(Circulation. 1995;92:1399-1407.)
© 1995 American Heart Association, Inc.
Articles |
Fibrinolysis Inhibits Shear Stress–Induced Platelet Aggregation
From the Houston VA Medical Center (S.G.K., M.H.K., A.I.S.) and Rice University (J.L.M., J.D.H.), Houston, Tex; and the University of Massachusetts Medical School (A.D.M., S.E.B.) and the Medical Center of Central Massachusetts (D.R.), Worcester, Mass.
Correspondence to Andrew I. Schafer, Chief, Medical Service, Houston Veterans Affairs Medical Center, 2002 Holcombe Blvd, Houston, TX 77030.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Shear stress–induced platelet aggregation may initiate arterial thrombosis at sites of pathological blood flow. Shear stress–induced platelet aggregation is mediated by von Willebrand factor (vWf) binding to platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa. Tissue-type plasminogen activator (TPA) induces thrombolysis in coronary arteries through the local generation of plasmin. Plasmin also proteolyses GP Ib and plasma vWf.
Methods and Results Because these effects could mitigate shear
stress–induced platelet aggregation, we investigated the effect of
fibrinolytic agents on platelet aggregation in response to a
pathological shear stress of 120 dynes/cm2 generated by a
cone-and-platen rotational viscometer. Plasmin inhibited shear
stress–induced aggregation of washed platelets, and this was
associated with a decrease in GP Ib. TPA, at concentrations 
2000
IU/mL, significantly inhibited shear stress–induced platelet
aggregation of platelet-rich plasma without a decrease in
platelet GP Ib. In plasma-platelet mixing experiments, we
determined that the TPA effect was localized to plasma. Purified vWf
multimer degradation by TPA (in the presence of exogenous
plasminogen) was associated with the loss of the capacity
of vWf to support shear stress–induced platelet aggregation.
Conclusions These results demonstrate that TPA inhibits platelet aggregation in response to pathological shear stress by altering the multimeric composition of vWf. This effect of TPA on shear stress–induced platelet aggregation may contribute, along with fibrinolysis, to the therapeutic effect of TPA in restoring blood flow during acute coronary artery thrombosis.
Key Words: stress, shear • enzymes • plasminogen activators • platelets
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Platelets aggregate in vitro when subjected to levels of fluid shear stress that are attained under pathological conditions in stenotic arteries.1 2 3 4 5 6 7 8 9 10 11 12 13 von Willebrand factor (vWf) is particularly important in mediating the formation of arterial platelet thrombi under these conditions.1 2 3 4 5 6 7 8 9 10 14 15 16 17 The platelet aggregation response to fluid shear stress occurs in the absence of exogenous agonists2 3 and requires intact vWf multimers, extracellular Ca2+, endogenous ADP contained in platelet granules, and metabolically active platelets with intact platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa.1 2 3 4 5 6 7 8 9 10 Arterial and pathological levels of shear stress (>30 dynes/cm2) initiate platelet aggregation by inducing the binding of vWf to platelet GP Ib.4 vWf must bind to both platelet GP Ib and GP IIb/IIIa to support full platelet aggregation at these elevated levels of shear stress.1 2 3 4 5 6 7 8 9 10 14 15 16 17
Thrombolytic agents (eg, tissue-type plasminogen activator [TPA]) are widely used to induce reperfusion of coronary arteries occluded by thrombi during acute myocardial infarction.18 19 These drugs generate plasmin, the major fibrinolytic protease, from the plasma zymogen plasminogen. Plasmin not only digests fibrin but also has complex actions on the function of platelets, which are the predominant constituents of coronary thrombi.20 21 22 23 24 Furthermore, fibrinolytic enzymes have been shown to degrade vWf in vivo and in vitro.25 26
The aim of this study was to determine the effects of fibrinolytic enzymes on shear stress–induced platelet aggregation. Previous studies have examined the actions of plasmin and TPA only on chemical agonist–induced platelet function in the absence of shear stress. The effects of these enzymes on platelet aggregation induced by the pathologically elevated levels of shear encountered in stenotic coronary arteries have not been investigated previously. The capacity of TPA/plasmin–modified vWf to support shear stress–induced platelet aggregation also has not been studied before.
Because plasmin has been shown to cleave platelet membrane GP Ib,27 28 29 30 31 we reasoned that it should inhibit shear stress–induced platelet aggregation and that this effect could be an important mechanism contributing to the dissolution of coronary thrombi by thrombolytic agents. We now report that plasmin is a potent inhibitor of the shear stress–induced aggregation of washed platelets and that this action is accompanied by the proteolysis of platelet GP Ib. In platelet-rich plasma (PRP), plasmin generated by TPA also inhibits the shear stress–induced aggregation of platelets; however, this effect occurs without associated loss of platelet membrane GPs. Instead, TPA inhibits shear stress–induced platelet aggregation through the proteolysis of large multimers of vWf, the cohesive ligand that mediates platelet aggregation under conditions of pathologically elevated shear stress levels.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Materials
Plasmin, plasminogen, chromogenic substrate S-2251, and cyanogen bromide–digested fibrin(ogen) fragments were obtained from KABI Pharmacia. TPA (recombinant human tissue-type plasminogen activator [alteplase]) was from Genentech, Inc. Glycyl-L-prolyl-L-arginyl-L-proline (Gly-Pro-Arg-Pro) was from Calbiochem. D-Phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK) was from Chemica Alta Ltd. Prostacyclin (PGI2) and aprotinin were from Sigma Chemical Co. Human thrombin was from Enzyme Research Laboratories.
Preparation of Platelets
Venous blood was collected from
healthy volunteers who had not
taken aspirin or other platelet-inhibiting drugs for at least 10
days before donation. The blood was anticoagulated with either (1) 1/10
volume of citrate (3.8% [wt/vol] trisodium citrate, pH 7.4) for the
PRP experiments or (2) 1/7 volume of acid/citrate/dextrose (trisodium
citrate 85 mmol/L, citric acid 71 mmol/L, dextrose 111 mmol/L, pH 4.5)
for the washed platelet experiments. PRP was prepared by
centrifugation of anticoagulated blood at
220g for 14 minutes.
