From bloodjournal.hematologylibrary.org by guest on October 28, 2011. For personal use only.

1988 71: 629-635

Evidence for tissue factor-dependent activation of the classic extrinsic coagulation mechanism in blood obtained from bleeding time wounds
HJ Weiss and B Lages

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From bloodjournal.hematologylibrary.org by guest on October 28, 2011. For personal use only.

Evidence

for

Tissue

Factor-Dependent

Activation

of

the

Classic

Extrinsic

Coagulation

Mechanism
By

in Blood
Harvey J. Weiss
mechanism bleeding platolot FPA of bleeding. administration, occurs increase bleeding. or VIII, was The in patients early throughout in was obtainod. moasuring (FPA). seconds)

Obtained
and Bruce

From
Lages
in suggest of patients in patients that factor early activation correlation PF4 FPA activation secretion event incisions.

Bleeding

Time

Wounds

The was time plasma factor

activation studiod incision 4 (PF4). in the markedly indicating blood. sampling. near in FPA of by

of at

platelets collecting 30-second of and first

and blood

the intervals

coagulation from a A B2 (TxB2). 60 to standard and

diminishod was studios the (activation occurs blooding and to and in platolot impaired consistent as an time delayed

with with tissue X)

factor factor factor

or VII

doficioncy. of of the

and Theso classic from was no the

deficiency.

activation coagulation the arrest of the because production. decreased

concentrations

fibrinopoptide thromboxano (30 aftor

extrinsic during The rolation clear TxB2

mechanism bleeding there FPA aforemontionod formation In fact,

obsorvod increased was thus incisional blood appeared increase deficiencies

samples until thrombin increased

progrossivoly diminished that PF4 the content eithor

cessation hoparin formation monotonically the major of IX normally

is

loss or

between

latter
TxB2 impaired initialand with platelet a 1988 classic

wore
thrombin. by Grune (direct

normal
formation. perhaps during

in

some
which the synergistically,

subjects
suggests may arrest primary that

with
both of contribute

the
to bleeding.

most

whereas cessation

collagen

occurrod factor

markedly

& Stratton,
activation of

Inc.
factor X) extrinsic coagulation

T IS GENERALLY bleeding after during performance

primarily to the

HELD

transection of the
development

arrest of of blood vessels, as occurs pathway. bleeding time test, is due

that the primary of a platelet hemostatic plug at

MATERIALS Time and Collection

AND

METHODS oflncision Blood and for

the largely

end

of from

transected bleeding

vessels. time incisions

These studies are

Evidence
evaluated have

for
by shown

this
light4 that

derives
or the

Bleeding Sequential

electron
ends of

microscopy.35
transected

Measurement

ofBlood

LOSS

vessels

covered by

hemostatic tissue5 that the

platelet as early impairment as

Radioimmunoassays After compressing the upper arm with a sphygmomanometer cuff to a pressure of4O mm Hg, two longitudinal incisions (perpendicular
to the antecubital by Corp. capillary using crease) a Simplate NC). Durham, tubes were II At made on the time volar intervals, Pittsburgh) of surface blood of the from bleeding 30-second Co, device (Organon-

plugs

adhering to perivascular connective 10 to 30 seconds after the incision3 and formation function accounts in these during early
in thrombocytopenia46

of platelet plug ders of platelet thrombasthenia observed mechanism

such for

as von the The

Willebrands prolonged role of

or in disordisease bleeding the is less coagulation

and forearm time

Teknika crit

conditions. stages

each of the two wounds

was collected
(Fisher Scientific

into heparinized

microhematoknown

clear. diameter. The incision and blood collection were performed by the Recent studies have shown the presence of small fibrin fibers same operator throughout the study. The height of the column of at 30 seconds, primarily along the margins of the wounds and blood was immediately measured, and from this the blood volume also at the periphery of the hemostatic plugs,3 which was determined. The blood was then gently expressed into microcentrifuge tubes containing either I 20L phosphate-saline z buffer extends the reports of earlier studies.78 The mechanism (0.025 mol/L NaH2PO4, 0.025 mol/L, Na2HPO4, 0.15 mob/L accounting for this apparent early activation of coagulation pH 6.8, containing 12 zmoI/L indomethacin and 5 mmol/L as well as its significance for the primary arrest of bleeding is NaCI), not clear.
In some disorders

