Differentiation of Certain Platelet Factors
Related to Blood Coagulation
By EHWIN DEUTSCH, M.D.,
SHIRLEY A. JOHNSON, P H . D . , AND WALTER H.
SEEGERS, P H . D . , S C . D .
Study of the platelets from the viewpoint of the blood coagulation mechanisms seems likely to
be helpful for increasing our knowledge of thrombosis, and bleeding tendencies in thrombasthenias
and thrombopathias. A logical approach to their study is the differentiation of their properties
and where possible to ascribe those properties to individual components. Platelet factors 1, 3 and 4
are considered from that viewpoint. The first accelerates the formation of thrombin in a manner
analogous to serum Ac-globulin. Factor 3 is required for the development of threone activity, and
factor 4 has antiheparin properties.
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
T
HE importance of platelets in several
phases of the blood coagulation mechanisms is now being generally recognized. In experiments by Patton, Ware and
Seegers1 blood was collected with special care
and centrifuged in silicone coated glass to
remove platelets. The plasma obtained in
this manner was allowed to stand in silicone
tubes for many hours and very little fibrin
formed, and practically none of the prothrombin changed to thrombin. The clotting mechanisms thus remained essentially static when
deprived of platelet materials. Mann, H u m
and Magath 2 found a restoration of the clotting
time of stored plasma if platelet suspensions
were added in addition to brain thromboplastin. Quick8 concluded that the amount of
thromboplastin supplied by platelets is very
small, and that platelets function by activation
of an inactive thromboplastinogen found in
plasma. This substance would correspond to
the antihemophilic factor and the mechanism
of its action is probably somewhat different
from the theory proposed by Quick. Ware,
Fahey and Seegers4 fractionated platelet extracts with ammonium sulfate and found two
different activities. One accelerated the conversion of purified prothrombin, in the presence of thromboplastin and calcium. Its action
was said to be similar to that of serum Acglobulin. It was called platelet factor 1 and
later also platelet accelerator or plateletAcG. The other showed fibrinoplastic activity
in hastening the interaction of thrombin and
fibrinogen in purified systems as well as in
oxalated plasma. This was designated platelet
factor 2. van Creveld and Paulssen5 confirmed
the existence of platelet factors 1 and 2, and
demonstrated another one designated as platelet factor 3. This factor improved prothrombin
consumption in platelet deficient plasma and
had antiheparin activity. These properties
were obtained in the phosphatide fraction of
the platelets. Jiirgens' placed platelet extracts
in the ultracentrifuge and found a fibrinoplastic and an antiheparin activity in the
supernatant, and iii the precipitate a thromboplastic and an antithromboplastic activity.
This paper is concerned primarily with
platelet factors 1 and 3. Their properties are
adequately described to distinguish one from
the other. Platelet factor 1 accelerates prothrombin activation in Ac-globulin deficient
stored plasma. It also functions together with
thromboplastin and calcium in the activation
of Ac-globulin free purified prothrombin.
Platelet factor 3 functions with platelet cofactor I, and plasma Ac-globulin in the activation of prothrombin. It may also function in
the same way with histamine and certain anti-
From the Department of Physiology and Pharmacology, Wayne University College of Medicine,
Detroit, Mich.
This investigation was supported by a research
grant H-1467 from The National Heart Institute,
National Institutes of Health, Public Health Service.
Funds for Research Fellowships in Physiology were
provided by Parke, DaviB and Company.
Received for publication Oct. 1, 1954.
110
Circulation ReKarck, VolumeIII, January 19SS
ERYVLV DEUTSCH, SHIRLEY A. JOHNSON, AND WALTER H. SEEGERS
histamine compounds. The antiheparin activity of plasma is associated with a substance
different from factor 3.
MATERIALS
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Purified Prothrombin was prepared by the method
of Seegers and associates7' '• • and was, when
necessary, freed of the last traces of Ac-globulin by
dissolving in water and heating at minimal ionic
concentration at 53 C for 2 hours.
Platelet Cofactor I was prepared by a modification
of the method of Lorand and Laki10. The residue
of the plasma after Mg(OH)i adsorption in the
above described method for the preparation of
purified prothrombin was used as stalling material.