To prepare washed platelets,
PGI2 (2.5 µg/mL) was
added and the PRP was centrifuged at 800g for 15
minutes at 22°C. The supernatant plasma was discarded, and the
platelet pellet was resuspended in 5 mL Tyrode's buffer (in
mmol/L: NaCl 130, sodium citrate 10, KCl 3, NaHCO3 9,
NaH2PO4 0.8, and 0.1% glucose, pH 6.5)
containing PGI2 (300 ng/mL). Centrifugation
was repeated, and platelets were resuspended in 5 mL Tyrode's
buffer (pH 7.3) supplemented with HEPES (0.2 mol/L) and 300 ng/mL
PGI2. The concentration of platelets was adjusted to
250 000/µL, and CaCl2 (1 mmol/L) was added.
Plasmin Experiments Using Washed Platelets
Aliquots of the
washed platelet suspension were incubated in
a shaker for 1 hour at 37°C without or with plasmin (1.0, 0.25, and
0.0625 casein units [CU]/mL). After the incubation period, aliquots
of platelets to be analyzed by flow cytometry were treated
with aprotinin (200 kallikrein inhibitory units
[KIU]/mL), fixed with 1% formalin, and kept on ice. Other aliquots
of aprotinin-treated platelet suspensions were centrifuged
at 10 000 rpm for 1 minute in a microcentrifuge. Supernatant
samples were then collected and frozen on dry ice in methanol for
flow-cytometric analysis of glycocalicin, the plasmin cleavage
product of GP Ib. The remaining platelet suspension was
subjected to shear stress, as described below.
TPA Experiments Using PRP
PRP was incubated in a shaker for 1
hour at 37°C without or
with different concentrations of TPA (20 000, 2000, and 200 IU/mL).
Cyanogen bromide–digested fibrin(ogen) fragments (to be referred to as
fibrin) (80 µg/mL) were added as a
cofactor.32 33 34 35
Samples for flow cytometry were prepared as described above. The
remaining PRP was subjected to shear stress, as described below.
TPA Conversion From IU/mL to ng/mL
One international unit of
TPA is equivalent to 1.724 ng. Thus,
200 IU/mL is 350 ng/mL, 2000 IU/mL is equivalent to 3500 ng/mL, and
20 000 IU/mL corresponds to 35 000 ng/mL.
Flow Cytometric Analysis of Platelet Surface
GPs
The following murine monoclonal antibodies were used. 6D1
(provided by Dr Barry S. Coller, Mount Sinai Hospital, New York, NY) is
directed against the vWf binding site on the 
-chain of GP
Ib.36 7E3 (provided by Dr Coller) is directed against the
GP IIb/IIIa complex near its fibrinogen binding site.37
S12 (provided by Dr Rodger P. McEver, University of Oklahoma, Oklahoma
City) is directed against P-selectin.38 39
P-selectin,
also referred to as GMP-140,38 PADGEM
protein,40 and CD62,41 is a component of the
-granule membrane of resting platelets that is expressed only on
the platelet plasma membrane after platelet
secretion.42
The method has been described
previously.43 Fixed, washed
platelets or PRP was incubated with a saturating concentration of
FITC-conjugated 6D1, biotinylated 7E3, or biotinylated S12. For assays
with 7E3 or S12, the samples were subsequently incubated with
phycoerythrin-streptavidin (Jackson ImmunoResearch). The samples were
then analyzed in an EPICS Profile flow cytometer (Coulter
Cytometry). In control experiments to determine the maximal
platelet surface exposure of P-selectin, washed platelets were
incubated, before fixation, for 4 minutes at 22°C with purified human
-thrombin (1 U/mL), and PRP was incubated, before fixation, with
purified human -thrombin (1 U/mL) together with 2.5 mmol/L
Gly-Pro-Arg-Pro (an inhibitor of fibrin
polymerization).44
Glycocalicin Assay
Plasma or supernatant glycocalicin
concentrations were
determined by a previously described competitive inhibition
assay.45 A subsaturating concentration (1.2 µg/mL) of
FITC-conjugated monoclonal antibody 6D1 (GP Ib specific) was incubated
for 20 minutes at 22°C with either (1) test plasma that had been
filtered through a 0.22-µm Acrodisc (Gelman) and the pH buffered to
7.4 or (2) various concentrations of purified glycocalicin (prepared as
previously described).46 Samples were then incubated for
20 minutes at 22°C with fixed, washed platelets (final
concentration, 100 000/µL) and diluted 20-fold in modified Tyrode's
buffer, pH 7.4, before the platelet binding of 6D1 was
analyzed by flow cytometry. Linear regression analysis
was used to generate a standard curve from 0 to 70 nmol/L from the
purified glycocalicin samples. The glycocalicin concentration of
unknown plasma or supernatant samples was derived from this standard
curve.
Shear Stress–Induced Platelet Aggregation
Platelet
suspensions (washed platelets or PRP) were
subjected to a fluid shear stress of 120 dynes/cm2 for time
periods of 0, 15, 30, 60, and 120 seconds at 22°C in a rotational
cone-and-platen viscometer (Ferranti Electric, Inc). "Singlet"
platelet particle numbers were measured as previously
described.2 3 4 The platelet counts were
then expressed
as a percentage of the platelet count at the initial 0-second time
point.
Plasmin Activity Assay
Plasmin activity was determined with
an S-2251
chromogenic substrate assay47 performed at
37°C in a spectrophotometer using an enzyme kinetics software package
(Pharmacia LKB Ultrospec III, Pharmacia Biotech Inc). The data sheet
supplied by the manufacturer48 indicates that an activity
of 
A/min=0.05 (37°C, 405 nm) is obtained with a substrate
concentration of 6x10-4 mol/L and a plasmin
concentration
of 0.01 CU/mL. Plasmin generated by TPA in plasma was determined by
serial sampling at various time points.
