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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3788-3795
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Sol Sherry Thrombosis Research Center and the Department of
Microbiology and Immunology, Temple University School of Medicine, and
the Department of Medicine, University of Pennsylvania School of
Medicine, Philadelphia, PA; and Pharmacia & Upjohn Inc, Kalamazoo, MI.
High molecular weight kininogen (HK) and its cleaved form (HKa) have
been shown to bind to neutrophils. Based on studies using monoclonal
antibodies (mAbs), we postulated that CD11b/CD18 (Mac-1) might be the
receptor on the neutrophils for binding to HK/HKa. However, the direct
interaction of HK/HKa and Mac-1 had not been demonstrated. We therefore
transfected HEK 293 cells with human Mac-1. Cell binding assays using
fluorescein isothiocyanate-labeled HKa showed increased binding to the
Mac-1 transfected cells compared with the control transfected cells.
The binding was specific because unlabeled HKa, Mac-1-specific
antibody, and fibrinogen can inhibit the binding of biotin-HKa to Mac-1
transfected cells. HKa bound to Mac-1 transfected cells (20 000
molecules/cell) with a Kd = 62 nmol/L. To
demonstrate directly the formation of a complex between HKa and Mac-1,
we examined the interaction of HKa and purified Mac-1 in a cell-free
system using an IAsys resonant mirror optical biosensor. The
association and dissociation rate constants (kon and
koff, respectively) were determined, and they yielded a
dissociation constant (Kd) of 3.2×10
High molecular weight kininogen (HK) is an abundant
plasma protein (670 nmol/L) coded for by a gene with 10 exons
containing 6 domains (Mr = 20 kd).1 HK, together with 2 other plasma proteins, prekallikrein and factor XII, are called the
contact system in the blood coagulation cascade because they have been
found to require contact with artificial, negatively charged surfaces
for activation of the zymogens in vitro. Besides its essential role in
the activation of coagulation when blood contacts foreign surfaces, such as in cardiopulmonary bypass,2 HK is a multifunctional protein. HK can interact with blood and vascular cells including platelets,3 neutrophils,4 monocytes, and
endothelial cells.5,6 On each cell type, HK serves a
discrete biologic function. It prevents the activation of platelets by
inhibiting the calcium-activated cysteine protease
calpain,7 and it prevents the binding of thrombin.8 Platelets have been shown to contain HK, which
can be expressed on the exposed membrane surface of activated
platelets.9-11 HK that circulates with plasma prekallikrein
in a binary complex serves as an acquired receptor for kallikrein on
the surfaces of neutrophils,12 allowing kallikrein to
stimulate neutrophil aggregation13 and
degranulation.14 Endothelial cell-bound HK is a substrate
for plasma kallikrein, which cleaves HK to a 2-chain disulfide-linked
molecule, HKa, and releases the nonapeptide bradykinin. It has been
shown previously that HK/HKa can compete with an adhesive protein,
fibrinogen, for binding to neutrophils,12 and that, as a
consequence, it inhibits the adhesion of neutrophils to
fibrinogen-coated surfaces under radial flow conditions.15 It has also been shown that HK/HKa binds specifically, saturably, and
reversibly to neutrophils in the presence of
Zn2+.4 However, the cell surface receptor for
HK/HKa binding to neutrophils is unclear. Although it is suggested to
be the integrin Mac-1 (CD11b/CD18) based on the results of antibody
blocking studies,12 a direct interaction between HK/HKa and
Mac-1 integrin has not been demonstrated. Moreover, the occurrence of
another receptor on neutrophils for HKa, the urokinase receptor, made
it important to examine further the hypothesis that HK/HKa directly
binds to Mac-1.
