COP feedback -LRB- FB condition -RRB- . As explained , the FB condition was introduced to provide an external functional context for postural control , creating a more external focus of attention . To this end , a 2.5 m × 2.5 m vertical screen was placed in front of the child at a distance of 1.3 m. On this screen the force plate was depicted as a 40 cm × 40 cm square onto which the COP was projected as a red dot . The children were asked to keep this dot within the target area , which consisted of a 4 cm × 4 cm square . Deprivation of visual postural sway information was effectuated by blindfolding the children in the EC condition . Trial duration was 60 s. Participants performed each condition once , with the proviso that , as part of a separate study , five CP children performed each condition twice -LRB- the first trial was used for further analyses -RRB- . Because in the latter set of recordings trial duration was 30 s , the first 3,000 samples of all trials were used in the analyses to avoid differences in the reliability of the dependent measures . One CP child did not want to be blindfolded and was therefore excluded from further analyses , which rendered the number of CP children equal to the number of control subjects . Data analysis Prior to all analyses , the mean was subtracted from mediolateral and anterioposterior COP trajectories , which transformed the original time series into x and y time series , respectively . Subsequently , x and y time series were bi-directionally filtered -LRB- second-order low-pass Butterworth filter , cut-off frequency 12.5 Hz -RRB- and the resultant distance time series r was constructed using -LRB- see Prieto et al. 1996 -RRB- , with i = 1 , 2 , 3 , ... , N and N indicating the total number of analysed samples in the COP time series -LRB- i.e. , 3,000 samples -RRB- . The ` amount of sway ' was quantified by means of two conventional , scale-dependent measures -LRB- see Prieto et al. 1996 -RRB- . First , the average COP distance to the origin of the mean-centred posturogram was determined by taking the mean of the r time series -LRB- i.e. , mean amplitude rmean in mm -RRB- . Second , sway path length -LRB- SP in mm -RRB- was determined by taking the sum of the distances between consecutive points in the conventional posturogram , using . To examine the structure of COP trajectories in more detail , independent of its size or scale , x and y were normalised to unit variance by dividing those time series by their respective standard deviations σx and σy , resulting in a normalised posturogram . Subsequently , two scale-independent COP measures were quantified . First , the sway path of the normalised posturogram -LRB- SPn -RRB- was determined in a similar manner as described above for the conventional posturogram . Because the posturograms were normalised to its size , differences in SPn could only be the result of changes in the structure of the posturogram , with a larger SPn indicating a larger amount of ` twisting and turning ' or ` curviness ' in the COP trajectory -LRB- cf. , Donker et al. 2007 -RRB- . Second , COP regularity was quantified by calculating the sample entropy -LRB- cf. , Richman and Moorman 2000 ; Lake et al. 2002 ; see also Roerdink et al. 2006 -RRB- , which is a slightly improved version of the approximate entropy -LRB- cf. , Pincus 1991 -RRB- . The sample entropy -LRB- SEn -RRB- is the negative natural logarithm of an estimate of the conditional probability that subseries -LRB- epochs -RRB- of length m -LRB- in our case , m = 3 -RRB- that match pointwise within a specific tolerance also match at the next point -LRB- for a more formal and detailed explanation see Richman and Moorman -LRB- 2000 ; Lake et al. 2002 -RRB- . In other words , smaller SEn values imply more regular COP time series , that is , a greater likelihood that sets of matching epochs in a time series will be followed by another match within a certain tolerance . On the other hand , highly irregular COP time series are characterised by the fact that sets of matching epochs tend to be followed by data samples of different values , resulting in larger SEn estimates . In the present study , SEn was quantified from the r time series , which was first normalised to unit variance . To optimise the choice of the tolerance for a given m -LRB- see also Roerdink et al. 2006 -RRB- we applied the approach of Lake et al. -LRB- 2002 -RRB- and selected the median value of the optimal tolerance over all trials -LRB- in our case , 0.05 -RRB- . The code used for calculating sample entropy was obtained from PhysioNet2 -LRB- Goldberger et al. 2000 -RRB- . To rule out the possibility that COP trajectories exhibited regular or deterministic patterns by chance , we compared the SEn estimates obtained for the COP trajectories to those obtained for surrogate data -LRB- cf. , Theiler et al. 1992 -RRB- . We used time-randomisation to preserve the probability distribution of the data -LRB- e.g. , mean , variance -RRB- while destroying the temporal correlations in the data , resulting in very large values for SEn. In contrast , phase-randomisation randomises the phases of the data in the Fourier domain but preserves the probability distribution of the data and its spectral power distribution -LRB- cf. , Kantz and Schreiber 2004 -RRB- . Consequently , estimates of SEn for phase-randomised COP data should be somewhat increased , although less pronounced as for the time-randomised COP data . Statistics A repeated-measures analysis of variance -LRB- ANOVA -RRB- was conducted on all dependent variables with group as between-subject factor -LRB- two levels : CP and TD children -RRB- and condition as within-subject factor -LRB- three levels : EO , EC and FB conditions -RRB- . The effect of randomisation was evaluated using a one-way repeated measures ANOVA with the factor randomisation -LRB- three levels : original , phase-randomised and time-randomised time series -RRB- , including each individual time series as a sample . Effect sizes for main and interaction effects are reported as partial eta squared -LRB- εp2 -RRB- . Besides significant effects -LRB- P < 0.05 -RRB- , also tendencies towards significance -LRB- P < 0.10 -RRB- are reported in view of the limited sample size . Paired-samples t-tests were used for post-hoc analyses of significant condition or randomisation effects . The analyses were performed using SPSS -LRB- SPSS , Inc. , Chicago IL , USA -RRB- . Results The results -LRB- F , P and εp2 values -RRB- of the group by condition ANOVA for the dependent COP variables are presented in Table 1 . In Fig. 1 , the group by condition effects are summarised for all dependent measures . CP children 's sway amplitude was greater than that of TD children -LRB- i.e. , rmean = 12.8 mm and 5.7 mm , respectively , although this difference was strictly speaking not significant , P = 0.062 -RRB- . Neither a significant main effect of condition nor a significant group by condition interaction effect was observed for rmean . Furthermore , no significant main or interaction effects were observed for the sway path in the conventional posturogram -LRB- SP ; mean 1,367 mm , standard error 108 mm -RRB- , whereas the sway path in the normalised posturogram -LRB- SPn -RRB- differed significantly between groups and conditions in the absence of a significant interaction -LRB- see Table 1 -RRB- . Specifically , SPn was significantly larger for the TD children -LRB- 328 -RRB- than for the CP children -LRB- 212 -RRB- . Moreover , post-hoc analysis revealed that concurrent visual feedback significantly increased SPn -LRB- 310 -RRB- compared to both standing with eyes open -LRB- SPn = 259 : t -LRB- 17 -RRB- = 2.41 , P < 0.05 -RRB- and standing with eyes closed -LRB- SPn = 243 : t -LRB- 17 -RRB- = 2.49 , P < 0.05 -RRB- . Table 1Main and interaction effects of group -LRB- between-subject factor , two levels : CP and TD children -RRB- and condition -LRB- within-subject factor , three levels : EO , EC and FB -RRB- on mean sway amplitude -LRB- rmean -RRB- , sway path of the conventional posturogram -LRB- SP -RRB- , sway path of the normalised posturogram -LRB- SPn -RRB- and sample entropy -LRB- SEn -RRB- GroupConditionInteractionF -LRB- 1 , 16 -RRB- aP-valueεp2F -LRB- 2 , 32 -RRB- aP-valueεp2F -LRB- 2 , 32 -RRB- aP-valueεp2rmean4.010 .0620.201.35 ns0 .081.11 ns0 .06 SP1 .86 ns0 .100.13 ns0 .010.13 ns0 .01 SPn11 .11 < 0.0050.415.12 < 0.020.242.04 ns0 .11 SEn13 .49 < 0.0050.465.53 < 0.010.263.230.0560.17 ns not significantaIn case the assumption of sphericity was violated , the number of degrees of freedom was adjusted using the Huynh -- Feldt method -LRB- corrected degrees of freedom are not listed here -RRB- Fig. 1Effects of group -LRB- CP and TD children , represented by black and grey bars , respectively -RRB- and condition -LRB- EO , EC and FB -RRB- on the ` amount of sway ' -LRB- upper panels , mean sway amplitude rmean and sway path of the conventional posturogram SP -RRB- and on the ` regularity of sway ' -LRB- lower panels , sway path of the normalised posturogram SPn and sample entropy SEn ; note that lower SEn values imply more regular posturograms -RRB- . Error bars represent the standard error As can be seen in Table 1 , the main effects of group and condition were both significant for sample entropy , while the group by condition interaction tended towards significance -LRB- P = 0.056 -RRB- . SEn was significantly larger for the TD children -LRB- 1.01 -RRB- than for the CP children -LRB- 0.71 -RRB- . Moreover , post-hoc analysis revealed that the FB condition significantly increased SEn -LRB- 0.98 -RRB- compared to both standing with eyes open -LRB- SEn = 0.86 : t -LRB- 17 -RRB- = 2.37 , P < 0.05 -RRB- and standing with eyes closed -LRB- SEn = 0.83 : t -LRB- 17 -RRB- = 2.42 , P < 0.05 -RRB- . The observed tendency of the group by condition interaction towards significance -LRB- see Fig. 1 , lower right panel -RRB- may be due to the fact that for CP children SEn increased gradually over EO , EC and FB conditions -LRB- 0.71 , 0.73 and 0.76 , respectively -RRB- , whereas in the TD children SEn first reduced in the EC condition -LRB- 0.93 -RRB- and then increased for the FB condition -LRB- 1.21 -RRB- as compared to standing with eyes open -LRB- 1.01 -RRB- . The surrogate analysis revealed that SEn differed significantly with randomisation -LRB- F -LRB- 2 , 106 -RRB- = 2915.90 , P < 0.001 , εp2 = 0.98 -RRB- . Post-hoc t-tests indicated that SEn differed significantly over all comparisons -LRB- all t -LRB- 53 -RRB- > 7.2 , all P < 0.001 -RRB- , indicating that the regular or deterministic patterns observed in the original data did not occur by chance . SEn was on average -LRB- standard error -RRB- 0.89 -LRB- 0.04 -RRB- , 1.06 -LRB- 0.04 -RRB- and 3.44 -LRB- 0.02 -RRB- for original , phase-randomised and time-randomised COP data , respectively . Discussion In the present study we compared posturograms of children with CP with those of TD children under three conditions . Across conditions , we expected the posturograms of CP children to reflect a greater amount of sway and more regular sway than those of TD children -LRB- Hypothesis 1 -RRB- . Furthermore , in line with the recently postulated relation between the amount of attention invested in posture and COP regularity , we expected that concurrent visual COP feedback , providing a functional external context for postural control leading to a more external focus of attention , would result in a decrease in both the amount of sway and sway regularity , whereas the EC condition , in which visual information is absent , would have opposite results -LRB- Hypothesis 2 -RRB- . The data were largely in agreement with both hypotheses , as will be discussed next . Furthermore , the finding that SEn estimates were significantly increased after time - and phase-randomisation of the original data ruled out the possibility that the regular or deterministic properties observed in the original data occurred by chance . CP children exhibit greater and more regular sway than TD children Posturogram characteristics of children with CP differed markedly from those of TD children -LRB- see Fig. 1 -RRB- . As expected -LRB- Hypothesis 1 -RRB- , the CP children exhibited a larger amount of sway than the TD children . In particular , a near significant -LRB- P = 0.062 , accompanied by a large effect size -RRB- increment in mean sway amplitude -LRB- rmean -RRB- was observed in the CP children compared to that in the TD children -LRB- see Table 1 and Fig. 1 -RRB- . Although , strictly speaking , not significant due to the large interindividual variations , this effect suggests that ` postural steadiness ' is reduced in CP children , consistent with previous results on balance control in CP children -LRB- e.g. , Cherng et al. 1999 ; Ferdjallah et al. 2002 ; Liao and Hwang 2003 ; Rose et al. 2002 -RRB- . An interesting and unexpected finding was that the total COP excursion -LRB- i.e. , SP -RRB- did not differ significantly between CP and TD children . In combination with the apparent increase in sway amplitude , this finding hints at the presence of marked differences in the dynamical structure of posturograms between CP and TD children . To expose those differences , we focused on the sway regularity using two scale-independent measures . Scale-independence was achieved by normalising the data to unit variance . As expected , sway path length of the normalised