TPA Mixing Experiments With Plasma
Aliquots of PRP were
incubated without or with 20 000 IU/mL
TPA. At the end of the incubations, PPACK (5 mmol/L) and aprotinin (200
KIU/mL) were added to both TPA-treated and control samples (to
neutralize TPA and plasmin, respectively). An aliquot of each sample
was centrifuged at 3000g for 10 minutes to recover
platelet-poor plasma (PPP). The remaining TPA-treated and control
PRP were each divided into two aliquots of equal volumes. Washed
platelets were then prepared from these preparations, as described
above.
Each aliquot of either TPA-treated or control washed platelets was resuspended in either TPA-treated or control PPP to obtain four combinations of reconstituted platelet suspensions: (1) TPA-treated platelets in TPA-treated plasma; (2) TPA-treated platelets in control plasma; (3) control platelets in TPA-treated plasma; and (4) control platelets in control plasma. These reconstituted platelet suspensions were then subjected to a shear stress of 120 dynes/cm2.
vWf Purification and Radioiodination
vWf multimeric forms
were purified from normal human
cryoprecipitate, as described previously, and the vWf antigen levels of
the purified fractions were quantified by solid-phase immunoradiometric
assay.2 49 50 51 vWf
multimers were separated by
SDS–1% agarose gel electrophoresis, overlaid with rabbit
[125I]anti-human vWf polyclonal antibody, and
analyzed by autoradiography.2 49
Unless otherwise specified, the final concentration of vWf was 100 U/dL
(100% antigen level).
Multimeric Analysis of TPA-Treated Purified
Radioiodinated vWf
Aliquots of purified vWf were incubated in a shaker
for 1 hour
at 37°C without or with different concentrations of TPA (20 000,
2000, and 200 IU/mL) in the presence of fibrin (80 µg/mL) and
plasminogen (180 µg/mL). Aliquots were also treated with
TPA (20 000 IU/mL) in the absence of exogenous plasminogen
and with plasminogen and/or fibrin alone. One fraction of
each sample was then mixed with 0.02 mol/L Tris-HCl, 2 mmol/L EDTA, 8
mmol/L urea, and 2% SDS, pH 8.0, and incubated for 20 minutes at
60°C. Unreduced vWf multimers were separated by SDS–1%
agarose gel electrophoresis and displayed by rabbit
[125I]anti-human vWf antibody overlay and
autoradiography.2
TPA Mixing Experiments With Purified vWf
Fibrin (80
µg/mL) and plasminogen (180 µg/mL)
were added to purified vWf in Tyrode's buffer and incubated without or
with 20 000 IU/mL TPA for 1 hour at 37°C. Aliquots of PRP were also
incubated without or with 20 000 IU/mL TPA. At the end of the
incubations, PPACK (5 mmol/L) and aprotinin (200 KIU/mL) were added to
each vWf buffer and PRP sample to neutralize TPA and plasmin,
respectively. The samples of TPA-treated and control PRP were divided
into two equal parts. Washed platelets were then prepared from each
part.
Each aliquot of either TPA-treated or control washed platelets was resuspended in either TPA-treated or control vWf buffer to obtain four combinations of reconstituted platelet suspensions: (1) TPA-treated platelets in TPA-treated vWf buffer, (2) TPA-treated platelets in control vWf buffer, (3) control platelets in TPA-treated vWf buffer, and (4) control platelets in control vWf buffer. These reconstituted platelet suspensions were then subjected to a shear stress of 120 dynes/cm2.
Statistics
Student's two-tailed unpaired t
test was used for
statistical analysis. However, in the experiments with a 2x2
factorial design (as in the case of the plasma-platelet and
vWf-platelet mixing studies performed without and with TPA
treatment), ANOVA was performed. Values are expressed as mean±SEM, and
statistical significance was determined at a level of
P<.05.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Preincubation of washed platelets with plasmin led to inhibition of shear stress–induced platelet aggregation. This effect of plasmin was concentration dependent, with partial inhibition of shear stress–induced platelet aggregation noted at 0.25 CU/mL and complete inhibition at 1.0 CU/mL (Fig 1
).
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Aliquots of the same washed platelet suspensions were
analyzed by flow cytometry before exposure to shear stress.
Plasmin treatment resulted in a concentration-dependent loss of
platelet surface GP Ib (Fig 2) that paralleled
the inhibitory effect of plasmin on shear stress–induced
aggregation (Fig 1
). Platelet surface GP IIb/IIIa was not
significantly affected by the same concentrations of plasmin, although
there was a decrease with 1.0 CU/mL (Fig 2). There was minimal
platelet surface expression of P-selectin with all concentrations
of plasmin tested (0.0625 to 1.0 CU/mL; Fig 2). The loss of
platelet surface GP Ib by plasmin treatment was associated with a
plasmin concentration–dependent increase in the levels of glycocalicin
(a proteolytic fragment of GP Ib) released into the supernatant (Fig
3).
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Incubation of PRP with 20 000 IU/mL TPA rapidly generated free plasmin
activity, with a peak of 0.375 CU/mL in 5 minutes and a plateau of
0.3 CU/mL for the remaining 55 minutes (Fig 4). TPA
at a concentration of 2000 IU/mL generated plasmin activity in PRP that
gradually increased to 0.15 CU/mL over a period of 60 minutes. TPA at
200 IU/mL did not lead to the generation of detectable plasmin activity
in PRP (Fig 4).
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In PRP, TPA inhibited shear stress–induced platelet aggregation in
a concentration-dependent manner (Fig 5). Complete
inhibition of aggregation was noted when PRP was incubated for 60
minutes at 37°C with 20 000 IU/mL TPA. (More than 50% of the
inhibitory effect of 60 minutes of preincubation with TPA
was seen after only 20 minutes of preincubation with the same
concentration; data not shown.) At 2000 IU/mL, TPA partially inhibited
shear stress–induced platelet aggregation, whereas incubation with
200 IU/mL had no effect.
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Flow cytometric analysis of the same TPA-treated PRP samples
before exposure to shear stress showed no loss of either platelet
surface GP Ib or GP IIb/IIIa, even after treatment with the highest
concentration of TPA (20 000 IU/mL; Fig 6). There was
likewise no P-selectin expression on the platelet surface at any of
these concentrations of TPA (200 to 20 000 IU/mL; Fig 6).