of

hemostasis

ofcoagulation normal

such

as

hemophilprolonga-

EDTA

for 50

the U/mL

measurement

of

TxB2

levels blood in

from

the

ia, the tion has formation findings

bleeding

time

is usually

(although

proximal
heparin,

wound

or 120 L
trasybol

phosphate
(aprotinin),

buffer

been described in some subjects)9#{176} and early fibrin zmol/L occurs within the bleeding time wound. The in other coagulation disorders are less clear. The time in humans is not usually prolonged by warfarin reported.3 occain other in rats has been factor V deficiency,6 and sporadically of
in samples

containing 100 U/mL 5 mmol/L EDTA, I2

and 2 mmol/L theo-

indomethacin,

I mmol/L

adenosine,

bleeding

From Medicine. University Submitted Supported Health

the St

Division

of ofPhysicians 17, by a grant requests

Hematology-Oncology, Hospital and accepted No. the Harvey from to /987; Grant Center Surgeons. October HL27346 Charles J.

Department and New /9, from Slaughter Weiss. MD. St Columbia York. /987. the US

of

therapy,2 although prolongation It is prolonged frequently in sionally coagulation

To evaluate

Lukes-Roosevelt August in part and

College

in factor VII deficiency,78 disorders.9#{176}

more carefully

Public

the activation hemostasis,

the coagulameasured
of bleeding

Service reprint

FoundaLukes-

tion

mechanism
A

during
(FPA)

early

we have

tion.
Address

fibrinopeptide time blood coagulation

tion, we also

sequentially

in normal disorders. measured 4 factor

subjects and In addition, levels (PF4). Our hemostasis

in patients with to study platelet B2 (TxB2) presented is activated by VII a mechanism of activation

various /0019. activaand

Roosevelt
The

Hospital
publication in costs

Center.
ofthis

428
article

West
were

59th

St.

New
in part

York.

NY

of thromboxane findings, mechanism

defrayed

by page

platelet

indicate
the
probably

that

the coagulation of primary

charge payment. herein, advertisement early in dicate this fact. that the

This

article
with

must
/8 Inc.

therefore
U.S.C.

be
1 734

hereby
solely

marked
to in-

accordance & Stratton,

course

I 988

by

Grune

involves

tissue factor-factor

0006-497//88/7103-0006$3.00/0

Blood,

Vol

71.

No

3 (March).

1988:

pp

629-635

629

From bloodjournal.hematologylibrary.org by guest on October 28, 2011. For personal use only.

630

WEISS

AND

LAGES

phylline for the measurement of PF4 and FPA levels in blood from PF4 the distal wound. The diluted wound blood samples were centrifuged PF4 levels were measured by radioimmunoassay using the assay at I 2,000 g for two minutes at room temperature, and sL of the 100 kit from Abbott Laboratories, Diagnostics Division (Chicago). 23I supernatants was removed. Abiquots of these supernatants were labeled PF4 was used as a tracer, and the separation of bound and processed for FPA radioimmunoassay (see the next section) or free fractions was achieved by ammonium sulfate precipitation. The

stored

frozen

at -80#{176}C before

assay

of

the

other

substances.

As

control levels,
directly parafilm

on the assay procedures for determining TxB2, PF4, and FPA milliliter several drops of unanticoagulated venous blood were taken
from and the then butterfly drawn up infusion into set capillary tubing onto tubes, a piece measured, of (SD) and

sensitivity ng/mL,

of

our and

assay the

procedure

expressed of variation

as

nanograms

per

of PF4 giving

10% displacement
interassay coefficient

of tracer,

was 3.5 1.0

was I I .0%.

