PLATELET-FUNCTION STUDIES IN THE BERNARD-SOULIER SYNDROME

PLATELET-FUNCTION STUDIES IN THE BERNARD-SOULIER SYNDROME*

Thomas C. Bithell Departments of Clinical Pathology and Internal Medicine

University of Virginia School of Medicine, Charlottesville, Virginia 22901

Sunil J. Parekh Division of Hematology. University of Utah School of Medicine, Salt Lake City, Utah

and the Christian Medical College, Vellore, India

Robert R. Strong University of Virginia School of Medicine, Charlottesville. Virginfa 22901

A hereditary bleeding disorder characterized by thrombocytopenia and “giant” morphologically abnormal platelets was first described by Bernard and Soulier in 1948.’ The major features of this rare syndrome are summarized in TABLE 1. The disorder is inherited as an incompletely recessive autosomal trait, and laboratory abnormalities are commonly demonstrable in clinically unaffected heterozygotes. The hemorrhagic diathesis frequently is severe and the bleeding time is markedly prolonged, both to a degree usually out of proportion to the mild-to- moderate thrombocytopenia. Platelet dysfunction presumably is the major cause of bleeding, and on the basis of abnormalities in prothrombin consumption and platelet factor 3 activity, the disorder has been classified among the thrombocyto- pathies2 However, abnormalities of platelet aggregation or other evidence of a defect in the release reaction have not been well documented, and in general, few studies of platelet function employing newer techniques have been reported. The present report summarizes studies of four kindreds affected with this syndrome and documents two new observations; i.e., the lack of platelet aggregation by bovine fibrinogen and a lack of ADP-induced platelet “swelling.” These abnormalities appear to be unique to this syndrome and provide new information concerning the nature of the platelet defect.

Materials and Methods

Silicone-coated glassware was used throughout. Platelet-rich plasma (PRP) was prepared by centrifuging a mixture of whole blood and 0.01 volume of 1 M buf- fered citrate (pH 4.7) or 0.1 volume of 0.1 M trisodium citrate for 12-20 minutes at 200 g. Red cells and leukocytes were enumerated in each suspension. Platelet- poor plasma (PPP) was prepared by spinning the remaining blood at 2,000 g for 30 minutes. Control platelet suspensions were diluted with isologous PPP so that the platelet concentration equaled that in the PRP of the patient.

Collagen was prepared from human breast fat as described by Zucker and B ~ r e l l i . ~ Other platelet aggregating agents tested were bovine Cohn Fraction I (Armour Pharmaceuticals, Kankakee, Ill.; coagulable protein approximately 60%, bovine fibrinogen twice purified by the method of Laki4 (coagulable protein 90%, adenosine-5’-diphosphate (Sigma Chemical Co., St. Louis, Mo.) ; epinephrine bitartrate ( K & K Laboratories, Plainview, N. J.) and bovine thrombin

Supported in part by U.S.P.H.S. General Research Support Grant No. 5501RR05431-09, and Research Grants AM44489 and AM0002 and Hematology Training Grant AM5098 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. Reprint Address: (Dr. Bithell) Department of Clinical Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22901.

145

Cli

nic

al F

eatu

res

Mod

erat

e to se

vere

ble

edin

g of

pur

puri

c typ

e

Aut

osom

al r

eces

sive i

nher

itanc

e

Lab

orat

ory

Fea

ture

s Z 2 J

Mild

to m

oder

ate t

hrom

bocy

tope

nia

“Gia

nt” m

orph

olog

ical

ly ab

norm

al p

late

lets

Con

sang

uini

ty co

mm

on

Het

eroz

ygot

es as

ympt

omat

ic; l

abor

ator

y ab

norm

aliti

es co

mm

only

pre

sent

b K CD

Prol

onge

d ble

edin

g ti

me

El w &

Prot

hrom

bin

cons

umpt

ion

and PF-3 a

ctiv

ity in

TG

T u

sual

ly d

efic

ient

No

ther

apy

of p

rove

n va

lue

Nor

mal

clo

t ret

ract

ion

and

bloo

d co

agul

atio

n.

1 $j

Bithell et al. : The Bernard-Soulier Syndrome 147

(Parke Davis Co., Detroit, Mich.) . These agents were dissolved in 0.05 M imidazole buffer, pH 7.2. ADP was dissolved in imidazole buffer of pH 6.8. In all experiments, the concentrations indicated refer to those in the final aggregating mixture.

The bleeding time was determined by a modification of the Ivy method,5 and platelets were enumerated as described by Brecher and Cronkite.s Mean platelet volume was determined in a Coulter model B electronic particle counter (Coulter Electronics, Hialeah, Fla.) as described by Bull and Zucker,’ and mean platelet diameter was determined with a Leitz ocular micrometer. Platelet aggregation was determined at 37°C in a Born Aggregometer, and tracings were made with a Vitatron linear recorder, No. UR 400. Results are expressed as change in optical density (O.D.) as a percentage of the initial O.D., or as percent aggregation8 (See FIGURE 4). The serum prothrombin time was determined by the method of Q ~ i c k , ~ employing Platelin (Warner-Chilcott, Morris Plains, N. J.), 0.1 m1/2 ml of blood, as a platelet substitute. Two-stage prothrombin consumption was determined by the method of Ware and Seegers.lo Tests for platelet factor 3 in the thromboplastin generation test (TGT) were performed as described by Eichel-

Numb.. 1

I

VELLORE KINOIEDS

m

Is!