Glycocalicin
was not released into the supernatant of TPA-treated PRP, confirming
that GP Ib was not cleaved (Fig 3; see Reference 52).
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Thus, under the conditions of TPA treatment of platelets in a
plasma milieu that completely blocked shear stress–induced
platelet aggregation, there was no detectable loss of the
platelet membrane GPs known to be required for shear
stress–induced platelet aggregation. Therefore, we hypothesized
that the inhibitory action of TPA on shear stress–induced
aggregation of PRP was due either to some other direct effect on
platelets independent of monoclonal antibody binding to GP Ib or GP
IIb/IIIa and/or to an effect of TPA on plasma vWf. We therefore
performed experiments in which PRP was reconstituted by mixing PPP with
washed platelets, both of which were obtained from PRP treated or
not treated with TPA. As shown in Fig 7, PRP
reconstituted by mixing control PPP with control platelets
supported normal shear stress–induced aggregation. Control PPP mixed
with TPA-treated platelets exhibited the same degree of shear
stress–induced platelet aggregation. In contrast, when TPA-treated
PPP was reconstituted with either TPA-treated or control platelets,
shear stress–induced aggregation was completely inhibited.
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Purified vWf (including the largest plasma-type multimeric
forms) in Tyrode's buffer containing a
physiological concentration of exogenously added
plasminogen and fibrin was treated or not treated with TPA
(20 000 IU/mL). Washed platelets obtained from PRP treated or not
treated with TPA (20 000 IU/mL) were then resuspended in TPA-treated
and control vWf multimeric preparations in four ways, as in the
plasma mixing experiments. When control or TPA-treated platelets
were resuspended with control vWf, shear stress–induced platelet
aggregation was normal (Fig 8). In contrast, when
control or TPA-treated platelets were resuspended with TPA-treated
vWf, shear stress–induced platelet aggregation was inhibited (Fig
8). Proteolytic products of plasmin-treated vWf (0.3 CU/mL for
1
hour at 37°C) did not inhibit aggregation of PRP in response to shear
stress (data not shown).
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These plasma and purified vWf mixing experiments suggested that TPA
treatment alters vWf multimers to produce inhibition of
platelet aggregation in response to shear stress. Fig 9 shows
that the treatment of purified vWf with TPA, in
the presence of exogenous plasminogen and fibrin, produced
a concentration-dependent proteolysis of vWf multimers as TPA
levels were increased from 200 to 20 000 IU/mL. Some proteolysis of
large vWf multimers was detected even at 200 IU/mL TPA. The
extent of this proteolysis increased considerably at 2000 IU/mL TPA and
was maximal in our experiments at 20 000 IU/mL TPA. In control
experiments, large vWf multimers were not proteolyzed in the
presence of (1) TPA (20 000 IU/mL) and fibrin in the absence of
exogenous plasminogen, (2) plasminogen and
fibrin in the absence of TPA, or (3) fibrin alone (Fig 9).
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Discussion |
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Under conditions of high shear stress, vWf binds to its platelet membrane receptors, GP Ib and GP IIb/IIIa, and induces platelet aggregation.1 2 3 4 5 6 7 8 9 10 14 15 16 17 Shear stress levels of at least 120 dynes/cm2, which was the level used in this study, are attained in stenosed coronary arteries.12 13 We have demonstrated that plasmin inhibits shear stress–induced aggregation of washed platelets, an effect that is associated with plasmin-induced cleavage of platelet membrane GP Ib. Although plasmin generated by exogenous TPA likewise inhibits shear stress–induced platelet aggregation in a plasma milieu, this effect occurs in the absence of GP Ib cleavage. These observations suggested that TPA exerts its inhibitory action in PRP on a plasma component rather than on platelets. This was confirmed by mixing studies, which demonstrated that TPA pretreatment of plasma inhibited shear stress–induced platelet aggregation, independent of platelet pretreatment with TPA. In addition, treatment of purified vWf with increasing concentrations of TPA led to progressive proteolysis of large vWf multimers and the concurrent loss of its capacity to support shear stress–induced platelet aggregation, likewise independent of platelet pretreatment with TPA. Thus, we conclude that TPA inhibits shear stress–induced platelet aggregation in association with degradation of the large vWf multimers that are most effective in mediating shear stress–stimulated aggregation.
Plasmin, the major fibrinolytic protease, is known to have important
effects on platelet function.24 At higher
concentrations (1.0 CU/mL), it causes activation and aggregation of
washed platelets,20 whereas at lower concentrations
(<0.5 CU/mL), it inhibits platelet aggregation and
thromboxane A2 production in response to
agonists.21 In washed platelet suspensions, plasmin,
either added
directly28 29 30 31 or
generated by the action of
TPA on plasminogen,27 31 degrades platelet
GP Ib. Glycocalicin, a heavily glycosylated fragment of the GP Ib
chain that contains the vWf binding site, is released into the
supernatant.27 28 29 30 This
effect of plasmin is associated
with a decrease in ristocetin-induced, vWf-dependent platelet
agglutination.27 28 29 30 We
have now shown that this
plasmin-induced cleavage of GP Ib from the surface of washed
platelets is associated with a plasmin concentration–dependent
inhibition of shear stress–induced platelet aggregation. Stricker
et al27 reported that TPA and plasminogen
incubated with washed platelets also degraded GP IIb/IIIa, although
the Ca2+ concentration in their platelet
suspensions was not stated. In contrast, Winters et al31
demonstrated that GP IIb/IIIa did not undergo plasmin-mediated
proteolysis in the presence of Ca2+ in a washed
platelet system. Our data, from washed platelet suspensions
containing 1 mmol/L Ca2+, are consistent with the
latter study, demonstrating that GP IIb/IIIa on the platelet
surface was intact after incubation with plasmin.