It was brought to pH 5.9 with M/5 phosphate
buffer and the factor was adsorbed on kaolin. The
kaolin was washed once with M/20 borate buffer
and then eluted with 0.1 per cent ammonium
carbonate at room temperature. The eluate was
neutralized and fractionated with ammonium
sulfate. The precipitate between 35 and 50 per cent
saturation was dissolved in distilled water and
dialyzed against 0.1 per cent ammonium carbonate.
After freeze-drying this preparation is stable for a
long time. It restores the prothrombin utilization of
hemophilic blood to normal.
Platelet Sitspenmni. We were guided by the work
of Johnson, Smathere and Schneider11. Bovine
platelets were obtained by differential centrifugation. They were washed thoroughly. Then the
suspension was frozen, thawed, and washed again
four times with saline. The platelet particles were
suspended in saline and put through a mechanical
homogenizer three times. Then the product was
placed in a refrigerator at 4° C.
Stored Oxalated Human Plasma was prepared by
the method of Deutsch and Schaden". Its one-stage
prothrombin time was longer than 60 sec., and
could be shortened, by addition of BaC0| adsorbed
bovine serum (Ac-globulin source) to a lower
value than the normal value was before storage.
Antiheparin Activity. A simple qualitative test
was set up. Bovine oxalated plasma was defibrinated
by heating at 53° C for 3 min. Then a mixture of
0.3 ml. defibrinated plasma, plus 0.1 ml. fibrinogen
solution, plus 0.1 ml. test material, plus 0.1 ml.
thrombin was made. The thrombin was of such
strength that clotting occurred in 15 sec. If the
test material contained heparin the clotting time
became longer in proportion to the amount of
heparin it contained. Consequently if the test
material previously had its heparin activity reduced,
for example by platelet materials, the clotting time
tended in the direction of 15 sec. from longer
clotting times.
Crude Lung Extract Tkromboplaslin was prepared
according to McClaughry and Seegers11.
Thrombin determination after Seegers and Smith14.
111
EXPERIMENTAL RESULTS
Platelet Factor 1 of Platelet Suspension and
Stored Human Plasma. After aging of plasma
its one stage prothrombin time was longer
than 60 seconds, and was shortened to 14
seconds with BaCOj adsorbed with serum.
The latter serves as a source of serum Acglobulin. With addition of fresh platelet suspensions to aged plasma the clotting time was
reduced to 25-30 seconds. This property of
the platelet suspensions was retained after
two weeks of storage. Sometimes the accelerating effect was most effective on the second or
third day of storage. In their study Mann,
Hum and Magath2 were the first to point out
that platelets can restore the deficiency found
in stored plasma and record a prothrombin
time of 13 seconds. That represented complete
restoration of the prothrombin time in their
experiments. In our work we were only occasionally able to get near a complete restoration
of the prothrombin time by testing the platelet
extracts after 2 or 3 days of storage when their
accelerating activity was at its peak. In the
experiments of Mann, Hum and Magath1
the platelets were not washed and might have
been contaminated with appreciable amounts
of plasma Ac-globulin. Despite these remarks
about their technical details we are in general
agreement with their conclusions, and add the
observation that the accelerator factor or
factor 1 is quite stable upon storage. We also
found that factor 1 activity is in the supernatant solution after low speed centrifugation
of platelet suspensions. By centrifuging for one
hour at 100,000 g. it is sedimented and can
completely be recovered by resuspending the
sediment in saline.
Platelet Factor 1 of Platelet Suspensiori and
the Activation of Purified Prothrombin. For
these experiments the purified prothrombin
was heated to remove the last traces of plasma
Ac-globulin. Then calcium + platelet suspension + lung thromboplastin yielded thrombin
rapidly (fig. 1). Next it was found that calcium
+ platelet suspension + rabbit braia thromboplastin yielded thrombin less rapidly and
there •\vas not a full yield. The results were,
however, very dependent upon the way in
which the brain thromboplastin was prepared.