P 6

I

n

m

Is!

P

=Oinuollv &.cud wrnhrs

0 p =Oinxdl(l und4-d n.mb.r, ,A Gone .looh

8 fl = C h d v unoHrf.d m.mb.rs rlmovi 0mni plm.l.i.

Numb-r, wahm i i i b d s #nd*al. nurnbar 01 uUawi d sam. I..

FIGURE 1. Pedigree of the Bernard-Soulier syndrome in two Indian kindreds. Key: Roman numerals denote generations; Arabic numerals below symbols denote individuals

referred to in text and tables; Arabic numerals within symbols indicate number of silbings of same sex.

db = nonidentical twins; 0 = clinically affected members a 0 = clinically unaffected members’studied without “giant” platelets w 0 = clinically unaffected members studied with “giant” platelets

0 = male; 0 = female; /r = proband; + = decreased, not studied; O=O = consanguineous marriage;

148 Annals New York Academy of Sciences

berger.” Kaolin-induced platelet factor 3 activity was determined by the methods of Hardisty and Hutton,12 and Weiss.13 Platelet “swelling” was determined photo- metrically, as described by Born,l4 and estimates of platelet adhesiveness were made by the methods of Salzman,15 Borchgrevink,lB Hardisty and coworkers,’’ and Weiss and colleagues.8

Case Reports

In FIGURE 1 are pedigrees of two kindreds studied in India by Dr. Parekh. Both probands were the offspring of consanguineous marriages. The clinical picture was one of purpura, severe epistaxis, and menorrhagia, and three siblings of proband No. 1 died of hemorrhage in childhood. Laboratory abnormalities were demonstrated in both pareats of proband No. 1, but not in the suniving parent of proband No. 2. Pedigrees of two kindreds recently studied in Charlottesville are seen in FIGURE 2. In both probands, bleeding began in early childhood, and menorrhagia of exsanguinating proportions has been the major problem. Both of these patients underwent splenectomy, which produced a slight increase in the platelet count but no diminution in the severity of bleeding. The parents of proband No. 3 and her grandfather had demonstrable laboratory abnormalities, but both parents of proband No. 4 were normal.

Laboratory Data

“Giant” platelets are the most’characteristic feature of this syndrome, and are seen in FIGURE 3. The large size and abnormal appearance of the platelets is ap- parent, and was similar in all four probands and in affected heterozygotes. Varia- tion in platelet size was marked, and in a minority of platelets the dense central granulation produced a “lymphocytoid” or “pseudonucleated” appearance.

VIRGINIA UINDPEDS

* I

I

m

Ip

= C l i n ~ d l y d k w d m.mb.rs

I 0 p = c l ~ ~ ~ d y unoH.o.d m.mb.n -8th “kant” 010t.l.li

0 ~ C l m ~ c m l l ~ u n d l r h d m.mb.n wlhoul “Giant” plahbu

I

m

lE

P

FIGURE 2. Pedigree of the Bernard-Soulier syndrome in two Virginia kindreds. The key is the same as in FIGURE 1.

Bithell er al. : The Bernard-Soulier Syndrome 149

FIGURE 3. “Giant” platelets in the Bernard-Soulier syndrome. Wright’s stain of a buffy coat smear, prepared from citrated blood of Proband number 3 ( x 950).

Basic laboratory data on the four probands and on the five identifiable heterozygotes are summarized in TABLE 2. The wide range of platelet counts indicated were obtained during prolonged followup. In probands No. 3 and No. 4, the platelet count has never been normal except for a brief period following splenectomy or acute hemorrhage. The bleeding time was markedly prolonged even when the platelet count was normal, and clot retraction was invariably normal even when the platelet count was as low as 25,000/mm3 in probands No. 1 and No. 2. “Giant” platelets, defined as those over 2.5 p in diameter in stained blood smears, comprise a high percentage of the circulating population in all probands. Mean platelet diameter and mean platelet “volume” were two to three times normal, but forms five to seven times larger than normal were seen.

All heterozygotes were asymptomatic. The parents of proband No. 1 had both mild thrombocytopenia and giant platelets. The heterozygotes in kindred No. 3 had only giant platelets, but in the case of the father these were striking and were nearly as numerous as in the proband. It is noteworthy that mean platelet diameter in the three heterozygotes in kindred No. 3 was normally distributed, whereas in the probands it was skewed, fitting a logarithmic normal distribution. Bone- marrow smears revealed normal numbers of megakaryocytes, in some cases surrounded by “giant” platelets; the results of detailed studies of blood coagulation were within normal limits.