TxB2 processed as described for the wound blood samples. The results on these samples are indicated as control values. All studies were TxB2 levels were measured by performed under a protocol approved by the Institutional Review standards obtained from the Upjohn Board of the St Lukes-Roosevelt Institute for Health Sciences. tracer obtained from NEN Products
specific antibody obtained from Dr

radioimmunoassay Co J.B. (Kalamazoo, Smith, MI), Temple

using 3H-TxB2

TxB2

(Boston),

and a sensitive
0.0041 1 2.8%

and

University,

FPA
Radioimmunoassay ofFPA was performed by a modification were incubated of (1:0.5,

Philadelphia. pmol TxB2

and interassay

PA. This assay had a sensitivity 0.0072 of (TxB2 was required for 10% displacement
and (P0), intraassay including coefficients of variation

of the tracer)
of all and

the method of Nossel et al.2 Briefly, samples vol/vol) for ten minutes at room temperature
mg/mL saline 0.1% at bentonite (Sigma Chemical Co. The at St

I I .0%, respectively.
0.05%.

The cross-reactivities

of this antibody
were

with

other
than

with
Louis)

a suspension pH
was

of 80 prostaglandins

6-keto-PGF,a,

less

in Tris-buffered

(0.1
chicken g

mob/L
for

NaCI,
ten minutes

0.05
(Sigma).

mmol/L
4#{176}C and

Tris),
mixture the

8.9, containing
then centrifuged plasma Coagulation Assays

ovalbumin

4,800

supernatant

removed and refrozen until radioimmunoassayed for FPA. Adsorbed plasmas were diluted in the same buffer, incubated with anti-FPA serum at 4#{176}C I 8 hours, and after the addition for of 251-desaminotyrosyl-FPA, incubated for an additional two hours. A charcoal

Coagulation factors were assayed by one-stage methods using as substrates plasmas from patients with severe congenital deficiencies of these factors or, in the case of factor V. artificially depleted plasma.23

suspension

at 4#{176}C. Subjects With Coagulation Defects Decanted supernatants were counted on a Beckman Gamma 4000 counter, and values were determined from a curve obtained by Ten patients, aged 24 to 47, had severe congenital deficiencies of testing serial dilutions of FPA standards. FPA antiserum, FPA one coagulation factor (VIII, IX, V, VII, or X), as indicated in Table standards, and desaminotyrosyl-FPA were obtained from IMCO 1 , and had not had a transfusion for at least months 2 before the Corp. Stockholm; desaminotyrosyl-FPA was labeled with I25I by the study. The four patients with factor VIII deficiency and the one chboramine-T method. patient with factor IX deficiency required periodic transfusions for

was added

and

the assay

mixtures

centrifuged

Table

1. Subjects

Studied
Platelets

PU

PT

Plasma

Level

(per

zL)

subject

Sex

(a)

(a)

(%)

x i0

Factor VIII
VIII-1 VIII-2 VIII-3
VIll-4 FactorlX

M M M
M M

120 115 134 141 103 161

126

12 14 13 13 14 36
89

<1 <1 <1 <1 <1 4

<1

230 135 137 153 270 212

293

FactorV
FactorX

F
M

Factor VII-1 VIl-2

Vll-3

VII M M F + heparin M F
13) M.F

36 37 38

30 33 33

2t 2t 3t

249 220 231

Normal
H-i H-2

700 470
35-45

20 29
12-14

317 222
215-351

>50

Normalsubjects(nAbbreviations: Deficient

PU.
coagulation and

partial

thromboplastin

time; PT. prothrombin

details.

time.

factor. Methods for further

tSee

Materials

From bloodjournal.hematologylibrary.org by guest on October 28, 2011. For personal use only.

