© 1996 American Heart Association, Inc.
Articles |
Plasma Fibrinogen Inhibits Platelet Adhesion in Flowing Blood to Immobilized Fibrinogen
From the Departments of Haematology and Internal Medicine (J.J.Z.), University Utrecht, Utrecht, Netherlands.
Correspondence to Ph.G. de Groot, University Hospital Utrecht, Department of Haematology, G.03.647, PO Box 85500, 3508 GA Utrecht, Netherlands. E-mail j.vd.velde@digd.azu.nl.
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Abstract |
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Abstract The influence of variations in plasma fibrinogen concentration on platelet adhesion to immobilized fibrinogen was investigated in a parallel-plate perfusion chamber. At a shear rate of 1600 s-1 platelet adhesion decreased when increasing concentrations of purified fibrinogen were added to the plasma (IC50=1.5±0.2 g/L fibrinogen, n=24). Washed platelets reconstituted in a human albumin solution with red blood cells were more sensitive for soluble fibrinogen (IC50=0.4±0.1 g/L, n=5, P<.05). When platelet activation during circulation of the blood was minimized by using a single-passage perfusion system, an IC50 of 2.0±0.2 g/L was found (n=9). To exclude the possibility that the inhibition of fibrinogen was caused by irreversible changes in the fibrinogen molecule during the purification procedure, normal plasma was mixed in different ratios with plasma from a patient with congenital afibrinogenemia. Under these conditions, the plasma fibrinogen IC50 was 1.5±1.1 g/L. Absence of endogenous fibrinogen in the platelets of the patient resulted in an IC50 of 1.2±0.5 g/L for plasma fibrinogen. These results demonstrate that increased plasma fibrinogen concentrations inhibit platelet adhesion to fibrinogen under flow.
Key Words: platelet adhesion • fibrinogen • shear • glycoprotein IIb:IIIa
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Introduction |
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Ample evidence suggests that high plasma fibrinogen concentration is a risk factor for cardiovascular disease.1 2 3 In vitro, platelet hyperreactivity to aggregating agents has been reported in patients with very high plasma fibrinogen levels,4 and studies on the effects of different fibrinogen levels on platelet aggregation show an enhanced aggregability with increasing fibrinogen levels.5 6 7 8 9 10
The receptor for fibrinogen on the platelet membrane is the activation-dependent GPIIb:IIIa complex.11 12 13 GPIIb:IIIa is essential for both platelet aggregation and platelet adhesion to fibrinogen. GPIIb:IIIa on the membrane of nonactivated platelets does not bind soluble fibrinogen, which occurs only after the platelets are stimulated.8 14 15 16 17 18 19 However, fibrinogen adsorbed on a surface supports adhesion of nonactivated platelets via GPIIb:IIIa under static20 21 as well as under flow22 23 24 conditions. In vivo, fibrinogen deposition is found in normal intima, and it is abundantly present in atherosclerotic plaques.25 26 Fibrinogen is also a part of a thrombotic fibrin network. Moreover, after adsorption from circulating plasma, surface-bound fibrinogen serves as a major ligand for platelet adhesion to artificial surfaces, such as prosthetic valves, dialysis membrane, and vascular grafts.27 28 29 30
The interaction of nonactivated platelets with
immobilized fibrinogen is mediated via GPIIb:IIIa and
involves the same binding epitopes in fibrinogen as the interaction of
soluble fibrinogen with GPIIb:IIIa on activated platelets.
Both interactions can be inhibited by RGD-containing peptides and
fibrinogen-specific peptides corresponding to the carboxy terminus
of the 
-chain of fibrinogen.31 32 33 34 35 36 37 38 Although much
information is available on the influence of fibrinogen concentration
on platelet aggregation,5 6 7 8 9 it is not known whether
the plasma fibrinogen concentration also influences platelet
adhesion to immobilized fibrinogen. In this study we
describe the influence of different plasma fibrinogen concentrations on
platelet adhesion to fibrinogen in flowing whole blood. Our results
indicate that increased plasma fibrinogen decreases platelet
adhesion to fibrinogen.