112
800
PLATELET FACTORS AND BLOOD COAGULATION
LUNG
Thromboplastin
• Freth Platelets
° Six-day-old Platelets
o "
«
" Supernatant
BRAIN
Thromboplastin
Storage of Platelet Suspension and Platelet
Factor 3 Activity. When the platelet suspension
was stored in the refrigerator platelet factor 3
activity began to decrease on the third day of
storage in the refrigerator (fig. 2) and disappeared almost completely after 6 to 8 days.
As indicated above the activity of platelet
factor 1 was not significantly changed under
the same conditions. Factor 3 activity was
associated with the platelet particles, since the
supernatant obtained by light eentrifugation
of the platelet suspensions had only a very
small amount of activity. Whether any
is found in the supernatant is very much de-
20
40
60
80
100
PROTHROMBIN ACTIVATION TIME In minutes
. FIG. 1. Activation of Ac-globulin free purified prothrombin. With lung thromboplastin and platelet
factor 1 as found in fresh platelets, in 6 day old platelets, and in 6 day old supernatant. With brain
thromboplastin and platelet factor 1 as found in
fresh platelets, in 6 day old platelets, and in 6 day
old supernatant. The controls were with brain thromboplastin or lung thromboplaatin and calcium alone.
Platelet factor 1 functions with either lung extract
thromboplastin or with brain thromboplastin. It is
stable. It substitutes for Ac-globulin.
For example: crude brain thromboplastin was
obtained from rabbit brain according to
Quick16. Two different extracts were then
made. The dried brain powder was suspended
in physiological saline solution, homogenized
and used with the particles in it. This extract
gave the results recorded on fig. 1. In another
experiment 0.5 gm. brain powder were extracted with 5 ml. saline 30 minutes at 37 C.
The extract was centrifuged at a low speed for
5 minutes to remove the particles. Together
with platelet suspension this extract almost
equals that of lung thromboplastin. It seems
likely, therefore, that platelet factor 1 can
substitute for Ac-globulin under appropriate
conditions.
A good activation rate could be obtained
not only with fresh platelet suspensions but
also with those stored up to 6 to 9 days.
Moreover, the activity remained in the supernatant solutions upon light eentrifugation of
IUW
Lung Extract+Platelets
900 ~
800
f ^
Fresh Platelet Preparation
-/
f'
T
/
4Day Old Platelet Prep.*
700
bin units per ml.
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Qro\no Thromboplastin)
fresh or stored platelet suspensions. Thus we
may conclude that platelet factor 1 is relatively stable at 4 C.
1
5 400
I
-
/
/
6 Day Old Platelet Prep.*
if/*"
/ / /
10 Day Old Platelet Prep.*
300
200
100
if yS
\1 Mp^ /A
* With Bovine Plasma
10
20
30
40
50
60
Prothrombin activation time in minutes
Fio. 2. Effect of storage on platelot factor 3. Rate
of activation of purified prothrombin (last traces of
Ac-globulin not removed) with bovine plasma plus
fresh platelet preparations or plus 4, 6 or 10 day
old platelet preparations. The defibrinated bovine
plasma served as source of platelet cofactor I. For
that purpose it was ether treated to reduce antithrombin-III concentration and diluted 12 times. For
control purposes the rate of activation of purified
prothrombin with lung extract calcium and platelets
is shown. Platelet factor 3 is not stable.
ERWIN DEUTSCH, SHIRLEY A. JOHNSON, AND WALTER H. SEEGERS
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
pendent upon the speed of centrifugation.
Even centrifugation at 2000 g. reduces its
concentration greatly.
Platelet Factor S Functions with Linadryl.
Murray, Johnson and Seegers16 discovered that
many purified prothrombin preparations activate with calcium, Ac-globulin, platelets and
certain antihistamine compounds. The activation takes place rapidly if the reaction is
started with a small amount of thrombin.
We confirmed their observations and tested
various platelet preparations in combination
with Linadryl (4-)2-(benzhydryloxy)ethylmorpholine). The purpose was to see whether
the factor in platelets which functions with
Linadryl is platelet factor 3 or another factor.