Integrins are a family of adhesion molecules serving functions involved
in cell-cell and cell-extracellular matrix interactions. Mac-1
(CD11b/CD18), LFA-1 (CD11a/CD18), and p150,95 (CD11c/CD18), are
leukocyte integrins. Mac-1, also known as complement receptor type 3 (CR3), is a heterodimeric receptor that is primarily expressed on
monocytes, macrophages, neutrophils, and natural killer cells. The
function of Mac-1 was initially described as the ability to bind iC3b
(a cleaved form of C3b), and therefore to mediate the phagocytosis and
lysis of iC3b-coated erythrocytes,16 and to contribute to
elevated natural killer cell activity against iC3b-coated target
cells.17 In addition to the function of Mac-1 in the immune
defense system through binding to its ligand, iC3b, Mac-1 mediates a
number of cell-cell and cell-extracellular matrix interactions in
which iC3b is not involved, such as neutrophil
aggregation18 and chemotaxis,19 and neutrophil
adhesion to human umbilical vein endothelial cells.20 These
observations suggested that Mac-1 is a multifunctional receptor.
Through its ability to recognize multiple and unrelated ligands,
including iC3b,16,17 fibrinogen,21 factor
X,22 and the counter-receptor intercellular adhesion molecule-1 (ICAM-1),23 Mac-1 plays critical roles in cell
adhesion, migration, and invasion, which are central to inflammation,
immune responses, vascular biology, hemostasis, and thrombosis. The
binding of Mac-1 to fibrinogen/fibrin results in the adhesion of
neutrophils/monocytes to the sites of fibrin deposition, and the
binding of Mac-1 to ICAM-1 causes the adhesion of neutrophils/monocytes
to the endothelium. After binding to the zymogen of factor X, Mac-1
coordinates the activation of factor X independent of tissue factor and
factor VII; this is followed by rapid fibrin formation.24
Therefore, the ability to interfere with Mac-1-mediated leukocyte
adhesion functions offers many opportunities for therapeutic
intervention in diseases as diverse as thrombosis, inflammation, and cancer.
In this report, we show, for the first time, that cleaved high
molecular weight kininogen, HKa, binds directly to Mac-1 both on cells
and in a purified system, and we demonstrate that HKa inhibits
Mac-1-mediated adhesion to fibrinogen and ICAM-1. This study may
provide additional information for drug design in anti-adhesion therapy.
Materials
Cell culture and transfection
Flow cytometry and cell sorting Cells were incubated with Mac-1-specific antibody LM2/1 (ascitic fluid, mouse IgG1, antihuman CD11b)25 for 45 minutes on ice. After they were washed 3 times with cold Hanks' balanced salt solution buffer with 1% bovine serum albumin (BSA), the cells were incubated with FITC-conjugated goat antimouse IgG for 30 minutes on ice. After they were washed 4 times, the cells were fixed with 1% paraformaldehyde in PBS overnight and then analyzed on a flow cytometer. For cell sorting, the cells were directly analyzed and sorted on a flow cytometer without fixation with paraformaldehyde.Labeling of kininogen For binding of cleaved human high molecular weight kininogen (HKa) to HEK 293 cells, HKa was labeled with FITC or biotin. Briefly, a total reaction volume of 1 mL containing 1 mg HKa, 1 mg celite-FITC (Sigma) or 1 mg NHS-LC-biotin was adjusted to pH 8.0 with 5% sodium bicarbonate solution. The reaction mixture was incubated for 20 minutes at room temperature with intermittent vortexing for labeling with FITC or for 2 hours on ice for labeling with biotin. The labeled HKa (FITC-HKa or biotin-HKa) was purified by Sephadex G-25 spin column (Bio-Rad Laboratories, Hercules, CA) and eluted with PBS. The labeled protein retained more than 95% of its procoagulant activity.Binding assays Unstimulated HEK 293 cells (4×106/mL), transfected with Mac-1 or control, were washed with binding buffer (10 mmol/L HEPES, pH 7.2, 137 mmol/L NaCl, 4 mmol/L KCl, 50 µmol/L ZnCl2, and 0.1% gelatin) and incubated with FITC-HKa at indicated concentrations for 30 minutes at room temperature. Nonspecific binding was measured by including 10 mmol/L EDTA in the binding assay. After they were washed, the cells were fixed with 1% paraformaldehyde in PBS and then analyzed on an Epics Elite flow cytometer (Coulter Diagnostics, Hialeah, FL). Alternatively, the binding reactions were carried out in wells of a filtration 96-well