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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:64-71

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:64-71.)
© 1996 American Heart Association, Inc.

Articles

Adhesion of Blood Platelets Is Inhibited by VCL, a Recombinant Fragment (Leucine504 to Lysine728) of von Willebrand Factor

Jan J. Sixma; Martin J.W. IJsseldijk; George Hindriks; G. Henrita van Zanten; Marian Gorecki; Amos Panet; Leonard I. Garfinkel; Philip G. de Groot

From the Department of Haematology, University Hospital Utrecht (Netherlands) (J.J.S., M.J.W.I., G.H., G.H. van Z., P.G. de G.), and Bio-Technology General (Israel) Ltd, Kiryat Weizmann, Rehovot, Israel (M.G., A.P., L.I.G.).

Correspondence to Dr J.J. Sixma, Department of Haematology, University Hospital Utrecht, PO Box 85500, 3508 GA Utrecht, Netherlands.


*    Abstract
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 *Abstract
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Abstract VCL, fragment Leu504 to Lys728 of von Willebrand factor (vWF) expressed in Escherichia coli, contains the glycoprotein (GP) Ib–binding domain of vWF. This fragment inhibited ristocetin-induced platelet aggregation with an IC50 of 0.2 µmol/L and botrocetin-induced aggregation with an IC50 of 0.08 µmol/L. We studied the antiadhesive profile of VCL by adding it to blood that was circulated over various adhesive surfaces. VCL inhibited adhesion to endothelial cell matrix, which served as a model of the vessel wall. Maximal inhibition at a high shear rate of 1600 s-1 was stronger (60%) than at a low shear rate of 300 s-1 (40%). Half maximal inhibition was found to be 1.5 µmol/L at both shear rates. The role of various adhesive molecules was investigated in more detail by coating glass coverslips with collagen type I, laminin, fibronectin, or vWF. Fibrinogen was studied as well. Platelet adhesion to laminin and vWF was not inhibited by VCL. Adhesion to collagen, fibronectin, and fibrinogen was particularly inhibited at a high shear rate. VCL coated to a coverslip caused a concentration-dependent adhesion that was blocked by antibodies against GPIb, which block interaction with vWF. Binding studies showed a nonsaturable ristocetin binding of VCL to platelets that was blocked by vWF or inhibitory antibodies against GPIb. Binding to collagen was weak, and VCL did not inhibit binding of vWF at a 5000-fold excess. From these data, we conclude that VCL inhibits adhesion in all cases in which adhesion is vWF dependent by competing for vWF binding to activated GPIb. The lack of inhibition of adhesion to vWF as a single molecule may be explained by assuming that this adhesion is determined by interaction of nonactivated GPIb with vWF that has been changed in conformation by adsorption. Studies investigating thrombus formation on the connective tissue of an atherosclerotic plaque in a human coronary artery showed that VCL was able to partially prevent this thrombus formation. VCL may be of value in preventing adhesion and thrombus formation under conditions in which these processes are dependent on vWF.


Key Words: von Willebrand factor • blood platelets • adhesion


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
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Platelet adhesion is a first and essential step in the development of a hemostatic plug or a thrombus. This process is mediated by adhesive proteins in the vessel wall and by receptors for them on the platelet membrane (for reviews see References 1 through 3). The most prominent proteins adhesive for platelets in flowing blood are vWF, different types of collagen, fibronectin, and to a lesser extent laminin4 and thrombospondin.5

vWF is particularly important at high shear rates. Lack of vWF causes a defect of adhesion at shear rates over 800 s-1.6 The platelet has two receptors for vWF: GPIb, which interacts with surface-bound vWF at all shear rates, and GPIIb-IIIa, which interacts with vWF when platelets are activated. GPIIb-IIIa is involved in platelet-platelet interaction, and vWF plays a role in this interaction, particularly at high shear rates.7 Interaction of vWF with GPIIb-IIIa is probably also important for platelet spreading.8 9 10 When this spreading is decreased by interference with GPIIb-IIIa, a defect in adhesion may become apparent at shear rates higher than 1500 s-1.8