In contrast to washed platelets, our studies show that GP Ib (and GP IIb/IIIa) remain intact on TPA-treated platelets in plasma. These results are consistent with previous reports indicating that incubation of platelet-rich plasma with TPA at 37°C does not cleave platelet surface GPs.22 31 Perhaps as a result of differences in the milieu that the platelets are exposed to, the results of our in vitro studies contrast with those of Michelson et al,53 who evaluated platelet GP Ib in patients with acute myocardial infarction who were treated with TPA. They reported that some GP Ib cleavage did occur after thrombolytic therapy (as determined by a decrease in total platelet GP Ib in conjunction with a corresponding increase in plasma glycocalicin), although, as in our experiments, there was no decrease in platelet surface GP Ib. This was most likely due to the concomitant translocation of GP Ib from intraplatelet stores to the platelet surface.
The difference between the effect of fibrinolytic enzymes in the washed platelet system and the PRP system may involve a number of factors, including the inaccessibility of platelet membrane GPs to enzymatic cleavage in the plasma milieu, the presence of plasminogen activator and plasmin inhibitors in plasma, and the presence of vWf, fibrinogen, or other preferred substrates for plasmin or TPA in plasma. Our findings suggest that plasmin-induced cleavage of the platelet membrane GP receptors for vWf is not involved in the inhibition of shear stress–induced aggregation in a plasma milieu.
The treatment of plasma with TPA, independent of TPA treatment of platelets, produced inhibition of shear stress–induced platelet aggregation. We further evaluated whether this plasma effect was due to proteolytic degradation of the ligand vWf. Mixing studies demonstrated that purified vWf multimers pretreated with TPA (in the presence of plasminogen and fibrin) are proteolyzed progressively as TPA concentrations are increased, with associated decreased capacity to support shear stress–induced platelet aggregation.
Degradation of vWf by fibrinolytic enzymes has been observed in humans. Federici et al25 analyzed plasma vWf in patients with acute myocardial infarction who received thrombolytic therapy and demonstrated a significant loss of higher-molecular-weight vWf multimers up to 24 hours after TPA therapy, associated with a loss of ristocetin cofactor activity. These authors25 also demonstrated that thrombolytic therapy induced plasmin-mediated cleavage of vWf. Furthermore, it was found that there were more plasmin-generated fragments of vWf in patients who had bleeding complications of thrombolytic therapy than in those who did not.25 Hamilton et al26 demonstrated that plasmin degraded vWf in vitro. In vivo, the treatment of patients and volunteers with streptokinase demonstrated a probable transient degradation of vWf, although the major effect was an increase in vWf antigen and platelet agglutinating activity. Furthermore, Eikenboom et al54 described a patient with a primary state of hyperfibrinolysis that resulted in proteolytic degradation of vWf and a clinical syndrome of acquired von Willebrand disease.
The proteolytic products of vWf present in the medium could effectively compete with large vWf multimers for binding sites on platelet GP receptors available during shear without being able to support aggregation. Mascelli et al55 demonstrated that a fragment of bovine vWf obtained by plasmin digestion can compete with intact vWf and bind to platelets, albeit with a 10 times lower affinity in vitro. Sugimoto et al56 and Alevriadou et al57 demonstrated that a reduced and alkylated recombinant fragment of the vWf monomer that includes the amino acids 445 through 733 and contains the GP Ib binding site is capable of attaching to GP Ib (even in whole blood) in the absence of any agonist, thereby blocking the binding of large vWf multimers and inhibiting shear stress–induced platelet aggregation. However, when purified vWf was treated with plasmin and added to PRP, shear stress–induced aggregation was normal. This argues against the presence of plasmin-generated vWf fragments that actively interfere with the binding of normal large multimeric vWf in plasma under elevated shear stress conditions. We therefore conclude that proteolysis of vWf (demonstrated directly in mixing experiments using purified vWf multimers, plasminogen, fibrin, and TPA) is most likely the major mechanism of TPA inhibition of shear stress–induced aggregation of platelets in plasma.
Recent studies have demonstrated the enhanced clinical efficacy of "front-loaded" or bolus regimens of TPA in the treatment of acute myocardial infarction.58 59 60 61 62 63 64 65 These regimens use more rapid administration of larger doses of TPA. The recently completed GUSTO trial demonstrated that accelerated dosing with TPA provides a survival benefit over previous standard thrombolytic protocols.59 In all of these more efficacious "front-loaded" or bolus TPA regimens, peak plasma TPA concentrations have considerably exceeded 2000 IU/mL.60 61 62 63 We observed significant, although incomplete, inhibition of shear stress–induced platelet aggregation at a concentration of 2000 IU/mL TPA. Peak mean plasma TPA concentrations with accelerated TPA schedules have been determined to be as high as >7000 IU/mL.60 61 62 63 On the basis of these considerations, the TPA concentrations used in our experiments are likely to be pharmacologically and clinically relevant. Nevertheless, the clinical implications of our findings should be interpreted with caution. Further studies will be needed to clarify the applicability of these in vitro observations to events that occur under in vivo shear stress conditions. Specifically, it will be important to determine whether there is a correlation among the extent of proteolysis of large plasma vWf multimers in vivo, inhibition of shear stress–induced aggregation, and both acute antithrombotic effects and the risk of reocclusion after thrombolytic therapy.
The relevance of our findings with regard to other thrombolytic agents (eg, streptokinase or urokinase) is not known. Because all thrombolytics generate plasmin, it may be reasonable to speculate that plasmin-induced vWf degradation could affect shear stress–induced platelet aggregation, at least in vitro. As discussed above,25 26 streptokinase does cause plasmin-induced vWf degradation.