plate containing polyvinylidene difluoride (PVDF) membrane (pore size, 1.2 µm; Millipore, Bedford, MA). After filtration, the fluorescence of cell bound FITC-HKa was measured on a Cytofluor 2350 fluorescence plate reader (Millipore) with a 485-nm excitation filter and a 530-nm emission filter.Inhibition of biotin-HKa binding to Mac-1-HEK 293 cells Mac-1-HEK 293 cells were eluted from a confluent monolayer culture dish with PBS containing 5 mmol/L EDTA and washed with binding buffer (10 mmol/L HEPES, pH 7.2, 137 mmol/L NaCl, 4 mmol/L KCl, 50 µmol/L ZnCl2, 1 mmol/L MgCl2, 1 mmol/L CaCl2, and 0.1% gelatin). Cells (4×106/mL) in the same buffer were incubated with biotin-HKa (10 nmol/L) for 45 minutes at room temperature in the absence or presence of unlabeled HKa, antibodies, or fibrinogen at indicated concentrations. After the addition of avidin-FITC and continued incubation for 10 minutes, 3 aliquots from each reaction were transferred to the wells of a filtration 96-well plate containing PVDF membrane (pore size, 1.2 µm; Millipore). The wells were pre-wet with binding buffer for 2 hours. After filtration, the fluorescence of cell bound biotin-HKa was measured on a Cytofluor 2350 system (Millipore). The relative amount of biotin-HKa bound to the cells in the presence of inhibitors was determined by comparison with biotin-HKa alone.Adhesion assays HEK 293 cells transfected with Mac-1 used for adhesion assays were labeled with 5-chloromethylfluorescein diacetate (CMFDA). Briefly, confluent cells were eluted from dishes and resuspended in serum-free medium containing CMFDA (10 µmol/L) and incubated at 37°C for 30 minutes. Free probes were removed by washing with buffer used for adhesion assays (10 mmol/L HEPES, pH 7.2, 137 mmol/L NaCl, 4 mmol/L KCl, 50 µmol/L ZnCl2, 1 mmol/L MgCl2, 1 mmol/L CaCl2, and 0.1% gelatin). Ligands, fibrinogen (50 µg/mL), ICAM-1 (10 µg/mL), or fibronectin (15 µg/mL) were immobilized on 96-well Immulon II microtiter plates (Dynatech Laboratories, Chantilly, VA) at room temperature for 2 hours. The wells were then blocked with gelatin. Labeled cells (3-4 × 105/well) were added to wells and incubated in the presence or absence of indicated HKa for 60 minutes. Unbound cells were removed by washing with adhesion buffer 3 times. Adherent cells were lysed in 0.1 N NaOH, 0.2% sodium dodecyl sulfate (SDS), and 0.5% Triton X-100. The plates were read on a Cytofluor 2350 system (Millipore).Purification of Mac-1 from human neutrophils Mac-1 was purified from human neutrophils with slight modifications to the immunoaffinity purification procedure previously described.26 Briefly, human granulocytes were obtained from normal, healthy volunteers by leukopheresis using a Fenwal CS3000 blood cell separator (Baxter Healthcare, Deerfield, IL) followed by dextran density gradient. The enriched neutrophil product was then lysed with 0.05 mol/L Tris, pH 8.0, 2 mmol/L MgCl2, 1.0% Triton X-100, 0.15 mol/L NaCl, 5 mmol/L DFP, and 0.2 TIU/mL aprotinin for 1 hour at 4°C by gentle stirring before the resultant lysate was centrifuged at 50 000g to remove insoluble material. The clarified lysate was loaded by batch onto LM2/1 immunoaffinity resin (10 mL CNBr-activated Sepharose (Pharmacia Biotech, Piscataway, NJ) coupled with 5 mg LM2/1/mL resin) and incubated 16 hours at 4°C with gentle rotation. The resin was then loaded into a column (1.0×13 cm), and sequentially washed with 20 column volumes (CV) lysis buffer, 10 CV lysis buffer containing 1.0 mol/L NaCl, and 10 CV lysis buffer substituting 1.0% n-octyl glucoside for the Triton X-100. Mac-1 was eluted with 5 CV 0.1 mol/L sodium acetate, pH 4.0, 2 mmol/L MgCl2, 0.15 mol/L NaCl, and 1.0% n-octyl glucoside by collecting 1.0-mL fractions in polypropylene microcentrifuge tubes containing neutralizing buffer (10% by volume of 2 mol/L Tris, pH 9.0). Aliquots of the eluate fractions were immediately assessed by SDS-polyacrylamide gel electrophoresis (4%-15% gradient gels), and they showed 2 bands of equal density (MWt, 160 000 and 95,000). Peak fractions were pooled and underwent dialysis against 10 mmol/L HEPES, 0.137 mol/L NaCl, 4 mmol/L KCl, 11 mmol/L D-glucose, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 0.005% Tween-20 for 16 hours at 4°C. The final protein preparation was sectioned into aliquots and stored at 80°C until used.