Studies of vWF have identified functional domains involved in various interactions of the molecule.2 11 12 Particularly the A1 repeat, one of three homologous repeats recognized after cDNA cloning and sequencing, is characterized by the presence of at least four functional domains: (1) a collagen-binding site probably localized between residues 542 and 622,13 (2) a heparin-binding domain probably localized between 565 and 587,14 (3) a sulfatidate-binding site localized between 512 and 673,15 and (4) the GPIb-binding domain. With use of synthetic peptides, a domain involved in ristocetin-induced binding has been localized to the residues 474 to 488 and 694 to 708. These two stretches of amino acids are located on both sides of a disulfide-bonded loop between residues 509 and 695.16 17 There is evidence that the binding of vWF to GPIb induced by botrocetin is localized elsewhere within the disulfide-bonded loop.18 19

Since platelet adhesion is the first step in formation of a thrombus, it is attractive to inhibit this step when trying to prevent thrombosis. An attractive approach is to use fragments of the ligands or receptors involved in adhesion at the molecular level. This approach has been used successfully for the inhibition of platelet aggregation and tumor metastasis. Peptides containing the RGD sequence present in many ligands of the family of adhesion receptors called integrins have been shown to work as potent inhibitors.20 21 Also, peptides based on the sequence of laminin have been shown to be potent inhibitors of tumor metastasis.22 23

In this article, we report on studies in which a fragment of vWF, VCL, which was previously shown to inhibit ristocetin- and botrocetin-induced platelet aggregation,24 was investigated for its ability to inhibit platelet adhesion. We found a dose-dependent inhibition in all situations in which vWF was involved as a ligand. The fragment itself was able to support adhesion when applied to a coverslip.


*    Methods
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 *Methods
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Construction, Synthesis, and Purification of Peptide
The sequence encoding Leu504 to Lys728 (VCL) was derived from a human endothelial cDNA library (Clontech). The coding sequence was placed into a vector under control of an inducible {lambda} PL promoter25 and the deo P1P2 RBS.26 The peptide was purified from Escherichia coli after induction by ion exchange chromatography with CM-Sepharose, yielding essentially pure material. The disulfide bond between Cys509 and Cys695 was formed by oxidation. Purity and oxidation were verified with SDS-PAGE under reducing and nonreducing conditions as well as by high-performance liquid chromatography with a Superose 12 column. Note that the peptide contains an extra N-terminal methionine.

Functional Studies
The functional activity of VCL was determined in aggregation studies. These studies were performed as follows. Platelet-rich plasma (250 000/µL) was incubated for 30 minutes at 20°C with various concentrations of VCL. Aggregation was performed in an aggregometer (Bio/Data Corp) with 500 µL platelet-rich plasma that was prewarmed for 1 minute at 37°C. The appropriate aggregating agents such as ADP (FC, 5 µmol/L), collagen (equine tendon collagen type I, Hormonchemie; FC, 4 µg/mL), botrocetin purified from Bothrops jararaca venom (Sigma Chemical Co) by DEAE chromatography as described by Fujimura et al27 (FC, 35 µg/mL), and ristocetin (H. Lundbeck & Co; FC, 1.0 mg/mL) were then added. Aggregation studies were performed at 37°C and 900 rpm stirring speed.

Perfusion Studies
Perfusion studies were carried out in a parallel-plate perfusion chamber with well-defined rheological characteristics devised to accommodate duplicate protein-coated glass microscope coverslips.28 29 Whole blood obtained by venipuncture from healthy volunteer donors was anticoagulated with 1/10 vol 110 mmol/L trisodium citrate (citrate blood) or with 1/10 vol 200 U/mL LMWH blood (Fragmin, Kabi Pharmacia). The LMWH blood was used for perfusion studies over laminin, since adhesion to laminin is dependent on the presence of divalent cations.4 VCL was added to whole blood as a 64-µmol/L stock solution in HBS in varying amounts to reach the appropriate concentration. The same amount of HBS was added to the controls. The blood (15 mL) was then prewarmed at 37°C for 5 minutes and recirculated through the perfusion chamber for 5 minutes at wall shear rates ranging from 300 to 1800 s-1. The coverslips were removed, rinsed with 10 mmol/L HEPES buffer, pH 7.4, containing 150 mmol/L NaCl (HBS), fixed in 0.5% glutardialdehyde in PBS, dehydrated in methanol, and stained with May-Grünwald-Giemsa as previously described.29 Platelet adhesion was evaluated with a light microscope, and the coverage was measured with an image analyzer (AMS 40-10). Platelet adhesion was expressed as the percentage of the surface covered with platelets.