We found a fast rate of activation and a maximum yield of thrombin, under the conditions
of the test, with platelet suspensions. Upon
light centrifugation of platelet suspensions
it was found that the activity remained with
the particles and not so much with the supernatant. In this respect, the results followed
the same pattern with Linadryl as with the
respective platelet preparations and platelet
cofactor I of plasma.
Importance of Thrombin. It can be shown
that thrombin is important for the activation
of prothrombin with, platelet factor 3, plus
diluted plasma. To demonstrate this (fig. 3) a
large amount of prothrombin was used as a
substrate. As source of platelet cofactor I
plasma (defibrinated, and ether treated to
reduce antithrombin-III) was diluted 1:12.
Six day old platelet suspensions were used as
a source of platelet factor 3. Without adding
thrombin only 600 units of thrombin were
obtained. With 8 units/ml, of thrombin added
to start the interactions the thrombin yield
was 1800 units. This yield is 100 per cent in
terms of two-stage prothrombin analysis and
equals the yield obtained when thrombin
and Linadryl, serve as activators.
Antiheparin Activity of Platelet Preparations.
With the use of methods described above an
antiheparin activity could be detected in the
platelet suspensions, and in the supernatant
after light centrifugation of platelet suspensions. When platelet suspensions are washed
much antiheparin activity is found in the
113
1800
1600
-
1400
• Plotelets + LINADRYL + Thrombin
o Platelets + PLASMA + Thrombin
Platelets + PLASMA
Platelets + LINADRYL
20
40
60
80
Prothrombin Activation Time in Minutes
100
FIG. 3. The effect of thronibin on platelet factor
3 and Linadryl or on platelet factor 3 and platelet cofactor I. The top curve was obtained with
thrombin (8 units/ml.) and the lower curves without thrombin. In all cases platelet factor 3 was supplied in the form of 6 day old platelet suspension.
Platelet cofactor I activity was supplied by defibrinated bovine plasma. This plasma was ether
treated to remove antithrombin-III and diluted 12
times. The purified prothrombin used as substrate
was not heated to remove the last traces of Ac-globulin. The results show that thrombin increases the
rate of the reactions and yield of thrombin when
Linadryl was used and also when plasma was used as
a source of platelet cofactor I.
first wash solutions. The 8 day old platelet
suspension still showed a fairly high activity,
but after 14 days of storage the activity had
disappeared. The distribution of the antiheparin activity does not parallel that of the other
two factors, and particularly not that of factor
3. It can be considered that the antiheparin
activity is not associated with factor 3, and
that it is a separate factor.
114
PLATELET FACTORS AND BLOOD COAGULATION
DISCUSSION
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
The platelets are of great importance for the
coagulation of blood. They have the potential
of function in the clotting process at different
points: They participate in the activation of
prothrombin, when in combination with
platelet cofactor I or with the plasma thromboplastin component (PTC)17> 18> 19; they accelerate the activation of prothrombin and
the interaction of thrombin and fibrinogen,
they inhibit heparin and fibrinolysin21 and
contain a substance that can be clotted with
thrombin. Moreover, they are necessary for
clot retraction, and exert a vasoconstrictor
action. There is as yet not enough information
to use as a basis for defining all these properties
in terms of different substances, but it is evident that the factor 1 and 3 functions are
associated with separate platelet substances.
Therefore, it seems convenient to speak about
these in terms of definite platelet factors.
Platelet factor 1 is much more stable when
stored at 4 C than is factor 3. The latter has
largely lost its activity after 6 to 8 days, while
at that time the platelet suspensions still have
a high factor 1 activity. Platelet factor 1 is
very easily extracted with water from a platelet homogenate, while factor 3 activity remains
with the platelet particles. A small amount of
factor 3 activity which may sometimes be
found in these extracts, is easily sedimented
at 2000 to 3000 g. Factor 3 is associated with a
phosphatide fraction of the platelets. Factor 1
can largely correct the one-stage prothrombin
time of aged oxalated human plasma and
would, therefore, represent the factor, whose
activity Mann, Hum and Magath were
measuring. In that respect it functions largely
like Ac-globulin. From the kinetics of prothrombin activation Ware, Fahey and Seegers
observed that the activity corresponds more
nearly to that of serum Ac-globulin than
plasma Ac-globulin. Our work amplifies these
findings. Platelet factor 1 activates Ac-globulin
free purified prothrombin in conjunction with
lung or certain brain extract thromboplastins.