Preliminary experiments established that mean platelet “volume” was the same in PRP prepared by centrifugation as in that obtained by allowing blood to settle without centrifugation. This excluded the possibility that centrifugation removes significantly more large than small platelets, producing a nonrepresentative platelet population in the final PRP. The yield of “giant” platelets free of contaminating leukocytes and erythrocytes was very low, and ranged from 40,000-1~00,00~/ mm3 of PRP.

Prob

ands

Kin

dred

no. 1

(IV

-4)

Kin

dred

no.

2 (

V-3

)

Kin

dred

no.

3 (

IV-1

)

Kin

dred

no.

3 (

V-1)

Het

eroz

ygot

es

Kin

dred

no.

1 (1

11-1

)

Kin

dred

(1

11-2

)

Kin

dred

no.

3 (

11-1

)

Kin

dred

(1

11-2

)

Kin

dred

(111-3)

Nor

mal

Sub

ject

s

(Mea

n f 2

S.D.)

TA

BL

E

2

PE

RT

INE

NT

LA

BO

RA

TO

RY

D

AT

A

ON

PRO

BAND

S A

ND

HE

TE

RO

ZY

GO

TE

S

Pla

tele

t Cou

nts.

( /

mm

3)

Ble

edin

g T

ime

( min

) “G

iant

” P

late

lets

M

ean

Clo

t R

etra

ctio

n (D

iam

eter

> 2.

5/~

) P

late

let

Dia

met

er

(%)

(ll)

(q

uali

tati

ve)

24,0

00-1

08,

000

> 30

N

orm

al

-80

25,0

00-2

00,0

00

> 30

N

orm

al

-80

90,0

0&16

7,00

0 >

30

Nor

mal

72

67,0

0&12

5,00

0 >

30

Nor

mal

75

-

-

3.5

3.7

108,

000

3.5

Nor

mal

-2

0

111,

000

5.5

Nor

mal

-2

0

240,

000

0.5-

4.5

Nor

mal

40

198,

000

0.5-

2.5

Nor

mal

28

175,

000

4.0-

6.5

Nor

mal

64

-

-

2.6

2.5

3.1

150,

000-

450,

000

0.5-

9.0

-

7-1

5 1.

5-2.

4

ul

C

* Ran

ge o

f m

ultip

le d

eter

min

atio

ns o

ver

2-4

year

per

iod.

Bithell et al. : The Bernard-Soulier Syndrome

Proband Kindrod #3 = ----- addod

I 1 1 1 10 4 a 12 16

TIME AFTER T o (mins)

O.D.@lo - O.D.(Minimum) ( OD. @ lo ) FIGURE 4.

ADP

A c

g %

Platelet aggregation by ADP.

Control 7 --i--- Proband Kindrod#O

151

3 U I U

I- 10 +I soc

-I5 I I TO 0.5

TIME AFTER 1, (min)

FIGURE 5. Initial change in optical density of platelet-rich plasma (platelet “swelling”) induced by ADP. Increase in optical density is indicated by an upward deflection, the reverse of conventional aggregometer tracings.


Studies of platelet aggregation by ADP are illustrated in FIGURE 4. Maximal aggregation of patients’ platelets produced by 5 micromolar ADP was normal, as was the rate of disaggregation with 0.25 micromolar concentrations. However, two subtle but significant abnormalities of patients’ platelets were consistently observed; i.e., first, the increase of O.D. that normally preceeds ADP-induced aggregation was absent, and second, the rate of aggregation was abnormally rapid.

The initial increase in O.D. of PRP was studied by the photometric method of BornI4 (FIGURE 5 ) . In this method, aggregation that normally follows the initial increase in O.D. is inhibited by the addition of 5 mM EDTA, the optical effect is magnified by increasing the sensitivity of the recording circuit to maximum and by using dilute platelet suspensions, and the optical artefact produced by dilution is eliminated my reducing the volume of ADP added to 20 ~ 1 , which was introduced by means of an Eppendorff micropipette. In this representative study, a rapid increase of approximately 14% in the O.D. of the control suspension is produced by ADP. By contrast, no increase in O.D. is seen in the PRP of the patient. Similar results were obtained when this experiment was repeated with suspensions of patients’ platelets diluted so that the initial O.D. was equal to that of the control, and with more concentrated suspensions of control platelets having an initial O.D. equal to that of the patients PRP.

Numerous studies of this phenomenon by the photometric method and by electronic particle sizing are summarized in TABLE 3 , With normal platelets, 1 micromolar ADP produced a reproducible increase of approximately 15 % in O.D. and of approximately 12% in mean platelet “volume.” With the patients’ platelets, neither parameter changed significantly, even when 100 times more ADP was used. However, the patients’ platelets are capable of swelling to a normal extent when chilled for 30 minutes. As with normal platelets, this change was reversible on rewarming.

The data summarized in FIGURE 6 illustrate the wide normal range of collagen- induced platelet aggregation obtained with dilute platelet suspensions. The maximal aggregation of platelets of the probands was nevertheless normal at various collagen concentrations (A) and at various platelet concentrations ( B ) , and in many experiments was greater than normal.