Protein concentration was determined using amino acid analysis.
Real-time bimolecular interaction assay Analysis of Hka-Mac-1 interaction was carried out with purified human Mac-1 on an IAsys resonant mirror optical biosensor (Affinity Sensors, Cambridge, UK). In this assay, biotinylated HKa was immobilized on the sensor chip surface by using an IAsys biotin cuvette (FCB-0401; Affinity Sensors) and the accompanying protocol. Briefly, the biotin surface of the cuvette was washed with PBS/Tween-20 buffer, and streptavidin was added at a concentration of 10 µg/mL. After streptavidin was captured on the surface, Mac-1 was added to test whether there was any nonspecific binding of Mac-1 to streptavidin. No binding of Mac-1 to streptavidin was observed. Biotinylated HKa was then added to the streptavidin-captured surface, and the amount of immobilized HKa was equivalent to a signal of 400 arc seconds. The maximum signal (Rmax) expected on Mac-1 binding for the saturation of 90% of HKa binding sites was calculated from the molecular weight ratio of analyte (Mac-1)/captured ligand (HKa) and a stoichiometry of 1. The calculated Rmax is 800 arc seconds. Because of the random biotinylation and steric hindrance factors that may reduce the stoichiometry of 1 to a fraction, a lower effective Rmax is expected (see "Results"). This cuvette immobilized with HKa was stored refrigerated in binding buffer and used in subsequent experiments for examining the interaction of Mac-1 and HKa. Purified Mac-1 was added at the indicated concentration, and the association was monitored. The binding buffer used was: 10 mmol/L HEPES, pH 7.2, 137 mmol/L NaCl, 4 mmol/L KCl, 50 µmol/L ZnCl2, and 0.05% Tween-20. In the dissociation phase, Mac-1 solution was replaced with the binding buffer, and the dissociation of the binding was monitored. The HKa surface was regenerated by the addition of 10 mmol/L EDTA and 0.5 mol/L NaCl and was followed by washing with binding buffer.
Binding of HKa to HEK 293 cells transfected with Mac-1 The direct interaction between HKa and Mac-1 was examined using HEK 293 cells stably transfected with a vector containing the cDNA for human Mac-1. Little signal was detected on control transfected HEK 293 cells with monoclonal antibody LM2/1, specific for Mac-1, as demonstrated by flow cytometry (Figure 1A). In contrast, the transfected HEK 293 cells contained 2 populations of cell surface Mac-1 expression (Figure 1B). HEK 293 cells expressing high and low levels of surface human Mac-1 were separated using FACSorting technique, as shown in Figure 1C,D. HEK 293 cells expressing high levels of surface human Mac-1 were used in subsequent experiments, and control transfected HEK 293 cells were used in control experiments. A concentration-dependent binding of FITC-Hka to the Mac-1 transfected HEK 293 cells was observed but was not found using the control transfected cells when varying concentrations of FITC-HKa were used in a cell-binding assay (Figure 2). FITC-HKa specifically bound 3 times more to the Mac-1 transfected HEK 293 cells than to the control transfected cells (Figure 2). As a control, FITC-labeled BSA showed no binding to HEK 293 cells transfected with Mac-1 in the same type of binding assays (data not shown). In the presence of 10 mmol/L EDTA, the binding of FITC-HKa to Mac-1 transfected HEK 293 cells was inhibited 70% (Figure 2), indicating that the binding was metal dependent. Using the Scatchard plot of bound/free versus bound, we found evidence for a single class of binding site with Kd = 62 nmol/L and Bmax = 3.1 fmoles/105 cells (20 000 molecules per cell) in the Mac-1 transfected HEK 293 cell system.
