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Inhibition of Intravascular Platelet Aggregation by Endothelium-Derived Relaxing
Factor: Reversal by Red Blood Cells
By Donald S. Houston, Pauline Robinson, and Jon M. Gerrard
Studies w e r e performed to determine whether endothelium-derived relaxing factor (EDRF) can inhibit platelet
aggregation within t h e vascular lumen. and if so. w h e t h e r
t h e inhibition persists in t h e presence of red blood cells
(RBCs). Canine femoral arteries mounted in a n organ bath
were perfused with physiologic saline solution to which
acetylsalicylic acid was added to block prostacyclin formation. During contraction with phenylephrine, addition of
acetylcholine to t h e perfusing solution to evoke EDRF
release relaxed t h e vessel wall. Washed human platelets
labeled with ''C-5-hydroxytryptamine w e r e added to t h e
perfusing solution, and activated by thrombin infused via a
branch vessel. T h e perfusate was collected downstream
and centrifuged; t h e fraction of '4C-5-hydroxytryptamine
appearing in t h e supernatant reflected t h e degree of
platelet activation. Stimulation of EDRF release with acetylcholine inhibited 14C-5-hydroxytryptamine release. Hemoglobin (Hb) (IO-' mol/L) blocked vascular relaxation and
platelet-inhibition. RBCs at a hematocrit of 10% (treated
with echothiophate to block erythrocyte cholinesterase)
did not prevent relaxation but reversed t h e platelet inhibition. Lower hematocrits did not completely block t h e
inhibition. Thus, erythrocyte Hb may modulate t h e inhibition of intraluminal platelet aggregation by EDRF.
E
land, OH) for measurement of isometric tension. At the beginning of
the experiment the vessel was stretched gradually to a resting tension
Of 4 g.
Platelet preparation. Blood was obtained by venupuncture from
healthy volunteers who had taken no aspirin or other drugs within
the preceding week. Blood, 48.6 mL, was collected into 11.4 mL of
citrate anticoagulant, and promptly centrifuged at 200g for 25
minutes at room temperature. The platelet-rich plasma thus obtained was incubated with ''C-5-hydroxytryptamine (740 Bq/mL)
for 30 minutes, then centrifuged at 800g for 10 minutes. The pellet
thus obtained was resuspended in wash solution (composition below)
to a volume of 25 mL. This platelet suspension was then centrifuged
at 800g for 30 seconds to sediment out contaminating RBCs and
white blood cells, and the platelet suspension decanted. Just before
use, aliquots of this platelet suspension were taken, one-fifth volume
of citrate anticoagulant added, and then centrifuged at 800g for 10
minutes; the pellet thus obtained was resuspended in 4 mL of the
perfusing solution drawn from the reservoir chamber currently
supplying the vessel (ie, gassed and maintained at 37OC, and
containing drugs, Hb, or RBCs).
RBC preparation. The packed RBCs obtained as above were
suspended in 3 vol of wash solution. The acetylcholinesterase
inhibitor, echothiophate; was added to a final concentration of
mol/L and incubated for 30 minutes. The cells were then centrifuged
at 800g for 10 minutes, and washed by resuspension in wash solution
and repeat centrifugation. The packed cells thus obtained were
diluted to the desired hematocrit in physiologic saline solution with 1
g/L albumin.
Drugs and solutions. Except as noted, drugs were dissolved in
distilled water and diluted in physiologic saline to the appropriate
concentrations. Acetylcholine chloride, acetylsalicylic acid (dissolved in 50 mmol/L Tris HCl), bovine albumin (fatty acid free),
NDOTHELIAL CELLS can release a nonprostanoid
vasodilator, known as endothelium-derived relaxing
factor (EDRF), in response to a variety of stimuli such as
acetylcholine and bradykinin.'.' A product of endothelial
cells, seemingly identical to EDRF, can also inhibit the
aggregation of platelets, as studied in ~ i t r o . ~Whether
.~
EDRF inhibits platelets in vivo is less certain. Free hemoglobin (Hb) can inhibit endothelium-dependent relaxation,
apparently by binding EDRF.' Hb contained within red
blood cells (RBCs) might also bind EDRF. Therefore, the
current experiments were undertaken to determine whether
EDRF released intraluminally in a perfused artery can
inhibit activation of platelets flowing through that vessel, and
whether RBCs can interfere with this inhibitory effect.