COAGULATION

MECHANISMS

IN

BLEEDING

WOUNDS

631

episodes

patients
methacin tion
in

of clinical bleeding. had recently ingested

(VIII-4) Results). bleeding and A samples third but were

Two of the factor VIII-deficient ples. The average FPA either ibuprofen (VIII-l) or indo- samples were 203, 547,
found to have (VIII-3) had not been impaired specifically TxB2 produc- 3.9

values
and I ,l07

in

the
ng/mL

1-,

2-,

and

3-minute
values,

(control

3.1 three

[SD]), minutes
in

and was
thrombin

the 491

average ng/mL
concentration.

concentration (Table
More

during thus by reflecting

confirdirect

the

(see his

patient had

no detectable

TxB2
questioned

first
an

2),

about drug ingestion. Patient VIII-l was restudied after abstaining mation for prothrombin activation was obtained from all medication for at least I week. The patients with factor V specific radioimmunoassay2526 for the factor and X deficiency had moderate bleeding symptoms occasionally prothrombin fragment F,#{247}2 (kindly performed requiring factor replacement. Among the three patients with factor VII deficiency,
disorder, to have factor (VIl-l, VII 2, two factor 3) all VII antigen by of whom VII-l <5% an levels using had and obtained symptoms VII-2) of were 12 and recently a moderate reported on and all 1.7% 16, respectively), three of immunosorbent bleeding previously24 patients normal, neth Bauer F,
+ 2

increase

using a

Xa-mediated by Drs KenoneIn normal over

nmol/L

and

Robert

Rosenberg,

Boston).

(patients levels

subject,

was
(1.0

found
nmol/L)

to

increase
to a value

progressively
of 30.2

the
in the

(patients enzyme-linked

andcontrol

final values
ng/mL

value sample

(FPA

concentration,

1,784

ng/mL)

obtained PF4 8 11 of an of

assay before

method have been found to be 1%, 50%, respectively (Dr Douglas A. Triplett, Muncie,
nication). Two (Liquaemin Subjects normal subjects (aged Organon, 24 and West

IN,
28)

personal
were 5,000 of studied units Ni).

commubefore of

cessation of bleeding at 3.5 minutes. The average in the first three minutes were 246, 456, and (control values, was 404 (Fig I) increase In the first most
of

29.6 ng/mL

20.2
(Table

[SD]),

and

the for the was

and

average PF4 and period undetectable linearly

then rapidly

and
heparin Normal

I 5 minutes

after

intravenous
sodium,

administration

concentration
the curves

2). Visual

inspection

Orange,

exponential
sampling. during

suggests a linear increase for FPA throughout normal after

the bleeding

subjects, which
time

TxB2 TxB2
curve

Controls for the study were normal hospital personnel = (n 13), in the midportion aged 25 to 50, who denied recent ingestion of drugs known to affect the cessation platelet function or hemostasis. Bleeding time and volume studies near were performed on I 3 subjects (five males, eight females) and
radioimmunoassay
females).

minute,

increased

of bleeding.

studies

on

nine

subjects

(four

males,

five Coagulation To study early

Defects
further burst of the thrombin mechanisms activity and PF4 and that might account for its possible relation

RESULTS Normal Subjects

the

to platelet
patients values variable values (SEM) in normal bleeding for times for subjects blood are volume, addition shown values Average sequential
I Because

activation
with

(increasing

TxB2),

we studied defects. In

well-characterized

coagulation 2 to 4 that in patients their average

FPA,
in Fig

PF4, and TxB2 obtained

.

to the curves in Figs for FPA, PF4,and TxB2

we have also tabulated

show the sequential and controls (gray concentration

minutes), minutes
of

the

of the average

were obtained from a progressively the total number of subjects studied, on the noSEM assay are curves shown). are the average

times (3.0 to 8.0 area), greater than 3.0 during lesser percentage bleeding and the last two FPA. of two values

the

first The

three
(Table

minutes
2).