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Methods |
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Fibrinogen-Coated Coverslips
One gram purified human fibrinogen (grade L) (KabiVitrum) was dissolved in 25 mL distilled deionized water at room temperature, dialyzed for 6 hours against distilled deionized water at room temperature, clarified by centrifugation, and stored at -20°C. The fibrinogen clottability was 95% to 99% when determined according to the method of Clauss.39 Fibrinogen purity was tested by using sodium dodecyl sulfate–polyacrylamide gel electrophoresis; the typical three-band pattern was present without visible contamination. The vWF antigen concentration, which was measured in the fibrinogen preparation, was <0.6 ng/mg fibrinogen. This vWF concentration did not influence platelet adhesion to immobilized fibrinogen. Fibrinopeptide A measurements (Malinckrodt Inc) in the purified fibrinogen solution revealed less than 0.2
mol FPA/1
mol fibrinogen. Eighteen-millimeter square glass coverslips were cleaned overnight in chromic acid, stored in 80% ethanol, and rinsed in distilled water. Fibrinogen was diluted to 0.1 g/L (determined by using absorbance measurements with an extinction coefficient of 1.6 mL·mg-1·cm-1)22 in 50 mmol/L ammonium acetate, pH 7.4, and sprayed onto coverslips with a Badger Model 100 airbrush. For experiments with a fibrin-coated surface, thrombin (Sigma) at a final concentration of 0.4 U/mL was added to the fibrinogen solution just before spraying.22 The coating density on the coverslip was 3 µg/cm2; at this concentration platelet adhesion did not change with increasing coating density. The fibrinogen-coated coverslips were blocked in 1% human serum albumin in phosphate-buffered saline (5 mmol/L phosphate buffer and 150 mmol/L NaCl, pH 7.4) for 30 minutes and kept in phosphate-buffered saline before use in perfusion experiments that were performed on the same day. No differences in reactivity were found between fibrin(ogen) coated on glass coverslips and fibrin(ogen) coated on Thermonox coverslips.
Perfusion Procedures
Perfusion experiments were performed in a parallel-plate
perfusion chamber.40 Whole blood obtained by
venipuncture from healthy volunteer donors who had taken no
medication within the preceding 10 days was anticoagulated with 1:10
(vol/vol) trisodium citrate (110 mmol/L). Perfusates were
prewarmed at 37°C for 10 minutes, and a total volume of 15 mL was
recirculated through the perfusion chamber for 3 or 5 minutes at wall
shear rates of 300 s-1 (34 mL/min) or
1600 s-1 (57 mL/min).
For single-passage perfusion experiments, blood (total volume, 25 mL) was drawn through a specially devised small perfusion chamber with a slit width of 2 mm and a height of 0.1 mm by using an infusion pump (Pump 22, Model 2400-004, Harvard Apparatus) placed distally to the perfusion chamber for 5 minutes at a shear rate of 1600 s-1.
After the perfusions, the system was rinsed with HBS (10 mmol/L HEPES and 150 mmol/L NaCl, pH 7.4). The coverslips were removed and rinsed again with HBS, then fixed in 0.5% glutaraldehyde/phosphate-buffered saline, dehydrated in methanol, and stained with May-Grünwald-Giemsa.40
To test the influence of platelet manipulation, platelets and
red cells were washed for perfusion experiments with reconstituted
blood samples.41 Platelets
(200x109/L) and red cells were suspended in a 4%
human albumin solution (Krebs-Ringer buffer, 5 mmol/L
-D-glucose, pH 7.4) at a total volume of 15 mL and a
hematocrit of 40%, vol/vol.
The plasma fibrinogen concentration of blood samples was increased by adding human SPF to the samples, after which the plasma fibrinogen concentration was determined.39 Control samples were corrected for the increase in volume by adding HBS. Platelet adhesion to fibrinogen revealed a monolayer of platelets with a morphology ranging from dendritic to fully spread platelets. The morphology of adherent platelets did not change after addition of fibrinogen or HBS compared with control samples of whole blood without the extra addition.