Adhesion to Cryostat Cross Sections of Coronary Arteries With Atherosclerotic Plaques
Glass coverslips (18x18 mm; Menzel Gläser) were cleaned by being soaked overnight in chromosulfuric acid and rinsed thoroughly with deionized water. They were air dried and coated with Denhardt's solution (0.02% Ficoll 70 [Pharmacia AB], 0.02% BSA fraction V, and 0.02% polyvinylpyrrolidone 360 [Sigma] in deionized water) for 3 hours at 68°C. The coverslips were rinsed with deionized water (20 seconds), fixed in ethanol/acetic acid (3/1; 20 minutes at room temperature), air dried, and baked for 3 hours at 180°C.

Frozen cross sections of postmortem (obtained within 24 hours after death) coronary arteries were cut at 6-µm thickness at 20°C and were mounted on Denhardt's solution–coated glass coverslips.30 The Denhardt's coating was used to prevent detachment of the tissue section in flow. Tissue sections stuck very well on this nonthrombogenic coating. After air drying (2 hours), the sections were incubated in a 4% human albumin (Behringwerke AG) solution in HBS (12 to 16 hours at 4°C) to block nonspecific binding. The coverslips were then rinsed with HBS and inserted into the perfusion chamber. A minimum of six serial sections per frozen artery segment was used for perfusions. After perfusion, the coverslips were removed from the chamber, rinsed with HBS, and fixed with 2% paraformaldehyde in PBS for 5 minutes. Platelet deposition was demonstrated with a biotinylated monoclonal antibody directed against GPIb that is specific for platelets, followed by a streptavidin–biotinylated horseradish peroxidase complex (Dakopatts) as described previously.30

Purified Proteins and Coating Procedure
Mouse laminin (A1,B1,B2; Gibco) was analyzed on a 6% SDS-PAGE gel. It yielded the characteristic single band of about 800 kD on unreduced gels and three bands of 400 000, 215 000, and 205 000 kD under reducing conditions. vWF was purified from control cryoprecipitates by gel filtration on Sepharose 4B (Pharmacia AB) as described earlier.31 32 vWF in the void volume was precipitated by dialysis against 1.9 mol/L ammonium sulfate, pH 7.0, at 4°C and stored as ammonium sulfate suspension at 4°C until use. Precipitated protein was collected by centrifugation (2 minutes, 10 000g). The pellet was dissolved in 0.05 mol/L Tris-HCl and 0.1 mol/L NaCl, pH 7.4 (TBS), and dialyzed against the same buffer. The characteristic multimer pattern of vWF was seen after agarose electrophoresis. The characteristic pattern of a band at 270 000 and two minor proteolytic bands at 170 000 and 140 000 kD was found in SDS-PAGE of reduced vWF. Fibronectin was isolated from human plasma by affinity chromatography on gelatin-Sepharose as previously described.32 SDS-PAGE after reduction showed the characteristic doublet at 220 000 kD. Fibrinogen was purchased from Kabi Pharmacia. SDS-PAGE after reduction yielded the characteristic triplet at 62 000, 58 000, and 51 000 kD. Small quantities of fibronectin and vWF were found by ELISA. These quantities gave no platelet adhesion by themselves when they were applied to a coverslip (experiments not shown). Collagen type I (calf skin) was purchased from Sigma and equine tendon from Hormonchemie. Both were dissolved at 1.4 mg/mL in 50 mmol/L acetic acid.

Glass coverslips (18x18 mm, Menzel Gläser) were cleaned by being soaked overnight in 80% ethanol and rinsed thoroughly with distilled water. They were coated by incubation for 1 hour at room temperature with laminin (100 µg/mL in 10 mmol/L phosphate buffer, pH 7.4, 0.15 mol/L NaCl [PBS]) and vWF (10 µg/mL in PBS). After coating, the glass coverslips were incubated with 1% human albumin in PBS for 2 hours. Fibronectin, fibrinogen, and collagen type I were sprayed onto glass coverslips (cleaned as described above) with a retouching airbrush (Badger Model 100 Il, Badger Air Brush Co) connected to a nitrogen cylinder operating at a pressure of 1.5 atm. The fine droplets dried instantaneously on the glass surface at room temperature.33 The collagens were coated at 30 µg/cm2, fibronectin at 20 µg/cm2, and fibrinogen at 20 µg/cm2.