of bleeding in FPA
within

and content
the range

for the in all

of

entire factor
normal

period

initial
patients

increases
were

points which

(for VIII-deficient

The volume of blood increased during the first two minutes and then declined until cessation of bleeding. Both FPA and PF4 were consistently detectable in the earliest blood sam- ng/mL)

values during the first three minutes (Fig 2A), and was this true whether or not TxB2was detectable in the incisional blood. In addition, the average plasma concentration (689
was similar to that observed in controls (491 ng/

0.? 0

241

.-.
A-6

0-c

PF4 TxB2
Volume

20 6
-J Ui

5 4

o.-o

5 2 8
!

a-

10j 2 I

Fig 1. Concentration of FPA. PF, and TxB2 in bleeding time blood from normal subjects. Blood from bleeding time incisions was collected at 30-second intervals. Mean values SEM) ( are shown for the volume (n 1 3, average oftwo incisions for each subject) and for levels of FPA (ng/mL). PF4 (ng/mL). and TxB2 (pmol/mL) (n - 9. see Results for an explanation).

4
00

5
0
-L-

45

TIME (MIN)

From bloodjournal.hematologylibrary.org by guest on October 28, 2011. For personal use only.

632

WEISS

AND

LAGES

Tab Ic 2.

Average

Blood

Con contra

tions

and

Bleeding

Time Subjects

Values

Coagulation-Deficient
Normal VII

Heparin 3 Mean 1 2 VIII IX

Subjects

Substances Assayed A. First 3 mm

FPA(ng/mL) PF4(ng/mL) TxB2 (pmol/mL) 491 404 (220-773) 4

86 77 2

ii 160 5

13 254 6

21 241 1 6

82 400 6

89 315 4

22 64 318 46 2

47 133 3

63 456 3

689 (278-i 373 17

268 .734)
150

841
483

(202-825)

(108-934)
1

13

(1-16) B. Entire

(0-75)

period
1,617 827
(504-i 13

FPA(ng/mL) PF4(ng/mL)

347 122
.409)

40 399 44

110 417 17

1.437 1.096 10

198 539 10

277 492 15

637 400

95 456 15

66 1.022

1.008

203

1.001 558
23

(727-4,221)

(626-1.734) 791 181

534 143
(158-934)

TxB2(pmol/mL)

212

20 14
(0-75)

(4-27)

Hemostasis
Bleeding time (mm) 99 6.0 0.5 (3-8.0) Totalbboodloss

15
337

7 59

12.5 261 37

4 83

7.5

2 8

14.5 120

7 115

6 (3-9) 75

1 31

3.5 40

ii

127 68

(MI.)
Values are the average

(17-181)
values for the first six (kminute) samples (A). or for all the samples (B) until cessation

(25-186)
of bleeding. For groups where three or

more subjects
mL, Table

were studied
2). In

(normal. factor subjects

VII,

and

factor

VIII),

SEM )( and the range

(in parentheses)

of values re also shown. a

two

(VIII-2

and

VIII-4),

the

the

abnormality of in FPA

was production.

less

pronounced (The total venous blood as in normal PF4 production in one

subject

than

was

the PF4,

impairdeter-

bleeding time values either rapidly (patient

was at the upper declined (patient VIII-2)

FPA

limit of normal, and the FPA ment VIII-4) or did not rise as mined three
was

releasable

from
production

minutes
normal

to the
in the

tion
with
As

of bleeding.
factor
seen V

patient

termina- was the shown].)

extent withbut PF4

serum after clotting same in this patient Heparin reduced

factor values

with thrombin, subjects [data not (to the same subject

remained

IX
in

deficiency.
Fig 2B, FPA concentrations in patients

as

V deficiency)
in the other

normal
(H-2)