Data comparing the rate of aggregation of giant platelets to that of normal platelets are summarized in TABLE 4. This was estimated by determining the time

TABLE 3

PLASMA AND MEAN PLATELET “VOLUME” EFFECT OF ADP AND CHILLING ON OPTICAL DENSITY OF PLATELET-RICH

Photometric Technique % increase in O.D. of PRP

(Mean -C 2 SD)

Adenosine Diphosphate (1 PM) Controls Proband No. 3 Proband No. 4

15 f 3.5 (n = 12) 0 (n = 8) 2 (n = 4)

Chilling (30 min at 4°C) Controls Proband No. 3 Proband No. 4

16 f 3.5 (n = 10) 14 (n = 4) 12 (n = 2)

Electronic Particle Sizing % increase in mean platelet

“volume” (Coulter B ) (Mean i 2 SD)

12 -C 3.0 (n = 12) 0 (n = 8) 2 (n = 4)

Bithell et al. : The Bernard-Soulier Syndrome

Adenosine-di-phosphate

Collagen (0.5-1 PM)

( 1 : 80 dilution)

153

2.59 2 0.5 (n = 22)

2.14 2 0.5 (n = 22)

1.4 f 0.4 (n = 14)

1.4 -C 0.4 (n = 14)

1.0 f 0.2 (n = 8)

1.1 f 0.2 (n = 8)

A. EFFECT OF COLLAGEN CONCENTRATION 8. EFFECT OF PLATELET CONCENTRATION (PlatoIolr=75,000/mm~) (Collagon=l-80 dilution)

I

I 0 1 I I I I I 1 I I I

1-160 I80 1.40 1.20 SO 15 100 121 IM

COLLAGEN DILUTION PLATELET COUNT 0 0 3 / ~ ~ 3 1 FIQWRE 6. Collagen-induced platelet aggregation.

elapsed from the addition of the aggregating agent until half the maximal aggregation had occurred. This “half aggregation time” of patients’ platelets was shorter, to a statistically significant degree (p<O.O1 ), than that of normal platelets, when induced by either ADP or collagen. Abnormally rapid aggregation was particularly striking in the case of proband No. 4.

FIGURE 7 illustrates the lack of platelet aggregation by bovine fibrinogen, a curious empirical observation first made in the Indian probands by Dr. Parekh. The rapid and irreversible aggregation of normal platelets produced by 1.5 mg of bovine fibrinogedml of PRP is seen in the upper tracing; the total absence of aggregation of patients’ platelets is seen below. This has been an absolutely consistent finding in all four probands on repeated testing (inset, FIGURE 7), whereas in 39 normal controls aggregation by bovine fibrinogen is consistent and highly reproducible. This marked difference between normal and “giant” platelets is unaffected by platelet numbers or by fibrinogen concentration over a wide range.

In an attempt to define the specificity of the test, the aggregating affects of bovine fibrinogen were tested on the platelets of patients suffering from various other disorders, as summarized in TABLE 5 . The results were entirely normal in all of these 29 cases, which include examples of various forms of thrombocytopenia and of many acquired and hereditary disorders of platelet function.

TABLE 4 RATE OF PLATELET AGGREGATION INDUCED BY COLLAQEN A N D ADP

“Half” Aggregation Time (min)

Proband Aggregating Agent I ~ ~ ~ ~ l ~ b & t ~ Kindred No. 3

Proband Kindred No. 4

154

I I I

Annals New York Academy of Sciences

PLATELETS = 100,000/mm3 Conlrol

Bovine

Fibrinogen

(1.5 mg/ml)

f

BOVINE FIBRINOGEN

Various additional aggregation studies were carried out. The platelets of these patients were aggregated by epinephrine ( 5 micromolar), and by bovine thrombin (0.1 N.I.H. u/ml). However, aggregation of dilute platelet suspensions by these agents proved difficult to quantitate, since minimal or no aggregation was frequently seen with normal platelets.

The results of various tests of platelet adhesiveness were normal (TABLE 6) . The normal range of adhesion to glass beads, as measured by the method of Hardisty and coworkers17 is very wide when dilute platelet suspensions are used. Platelet adhesion to collagen fibers in EDTA plasma could readily be seen microscopically but was difficult to quantitate by standard methods. The method of Weiss and colleagues* was modified by the use of high concentrations of

TABLE 5 DISORDERS IN WHICH BOVINE FIBRINOGEN-INDUCED PLATELET AGGREGATION IS NORMAL

Number of Cases

Hereditary von Willebrand’s disease Abnormalities of “release” reaction (Thrombopathia)

6 2

Acquired Normal subjects following aspirin ingestion Thrombocytopenia

6

Acute leukemia 2 Idiopathic ( U P ) 2 Macroglobulinemia (Postsplenectomy) 1

Multiple myeloma (with azotemia) 2 Macroglobulinemia 3 Myelofibrosis with “giant” platelets 1 Hemorrhagic thrombocythemia 2 Acquired “thrombasthenia” 1 Laennec’s cirrhosis with high levels of FDP 1