MATERIALS AND METHODS
Tissue preparation. Femoral arteries were obtained from mongrel dogs of either sex, 15 to 30 kg, anesthetized with sodium
pentobarbital (30 mg/kg intravenously [IV]), given heparin 5,000 U
IV, and exsanguinated. The entire vessel from abdominal wall to
popliteal region was removed carefully. The artery was placed in
cold physiologic saline solution (composition below) and cleaned of
loose adherent connective tissue. One side branch was selected about
2 cm from the proximal end, and was cannulated for infusion of
thrombin. All remaining side branches were ligated. At a branchfree site in the distal half of the vessel, two fine wire hooks were
pierced through the vessel wall on opposite sides (see Fig 1).
The vessel was placed in a chamber containing 600 mL of
physiologic saline solution, maintained at 37OC by immersion in a
heating bath and gassed with 10%0 , / 5 % C0,/85% N,. Each end of
the vessel was cannulated; the proximal end was connected to a roller
pump that perfused the vessel at a rate of 2 mL/min. The roller
pump drew from a reservoir of physiologic saline solution with 1 g/L
bovine serum albumin (also maintained at 37OC and gassed with
10% 0 , / 5 % C 0 2 / 8 5 %N2),to which drugs, Hb, or RBCs could be
added. The pump could be switched to draw from a syringe
containing platelets suspended in the same perfusing solution. The
effluent was collected after a 2-minute delay downstream from the
vessel. Bovine thrombin (2.5 U/mL in physiologic saline solution)
could be infused through the cannulated side branch at a variable
rate corresponding to about 0.25 to 1 U/mL after dilution in the
perfusate flow.
One of the hooks placed through the vessel wall was connected to a
clip anchored on the bottom of the chamber, while the other was
connected to a force transducer (model UTC3; Gould Inc, Cleve-
Blood, Vol 76, No 5 (September 1). 1990: pp 953-958
0 1990by The American Society of Hematology.
From the Departments of Medicine and Pediatrics, University of
Manitoba, Winnipeg, Canada.
Submitted August 14, 1989; accepted May 15,1990.
Supported by an award from the Sellers Foundation.
Address reprint requests to Jon M. Gerrard. MD, PhD. Manitoba
Institute of Cell Biology, 100 OIivia St. Winnipeg, Manitoba R3E
OV9, Canada.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7605-0025$3.00/0
953
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954
HOUSTON. ROBINSON, AND GERRARD
ForceTransducer
F i g l . &hunotkdpuiu.ion oppmrotur. Flow is from
right to left in this d i n g " . Tho
reservoir of ohwiolook soline
4 I C 4IN2
bovine Hb. and phenylephrine hydrochloride w m obtained from
Sigma Chemical Co (SI Louis, MO). Bovine thrombin (Thrombostat) was obtained from Parke-Davis (Scarborough. Ontario,
Canada). Fxhothiophate iodide (Ayerst) was dissolved in the s u p
plied diluent and stored refrigerated in this stock solution. I2-I'Cl-Shydroxytryptamine binoxalate (2.2 GBq/mmol; 3.7 MBq/mL) was
obtained from New England Nuclear (Boston MA).
W i n e H b (which is largely in the o x i d i d or methemoglobin
form) was prepared in the reduced form after the method of Martin
et al.' A I O ' mol/L solution of H b was prepared fresh daily in 5 mL
distilled water. and a 40-fold molar excess of sodium dithionite
(NaS?O,) added. To remove the dithionite. the solution was
dialy7ed in 2 L distilled water with .0019 EDTA at 4% for 2 hours.
For 30 minutes before and throughout the dialysis. the water was
gasscd with N,. The content of reduced H b thus obtained (55% to
80% of the total) was determined indirectly by spectrophotometry
(Reckman DU-R) a t 632 nm. The amount of H b used was adjusted
accordingly.
Physiologic saline solution contained the following (except as
noted, all concentrations given in millimolar): NaCl I 18.3. KCI 4.7,
CaCI, I .2. MgSO, I .2. KH:PO, 1.2. NaHCO, 25. Na,EDTA 0.026.
and glucose 1 I . I; pH was adjusted to 7.4 after gassing with 5% CO,.