(H-i),
within

factor
out first the

or X

entire

deficiency period of (1 1 and

were strikingly bleeding (Fig I 3 ng/mL, were

generation

decreased through2B), nd the average a respectively) during

2% also

the normal range. patients with factor

the Fig TxB2. 4A,
Results are

PF4 VII

production deficiency.
in Fig

was
4 and

normal all in
Table

three
seen in

concentrations three minutes

shown

2. As

of bleeding

approximately
was

of the
abnormal

TxB2 to

production

was

decreased ingested (or

in
and

three
VIII-4)

factor

normal
in the strikingly
the range

value

(Table

2).FPA
with Each of
the

VIII-deficient

patients,
have It
agents.

two
recently was
VIII-2,

of whom(VIII-l

three patients so (Fig 2C). three

(Fig 2C),

factor VII deficiency ofthe six values obtained bleeding was

average 202 to

but less were known during flammatory

nonsteroidal antiinsomewhat increased) he abstained had from

patient normal with in the factor patients IX

normal

first

minutes
as were (normal

outside
825;

the
Table

normal of 2 I , 82,
2). In

when
these with

repeated
drugs), factor
TxB2

in patient
patient production

VIII-l
and was

(after
the also

concentrations

and

89 ng/mL

range,

all deficiency.

three subjects, portion of the

FPA

values

increased

rapidly

thein terminal

Vand

factor

X deficiency

(Fig

4B)

and

in two

of

bleeding time normal values. Administration two normal subjects markedly

expected (Fig 2D

and approached heparin of (5,000 inhibited FPA PF4

in

or
units)

achieved
to

the
ciency

three
(Fig

patients
4C). In

(VII-2
the

and
one

VII-3)
factor but

with

factor

VII
was

defi-

Vil-deficient
production

patient in the later but were of

production are
VIII

as (Vu-i)

and Table for

was

2). sequential
normal

normal
values in with was one shown
deficiency,

with a prolonged bleeding during the first 8.5 minutes

(9 to 2.5 I

time, TxB2

decreased

PF4.
Fig except (VIII-4), 3.

The
PF4 for a

findings
generation

insamples heparin,
in subject generally

factor

the

initial H-2 the

minutes). In rate of TxB2 curve decreased

normal subjects production was at during values the

receiving normal, that

moderately
was

delayed production
normal

subject factor IX normal

flattened

and

the in of PF4

patient production

somewhat

remainder

deficiency.

The

initial rate

in bleeding. Blood In
In

factor X deficiency (Fig the most part, near

contrast, patient

PF4
with

3B and Table 2) and remained, for the lower limit of normal values. generation was somewhat decreased in the V deficiency (Fig 3B and Table 2),

Loss
patients

After
with

Bleeding

Time

Incisions the were blood loss essentially

factor VIII

factor

but (volume)-v-time

curve

and the

deficiency, bleeding time

From bloodjournal.hematologylibrary.org by guest on October 28, 2011. For personal use only.

COAGULATION

MECHANISMS

IN

BLEEDING

WOUNDS

633

6500
4500

2000

.4
B

4000
I500 3000 I000 2000
=

VIU
.-

I 000
p.X

E
a

500

0
Ui

u0

0 2000 C

6500
aUi 0

c_
-

(-)