TPA is synthesized by endothelial cells and released into the circulation in response to the same flow-generated arterial wall shear stresses that are responsible for triggering platelet activation.66 Shear stress also stimulates vessel wall production of the platelet inhibitory molecules PGI267 and nitric oxide68 and may enhance the capacity of various proteolytic enzymes to degrade large plasma vWf multimers.69 A model emerges, therefore, in which the proaggregatory effects of shear stress are counterbalanced by vessel wall production of TPA, PGI2, and nitric oxide, as well as shear stress–induced heightened susceptibility of vWf to proteolysis, to maintain blood fluidity and vessel patency in areas of atherosclerotic stenosis with intact endothelium. When the endothelium is damaged by plaque fissuring or rupture, this balance is tipped in favor of platelet activation, resulting in platelet adhesion, secretion, and aggregation leading to vaso-occlusion and ischemia or infarction. In addition, pathologically increased levels of shear stress in stenotic arteries may lead to direct platelet aggregation,1 2 3 4 even in the absence of endothelial damage.
Results of experiments presented here suggest that therapeutic doses of TPA could enhance arterial reperfusion and lessen reocclusion by inhibiting platelet aggregation in response to pathological shear stress. This effect of TPA/plasmin may be supplementary to fibrinolysis in restoring blood flow during acute coronary thrombosis. Further investigation will be required to determine whether inhibition of shear stress–induced aggregation is a clinically relevant in vivo effect of TPA administered for the treatment of arterial thrombosis.
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Acknowledgments |
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The authors received the following grant support: VA Merit Review grants (Drs Schafer and Kroll), National Institutes of Health grants HL-18584 (Drs Moake and Hellums), HL-36045 (Dr Schafer), and HL-02311 (Dr Kroll); and grants from the American Heart Association/Bugher Foundation Center for Molecular Biology (86-2216) (Dr Kamat), the Texas Affiliate of the American Heart Association (Dr Kroll), and The Methodist Hospital Foundation (Dr Kroll). The authors thank Drs Barry S. Coller and Rodger P. McEver for generously providing monoclonal antibodies. The authors also thank Nancy Turner and Leticia Nolasco for their expert technical assistance.
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Footnotes |
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Guest editor for this article was Joseph Loscalzo, MD, Boston University Medical Center Hospital, Boston, Mass.
Received February 14, 1995; accepted March 26, 1995.
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TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Peterson DM, Stathopoulous NA, Giorgio TD, Hellums JD, Moake JL. Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa. Blood. 1987;69:625-628.
2. Moake JL, Turner NA, Stathopoulous NA, Nolasco LH, Hellums JD. Involvement of large plasma von Willebrand factor (vWf) multimers and unusually large vWf forms derived from endothelial cells in shear stress-induced platelet aggregation. J Clin Invest. 1986;78:1456-1461.
3.
Moake JL, Turner NA, Stathopoulous NA, Nolasco LH,
Hellums JD. Shear-induced platelet aggregation can be
mediated by vWf released from platelets, as well as exogenous large
or unusually large vWf multimers, requires adenosine
diphosphate, and is resistant to aspirin.
Blood. 1988;71:1366-1374.
4.
Chow TW, Hellums JD, Moake JL, Kroll MH. Shear
stress-induced von Willebrand factor binding to platelet
glycoprotein Ib initiates calcium influx associated with
aggregation. Blood. 1992;80:113-120.
5. Sakariassen KS, Nievelstein PF, Coller BS, Sixma JJ. The role of platelet membrane glycoproteins Ib and IIb-IIIa in platelet adherence to human artery subendothelium. Br J Haematol. 1986;63:681-691. [Medline] [Order article via Infotrieve]
6. Weiss HJ, Hawiger J, Ruggeri ZM, Turitto VT, Thiagarajan P, Hoffmann T. Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J Clin Invest. 1989;83:288-297.
7.
Nichols TC, Bellinger DA, Johnson TA, Lamb MA, Griggs
TR. von Willebrand's disease prevents occlusive
thrombosis in stenosed and injured porcine coronary
arteries. Circ Res. 1986;59:15-26.
8.
Coller BS, Folts JD, Scudder LE, Smith SR.
Antithrombotic effect of a monoclonal antibody to the
platelet glycoprotein IIb-IIIa receptor in an
experimental animal model. Blood. 1986;68:783-786.
9.
Bellinger DA, Nichols TC, Read MS, Reddick RL, Lamb
MA, Brinkhous KM, Evatt BL, Griggs TR. Prevention of occlusive
artery thrombus by a mural monoclonal antibody to porcine von
Willebrand factor. Proc Natl Acad Sci
U S A. 1987;84:8100-8104.
10. Ikeda Y, Handa M, Kawano K, Kamata T, Murata M, Araki Y, Anbo H, Kawai Y, Watanabe K, Itagaki I, Sakai K, Ruggeri ZM. The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J Clin Invest. 1991;87:1234-1240.
11. Turitto VT. Blood viscosity, mass transport, and thrombogenesis. Prog Hemost Thromb. 1982;6:139-177. [Medline] [Order article via Infotrieve]
12. Back LD, Radbill JR, Crawford DW. Analysis of pulsatile viscous blood flow through diseased coronary arteries in man. J Biomech. 1977;10:339-353. [Medline] [Order article via Infotrieve]
13. Lipowski HH, Usami S, Chien S. In vivo measurements of "apparent viscosity" and microvessel hematocrit in the mesentery of the cat. Microvasc Res. 1980;19:297-319. [Medline] [Order article via Infotrieve]
14.
Roth GJ. Developing relationships:
arterial platelet adhesion, glycoprotein
Ib, and leucine-rich glycoproteins.
Blood. 1991;77:5-19.
15. O'Brien JR. Shear-induced platelet aggregation. Lancet. 1990;335:711-713. [Medline] [Order article via Infotrieve]
16. Weiss HJ, Turitto VT, Baumgartner HR. Effect of shear rate on platelet interaction with subendothelium in citrated and native blood, I: shear rate-dependent decrease of adhesion in von Willebrand's disease and Bernard-Soulier syndrome. J Lab Clin Med. 1978;92:750-764. [Medline] [Order article via Infotrieve]
17. Baumgartner HR, Tschopp TB, Meyer D. Shear rate dependent inhibition of platelet adhesion and aggregation on collagenous surfaces by antibodies to human factor VIII/von Willebrand factor. Br J Haematol. 1980;44:127-139. [Medline] [Order article via Infotrieve]
18. Marder VJ, Sherry S. Thrombolytic therapy: current status. N Engl J Med. 1988;318:1512-1520. [Medline] [Order article via Infotrieve]
19. Loscalzo J, Braunwald E. Tissue plasminogen activator. N Engl J Med. 1988;319:925-931. [Medline] [Order article via Infotrieve]
20. Schafer AI, Maas AK, Ware JA, Johnson PC, Rittenhouse SE, Salzman EW. Platelet protein phosphorylation, elevation of cytosolic calcium, and inositol phospholipid breakdown in platelet activation induced by plasmin. J Clin Invest. 1986;78:73-79.