posturogram was significantly reduced in CP children , indicating less ` twisting and turning ' in the posturogram -LRB- see also Donker et al. 2007 -RRB- . Relatedly , resultant COP trajectories of the CP children were more regular than those of the TD children , as indexed by significantly lower sample entropy values . Recently , increased regularity in COP trajectories has been reported for a variety of pathological conditions , including athletes with a sports-related cerebral concussion -LRB- Cavanaugh et al. 2005 using approximate entropy -RRB- , patients with Parkinson 's disease -LRB- Schmit et al. 2006 using recurrence quantification analysis -RRB- and stroke patients -LRB- Roerdink et al. 2006 using sample entropy -RRB- . These findings are in line with the more general notion of ` dynamical diseases ' -LRB- Glass and Mackey 1988 ; Belair et al. 1995 -RRB- and the ` pathological regularity versus healthy complexity ' hypothesis -LRB- Goldberger 1996 , 1997 ; Goldberger et al. 2002 ; Kyriazis 2003 ; Lipsitz 2002 -RRB- , according to which less ` complex ' or more ` regular ' physiological time series reflect less effective physiological control . In a similar vein , the observed increased COP regularity in CP children may be viewed as the dynamical signature of poor postural control . Effect of visual information We further expected that the ` amount of sway ' would be reduced in the presence of concurrent visual COP feedback , providing a supposedly beneficial functional external context for postural control -LRB- cf. , Van der Weel et al. 1991 ; Volman et al. 2002 ; Wulf and Prinz 2001 -RRB- , as opposed to deprivation of visual information in standing with eyes closed . However , no significant main effects of condition were found for the scale-dependent COP measures -LRB- i.e. , mean amplitude and sway path , see Table 1 -RRB- . This unexpected result is in line with some recent studies showing that the use of visual COP feedback to facilitate quiet standing has no effect on conventional COP parameters in TD children -LRB- Lebiedowska and Syczewska 2000 -RRB- and healthy elderly and stroke patients -LRB- Dault et al. 2003 -RRB- alike . These findings suggest that participants may require some practice to benefit from visual COP feedback . This may explain why , in spite of the apparent lack of immediate effects , Ledebt et al. -LRB- 2005 -RRB- found that balance training with visual feedback improved CP children 's quiet standing ability as well as their functional limits of stability as assessed by a weight-shifting task . In contrast to the results obtained for the ` amount of sway ' under the various conditions , the scale-independent measures pertaining to the ` regularity of sway ' did show significant differences between the FB condition and the EO and EC conditions . This indicates that the dynamical structure of the posturogram must have been different in the FB condition compared to the EO and EC conditions , which was precisely what we anticipated when formulating our expectation that sway would be less regular in the FB condition -LRB- i.e. , longer sway path of the normalised posturogram and higher sample entropy -RRB- and more regular in the EC condition -LRB- Hypothesis 2 -RRB- . Sway path of the normalised posturogram was indeed significantly longer with visual feedback than for standing with eyes open or closed , indicating more twisting and turning -LRB- see also Donker et al. 2007 -RRB- . In parallel , sample entropy increased with concurrent visual feedback compared to standing with eyes open and eyes closed . Thus , in spite of the fact that the ` amount of sway ' did not change significantly over conditions , the observed changes in ` regularity of sway ' were as expected : sway was more regular in the EC condition and less regular in the FB condition . Our expectations concerning the ` regularity of sway ' with conditions were derived from the proposed direct relation between the amount of attention invested in posture and the regularity of COP trajectories -LRB- cf. , Donker et al. 2007 ; Roerdink et al. 2006 -RRB- . This interpretation is in line with the insight of Wulf and colleagues that adopting an external focus of attention , i.e. , a focus on the consequences of one 's movement in the external world , is beneficial for the performance and learning of motor skills -LRB- McNevin et al. 2003 ; McNevin and Wulf 2002 ; Wulf and Prinz 2001 ; Wulf et al. 2001 -RRB- . Specifically , we expected