a-

4500 4000

ILU -J
LU

r
.7

U-

3000

a-

-J

I500

2000

I
HEPARIN

1000

000

1))
0 2 4 6 8 10 TIME 12 0 (MINI 2 4 6 8 10 12 4

500

VII

2468I0I2

024

I0I2I4

Fig 2. FPA concentration in bleeding time blood in subjects TIME (MINI with coagulation defects. (A) Factor IX (0) deficiency = 1) and (n factor VIII (#{149}) deficiency (n 4). Factor VIll-deficient patients in Fig 3. PF4 concentration in bleeding time blood in subjects whom no TxB3 was detectable (TxB2-negative. see Fig 4) are with coagulation defects. Same legend as in Fig 2. Blood samples distinguished (dashed lines) from those with measurable TxB2were obtained from the same incision that was used for FPA (TxB2-positive. solid lines). Patient VlII-1 was studied under both determination. conditions. (B) Factor V (#{149}) and X (0) deficiency. (C) Factor VII deficiency (n = 3). (D) Two normal subjects (H-i and H-2) 15 minutes after receiving 5.000 units of heparin. Subjects are strating an early increase in FPA levels of the same order of identified by number in Table . Values 1 obtained in normal magnitude as observed herein. In addition, in one normal subjects were within the area shown in gray. subject studied, the F,#{247}2fragment of prothrombin2526

increased

progressively

normal to nine
ingested VIII-2.
IX

except
minutes

fora slight
in

prolongation VIII-2 and an increase and volume

of the and

bleeding VIII-4 (who

time

patients time

indomethacin)

The
in Fig
loss

bleeding
These

in volume in patient were normal in factor in Table -time curves

factor one

providing more direct had incisional blood. To determine the

generating thrombin

in evidence

bleeding time for prothrombin

samples, thereby activation in

mechanism
in bleeding

that
time

might

be operative
we studied

in

incisions,

deficiency.

results are

seen
blood X

5 and
were These

Table
strikingly were subjects

summarized 2, the volume-v

abnormal abnormal (Fig 5). in

2. As patients with severe congenital deficiencies of coagulation and total factors. The normal generation of FPA that we observed V but not factor all patients with factor VIII deficiency during first three the
(VII-i) of the minutes

in

deficiency.

in only

of
fibrin

bleeding
three

factor

VIl-deficient

that time

can

be observed

is consistent with atthe periphery

after an incision

recent observations of the bleeding

in patients with

wounds

minutes

DISCUSSION The FPA, method described herein for sequentially measuring

hemophilia. normal in

The
factor

additional
IX abnormal

findings
delayed

that
in

FPA
factor

generation
VII defi-

is

deficiency,
with thrombin

PF4,

and

TxB2

levels

in blood

emerging

from

bleeding

ciency,

and markedly

in patients
a mechanism are

deficient
in which generated

in either
factor by initially

time
ing.

incisions platelets and

was developed to study the activation coagulation during the primary arrest

The

early
by

appearance
heparin

of FPA
strongly

in normal ubjects s
suggest that 60 both

of both factor V or X is consistent of bleed- Xa and, subsequently, and its direct tissue factor VII thrombin sic system) and not through

activation of factor

X (classic

extrinVIII
of other

suppression

either
pathway results

the
and

intrinsic
conclusions

pathway2829
factor

and fibrin production occur within 30 to incision and are consistent with observations cal

seconds the of from histologithe demon-

or
and

the

alternate Factor

tissue factor IX.3#{176}33 The

involving

examination of bleeding time wounds78 and with results of a recently published study27 independently

studies incisional

suggest that the wound may

type, strongly

depth, and influence

location of an the coagulation

From bloodjournal.hematologylibrary.org by guest on October 28, 2011. For personal use only.

634

WEISS

AND

LAGES

h/\,%/v

TIME

(MIN)

I
c,J

Fig 5. Blood loss after bleeding time incision. Left. patients with factor V (#{149}) X (0) deficiency; or right. patients with factor VII deficiency (S. Vll-1 ; U. Vll-2; A. Vll-3). The range of normal values (n = 13) were within the gray area.