21. Schafer AI, Adelman B. Plasmin inhibition of platelet function and of arachidonic acid metabolism. J Clin Invest. 1985;75:456-461.
22. Loscalzo J, Vaughan DE. Tissue plasminogen activator promotes platelet disaggregation in plasma. J Clin Invest. 1987;79:1749-1755.
23. Pasche B, Loscalzo J. Platelets and fibrinolysis. Platelets. 1991;2:125-134.
24. Coller BS. Platelets and thrombolytic therapy. N Engl J Med. 1990;322:33-42. [Medline] [Order article via Infotrieve]
25.
Federici AB, Berkowitz SD, Zimmerman TS, Mannucci PM.
Proteolysis of von Willebrand factor after
thrombolytic therapy in patients with acute myocardial
infarction. Blood. 1992;79:38-44.
26. Hamilton KK, Fretto LJ, Grierson DS, McKee PA. Effects of plasmin on von Willebrand factor multimers: degradation in vitro and stimulation of release in vivo. J Clin Invest. 1985;76:261-270.
27.
Stricker RB, Wong D, Shiu DT, Reyes PT, Shuman MA.
Activation of plasminogen by tissue
plasminogen activator on normal and
thrombasthenic platelets: effects on surface proteins and
platelet aggregation. Blood. 1986;68:275-280.
28.
Adelman B, Michelson AD, Loscalzo J, Greenberg J,
Handin RI. Plasmin effect on glycoprotein Ib-von
Willebrand factor interactions. Blood. 1985;65:32-40.
29.
Adelman B, Michelson AD, Greenberg J, Handin RI.
Proteolysis of platelet glycoprotein Ib by
plasmin is facilitated by plasmin lysine-binding regions.
Blood. 1986;68:1280-1284.
30.
Michelson AD, Barnard M. Plasmin-induced
redistribution of platelet glycoprotein Ib.
Blood. 1990;76:2005-2010.
31.
Winters KJ, Eisenberg PR, Jaffe AS, Santoro SA.
Dependence of plasmin-mediated degradation of platelet
adhesive receptors on temperature and Ca2+.
Blood. 1990;76:1546-1557.
32.
Hoylaerts MD, Rijken C, Lijnen HR, Collen D.
Kinetics of the activation of plasminogen by human
tissue plasminogen activator: role of
fibrin. J Biol Chem. 1982;257:2912-2919.
33.
Zamarron C, Lijnen HR, Collen D. Kinetics of the
activation of plasminogen by natural and recombinant
tissue-type plasminogen activator.
J Biol Chem. 1984;259:2080-2083.
34. Chandler WL, Schmer G, Stratton JR. Optimum conditions for the stabilization and measurement of tissue plasminogen activator activity in human plasma. J Lab Clin Med. 1989;113:362-371. [Medline] [Order article via Infotrieve]
35. Verheijen JH, Nieuwenhuizen W, Wijngaards G. Activation of plasminogen by tissue activator is increased specifically in the presence of certain soluble fibrin(ogen) fragments. Thromb Res. 1982;27:377-385. [Medline] [Order article via Infotrieve]
36.
Coller BS, Peerschke EI, Scudder LE, Sullivan CA.
Studies with a murine monoclonal antibody that abolishes
ristocetin-induced binding of von Willebrand factor to
platelets: additional evidence in support of GP Ib as a
platelet receptor for von Willebrand factor.
Blood. 1983;61:99-110.
37. Coller BS. A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb/IIIa complex. J Clin Invest. 1985;76:101-108.
38.
Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton
DF. A platelet alpha-granule membrane protein (GMP-140) is
expressed on the plasma membrane after activation.
J Cell Biol. 1985;101:880-886.
39. Bevilacqua M, Butcher E, Furie B, Furie B, Gallatin M, Gimbrone M, Harlan J, Kishimoto K, Lasky L, McEver R, Paulson J, Rosen S, Seed B, Siegelman M, Springer T, Stoolman L, Tedder T, Varki A, Wagner D, Weissman I, Zimmerman G. Selectins: a family of adhesion receptors. Cell. 1991;67:233. Letter. [Medline] [Order article via Infotrieve]
40.
Hsu-Lin SC, Berman CL, Furie BC, August D, Furie
B. A platelet membrane protein expressed during platelet
activation and secretion: studies using a monoclonal antibody
specific for thrombin-activated platelets.
J Biol Chem. 1984;259:9121-9126.
41.
Knapp W, Dorken B, Rieber P, Schmidt RE, Stein H, von
dem Borne AE. CD antigens. Blood. 1989;74:1448-1450.
42. McEver RP. Properties of GMP-140, an inducible granule membrane protein of platelets and endothelium. Blood Cells. 1990;16:73-80. [Medline] [Order article via Infotrieve]
43.
Yamamoto N, Greco NJ, Barnard MR, Tanoue K, Yamazaki H,
Jamieson GA, Michelson AD. Glycoprotein Ib (GP
Ib)-dependent and GP Ib-independent pathways of thrombin-induced
platelet activation. Blood. 1991;77:1740-1748.
44.
Michelson AD, Ellis PA, Barnard MR, Matic GB, Viles AF,
Kestin AS. Downregulation of the platelet surface
glycoprotein Ib-IX complex in whole blood stimulated by
thrombin, ADP or an in vivo wound. Blood. 1991;77:770-779.
45.