Blood samples of the perfusate were taken before and after perfusion experiments to measure single-platelet disappearance as a check on platelet microaggregate formation in the circulation.42 Platelets were counted in a Platelet Analyzer 810 (Baker Instruments) with apertures set between 3.2 and 16 µm3.42 Additional samples for ß-TG measurements in plasma were taken before and after perfusions as a check on platelet activation. The ß-TG concentration was measured by using an ELISA kit (Diagnostica Stago) and determined according to the instructions of the manufacturer. For citrated blood, the ß-TG concentration 15 minutes after the blood was drawn was 30 ng/mL; this concentration increased during storage up to 200 ng/mL within 2 hours but did not increase further during the next 2 hours, the time necessary for the experiments.
Mixing Experiment With Blood From an Afibrinogenemic
Patient
Blood from a patient with congenital afibrinogenemia
(225x109 platelets/L) and from a control donor
(165x109 platelets/L) with the same ABO blood group
was used. The patient's plasma fibrinogen concentration was 
2
µg/mL and platelet fibrinogen concentration, 10
µg/109 platelets, as measured by ELISA with a
peroxidase-conjugated rabbit anti-human fibrinogen antibody
(Dakopatts). 1,2-Diphenylenediamine (Merck) was
used as a substrate with absorbance measurements at 490 nm in a Vmax
microtiter plate reader (Molecular Devices). The control subject's
plasma fibrinogen concentration was 4.1 g/L, and the platelet
fibrinogen concentration was within the normal range. The vWF:Ag
concentrations of the patient's and control subject's plasma were 15
and 13 µg/mL, respectively, as measured by ELISA with a
peroxidase-conjugated rabbit anti-human vWF antibody
(Dakopatts). Platelets from the afibrinogenemic patient contained
vWF within the normal concentration range.
Platelet-poor plasma samples from the patient and the control donor were obtained after centrifugation of the blood at room temperature for 10 minutes at 3000 rpm, after which the samples were mixed in different ratios. The mixed plasma samples were then returned to the blood cells of the afibrinogenemic patient or the control donor to obtain blood samples with different plasma fibrinogen concentrations. The plasma fibrinogen concentration of the reconstituted blood samples was determined according to the method of Clauss.39 The reconstituted blood samples were gently mixed, and perfusions were performed for 3 minutes over a fibrinogen-coated surface at a shear rate of 1600 s-1. A perfusion time of 3 instead of 5 minutes was chosen due to the very high platelet surface coverages obtained with the patient's blood samples.
Evaluation of Platelet Adhesion
The extent of platelet adhesion was determined by light
microscopy of the stained coverslips at a magnification of x1000 with
the aid of an image analyzer (AS 40-10) that was interfaced to
the microscope.22 The results are expressed as extent of
relative adhesion or as percentage surface coverage by adherent
platelets.
The fibrinogen concentration at which half-maximal inhibition occurred, the IC50, was calculated by the competitive inhibition model22 : F(I)=IC50/(IC50+[Fgn]), where F(I) is the relative extent of adhesion in the presence of extra soluble fibrinogen (Fgn) compared with the control value in the absence of extra soluble fibrinogen. The statistical significance of the difference between means in the presence or absence of fibrinogen was calculated by using the t test.
Viscosity Measurements
The viscosities of the plasma and whole blood samples with
increased fibrinogen concentrations were determined in a Contraves Low
Shear 30 viscosimeter (Contraves AG) at 11 different shear rates
ranging from 6.6 to 125.5 s-1 at
37°C.41
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Results |
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Increasing Plasma Fibrinogen Levels With Purified Fibrinogen
The influence of soluble fibrinogen on platelet adhesion to immobilized fibrinogen was investigated after adding human SPF to whole blood followed by a 5-minute perfusion at a shear rate of 1600 s-1. Increasing concentrations of SPF added to whole blood resulted in a progressive decrease in platelet adhesion to immobilized fibrinogen (Fig 1
). An average of 1.5±0.2 g/L (n=24) extra fibrinogen
added to whole blood inhibited platelet adhesion by 50%
(IC50) (Table 1). When fibrin was used as
the adhesive surface an IC50 of 1.8±0.2 g/L (n=7) for
soluble fibrinogen was found (Table 1). When perfusions were performed
at a shear rate of 300 s-1, an
IC50 of 2.1±0.3 g/L was found (not shown). No significant
inhibition was found when the extracellular matrix of
endothelial cells was used as the adhesive surface (not
shown).