Bithell et al. : The Bernard-Soulier Syndrome TABLE 6

155

TESTS OF PLATELET ADHESIVENESS

Test Normal Range (+- 2 SD)

Robands Kindred Kindred No. 3 No.4

To glass beads in % adhesive no. adhesive x 103/mm3

To collagen in % adhesive no. adhesive x 103/mm3

To glass beads in % adhesive no. adhesive x 103/mm3

in vivo (Borchgrevink) % adhesive no. adhesive x 103/mm3

citrated PRP (Hardisty)

EDTA PRP ( Weiss)

whole blood (Salzrnan)

30- 70t 30-100 80- 95t 40-106 18- 85’ 25-340 20- 46. 24-156

59,69 54 85,52 39 88,90 85 95,69 52

30 20 30 22

32,23 51 31,21 44

Determined on subjects with normal venous platelet counts. t Platelets in PRP = 50-112,000/mma.

collagen, since with smaller amounts the number of adherent platelets was usually less than the counting error. Results obtained with the methods of Borchgrevink16 and Salzman15 were on the low side when expressed as absolute numbers of adherent platelets, but these data are difficult to interpret, since we do not have a significant number of control subjects with comparably reduced venous platelet counts.

TABLE 7 summarizes the results of several tests of platelet factor 3 activity. Prothrombin consumption, as determined by the two-stage method was entirely normal. However, an abnormally short one-stage serum prothrombin time, which could be normalized by added platelet substitute, was repeatedly observed. The venous platelet counts that correspond to these determinations suggest that thrombocytopenia is an unlikely explanation for this abnormality. Platelet factor 3 activity was also measured in the thromboplastin generation test. Platelet SUS- pensions were standardized by platelet volume, by platelet number, and by optical density. High and low concentrations of platelets ( 1 % to 0.008% V/V) l1

and intract and lysed suspensions were tested. The results in each case were normal. Platelet factor 3 activity was also normal when assayed by the methods of Hardisty and Huttonl2 and of Weiss and associates,8 in which the release of platelet factor 3 is induced by kaolin. In many cases, the platelet factor 3 activity of the patients’ platelets was significantly greater than normal, when standardized on the basis of platelet number.

Discussion

The clinical and laboratory features of the Bernard-Soulier syndrome are relatively clear-cut, and the exclusion of other hereditary forms of thrombocytopenia associated with “giant” platelets and platelet dysfunction is seldom difficult. Certain differences, however, have been noted between the original cases1J8,26 and those subsequently reported.lE32 In our patients, the mean platelet diameter and volume varied from 2.5 to 3 times normal, and the ‘‘giant’’ platelets would thus appear to be somewhat smaller than those described in other cases. A second difference is the normal results obtained with various tests of platelet factor 3 activity in our cases. The seruh prothrombin time was abnormal in our patients, as in virtually all other reported cases, but our data would suggest that this is not due to deficient prothrombin consumption. Discrepancies between platelet factor 3 activity measured in the thromboplastin generation test, the serum prothrombin

TABLE

7 Px

oTaa

ot.m

m C

ON

SU

MP

~ON

A

ND

F'LA

TELE

T FA

CT

VB

3

Acr

rvrr

~

c

u

m

Test

Prot

hrom

bin

Con

sum

ptio

n

Seru

m p

roth

rom

bin

time-

sec

Serum

prot

hrom

bin time w

ith a

dded

pla

tele

t sub

stitu

te-s

ec

2- s

tage

met

hod-

% c

onsu

med

@

15m

in

@

30m

in

@

45m

in

@

60m

in

@ 1

80 m

in (S

ilico

ned

tube

)

Ven

ous P

late

let c

ount

-( 1

0a/m

ms)

Plat

elet

Fac

tor

3 A

ctiv

ity

Thr

ombo

plas

tin g

ener

atio

n te

st e

mpl

oyin

g pa

tient

's pl

atel

ets

1-st

age k

aolin

-ind

uced

PF-

3 ac

tivity

(H

ardi

sty)

-sec

2-st

age k

aolin

-ind

uced

PF-

3 ac

tivity

(W

eiss

) ka

olin

onl

y-se

c

kaol

in +

ADP-

sec

Prob

ands

N

~~

~l

&~

ge

K

indr

ed No. 1

K

indr

ed No. 2

Kio

dred

No. 3

Kio

dred

No. 4

&

>25*

18

16

14

-17

19-2

3 LY

>25*

>2

5 >2

5 >2

5 >2

5 LY

L2 10

-75.

-

-

45,6

5 55

74-9

9. -

-

91,9

2 as

90

-99.

-

-

95,9

9 95

95-9

9.

-

-

95,9

7 -

42-9

9. -

-

73,8

1 71

z G CD tp

c) E. 8 Y

-

108

110

90-1

20

95-1

1.

s?Y

8.