Inhibition of biotin-HKa binding to Mac-1 transfected HEK 293 cells The specificity of biotin-HKa binding to Mac-1 transfected cells was examined using nonbiotinylated HKa in a competition binding assay. Figure 3 showed a concentration-dependent inhibition of biotin-HKa binding to Mac-1 transfected cells by unlabeled HKa. In the presence of 50-fold excess of unlabeled HKa, the binding of biotin-HKa to Mac-1 transfected cells was reduced to 20%. Fibrinogen is a known ligand for binding to Mac-1.21 A concentration-dependent inhibition of biotin-HKa binding to Mac-1 transfected cells by fibrinogen was observed (Figure 4), indicating that fibrinogen competed with biotin-HKa in binding to Mac-1 transfected cells. Antibody 2LPM19c, a monoclonal antibody against the µ
subunit of Mac-1, inhibited the binding of biotin-HKa (10 nmol/L) to
Mac-1-HEK cells by more than 90% with a 50% inhibitory concentration
of 10 ± 3 µg/mL (Figure 4), indicating that the interaction
between HKa and Mac-1 transfected cells was mediated primarily by the µ subunit of Mac-1.
Effect of divalent cations on the HKa binding to Mac-1 To determine the effect of divalent cations on the HKa and Mac-1 interaction, binding assays of FITC-HKa to Mac-1 transfected or untransfected HEK 293 cells were performed in the presence of different divalent cations as indicated. In each binding assay, cells were preincubated with buffer containing 10 mmol/L EDTA for 10 minutes at 37°C and then washed with binding buffer without any divalent cation. As shown in Figure 5, in the presence of Zn2+, the binding of HKa to Mac-1 transfected HEK 293 cells increased 2-fold. In the presence of Mn2+, the binding of HKa to Mac-1 transfected HEK 293 cells was increased; however, the binding of HKa to untransfected cells was also increased. There is no significant difference in HKa binding to Mac-1 transfected cells with or without the presence of Mg2+.
Interaction of HKa and purified Mac-1 We have examined the interaction of HKa and purified Mac-1 using an IAsys resonant mirror optical biosensor (Affinity Sensors). In this type of binding assay, biotinylated HKa was immobilized on the sensor chip surface by binding to streptavidin that had been pre-captured on the surface of an IAsys biotin cuvette (Affinity Sensors). Before HKa was immobilized on the surface of the cuvette, a control experiment was performed in which purified Mac-1 was added to the streptavidin surface. No interaction was detected between the streptavidin surface and Mac-1 at the highest concentration used in this experiment (data not shown). Purified Mac-1 was then added at indicated concentrations to the Hka-captured surface, and the response (in arc seconds) versus time (in seconds) was recorded. An overlay of sensor-grams for the binding of Mac-1 to the HKa surface is shown in Figure 6A. Sensor-grams show 2 phases: an association phase, detected when purified human Mac-1 was added and was allowed to bind to the immobilized HKa (20-300 seconds), and a dissociation phase, in which the Mac-1 solution was replaced with buffer (300-400 seconds). The association phase was linearized according to equation 3, and a plot of dR/dt versus R is shown in Figure 6B. A replot of the slope of these lines (ks) against Mac-1 concentration gives a straight line (Figure 6C). The slope of this straight line equals the association rate constant (kon) of 5.6 × 106 M 1s1. The
dissociation phase was analyzed according to equation 5. Figure 6D
shows a plot of ln(R1/Rn) versus time using the
dissociation phase data of the highest concentration of Mac-1. This
plot did not fit the model of single exponential decay. To estimate the dissociation rate constants, the plot ln(R1/Rn)
versus time was divided into fast and slow phases (phases 1 and 2, respectively). The dissociation rate constant (koff)
calculated from the slope of the first 20 seconds of this plot (phase
1) was 18.1×103
s1. Then the equilibrium dissociation
constant (Kd), calculated from the ratio of the rate
constants, was 3.2 nmol/L. Independent evaluation of Kd
from the steady state binding signal resulted in an estimated value of
5.26 nmol/L (R2 = 0.95; Scatchard plot not shown).