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, ——), with normal blood in the single-passage perfusion
system (
, ----), and with washed
platelets and red blood cells reconstituted in a human
albumin solution in the recirculating perfusion system
(
, ----). Perfusions were performed at a
wall shear rate of 1600 s-1 for 5 minutes
at 37°C. Results are expressed as the percent fibrinogen
surface covered by adherent platelets normalized by control values
without added fibrinogen. Lines were calculated from the equation for
the competitive inhibition model described in "Methods."
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The initial plasma fibrinogen concentration in whole blood from control
donors ranged between 1.7 and 3.4 g/L. Besides fibrinogen, other
GPIIb:IIIa binding proteins are also present in plasma. To study
the effect of fibrinogen alone on platelets we used a system
without plasma proteins. For this purpose, platelets and red blood
cells were washed and suspended in a buffer with 4% human
albumin and a hematocrit of 40%, vol/vol. Human SPF was then
added to the reconstituted blood samples to obtain different fibrinogen
concentrations (range, 0 to 2.9 g/L). Platelet adhesion was
strongly inhibited when fibrinogen was present (Fig 1
), and a 50%
inhibition of platelet adhesion by SPF was found at 0.4±0.1 g/L
fibrinogen (n=5, P<.05) (Table 1). Apparently washed
platelets were more sensitive to SPF than platelets in whole
blood; this difference was statistically different from the whole blood
system (Table 1). The IC50 of soluble fibrinogen for the
adhesion of washed platelets to a fibrin surface was 0.9±0.2 g/L
fibrinogen (n=3, P<.05), which was also significantly
different from the whole blood system (Table 1).
When platelets become activated ß-TG is secreted into
plasma. The ß-TG levels doubled when SPF was added to whole blood,
and the addition of fibrinogen resulted in a small increase in
single-platelet disappearance in the blood samples when
measured after perfusion (not shown). Thus, the addition of fibrinogen
might induce some platelet activation in the recirculating
perfusion system. To prevent platelet activation and microaggregate
formation as much as possible, we performed experiments with a
single-passage perfusion system. With this system the addition of
SPF to the blood also decreased platelet adhesion (Fig 1).
Platelet activation and microaggregate formation during perfusion
with and without added fibrinogen were negligible as determined by
ß-TG measurements and the number of single platelets,
respectively (not shown). A 50% inhibition was found when 2.0±0.2 g/L
extra fibrinogen was added to nonrecirculating whole blood (n=9) (Table 1); this result was not statistically different from the recirculating
whole blood system (IC50=1.5±0.2 g/L).
High-molecular-weight proteins such as fibrinogen play an
important role in determining plasma viscosity. An impaired transport
of platelets to the boundary layer might in this respect result in
decreased platelet adhesion. At low plasma viscosities platelet
adhesion decreases with increasing plasma viscosity, while at higher
plasma viscosities platelet adhesion increases.41 The
latter phenomenon is explained by an effect of the plasma viscosity on
the red blood cell rigidity influencing platelet transport. The
addition of soluble fibrinogen increased the whole blood viscosity and
the plasma viscosity (>1.12 mPaxs) (Table 2) in a
viscosity range at which adhesion should increase,41 but
in our experiments the platelet adhesion decreased (Fig 1). Thus,
the observed inhibition of platelet adhesion by soluble fibrinogen
was more likely the result of a direct effect of fibrinogen on
platelets.
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Platelet Adhesion With Blood From an Afibrinogenemic
Patient
To exclude the possibility that the influence of fibrinogen found
on platelet adhesion was due to changes in the fibrinogen molecule
as a consequence of the purification procedure, the blood of a patient
with congenital afibrinogenemia was used. To obtain plasma fibrinogen
levels, plasma from this patient was mixed with control subject plasma
in different ratios and added back to the blood cells. Perfusions at
1600 s-1 over fibrinogen-coated
coverslips with whole blood from only this patient resulted within 5
minutes in a very high surface coverage of 90±1%. The platelets
adhering to the fibrinogen-coated surface were extensively spread
with frequently dendritic platelets for both patient as well as
control subject platelets.