When used for perfusion. I g/L bovine albumin was added. Wash
solution (modified Hanks' balanced salt solution) contained: NaCl
137. KCI 5.36. KH:PO, 0.441. Na,HPO, 0.177. NaHCO, 3.35.
MgCI, 0.5. glucose 10. and I g/L bovine albumin; pH was adjusted
to 7.35. Citrate anticoagulant contained: trisodium citrate 85. citric
acid 65. glucose I1I. Glutaraldehyde was diluted to 0.1% (vol/vol)
in White's saline (NaCI 120. KCI 5. MgSO, 2.3, Ca [NO,]2 3.2.
NaHCO, 6.5. Na,HPO, 0.41. KH,PO, 0.19. Phenol Red 0.014;pH
7.4).
A~~tcgomcrry
studies. Platelets prepared a s above and suspended in physiologic saline solution with 1 g / L bovine albumin w m
studied in a light-transmission aggregometer (Payton Associates.
Scarborough. Ontario. Canada). Aggregometry was performed at
37% in I-mLsiliconi7. glasscuvettes. with magnetic stirring of the
platelets. The baxline of the recording p n was set using the platelet
suspension. and 100% aggregation was sct using the physiologic
solution im oiso' bubbled with
the gar mixture (not rhownl.
S w Met.riolm end Methods for
detailed OxpkMtion.
saline solution. Light transmission was measured and percent
aggregation recorded at 2 minutes after addition of bovine thrombin
(0.25 U/mL). Aggregation was stopped 3 minutes after addition of
the agonist by addition of an equal volume of 0.1% glutaraldehyde.
In ulxs where RRCs were incorporated in the suspension. the
volume of physiologic saline solution was decreased accordingly so
that the number of platelets was constant. Rocause of their opacity.
when RBCs were present light transmission could not be recorded.
After aggregation was stopped. the samples were centrifuged a t
SOOR for 10 minutes. Supernatant. 0.5 mL. was removed and 3.0 mL
of scintillation fluid (Ecolite. ICY. Irvine. CA) added for scintillation counting. The pellet was suspended in 0.5 mL Tris HCI with IS
scdium dodccylsulphate and counted in the same way. 5-Hydroxytryptamine release was expmscd a s the number of counts in the
supernatant divided by the total counts (pellet plus supcmatant). In
cases where the pcllet contained RRCs. it was resuspended in 20 mL
of physiologic saline solution to dilute the RBCs, which would
otherwise interfere with the scintillation counting. An aliquot of 0.5
mL of this suspension was taken for counting; the total counts thus
obtained were only slightly lcss than thoze obtained when no RRCs
were included. indicating that there was no substantial quenching
due to t he R RCs.
Perfusion studies. After mounting in the tissue chamber, the
arteries were perfuscd with physiologic saline solution with I g/L
bovine albumin. Acetylsalicylic acid (IO-' mol/L) was added to the
perfusion fluid for the initial 30 minutes to block endothelial
production of pmtacyclin. The vessels were then contracted by
adding phenylephrine (3 x IO 'to I O ' mol/L) to the bath solution
surrounding the vercel; the vesscl remained in contact with phenylephrine for the remainder of the experiment. After a stable contraction had been obtained. ptaence of functional endothelium was
demonstrated in each case by adding acetylcholine (10 'mol/L) to
the perfusion fluid and observing a relaxation in response to this
endothelium-dependent vasodilator (Fig 2). Thrombin was then
infused via the side branch at an initial rate of I U/mL (expressed as
the final concentration after dilution in the perfusing stream). The
response to thrombin was variable. but was consistently tachyphylactic; ie. when relaxation occurred. the tension would spontaneously
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955
EDRF, PLATELETS, AND ERMHROCMES
Acetylcholine
Phenylephrine 0
Acetylcholine
Thrombin
0
Hemoglobin
Acetylcholine
lg
t
2min
Fig 2. Isometric tension recording from a representative
perfusion experiment. Phenylephrine (3 x lo-’ mol/L) is added to
the bath surrounding the vessel; it remains in contact with the
vessel wall for the remainder of the experiment. Acetylcholine
(lo-’ mol/L) is added to the perfusing solution: “Wash” denotes
the change back to drug-free perfusing solution. Thrombin (1
U/mL) is infused via a proximal side branch, which continues
throughout the remainder of the experiment (the dose is reduced
to 0.25 U/mL after the second wash). Note that whereas the
responseto thrombin is tachyphylactic, the responseto acetylcholine is not. The heavy double bars represent elision of time. Hb
(lo-’ mol/L) is added to the perfusing solution, and acetylcholine
(lo-‘ mol/L) is added to this mixture once the response to Hb is
stable.