Coverslips were coated with VCL by spraying quantities ranging between 10 and 100 µg per coverslip with the retouching airbrush. The best results were obtained with VCL in HBS. All spray-coated coverslips were blocked with a 1% human albumin solution as described above. Control studies showed no platelet adhesion to glass coverslips incubated with 1% human albumin solution in PBS for 2 hours.

The roles of GPIb and GPIIb-IIIa were investigated with monoclonal antibodies. AK1 and AK2 directed against GPIb were a kind gift from Dr M. Berndt, Melbourne, Australia34 ; 6D1 directed against GPIb was a kind gift from Dr B. Coller, New York, NY35 ; and C17 directed against GPIIIa inhibiting platelet adhesion and aggregation via GPIIb-IIIa was a generous gift from Dr A. von dem Borne, Amsterdam, Netherlands.36 CLB-RAg 201 directed against vWF and inhibiting binding to collagen37 38 was a kind gift from Dr Jan van Mourik, Amsterdam, Netherlands.

Preparation of Endothelial Cell and Fibroblast Matrices
Human endothelial cells derived from umbilical veins were isolated and cultured as described before.39 40 For the experiments described, endothelial cells were cultured on glass coverslips previously coated with gelatin. To isolate the extracellular matrix, cells were grown to confluence and exposed to 0.1 mol/L NH4OH for 30 minutes at room temperature. The cell layer was completely removed by this procedure, leaving the extracellular matrix intact.41 The extracellular matrix was washed three times with PBS before use. Fibroblasts derived from human lung were cultured as described.42 The matrix was prepared as described above for the endothelial cells.

Binding Studies With VCL
VCL was radiolabeled with 125I using the lactoperoxidase–glucose oxidase beads (Enzymobeads, Bio-Rad) according to the instructions of the manufacturer. Noncovalently linked 125I was removed by dialysis against PBS. The specific activity ranged between 20 and 50 cpm/ng. Trichloroacetic acid (10%) precipitated 93% to 95% of the radioactivity. The effect of radiolabeling on VCL was studied by diluting the 125I-labeled VCL with various concentrations of unlabeled material and studying it in relevant binding assays. Radiolabeling did not affect binding of VCL. vWF was labeled with 125I using the lactoperoxidase–glucose oxidase beads (Enzymobeads) according to the instructions of the manufacturer. Radiolabeled 125I-vWF contained 14% free radiolabel. It was labeled to a specificity ranging between 250 and 300 cpm/ng. Radiolabeled vWF showed the same binding characteristics as unlabeled vWF in mixing experiments.

Platelet binding studies were performed with platelets washed with Krebs-Ringer buffer at pH 6.5.43 Platelets were resuspended to 200 000/µL in PBS with 1% human serum albumin and varying amounts of radiolabeled VCL. Ristocetin (FC, 1 mg/mL) or buffer was added to a volume of 650 µL. This was layered on top of 25% sucrose in PBS and incubated for 1 hour at room temperature. The tube was then spun for 2 minutes at 10 000g at room temperature, and the pellet was counted in a gamma counter. All assays were performed in triplicate. In some experiments, anti-GPIb antibody (AK2 or 6D1) was added as ascites in the indicated dilution and incubated for 1 hour at room temperature before the radiolabeled VCL was added.

Binding of radiolabeled VCL or vWF to collagen type III was studied with the following technique. Fibrillar collagen type III from human placenta (Sigma) was dissolved in 50 mmol/L acetic acid at 1 mg/mL, dialyzed against PBS for 48 hours at 4°C, and diluted with PBS to a concentration of 100 µg/mL. This suspension (125 µL) was pipetted into wells of a 96-well ELISA tray (Costar) and spun at 1500 rpm for 15 minutes in an ELISA tray rotor to coat the wells. Supernatants were carefully removed, and the wells were cautiously rinsed and blocked for 1 hour at room temperature with TBS containing 3% BSA and 0.1% Tween 20. Samples for binding were added in 100 µL TBS and incubated for 2 hours at room temperature. The supernatant was then removed, and the wells were rinsed three times with 200 µL TBS. The tips of the wells were cut off and counted for 5 minutes in a gamma counter. All assays were performed in triplicate. Collagen type III was used for technical reasons. Previous studies have shown similar binding characteristics of vWF to fibrillar collagen types I and III. Nonspecific binding of VCL was determined in the presence of a 10-fold excess of unlabeled VCL and subtracted. Nonspecific binding of vWF was determined in the presence of 5 µmol/L recombinant leech antiplatelet protein,44 45 a specific inhibitor of vWF binding to collagen.46