Plasma samples from a control donor and the afibrinogenemic patient
were mixed to obtain various plasma fibrinogen levels and returned to
the blood cells of both subjects. Experiments in which plasma from the
control subject was mixed with the subject's own cells showed that
this reconstitution procedure had no effect on the level of
platelet adhesion (not shown). The reconstituted blood samples of
control subject and patient cells with different plasma fibrinogen
concentrations were perfused for 3 minutes over a fibrinogen-coated
surface. Platelet adhesion of the patient's platelets
decreased from 79±3% to 9±4% surface coverage when the plasma
fibrinogen concentration increased from 0 to 3.7 g/L (Fig 2). Platelet adhesion of platelets from the
control subject decreased from 24±7% to 7±4% surface coverage when
plasma fibrinogen increased from 0.7 g/L to 4.1 g/L (Fig 2). These
results correspond to an IC50 for plasma fibrinogen of
1.2±0.5 and 1.5±1.1 g/L for the patient's and control subject's
platelets, respectively.
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) and afibrinogenemic platelets
(
) at different plasma fibrinogen concentrations. Perfusions were
performed at a wall shear rate of 1600
s-1 for 3 minutes at 37°C.
Results are expressed as percent fibrinogen surface covered by adherent
platelets (top) or as relative adhesion (percent of the surface
covered with platelets compared with the coverage at the lowest
plasma fibrinogen concentration) (bottom). See "Methods" for a
detailed description of the procedure used to obtain different plasma
fibrinogen concentrations.Addition of 2.4 g/L purified fibrinogen resulted in a decrease in platelet adhesion to a surface coverage of 7±1%. When 2.4 g/L purified fibrinogen was added to blood from a control donor with an initial plasma fibrinogen concentration of 2.3 g/L, platelet adhesion decreased from 31±8% to 5±2% surface coverage. No change in platelet morphology of the adherent platelets was found after addition of soluble fibrinogen compared with the control condition.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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A possible mechanism by which an elevated plasma fibrinogen concentration increases the risk of cardiovascular disease may be related to its influence on platelet function. High plasma fibrinogen concentrations increase the sensitivity of platelets to agonists.4 5 7 8 10 However, no information is available on the influence of plasma fibrinogen levels on the adhesion of platelets. Here we have studied the influence of plasma fibrinogen on platelet adhesion by using fibrinogen as an adhesive surface. The adhesion of platelets to immobilized fibrinogen is completely dependent on the presence of platelet membrane receptor GPIIb:IIIa and at high shear rates on a secondary interaction between GPIb and vWF. Here we show that platelet adhesion to immobilized fibrinogen or fibrin is strongly inhibited at both low and high shear rates when the plasma fibrinogen concentration is increased with purified fibrinogen (Fig 1
). A 50% inhibition was observed at 1.5±0.2 g/L fibrinogen
(Table 1). Because the inhibition by fibrinogen is seen at both low and
high shear rates, the inhibition must be the result of a direct effect
of plasma fibrinogen on GPIIb:IIIa or an influence on plasma viscosity.