return to its previous level after several minutes despite ccntinued
infusion of thrombin. The infusion rate of thrombin was then
decreased to 0.25 U/mL and continued at that rate throughout the
remainder of the experiment, except when platelets were studied
without an aggregatory stimulus. Relaxations to acetylcholine, on
the other hand, were not tachyphylactic, even after tachyphylaxis of
the response to thrombin.
When RBCs, acetylcholine, or Hb were added to the perfusing
solution, they were allowed to infuse long enough for the contractile
or relaxant response to stabilize before platelets were infused.
Platelets (suspended in 4 mL of the perfusion solution) were
infused for 2-minute periods. The effluent was collected into an equal
volume of iced 0.1% glutaraldehyde to stop aggregation. The
samples collected were processed for scintillation counting as described above under “Aggregometry” to determine fractional 5-hydroxytryptamine release.
Statistical analysis. All results are expressed as mean t SE.
Statistical testing was by two-way analysis of variance, using a
random-block design to account for repeated measurements.Specific
intergroup comparisons were made by Scheffe’s test.’
RESULTS
Aggregometry studies. Thrombin (0.25 U/mL) was an
effective agonist of platelet aggregation, as demonstrated by
light transmission aggregometry or by the release of I4C-5hydroxytryptamine measured simultaneously (Table 1). Neither aggregation nor 5-hydroxytryptamine release from platelets was inhibited by acetylcholine
mol/L) (n = 5).
Hb
mol/L) likewise did not affect aggregation or
release (n = 4), nor did the combination of H b plus acetylcholine. RBCs added to the platelet suspension with hematocrits from l% to 25%did not allow light transmission to be
recorded, but platelet activation as judged by 5-hydroxytryptamine release was inhibited slightly (by 18.7% with a
hematocrit of 1076, n = 7). This may have been due to
interference with stirring, which would not occur in the
perfused vessel. RBCs treated with echothiophate did not act
differently from untreated RBCs in this regard.
Perfusion studies. When thrombin was infused via the
cannulated branch vessel, platelet activation occurred as
shown by the release of I4C-5-hydroxytryptamine (70% to
80%). However, when no thrombin was infused as a platelet
agonist, the degree of platelet activation on transit through
the perfused artery was minimal (I4C-5-hydroxytryptamine
release: 8.3% f 2.4%,n = 5).
When acetylcholine was added to the perfusion solution,
prompt relaxations of the vascular smooth muscle ensued.
Additionally, platelet activation was significantly inhibited
(‘4C-5-hydroxytryptamine release reduced by 17.5% % 3.7%
of the response to thrombin, n = 20 total experiments). This
inhibitory effect was consistent and repeatable over the
duration of an experiment for a given vessel (Fig 3).
When Hb (lo-’ mol/L) was added to the perfusion
solution, a modest contraction of the vascular smooth muscle
would usually occur. When acetylcholine was added in the
presence of Hb, the relaxation evoked by acetylcholine was
prevented (Fig 2). H b caused no change in the degree of
platelet activation by thrombin. However, H b did abolish the
inhibitory effect of acetylcholine on platelet activation
(P< .05, n = 5) (Fig 4).
Echothiophate-treated RBCs, when added to the perfusion
solution a t a hematocrit of lo%, did not block the relaxation
that occurred on the addition of acetylcholine, but did
abolish the significant inhibitory effect exerted by acetylcholine on platelet aggregation (P< .05, n = 6) (Fig 4). In
preliminary experiments, lower concentrations of RBCs ( 1%
or 5% hematocrit) were insufficient to completely block
acetylcholine’s inhibitory effect (data not shown). The supernatant obtained after centrifugation of the perfusate containing RBCs was clear (in comparison with the marked red
color imparted by lo-’ mol/L purified Hb), indicating that
no major degree of hemolysis had occurred.