z .
LU

,IXIIIt.JI

The

role the The to

of

thrombin

in

the

early

activation

of

platelets

0 0

and 70 60
50

permanent increase

arrest in TxB2

of

bleeding content for

also that

requires we and

further others2739 PF4

cc

.0

study. observed
(similar

I-

and the rapid that recently platelet

appearance reported may by

of both FPA and 13-thromboglobulin27) could be, in part, be the results although after factor with sole mechaobtained the V

suggest
HEPARIN

that

activation/secretion not the First, patients

40 30 .A\VIi 20
lO 0

__
0 2 4 60 82 14

thrombin
nism,

mediated. however, defects in

That this is suggested defects. production in the FPA and

in

subjects
severe rin

with coagulation

most and

were observed either (factor X),

hepa-

administration

factor
0 2 4 6 8 lO TIME 2 (MINI subject

X deficiency,
2), marginally

PF4 values were

reduced

normal (heparin or reduced to a 1). patients

for

lesser
addition,

extent
TxB2
and

than
X

FPA
production

(factor
was
One

V and
normal
possible

heparin atient p
in FPA of the from
than explanation

In with
these

Fig 4. TxB2 concentration in bleeding time blood in subjects factor V with coagulation disorders. Same legend as in Fig 2. BlOOd samples were obtained from a different incision from that used for FPA and findings PF4 determination. requires

deficiency.

is

that

the

formation
of

of

fibrinogen
is required

a higher

concentration

thrombin

pathway
our study

that
using

is activated,
a device

and
widely

this applies
used

to the results at present performfor

in

for TxB2 substances

because

production such
in

as

or the secretion PF4. This appears

in vitro

platelet unlikely,
this

granule however,
never

previous

studies#{176}

was

ing bleeding factor V1135

time
appears

tests.
to be

For

example,
for

factor
the arrest

VIIIM

but

necessary

of bleeding

notobserved. The conclusions to our findings: in areas

by collagen

of where

Kaplan collagen

et al could is exposed, to precede

be relevant platelet of evidence evidence from

from an incision into the case for bleeding

in these animals.36 In

the cuticle of dogs, and this may from incisions into mesenteric
contrast, a previous study in

also berelease arteries fibrin

which

might be expected

formation. the previous in vitro

may

Thus, our findings and the studies#{176} suggest that thrombin

dual arrest technique mechanisms of bleeding. reported and the might that re not a
example,

blood the ing

volumes present factor VII

(and study
wounds

hence suggested
was

wound that
activated

size) blood
by not

were

larger

coagulation
a mechanism IX.

in lagen in human during

requirit is

than

provide early the of

for

activating for

and colplatelets studying system in the vivo

the

bleeding

time

Finally, activation

here coagulation

and

factor

VIII but

factor

Thus,

platelets

highly likely that activation of coagulation is influenced by the shear rate3#{176} (which exceeds 1,000 s in arterioles and is
probably
personal

after bleeding abnormalities

vitro in

time wounds of hemostasis

For factor

be useful in detecting possible by current

abnormalities in tissue by this be detected

less

than

20

s
and

in

the
by

wound,
the

Dr

V.T.
of

Turitto, in
the

techniques. tissue

communication)

composition

tissue to emphasized

which that in

the blood is our conclusions to the where at of later time bleeding study.

exposed. concerning earliest normal periods, time

Finally, it thrombin

must for-

factor or be method.

expression

might

mation
events observed variation

apply
in all

particularly
hemostasis hemophilic the further

(first three FPA reason for in perhaps

minutes)
values the wider related of were
The authors wish ACKNOWLEDGMENT to thank Lorraine Rosanne Rosano, Rabinowitz, and Thomas Carol Kruger, for

patients. The

observed

to Yolanda

Hernandez,

Hoffman

the prolongation patients,9#{176} requires

a subset

theseexpert technical asistance and, for referral of patients, Triplett, Barry Coller, and Margaret Johnson.

Drs Douglas

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Hematol
normal

persons. I. The histological appearance of the plug 20 minutes 24 hours after the bleeding and its role in the capillary haemostasis.
Acta Pathol Microbiol Scand 57:40, 1963

and

Borchgrevink

From bloodjournal.hematologylibrary.org by guest on October 28, 2011. For personal use only.

COAGULATION

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