-

Nor

mal

N

orm

al

Nor

mal

N

orm

al

z !2 c4

CD

45-5

6?

45

42

31,3

5 39

-

31

35

-

30

35

4-t -

-

Det

erm

ined

on

subj

ects

with n

orm

al v

enou

s pla

tele

t counts.

t Pla

tele

ts in

PR

P =

75,0

00/m

ma.


time, and kaolin-induced platelet factor 3 activity have been encountered by others,28,31,33 and the last method, in which platelet manipulation is minimized, usually has yielded normal r e s ~ l t s . ~ ~ . ~ ~ Normal platelet factor 3 activity is consistent with normal platelet aggregation by dilute collagen, normal glass adhesiveness, and the lack of disaggregation at low ADP concentrations. These tests are abnormal in most well-defined forms of thrombopathia, in which a defect in the release reaction presumably underlies deficient platelet factor 3 activity.34

There is no obvious explanation for these differences. It is possible that they result from variations in technique, or from variable expressivity of the genetic abnormality. Furthermore, the possibility that this syndrome encompasses a variety of fundamentally different disorders cannot be excluded on the basis of extant data. Tests for platelet aggregation by bovine fibrinogen and for ADP- induced “swelling” are simple to perform, and may help clarify this problem.

The nature of the platelet abnormality in this syndrome remains unclear, but most data suggest that the fundamental defect is in the platelet membrane. Grottom and SoIum22 demonstrated reduced electrophoretic mobility of the platelets in two cases, which was apparently due to an abnormally low platelet content of sialic acid. ADP produced normal aggregation but did not change the electrophoretic mobility. These workers suggested that the reduced negative charge on the platelet membrane could underlie both platelet dysfunction and reduced platelet survival. The absence of aggregation by bovine fibrinogen, the lack of ADP-induced “swelling,” and the abnormally rapid rate of aggregation demonstrated in the present study are consistent with such a hypothesis.

There is good evidence that platelet aggregation by bovine fibrinogen is the result of the electrostatic adsorption of this protein by the platelet membrane, and the consequent reduction in negative ~ h a r g e . ~ ” ~ ’ , ~ @ Aggregation by bovine fibrinogen is deficient in hereditary afibrinogenemia3@,M but can be restored by infusions of homologous f i b r i n ~ g e n , ~ ~ and is subnormal in Glanzmann’s throm- b a ~ t h e n i a , ~ ~ a disorder in which platelet fibrinogen may be deficient.38 These observations also provide indirect evidence that bovine fibrinogen-induced platelet aggregation also is dependent on the presence of homologous fibrinogen. A deficiency of sialic acid, which is the primary determinant of negative platelet charge and is known to play an important role in various adsorptive functions of the platelet membrane, could well diminish adsorption of homologous or hetero- logous fibrinogen or both, and thus impair bovine fibrinogen-induced platelet aggregation.

Knowledge concerning ADP-induced platelet “swelling” is fragmentary. The studies of Born suggest that this phenomenon is the result of altered platelet shape rather than increased volume,14 and there is good evidence that this change and the subsequent aggregation phase, although normally interdependent, are fundamentally discrete p r o c e s s e ~ . ~ ~ , ~ ~ * 4 ~ Thus, platelet “swelling” occurs without aggregation in Glanzmann’s thrombasthenia46 and in normal platelets in the presence of EDTA or cocaine.’ Aggregation in the absence of “swelling” was demonstrated in the present cases, and can be produced experimentally by low concentrations of epinephrine and by suspending platelets in hyperosmolar sucrose.42 Numerous processes could explain this abnormality, including a defect in the platelet membrane. Sialic acid is frequently involved in receptor sites for biologically active subs t an~es ,4~ .~~ and serotonin-induced smooth-muscle contrac- tion, a process which bears many similarities to platelet “swelling” induced by ADP, is mediated by glycoprotein receptors.47

The physiologic significance of platelet “swelling” induced by ADP remains


uncertain, but it has been suggested that it facilitates intimate contact between platelets, collagen, and subendothelial components, and thus may be important in producing a stable platelet thr0mbus.~4 The cause of defective hemostasis in the Bernard-Soulier syndrome remains obscure, but it is possible that the absence of platelet “swelling” and of the associated rigid cytoplasmic extr~sions,’~ to- gether with the abnormally large size of the platelets, results in a functionally inadequate platelet thrombus, or impairs and subsequent cohesion of the thrombus, despite normal adhesion and aggregation.

Summary

Studies of platelet function in four kindreds affected with the Bernard-Soulier Syndrome are summarized. Platelet aggregation induced by ADP and by dilute collagen suspensions was normal, but the increase in optical density and platelet “volume” that normally proceeds aggregation was lacking. Platelet aggregation by bovine fibrinogen was totally deficient, and the rate of aggregation by both ADP and collagen was abnormally rapid. The serum prothrombin time was abnormal, but other tests of platelet factor 3 activity were normal, as were estimates of platelet adhesiveness. The lack of initial platelet “swelling” and deficient bovine fibrinogen-induced platelet aggregation are heretofore unrecognized features of this disorder that may provide information as to the nature of the under- lying abnormality.