*    Results
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 up arrowIntroduction
 up arrowMethods
*Results
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Characteristics of VCL
On high-performance liquid chromatography Superose 12 analysis, VCL showed a single sharp peak indicative of homogeneous material. SDS-PAGE gave a single band with an Mr of 24 kD reduced and 25 kD unreduced. The functional activity of VCL was studied in aggregation tests. No effect was observed on ADP- or collagen-induced aggregation. Ristocetin-induced aggregation was completely inhibited at 0.5 µmol/L with half maximal inhibition at 0.2 µmol/L. Inhibition of botrocetin-induced aggregation has been published before by one of us (L.I.G.) in a study by Gralnick et al.24 It was completely inhibited at 0.5 µmol/L with half maximal inhibition at 0.08 µmol/L. Reduced VCL was not studied because it precipitates as soon as it is reduced at neutral pH.

Inhibition of Adhesion to ECM
The effect of VCL on adhesion to ECM was studied at 300 and 1600 s-1 (Fig 1Down). Maximal inhibition of 60% was found at concentrations of 5 µmol/L, with half maximal inhibition at 1.5 µmol/L at 1600 s-1. Maximal inhibition of 40% was found at 5 µmol/L, with half maximal inhibition at 1.5 µmol/L when the perfusions were performed at 300 s-1. Because hemostasis is determined by adhesion to the matrix of perivascular fibroblasts as well as to ECM, we also studied the inhibition of adhesion to a fibroblast matrix. At a final VCL concentration of 2 µmol/L, platelet adhesion was not inhibited at a shear rate of 300 s-1 but was inhibited by 40% at a shear rate of 1600 s-1 (Table 1Down). The inhibition at 1600 s-1 was of the same order of magnitude as that to the ECM.



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  		Figure 1. Graph shows inhibition of platelet adhesion to ECM by VCL. The final concentration of VCL in whole citrated blood (x axis) was plotted against the percent inhibition of platelet adhesion after 5 minutes of perfusion (y axis). Solid line indicates shear rate of 300 s-1; dotted line, shear rate of 1600 s-1. Values are the mean of three separate experiments for each two perfusions with each two coverslips per concentration of VCL. Platelet adhesion at 300 s-1 was 35.8±7.2 and at 1600 s-1 45.5±5.3 (mean±SEM).


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  		Table 1. Inhibition of Platelet Adhesion by VCL

Inhibition of Adhesion to Matrix Proteins
Platelet adhesion to the vessel wall or ECM is determined by a series of ligand-receptor interactions. The most important adhesive ligands are collagen, fibronectin, vWF, and laminin. To clarify further the inhibitory effect of VCL, we studied its effect on the adhesion to these proteins. We included fibrinogen, which is not a proper matrix protein but is also important, because platelet adhesion to fibrinogen/fibrin is essential for the propagation of a thrombus. The results are summarized in Table 1Up. Adhesion to laminin was studied only at a shear rate of 300 s-1, because this adhesion is strongly dependent on shear and very little or no adhesion is observed at 1600 s-1. VCL had no effect on adhesion to laminin. Both the adhesion to fibrinogen and fibronectin were inhibited more strongly at 1600 s-1 than 300 s-1. These results are in agreement with our previous observation that adhesion to these ligands at 1600 s-1 is supported by vWF.46 47 The adhesion to vWF was not inhibited at either 300 or 1600 s-1. This lack of inhibition might have been caused by the high density of vWF on the coverslip. We therefore also studied the effect of 2 µmol/L VCL on adhesion to glass coverslips incubated with either 3 or 6 µg of vWF and compared that with the effects on coverslips incubated with 10 µg vWF. Platelet adhesion increased as expected at higher vWF concentrations, but VCL had no significant effect whether at high or low surface density.