Table 1. Aggregometry Experiments
Thrombin
0.25 U/mL
(n = 5)
Thrombin
Unstimulated
In = 5 )
+ Acetylcholine
1oF mol/L
In
= 51
Thrombin
+ Hb
10-5
(n = 4)
Thrombin
+ Acetylcholine + Hb
(n = 4)
Thrombin
(n = 5)
Aggregation
58.2 k 3.7
C-5HT release
83.7 i 3.8
0
56.4
+ 3.1
58.5 f 3.7
60.5
k
3.0
85.2
+ 2.7
4.1
56.1
+ 3.0
14
5.9
*
1.2
Responses are expressed as percentages.
81.5 f 4.8
84.2
k
81.3 f 4.4
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956
HOUSTON, ROBINSON, AND GERRARD
Thrombin
0.25 Ulml
0Thrombin
+
Unstimulated
ACh 1uM
DISCUSSION
The physiologic role of EDRF is not yet fully known. From
observations that adenosine diphosphate and 5-hydroxytryptamine released from aggregating platelets can provoke
endothelium-dependent relaxation in coronary arteries:”
it
seems that one such role may be to promote vasodilatation at
sites of platelet thrombus formation, and thereby protect
arteries from occlusion.” Inhibition of platelet aggregation
by EDRF would further preserve blood flow. However, if the
platelet-inhibitory action of EDRF were unregulated, it
could cause a bleeding diathesis.
The current studies demonstrate that the endothelial cells
of a perfused artery can inhibit the activation of platelets
passing through the lumen. This inhibitory activity (triggered by acetylcholine) is almost certainly due to E D R F the
production of the other known endothelially derived platelet
inhibitor, prostacyclin, is prevented by acetylsalicylic acid;
and the inhibitory activity is blocked by Hb, a specific
antagonist of EDRF activity.’ Experiments in the aggregometer confirm that acetylcholine does not directly inhibit
platelet activation, nor does Hb augment it.
Several investigators have previously demonstrated that
endothelium-derived relaxing factor can inhibit platelet
a g g r e g a t i ~ n ’ * ~and
* ’ ~adhesion
-~~
in v i t r ~ ~ ’or- *in~ situ in a
buffer-perfused lung.24However, many of those studies have
used cultured endothelial cells rather than intact vascular
endothelium as the source of EDRF, and most have studied
the platelets in the aggregometer. Two studies have demonstrated that systemic exposure to a large dose of carbamylcholine to release EDRF in vivo can inhibit platelet function
(assessed by pulmonary platelet trapping” or in vitro aggregometryZ6);however, in those experiments one presumes
that the entire body’s endothelium has been activated. Thus,
until now it has remained moot whether a conduit artery’s
endothelium in situ could release enough EDRF locally to
inhibit platelets flowing through that artery. The present
experiments confirm that it can.
Free Hb can block the platelet-inhibiting action of locally
released EDRF. Whole blood contains about 200 to 250
Fig 3. Results of a perfusion experiment, in
which platelet activation in transit through the
vessel is reflected by the percentage of 14C label
released into the supernatant of the perfusate
collected downstream. In all but the last column
(“Unstimulated’l, thrombin is infused via a proximal side branch to activate the platelets. “ACh”
signifies the addition of acetylcholine (lo-’ mol/L)
to the perfusion solution (light columns) during
thrombin infusion.
times as much Hb as was required, in cell-free form, to
abolish the platelet inhibition. However, it has not been clear
whether the Hb contained within erythrocytes is available to
bind EDRF. One study” using a cascade bioassay system
found that a concentration of rat RBCs “equivalent to
mol/L hemoglobin” could abolish endothelium-dependent
relaxation when added downstream from the donor but
upstream from the assay tissue. This number of RBCs would
amount to a hematocrit of only about 0.02%. On the other
hand, two
have been able to demonstrate inhibition, presumed to be due to EDRF, of platelet aggregation in
whole blood using electrical impedence-aggregometry.