References

1. BERNARD, J. & J. P. SOULIER. Sur une nouvelle variete de dystrophid thrombocytaire hdmorragipare congdnitale. Sem. Hap. Paris 24: 3217.

2. LARRIEU, M. J. Congenital haemorrhagic disorders with normal platelet count and prolonged bleeding time. Scand. J. Haematol. Series Haematologica 7: 39.

3. ZUCKER, M. B. & J. BORRELLI. 1962. Platelet clumping produced by connective tissue suspensions and by collagen. Proc. SOC. Exp. Biol. Med. 109: 779.

4. LAKI, K. The polymerization of proteins. The reaction of thrombin on fibrinogen. Arch. Biochem. Biophys. 32: 317.

5. CARTWRIGHT, G. E. 1968. Diagnostic Laboratory Hematology. 4th edit. Grune & Stratton. New York, New York.

6. BRECHER, G. & E. P. CRONKITE. Morphology and enumeration of blood platelets. J. Appl. Physiol. 3: 365.

7. BULL, B. S. & M. B. ZUCKER. Changes in platelet volume produced by temperature metabolic inhibitors, and aggregating agents. Proc. SOC. Exp. Biol. Med. 120: 296.

8. WEISS, H. J., L. M. ALEDORT & S. KOCHWA. 1968. The effect of salicylates on the hemo- static properties of platelets in man. J. Clin. Invest. 47: 2169.

9. QUICK, A. J. 1966. The prothrombin consumption time test. Amer. J. Clin. Path. 43: 105. 1949. Two-stage procedure for the quantitative deter-

mination of prothrombin concentration. Amer. J. Clin. Path. 19: 471. Laboratory Methods in Blood Coagulation. Harper &

Row. New York, New York. 1965. The kaolin clotting time of platelet rich

plasma: a test of platelet factor-3 availability. Brit. J. Haematol. 11: 258. Platelet aggregation, adhesion, and adenosine diphosphate release in

thrombopathia (Platelet factor 3 deficiency). Amer. J. Med. 43: 570. Observations on the change in shape of blood platelets brought

about by adenosine diphosphate. J. Physiol. 209: 487. Measurement of platelet adhesiveness: a simple in vitro technique

demonstrating an abnormality in von Willebrand’s disease. J. Lab. Clin. Med. 62: 724. Platelet adhesion in vitro in patients with bleeding disorders.

Acta Med. Scand. 170: 231. 1964. Thrombasthenia, studies

on three cases. Brit. J. Haematol. 10: 371. 1957. La dystrophie thrombocytaire hhorrag i -

pare congdnitale. Rev. Hematol. 12: 222.

1948.

1965.

1951.

1950.

1965.

10. WARE, A. G. & W. H. SEEGERS.

11. EICHELBERGER, J. W., JR. 1965.

12. HARDISTY, R. M. & R. A. HUTTON.

13. WEISS, H. J.

14. BORN, G. V. R.

15. SALZMAN, E. W.

16. BORCHGREVINK, C. F. 1961.

17. HARDISTY, R. M., K. M. DORMANDY & R. A. HUTTON.

18. BERNARD, J., J. CAEN & P. MAROTEAU.

1967.

1970.

1963.


19. HIRSCH, E. O., J. FAVRE-GILLY & W. DAMESHEK. 1950. Thrombopathic thrombocyto-

20. ULUTIN, 0. N. 21. BRAUNSTEINER, H. & F. PAKESCH. 1956. Thrombocytoasthenia and thrombocytopathia

- old names and new diseases. Blood 11: 965. 22. GROTTUM, K. A. & N. 0. SOLUM. 1969. Congenital thrombocytopenia with giant platelets:

a defect in the platelet membrane. Brit. J. Haematol. 16: 277. 23. CULLUM, C., D. P. COONEY & S. L. SCHRIER. 1967. Familial thrombocytopenic thrombo-

cytopathy. Brit. J. Haematol. 13: 147. 24. KANSKA, B., S. NIEWIAROWSKI, L. OSTROWSKI, A. POPLOWSKI & J. PROKOPOWICZ. 1963.

Macrothrombocytic thrombopathia. Clinical coagulation and hereditary aspects. Thromb. Diath. Haemorrhag. 10: 88.

25. NIEWIAROWSKI, S., A. POPLAWSKI, J. PROKOPOWICZ, B. KANSKA, K. LECHNER & L. STOCKINGER. 1969. Abnormalities of platelet function and ultrastructure in macrothrombocytic thrombopathia. Scand. J. Haematol. 6: 377.

26. ALAGILLE, D., F. Josso, J. L. BINET & M. L. BLIN. 1964. La dystrophie thrombocytaire htmorragipare. Discussion nosologique. Nouvelle Rev. Franc. Hematol. 4: 755.