This may be indicated as follows:
Ca+ +
Thromboplastin
Platelet factor 1
Prothrombin
Thrombin
For these reasons factor 1 can also be referred
to as platelet accelerator, or platelet Ac-G.
The chemical differences between Ac-globulin
(a plasma factor) and platelet factor 1 are so
marked that they can hardly be one and the
same chemical substance.
It is evident that these observations may be
used for the development of quantitative assay
procedures. In fact two possibilities are presented. The activity of platelet factor 1 may
be tested on stored oxalated human plasma or
by its ability to activate purified prothrombin
with lung or brain thromboplastin and calcium.
Factor 3 and platelet cofactor I together
are able to substitute for tissue thromboplastin
in the activation of purified prothrombin in
the presence of Ac-globulin and calcium.
Following the suggestion of Murray, Johnson
and Seegers18 the activity represented by the
combined action of platelet factor 3 and platelet cofactor I is called threone activity. It is not
proposed that platelet factor 3 and platelet
cofactor I of plasma combine to form a new
compound. The importance of thrombin for
threone activity is ascribed to details related
to the change of plasma Ac-globulin to serum
Ac-globulin. These concepts may be represented by the following equations:
Thrombin
(1) Plasma Ac-globulin
> Serum Acglobulin
(2) Platelet Factor 3 + Platelet Cofactor I =
Threone
C+-IThreone
Serum Ac-G
(3) Prothrombin
• Thrombin
In considering equation 3 above from the
viewpoint of the quantitative determination
of either platelet factor 3 or platelet cofactor I
the following rule applies: When cofactor I
is in excess limited amounts of platelet factor 3
yield limited amounts of thrombin and thus
the quantity of thrombin formed may be
taken as a measure of the quantity of platelet
factor 3 (cf. fig. 2); and, conversely as observed by Johnson, Smathers and Schneider11,
when platelet factor 3 is in excess limited
amounts of platelet cofactor I yield limited
amounts of thrombin. Thus the quantity of
thrombin formed may be taken as a measure
ETtWIN DEUTSCH, SHIRLEY A. JOHNSON, AND WALTER H. SEEGERS
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
of the quantity of platelet cofactor I. Any
platelet cofactor II would first be removed
from plasma by adsorption on BaSO< or BaCO3.
Jiirgens9' 50 has recently come to the conclusion that the antiheparin activity of platelets can be ascribed to a factor distinctly
different from factor 3. This distinction was
not made in the work of van Creveld and Paulssen6, but could be confirmed in several experiments performed by us. For example, the
first wash solution of platelet suspension has a
very high antiheparin activity (factor 4),
while the third and fourth wash solutions are
without activity. After high speed centrifugation of the first wash solution the total antiheparin activity is obtained in the lower layer,
which shows practically no factor 3 activity.
The latter is found in the sediment.
SUMMARY
Platelet factor 1 accelerates the activation
of Ac-globulin free prothrombin preparations
in the presence of thromboplastin and calcium.
It remains stable when platelet "extracts"
are stored at 4 C, is extracted from the platelet particles with water, and is easily sedimented by ultracentrifugation. It can almost
restore the prothrombin time of oxalated stored
human plasma to normal.
Platelet factor 3 functions with platelet
cofactor I to give threone activity, and the
latter can replace tissue thromboplastin in
the activation of purified prothrombin. It is
not extracted with water, remains in the platelet fragments and is easily sedimented by centrifugation at medium speed. With storage this
factor is not stable. It can function together
with certain antihistamine compounds in the
activation of purified prothrombin.
Platelet factor 4 has antiheparin activity.
It is found in the bottom layer after high
speed centrifugation of platelet suspensions.
REFERENCES
1
PATTON, T. B., WAEE, A. G., AND SEEGERS, W. H.:
Clotting of plasma and silicone surfaces.
Blood 3: 656, 1948.