The present experiments show that RBCs can block
platelet inhibition by EDRF. Because a 10% hematocrit is
sufficient to do so, it would appear that the endothelium of a
large artery perfused in vivo by whole blood with a normal
hematocrit would not be able to release enough EDRF to
abolish platelet thrombus formation intraluminally. Even so,
EDRF may still be able to inhibit platelet adhesion to the
endothelial surface (and hence prevent initiation of thrombus
formation): as erythrocytes tend to stream to the center of
the lumen, platelets in the immediate vicinity of the endothelial cell may be exposed to inhibitory concentrations of
EDRF. Furthermore, synergistic interaction between the
antiaggregatory effects of EDRF and p r ~ s t a c y c l i n ’ ~may
*’~~’~
amplify the effect of EDRF.
In contrast to their effect on platelet inhibition, RBCs do
not block the relaxation of arterial smooth muscle evoked by
EDRF release in response to acetylcholine. This is in keeping
with in vivo observations of endothelium-dependent vasodilatation in blood-perfused arteries?* In contrast, the fact that
free Hb can block smooth muscle relaxation may indicate
that Hb can gain access to the subendothelial space and bind
EDRF there; intact RBCs, needless to say, cannot do so.
The lack of inhibition of relaxation by intact RBCs
demonstrates two other points. First, the blockade of acetylcholine’s inhibitory effect is not merely an artifact due to
breakdown of acetylcholine by the RBCS~~.”;
this confirms
that treatment with echothiophate has effectively blocked
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EDRF, PLATELETS, AND ERYTHROCYTES
957
n = 5
Hemoglobin 1OuM
*
*
1""1 Thrombin
A
-
0Thrombin
+ ACh luM
0.25 Ulml
RBC's
n = 6
I
Fig 4. Platelet activation during perfusion. In
all columns, thrombin is infused via a proximal side
branch. "ACh" signifies the addkion of acetylcholine t o the perfusion solution (light columns). (A)
"Hemoglobin" represents the addition of Hb (lo-'
mol/L) to the perfusion solution (overlying bar).
The inhibitory effect of acetylcholine is blocked in
the presenceof Hb (P< .05L but recovers after Hb
is withdrawn from the perfusion solution. (6)
"RBC's" represents the addition of erythrocytes
(10% hematocrit) to the perfusion solution (overlying bar). Platelet activation is significantly inhibited
by acetylcholine ( P< .05). and this effect is abolished by the presenceof RBCs.
T
0
d
20
0
B
!??!!!f
erythrocyte acetylcholinesterase. Second, the effect of RBCs
is not due simply to hemolysis and release of free Hb
(although we cannot rule out the possibility that a slight
degree of hemolysis may have occurred, which may have
contributed somewhat to antagonizing the EDRF effect).
It is of interest that a hematocrit on the order of 10%
appears necessary to block completely the platelet inhibition
by EDRF. This is 40 to 50 times the concentration of free Hb
needed to do so. This would suggest that the Hb contained
within RBCs is in some way unable to freely interact with
EDRF, even though it is generally supposed that nitric oxide
(the putative chemical identity of EDRF") should be freely
permeable through cell membranes. Streaming of RBCs to
the center and platelets to the periphery of the lumen may
explain this apparent discrepancy.
An intriguing speculation arises from the current result
Thrombin
0.25 Ulml
0Thrombin
+
ACh l u M
that a relatively large concentration of RBCs is needed to
block the platelet-inhibiting action of EDRF. In anemia,
platelets may be exposed to an increased tonic inhibitory
effect of EDRF, and the bleeding time thereby prolonged.
Such an inverse correlation between hematocrit and bleeding
time has long been recognized ~ l i n i c a l l y ~although
* - ~ ~ it has
never been fully explained.
In summary, EDRF released from a conduit artery's
endothelium can inhibit the activation of platelets in transit
through the vessel. This effect, at least in large-caliber
vessels, is blocked by the presence of RBCs.
ACKNOWLEDGMENT
The authors acknowledge the technical assistance of Esther
Taylor, and thank Dr Luis Oppenheimer and Michael Hoppensack
for provision of the arteries.
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HOUSTON, ROBINSON, AND GERRARD
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1990 76: 953-958
Inhibition of intravascular platelet aggregation by endothelium-derived
relaxing factor: reversal by red blood cells
DS Houston, P Robinson and JM Gerrard
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