that providing CP children with concurrent visual feedback about postural sway would help shift the focus of attention to the external consequences of postural sway -LRB- i.e. , cursor movements -RRB- by constituting a functional external context for postural control -LRB- e.g. , minimise cursor movements -RRB- . Providing a functional context for the task at hand generally enhances its performance -LRB- e.g. , Lin et al. 1998 ; Van der Weel et al. 1991 ; Volman et al. 2002 -RRB- . In line with these notions , we expected that standing with concurrent visual feedback , a functional task inducing a more external focus of attention , would lead to less regular sway compared to the EO condition , whereas standing with eyes closed would lead to more regular sway in the EC condition compared to the EO condition because task difficulty increased due to the absence of visual information . This predicted pattern of results was only found for the TD children and not for the CP children -LRB- see lower right panel of Fig. 1 -RRB- . As it turned out , COP regularity in the CP children gradually decreased from the EO condition -LRB- most regular -RRB- , via the EC condition , to the FB condition -LRB- least regular -RRB- . As a result of this difference in group behaviour , a strong tendency towards a significant group by condition interaction on sample entropy was found . From this latter observation one could tentatively infer that CP children 's posture actually improved when standing with eyes closed compared to standing with eyes open . In support of this somewhat counter-intuitive inference , Rose et al. -LRB- 2002 -RRB- showed that the percentage of CP children with abnormal sway characteristics did not differ between EO and EC conditions , while the number of sway path outliers even reduced when standing with eyes closed in that particular CP group . Following a similar line of interpretation , Newell et al. -LRB- 1997 -RRB- suggested that very young children may create self-induced perturbations when using visual information in controlling posture , a phenomenon which may be particularly prominent in CP children given that the development of proper neural control mechanisms is strongly delayed compared to TD children -LRB- viz. , Forssberg 1999 -RRB- . From the opposite SEn results between CP and TD children with eyes open and eyes closed it is fair to conclude that postural control in CP children is organised differently than in TD children , especially with regard to the use of normal sway-induced visual information . It has been suggested that creating an external focus of attention would allow for a more automatic control of movements -LRB- e.g. , Donker et al. 2007 ; McNevin and Wulf 2002 ; Roerdink et al. 2006 -RRB- , which is often found to be more efficient -LRB- e.g. , Blanchard et al. 2005 ; McNevin et al. 2003 -RRB- . Although no significant main effect of condition on mean amplitude was found , the finding that during quiet standing with visual COP feedback sample entropy increased -LRB- i.e. , less regularity in the posturogram -RRB- in both TD and CP children supports this idea . Moreover , it corroborates the suggestion that visual COP feedback may be advantageous when seeking to improve postural control , although it must be emphasised again that it requires training -LRB- see Ledebt et al. 2005 -RRB- . Conclusion The findings of the present study indicate that postural control in CP children is deteriorated compared to TD children , most likely due to a slowed development of neural control mechanisms in CP children . Moreover , visual deprivation -LRB- EC condition -RRB- influenced sway characteristics differently in CP and TD children , testifying to the altered nature of postural control in CP children . In line with the proposed relation between the amount of attention invested in posture and COP regularity , we found in CP and TD children alike that , compared to the EO and EC condition , COP trajectories were less regular in the presence of visual COP feedback , corresponding to a functional context mediated external shift in the focus of attention . Future studies should examine the merits of adopting an external focus of attention to enhance postural control in CP children in training or therapy , for example by providing visual COP feedback leading to a posture specific functional task or by introducing an attention-demanding secondary cognitive task .

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