Inhibition of Adhesion to Collagen
Perfusion studies were performed with calf skin and equine tendon collagen type I. The results of these studies are summarized in Table 1Up. The inhibition of adhesion to calf skin collagen type I was 35% at 300 s-1 and 75% at 1600 s-1. Adhesion to equine tendon collagen was inhibited by 15% at 300 s-1 and 25% at 1600 s-1. This inhibition is in agreement with earlier observations, showing that adhesion to collagen is dependent on vWF.32 The lower inhibition of adhesion to equine tendon collagen type I may indicate that the effect of VCL is less when the collagen is very reactive and when platelet vWF is thus released from {alpha}-granules.48 Because platelets adhere to VCL itself (see below) and VCL has a binding site for collagen, it was of interest to study the effect of preincubation of collagen with VCL. Collagen type I was sprayed on a coverslip, and this was incubated for 60 minutes at room temperature with VCL in a concentration of 2 µmol/L in PBS. Preincubation of collagen type I caused a slight but insignificant increase in adhesion of about 15% for equine tendon collagen type I. Preincubation of calf skin collagen type I caused no increase in adhesion, and preincubation of ECM also had no effect on adhesion.

Effect of VCL on Adhesion to an Atherosclerotic Plaque
Perfusion studies of atherosclerotic plaques were performed at 2250 s-1, which caused formation of platelet thrombi on the atherosclerotic lesion.30 VCL at 5 µmol/L concentration caused a decrease in thrombus size (Fig 2Down). This effect was reproducible, but there was considerable variation in the degree of inhibition among different blood donors.



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  		Figure 2. Photomicrographs show inhibition of thrombus formation on an atherosclerotic plaque. Cryostat sections of atherosclerotic coronary arteries were mounted on glass coverslips and exposed to circulating citrated whole blood with and without 5 µmol/L VCL at a shear rate of 2250 s-1 for a perfusion time of 5 minutes. Thrombi were stained with biotinylated anti-GPIb followed by streptavidin-peroxidase.30 Top, Control: large thrombi are present on the connective tissue of an atherosclerotic plaque. Bottom, 5 µmol/L VCL: a pronounced reduction in thrombus size was observed after perfusion in the presence of 5 µmol/L VCL.

Adhesion to VCL
Because VCL possesses the GPIb-binding domain of vWF, it was of interest to see whether platelets adhere to this fragment itself. VCL was sprayed onto glass coverslips in concentrations varying between 10 and 100 µg per coverslip and then blocked by human serum albumin as described in "Methods." The results are shown in Fig 3Down. Platelet adhesion increased with increasing surface concentrations of VCL. The results often fluctuated from one concentration to another without evident explanation. The effect of monoclonal antibodies against GPIb and GPIIb-IIIa on adhesion to VCL was tested in separate experiments. The inhibitory antibodies against GPIb, AK2, and 6D1 caused complete inhibition, whereas the noninhibitory antibody AK1 had much less effect; anti–GPIIb-IIIa (C17) had no effect at all. VCL also has the heparin-binding site, which is essential for the binding of vWF to heparin.14 49 The effect of heparin preincubation on the adhesion to VCL was therefore studied in separate experiments. Glass coverslips were coated with VCL, and these coverslips were preincubated with UFH (Thromboliquine, Organon) and LMWH (Fragmin, Kabi Pharmacia) at concentrations of 0.1, 0.3, and 1.0 U/mL for 2 hours at room temperature. Adhesion was already strongly inhibited by 0.1 U/mL UFH (90%) or LMWH (80%) and completely inhibited by 0.3 U/mL or higher UFH or LMWH.



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  		Figure 3. Graph shows platelet adhesion to VCL. Adhesion was studied at shear rates of 300 (solid line) and 1600 (dotted line) s-1. The curves are representative for three similar experiments. Every single point is the mean of two perfusions each with two coverslips in the chamber. Percent adhesion indicates the percentage of the coverslip covered by platelets.

Preincubation of a VCL-coated coverslip with UFH completely inhibited adhesion. This raised the question of whether UFH might influence the inhibitory action of VCL. For this purpose we studied the inhibitory action of VCL on platelet adhesion in flowing heparinized and citrated blood to ECM, calf skin collagen, and fibrinogen. VCL was also a good inhibitor in blood anticoagulated with UFH (results not shown).

Binding Studies With VCL
To improve our understanding of the mechanism of action of VCL, we radiolabeled it with 125I and performed binding studies. VCL binding to platelets was studied in the presence and absence of ristocetin. The results of a typical experiment are presented in Fig 4Down, top. Ristocetin-induced binding was not saturable up to a concentration of 10 µmol/L VCL. vWF competed for this binding (Fig 4Down, middle). VCL binding in the presence of ristocetin was inhibited by anti-GPIb (Fig 4Down, bottom). Binding in the absence of ristocetin was much lower and not inhibited by vWF and only weakly by anti-GPIb.