S
MANN, F. D., HURN, M., AND MAGATH, T. B.:
Observations on the conversion of prothrombin
to thrombin. Proc. Soc. Exper. Biol. & Med.
66:
33, 1947.
3
QUICK, A. J.: Studies on the enigma of the hemostutic dysfunction of hemophilia. Am. J. M.
Sc. 214: 272, 1947.
115
* WARE, A. G., FAHEY, J. L., AND SEEGERS, W. H.:
Platelet extracts, fibrin formation and interaction of purified prothrombin and thromboplastin. Am. J. Physiol. 164: 140, 194S.
'VAN CREVELD, S., AND PAULSSEN, M. M.
P.:
Isolation and properties of the third clotting
factor in blood platelets. Lancet. 1: 23, 1952.
•JURGENS, R.: Die Blutplattchen und ihre Bedeutung fur Blutungsneigung und Thrombusbildung. Deutsche med. Wclmsehr. 77:
1265, 1952.
7
SEEGERS, W. H.: The purification of prothrombin.
Record of Chem. Progress. 13: 143, 1952.
8
— , LOOMIS, E. C ,
AND VANDENBELT, J.
M.:
Preparation of prothrombin products: Isolation of prothrombin and its properties.
Arch. Biochem. 6: S5, 1945.
•WARE, A. G., AND SEEGERS, W. H.: Studies on
prothrombin: Purification, inactivation with
thrombin, and activation with thromboplastin
and calcium. J. Biol. Chem. 174: 565, 1948.
10
LORAND, L., AND LAKI, K.: A simple method for
purifying an activator of prothrombin (antihemophilic factor?). Biochim. et biophys.
acta 13: 44S, 1954.
"JOHNSON, S.
SCHNEIDER,
A.,
SMATHERS,
W.
M.,
AND
C. L.: Platelets and their plasma
cofactor activity in the activation of purified
prothrombin. Amer. J. Physiol. 170: 631, 1952.
11
DEUTSCH, E., AND SCHADEN, W.: Zur Reinigung
und Charakterisierung des VII Blutgerinnungsfoktors. Biochem. Ztschr. 324: 266, 1953.
a
MCCLAUGHRY, R.
I.,
AND SEEGERS, W.
H.:
Prothrombin, thromboplastin, Ac-globulin and
platelet accelerator: Quantitative interrelationships. Blood 5: 303, 1950.
14
SEEGERS, W. H., AND SMITH, H. P.: Factors which
influence the activity of purified thrombin.
Am. J. Physiol. 137: 3S4, 1942.
"QUICK, A. J.: The thromboplastin reagent for the
determination of prothrombin. Science 92:
113, 1940.
"MURRAY, M., JOHNSON, S. A., AND SEEGERS,
W. H.: The relationship of certain antihistamine drugs to the activation of purified
prothrombin. Am. J. Physiol. 178: 10, 1954.
17
AGGELER, P. M., WHITE, S. G., GLENDENING,
M. B., PAGE, E. W., LEAK, T. B., AND BATES,
G.: Plasma thromboplastin component (PTC)
deficiency: new disease resembling hemophilia.
Proc. Soc. Exper. Biol. & Med. 79: 692, 1952.
18
BIGGS, R., DOUGLAS, A. S., AND MACFARLANE,
R. G.: The formation of thromboplastin in
human blood. J. Physiol. 119: S9, 1953.
19
— AND —: The initial stages of blood coagulation.
J. Physiol. 122: 53S, 1953.
M
JUROENS, R.: Pharmakologische Beeinflussung der
Blutgerinnung. Archiv. axper. Path. u.
Pharmakol. 222: 107, 1954.
11
JOHNSON, S. A., AND SCHNEIDER, C. L.:
The
existence of antifibrinolysin activity in platelets.
Science 117: 229, 1953.
Differentiation of Certain Platelet Factors Related to Blood Coagulation
ERWIN DEUTSCH, SHIRLEY A. JOHNSON and WALTER H. SEEGERS
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Circ Res. 1955;3:110-115
doi: 10.1161/01.RES.3.1.110
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1955 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/3/1/110
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/