Kestin AK, Valeri CR, Khuri SF, Loscalzo J, Ellis PA,
MacGregor H, Birjiniuk V, Ouimet H, Pasche B, Nelson MJ, Benoit SE,
Rodino LJ, Barnard MR, Michelson AD. The platelet function
defect of cardiopulmonary bypass.
Blood. 1993;82:107-117.
46.
Michelson AD, Loscalzo J, Melnick B, Coller BS, Handin
RI. Partial characterization of a binding site for von
Willebrand factor on glycocalicin.
Blood. 1986;67:19-26.
47. Friberger P, Knoss M, Gustavsson S, Aurell L, Claeson G. Methods of determination of plasmin, antiplasmin and plasminogen by means of substrate S-2251. Haemostasis. 1978;7:138-145. [Medline] [Order article via Infotrieve]
48. Kabi Diagnostica data sheet for S-2251. Franklin, Ohio: Kabi Pharmacia; 1989.
49. Moake JL, Rudy CK, Troll JH, Weinstein MJ, Colannino NM, Azocar J, Seder RH, Hong SL, Deykin D. Unusually large plasma factor VIII, von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med. 1982;307:1432-1435. [Medline] [Order article via Infotrieve]
50. Kroll MH, Harris TS, Moake JL, Handin RI, Schafer AI. von Willebrand factor binding to platelet GP Ib initiates signals for platelet activation. J Clin Invest. 1991;88:1568-1573.
51. Moake JL, Olson JD, Troll JH Jr, Weinger RS, Peterson DM, Cimo PL. Interaction of platelets, von Willebrand factor, and ristocetin during platelet agglutination. J Lab Clin Med. 1980;96:168-184. [Medline] [Order article via Infotrieve]
52. Coller BS, Kalomiris E, Steinberg M, Scudder LE. Evidence that glycocalicin circulates in normal plasma. J Clin Invest. 1984;73:794-799.
53. Michelson AD, Gore JM, Rybak ME, Cola CA, Barnard MR. Effect of in vivo infusion of recombinant tissue-type plasminogen activator on platelet glycoprotein Ib. Thromb Res. 1990;60:421-424. [Medline] [Order article via Infotrieve]
54. Eikenboom JC, van der Meer FJM, Briet E. Acquired von Willebrand's disease due to excessive fibrinolysis. Br J Haematol. 1992;81:618-620. [Medline] [Order article via Infotrieve]
55. Mascelli MA, Edington TS, Kirby EP. Characterization of a fragment of bovine von Willebrand factor that binds to platelets. Biochemistry. 1986;25:6325-6335. [Medline] [Order article via Infotrieve]
56. Sugimoto M, Ricca G, Hrinda ME, Schreiber AB, Searfoss GH, Bottini E, Ruggeri ZM. Functional modulation of the isolated glycoprotein Ib binding domain of von Willebrand factor expressed in Escherichia coli. Biochemistry. 1991;30:5202-5209. [Medline] [Order article via Infotrieve]
57.
Alevriadou BR, Moake JL, Turner NA, Ruggeri ZM, Folie
BJ, Phillips MD, Schreiber AB, Hrinda ME, McIntire LV. Real-time
analysis of shear-dependent thrombus formation and its blockade
by inhibitors of von Willebrand factor binding to
platelets. Blood. 1993;81:1263-1276.
58. Vaughan DE, Braunwald E. Front-loaded accelerated infusions of tissue plasminogen activator: putting a better foot forward. J Am Coll Cardiol. 1992;19:1076-1078. [Medline] [Order article via Infotrieve]
59.
The GUSTO Investigators. An international
randomized trial comparing four thrombolytic strategies for
acute myocardial infarction. N Engl J
Med. 1993;329:673-682.
60. Lew AS, Cercek B, Lewis BS, Hod H, Shah PK, Ganz W. Efficacy of a two-hour infusion of 150-mg tissue plasminogen activator in acute myocardial infarction. Am J Cardiol. 1987;60:1225-1229. [Medline] [Order article via Infotrieve]
61. Tebbe U, Tanswell P, Seifried E, Feuerer W, Scholz K-H, Herrmann KS. Single-bolus injection of recombinant tissue-type plasminogen activator in acute myocardial infarction. Am J Cardiol. 1989;64:448-453.[Medline] [Order article via Infotrieve]
62.
Gemmill JD, Hogg KJ, MacIntyre PD, Booth N, Rae AP,
Dunn FG, Hillis WS. A pilot study of the efficacy and safety of
bolus administration of alteplase in acute myocardial
infarction. Br Heart J. 1991;66:134-138.
63. Tanswell P, Tebbe U, Neuhaus K-L, Glasle-Schwartz L, Wojcik J, Seifried E. Pharmacokinetics and fibrin specificity of alteplase during accelerated infusions in acute myocardial infarction. J Am Coll Cardiol. 1992;19:1071-1075. [Abstract]
64. Purvis JA, McNeill AJ, Siddiqui RA, Roberts MJD, McClements BM, McEneaney D, Campbell NPS, Khan MM, Webb SW, Wilson CM, Adgey AAJ. Efficacy of 100 mg of double-bolus alteplase in achieving complete perfusion in the treatment of acute myocardial infarction. J Am Coll Cardiol. 1994;23:6-10. [Abstract]
65. Agnelli G. New strategies for enhancing the speed and rate of coronary reperfusion. Am J Cardiol. 1993;72:51G-58G. [Medline] [Order article via Infotrieve]
66.
Diamond SL, Eskin SG, McIntire LV. Fluid flow
stimulates tissue plasminogen activator
secretion by cultured human endothelial cells.
Science. 1989;243:1483-1485.
67.
Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow
effects on prostacyclin production by cultured human
endothelial cells. Science. 1985;227:1477-1479.
68.
Buga GM, Gold ME, Fukuto JM, Ignarro LJ. Shear
stress–induced release of nitric oxide from
endothelial cells grown on beads.
Hypertension. 1991;17:187-193.
69.
Tsai H-M, Sussman II, Nagel RL. Shear stress
enhances the proteolysis of von Willebrand factor in normal
plasma. Blood. 1994;83:2171-2179.
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