High-molecular-weight proteins such as fibrinogen play an
important role in determining plasma viscosity,43 44 45 and
increasing the plasma fibrinogen concentration would result in
decreased platelet adhesion. However, a study describing the effect
of different plasma viscosities on platelet adhesion has shown that
at a plasma viscosity >1 mPaxs the platelet adhesion increased
due to changes in the red blood cell rigidity.41 In our
experiments the plasma viscosity was >1 mPaxs and increased after
addition of fibrinogen, while platelet adhesion decreased. The
inhibition of platelet adhesion was therefore likely to be due to
direct effects of soluble fibrinogen on platelets rather than an
effect of the changes in viscosity. When the extracellular matrix of
endothelial cells was used as the adhesive surface, no
inhibition was found with extra added fibrinogen. At the shear rates
used, platelet adhesion to the extracellular matrix was not
mediated by GPIIb:IIIa. This observation also supports the concept that
plasma fibrinogen directly influences the availability of GPIIb:IIIa on
the platelet membrane for interaction with immobilized
fibrinogen. This conclusion is surprising, because it is generally
accepted that plasma fibrinogen (2.5 g/L) does not interact with
GPIIb:IIIa on nonactivated platelets. One possibility
might be that the effects seen were due to impurities in the soluble
fibrinogen preparation. Experiments in which plasma from an
afibrinogenemic patient was mixed with normal plasma to vary the
fibrinogen concentration without the use of purified fibrinogen,
however, showed similar results as with purified fibrinogen. Another
possible explanation for the inhibition of platelet adhesion by
plasma fibrinogen is that platelet adhesion under flow conditions
requires a preactivation of the platelets before they can interact
via GPIIb:IIIa with immobilized fibrinogen. This is
unlikely because prostaglandin E2–treated
platelets also adhere to fibrinogen via GPIIb:IIIa.46
Collecting the blood in citrate, without the presence of other
platelet-activation inhibitors, always results in
slightly activated platelets as measured by the release of
ß-TG. It should be noted that for platelet aggregation studies
blood is also collected using citrate as anticoagulant and that under
these experimental conditions no significant binding of fibrinogen to
nonactivated platelets occurs.8 17
Nevertheless, we cannot completely exclude the possibility that in
whole-blood perfusion experiments trace amounts of ADP, which
modulates GPIIb:IIIa, are released from red cells. The mechanism of
GPIIb:IIIa-mediated inhibition by soluble fibrinogen is not
completely clear. The plasma fibrinogen concentration is relatively
high (2.5 g/L), and although the affinity of
nonactivated platelets for soluble fibrinogen is very
low,17 a small part of GPIIb:IIIa may already be occupied
by plasma or platelet-derived fibrinogen, thereby inhibiting
the availability of GPIIb:IIIa for adhesion. This hypothesis is
supported by the observation that the effect of increased plasma
fibrinogen concentrations was stronger when washed platelets were
resuspended in a human albumin solution with red blood cells
(Table 1). Additional platelet activation due to washing resulted
in a stronger inhibition of platelet adhesion by SPF.
When experiments were performed with blood from an afibrinogenemic
patient, platelet adhesion to fibrinogen was very high. Adding
fibrinogen to the plasma also decreased platelet adhesion of the
patient's blood samples, but the adhesion was still higher than when
perfusions were performed with the control donor's blood with
comparable plasma fibrinogen concentrations (Fig 2). The difference
between the two lies most likely in the absence of
endogenous fibrinogen in the patient's platelets.
These observations suggest that in addition to plasma fibrinogen,
endogenous platelet fibrinogen also influences the
absolute platelet adhesion to fibrinogen. Indeed, for
agonist-activated platelets, endogenous
fibrinogen will be expressed on the platelet
surface.47 48 49 50 In a similar way, platelet vWF
influences platelet adhesion to fibrin.51
In conclusion, the experiments presented here show that platelet adhesion to fibrinogen and fibrin is inhibited by soluble fibrinogen and that this inhibition is most likely due to competition between soluble fibrinogen and surface-bound fibrin(ogen) for GPIIb:IIIa. The significance of this observation remains to be established. After vessel injury platelets adhere to the collagen and vWF present in the subendothelium, a process dependent on GPIIb:IIIa only at very high shear rates. However, adhesion of circulating platelets to the strands of a developing fibrin clot would contribute to the formation of a life-threatening thrombus. Moreover, platelet adhesion to grafts is predominantly mediated via fibrinogen.52 53 Platelet adhesion plays a significant role in thrombosis on medium- and small-diameter prosthetic vascular grafts and is responsible for the poor outcome of small-diameter graft transplantation. The observations presented here add a new element to the regulation of GPIIb:IIIa-mediated platelet adhesion. Besides the levels of plasma and platelet vWF, platelet number, blood viscosity, hematocrit, and plasma [Mg2+], plasma fibrinogen concentration also seems to determine the level of platelet adhesion to fibrin strands and fibrinogen adsorbed to prosthetic grafts.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by a research grant (89.206) from the Netherlands Heart Foundation.
Received July 3, 1995; accepted January 30, 1996.
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