Mac-1 transfected HEK 293 cells binding to HKa-immobilized surface Specific binding of HKa to Mac-1 transfected HEK 293 cells was also observed using the IAsys resonant mirror optical biosensor (Affinity Sensors) (Figure 7). In this assay, biotinylated HKa was immobilized on the surface of a cuvette as described in "Materials and methods." The binding of Mac-1 transfected HEK cells to HKa was significantly greater than that of untransfected cells to HKa at equivalent cell numbers. The binding was Zn2+ dependent because, in the presence of 10 mmol/L EDTA, the binding of Mac-1 transfected HEK cells to HKa was decreased by 50%.
HKa inhibits Mac-1-mediated cell adhesion to fibrinogen and ICAM-1 The functional relevance of HKa directly interacting with Mac-1 was investigated by examining the effect of HKa on cellular adhesion to fibrinogen and ICAM-1, molecules abundant in the injured vessel wall. As shown in Figure 8, HKa blocked adhesion of Mac-1 transfected HEK cells to fibrinogen and ICAM-1 by 62% and 85%, respectively. HKa has no effect on the adhesion of Mac-1 transfected HEK cells to fibronectin, which is mediated by 1
integrin, indicating that HKa specifically inhibits the Mac-1-
mediated adhesion. Moreover, untransfected HEK cells failed to adhere
to fibrinogen or ICAM-1.
Plasma kallikrein activates human neutrophils,14,15 and, in plasma, prekallikrein circulates in a binary complex with high molecular weight kininogen (HK).29 Bradykinin is released from HK by plasma kallikrein cleavage. Cleaved HK (HKa) consists of a heavy chain and a light chain that remain linked by a single interchain disulfide bond. On the basis of studies of a patient with HK deficiency,12 we suggested that HK might serve as a cofactor for kallikrein binding to neutrophils. Later, we demonstrated that human neutrophils contain and bind HK,4 and further studies of HK binding to neutrophils indicated that both heavy chain (domain 3) and light chain (domain 5) of HK were involved in the binding to neutrophils.30 We have shown that fibrinogen acts as a noncompetitive inhibitor of HK binding to neutrophils.12 Inhibition studies with monoclonal antibodies suggested that Mac-1 might be the HK binding site on neutrophils. However, a direct interaction between HK/HKa and Mac-1 has not been demonstrated. Although both HK and HKa have been used previously for studies of the binding of kininogen to neutrophils, the contamination of HKa in HK preparation is not excluded. In this study, we used HKa and showed for the first time that HKa binds directly to cell surface human Mac-1 integrin and, consistent with this, forms a complex with Mac-1 in a purified system.
Submitted July 21, 1999; accepted February 7, 2000.
Supported by National Institutes of Heart Lung and Blood training grant 5T32HL07777 (R.W.C.) and project 2 of PO1 HL 56914 (R.W.C.) and by American Heart Association grant 9730241N (N.S.).
Reprints: Nijing Sheng, Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140; e-mail: nsheng{at}thunder.ocis.temple.edu.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Abstract
Introduction
![d[AB]/dt = [A][B]k<SUB>on</SUB> − [AB]k<SUB>off</SUB>](/content/vol95/issue12/fulltext/3788/img002.gif)
![dR/dt = k<SUB>on</SUB>[A]R<SUB>max</SUB> − (k<SUB>on</SUB>[A] + k<SUB>off</SUB>)R](/content/vol95/issue12/fulltext/3788/img003.gif)
) HKa
binding to Mac-1 transfected HEK 293 cells. (
) HKa binding to
untransfected HEK 293 cells. (
) HKa binding to Mac-1 transfected HEK
293 cells in the presence of 10 mmol/L EDTA. (
) HKa
binding to untransfected HEK 293 cells in the presence of 10 mmol/L
EDTA. The data are the mean ± SE of triplicate reactions.
)
or ICAM-1-coated (
) wells. After they were washed 3 times, adherent
cells were lysed. Plates were read on a Cytofluor 2350 system.
Percentage of adherent cells to each ligand in the presence of HKa was
determined by comparing that with in the absence of HKa, which was
calculated as 100%. Data are the mean ± SE of 3 separate
experiments.