27. D E B R ~ , R., J. BERNARD, J. P. SOULIER, S . BUHOT, J. L. BEAUMONT ET LAGRUE. 1952. Une nouvelle observation de dystrophia thrombocytaire htmorragique congenitale. La Presse Medicale 60: 1271.

28. MORAES-REGO, S. F. 1965. Descripcidn de una nueva plaquetopathia: la distrofia megacariocito-thrombocitaria congtnita. Sangre 10: 227.

29. MORAES-REGO, S. F. & D. A. LOMONACO. 1959. Observacao de um caso de distrofia thrombocitaria hemorragipara congenita, com estudo da funcao thromboplastica das plaquetas. Rev. Paul. Med. 54: 198.

30. ASCARI, E., F. GOBBI, U. BARBIERI & G. GROSSI. 1965. La distrofia thrombocitaria emorragipara (Malattia di Bernard e Soulier). I1 Progress0 Medico (Napoli) 21: 609, 737.

A propos d u n cas de thrombopathie hemorragique conghitale. (Etude au microscope Clectronique). Nouvelle Rev. Franc. Hematol. 7: 559.

32. JACKSON, D. P., R. C. HARTMANN & C. L. CONLEY. 1953. Clot retraction as a measure of platelet function. 11. Clinical disorders associated with qualitative platelet defects (Thrombocytopathic purpura). Bull. Johns Hopkins Hosp. 93: 370.

penia: successful transfusion of blood platelets. Blood 5: 568. 1965. Primary thrombocytopathy. Israel J. Med. Sci. 1: 857.

31. PAVLOVSKY, A., M. PINTO, A. CANAVERI & S . PAVLOVSKY. 1967.

33. CAEN, J. 34. WEISS, H. J., P. A. CHERVENICK, R. ZALUSKY & A. FACTOR.

1968. Thrombopathie et thrombasthtnie. Schweiz. Med. Wochschr. 98: 1641. 1969. A familial defect in

platelet function associated with impaired release of adenosine diphosphate. New Eng. J. Med. 281: 1264.

1968. Aggregation of human platelets by bovine platelet fibrinogen. Scand. J. Haematol. 5 : 474.

1969. Platelet surface charge and aggregation effects of polyelectrolytes. Thromb. Diath. Haemorrhag. 21: 450.

1970. ADP-induced aggregation of washed platelets effect on platelet and plasma fibrinogen. Scand. J . Haematol. 7: 236.

Immunological studies of proteins associated with the subcellular fractions of thrombasthenic and afibrinogenaemic platelets. Brit. J. Haematol. 15: 181.

1966. Studies on the mechanism of interference by fibrinogen degradation products (FDP) with the platelet function. Role of fibrinogen in the platelet atmosphere. Thromb. Diath. Haemorrhag. 15: 476.

40. PEPPER, D. S. & G. A. JAMIESON. 1969. Studies on glycoproteins. 111. Isolation of sialylglycopeptides from human platelet membranes. Biochemistry 8: 3362.

41. MADOFF, M. A,, S. EBBE & M. BALDINI. Sialic acid of human blood platelets. J. Clin. Invest. 43: 870.

42. SALZMAN, E. W., T. P. ASHFORD, D. A. CHAMBERS, L. L. NERI & A. P. DEMPSTER. 1969. Platelet volume: effect of temperature and agents affecting platelet aggregation. h e r . J. Physiol. 217: 1330.

43. WHITE, J. G. & W. KRIVIT. 1967. A n ultrastructural basis for the shape changes induced in platelets by chilling. Blood 30: 625.

44. SINAKOS, Z., Y. SULTAN, J. CAEN & J. BERNARD. 1967. Le comportement des plaquettes dans les maladies htmorragiques. La Presse Medicale 75: 269.

45. FALCAO, L., M. PROBST, A. GAUTIER, H. VAINER, H. MICHEL & J. P. CAEN. 1967. L'ultrastructure des plaquettes thrombasthtniques. (Effects de I'ADP et du fibrinogkne bovine). Thromb. Diath. Haemorrhag. 18: 691.

35. SOLUM, N. 0.

36. GROTTUM, K. A.

37. SOLUM, N. 0.

38. NACHMAN, R. L. & A. J. MARCUS.

39. KopBc, M., A. BUDZYNSKI, J. STACHURSKA, Z. WEGRZYNOWICZ & E. KOWALSKI.

1968.

1964.

160 Annals New York Academy of Sciences 46. ZUCKER, M. B., J. H. PERT & M. W. HILGARTNER. 1966. Platelet function in a patient

47. WESEMANN, W. & F. ZILLIKEN. 1968. Serotoninrezeptor und neuraminsaurestoffwechsel with thrombasthenia. Blood 28: 524.

der glatten muskulatur. Hoppe-Seyler’s Z. Physiol. Chem. 349: 823.

PLATELET-FUNCTION STUDIES IN THE BERNARD-SOULIER SYNDROME

Documents

Transcript of PLATELET-FUNCTION STUDIES IN THE BERNARD-SOULIER SYNDROME