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  		Figure 4. Graphs show binding of 125I-VCL to platelets. Top, Binding of 125I-VCL to washed platelets was performed as described in "Methods." Binding in the presence of ristocetin ({triangleup}——{triangleup}) was much higher than binding in its absence ({blacktriangleup}——{blacktriangleup}). Middle, Dose-dependent inhibition of VCL binding to washed platelets by vWF was observed in the presence of ristocetin ({triangleup}——{triangleup}) but not in its absence ({bullet}——{bullet}). The VCL concentration was 0.5 µmol/L in the presence of ristocetin and 2 µmol/L in the absence of ristocetin (to obtain enough counts). The binding was expressed as a percentage of the value with no added vWF (control). Bottom, Inhibition of 125I-VCL binding with anti-GPIb, which was expressed as a percentage of the binding without addition of anti-GPIb (AK2) in the presence or absence of ristocetin. Similar data were obtained with monoclonal antibody 6D1.

Collagen binding studies were performed using collagen type III. VCL bound much less to collagen than to vWF itself, with only 12 fmol bound at 0.07 µmol/L VCL versus 230 fmol vWF at 0.07 µmol/L vWF (Fig 5Down, top). The binding of VCL was inhibited by VCL itself, whereas vWF had about five times less effect (Fig 5Down, middle). vWF binding was inhibited by excess nonlabeled vWF or a specific monoclonal antibody, CLB-RAg 201, which has been described before,37 38 but not by a 5000-fold excess of VCL, which is in agreement with the fact that the collagen-binding site in the A3 domain of vWF is essential for the binding of vWF to collagen46 (Table 2Down).



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  		Figure 5. Graphs show binding of VCL and vWF to collagen type III. Top, 125I-VCL binding was performed as described in "Methods." Data on the y axis describe the amount bound per well. The binding curve shows the specific binding. Middle, 125I-vWF binding was performed as described in "Methods." Note the difference in units on the x axis with Fig 5Up, top. Bottom, Competition of binding of 125I-VCL (0.038 µmol/L) by VCL ({blacktriangleup}——{blacktriangleup}) or vWF ({triangleup}——{triangleup}).


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  		Table 2. Inhibition of 125I-vWF Binding to Collagen


*    Discussion
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 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
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Adhesion of platelets to the vessel wall is the first step in hemostasis and thrombosis. This makes it an attractive target for the development of an antithrombotic agent. Most drugs interfering with platelet function do not affect its adhesion. An approach based on functional domains of adhesive ligands or receptors should therefore be considered seriously.

In this article, we report on the antiadhesive profile of VCL, a recombinant fragment of vWF expressed in E coli and purified to homogeneity. This fragment, the activity of which was first established in aggregation studies with ristocetin and botrocetin, was found to be a potent inhibitor of platelet adhesion to subendothelium of umbilical arteries at 2600 s-1.24 We studied the adhesion to the ECM of cultured endothelial cells. Previously, we have demonstrated that the matrix of cultured human umbilical endothelial cells may serve as a good model for the subendothelium.41 The inhibition was shear rate dependent, with a more potent inhibition at a higher shear rate. This difference in inhibition related to shear rate was even more pronounced when adhesion to fibroblast matrix was studied. No inhibition was found at 300 s-1, whereas inhibition at 1600 s-1 was in the same range as that for ECM. This difference may reside in the requirement for vWF in supporting adhesion to the different matrices, which may be higher for ECM than fibroblast matrix at 300 s-1.

A more detailed study of the inhibitory profile of VCL revealed that it inhibited platelet adhesion in situations in which vWF was required as a second adhesive molecule. Previously, we and others have demonstrated that adhesion to collagen type I requires platelet vWF or exogenous vWF,32 50 51 and the same was shown for adhesion to fibrinogen/fibrin52 and fibronectin.46 47 The adhesion to laminin has no requirement for vWF,4 and this adhesion was indeed not inhibited by VCL. Curiously enough, adhesion to vWF coated on a surface was not inhibited at relatively low vWF densities. VCL can perhaps not compete in this case because vWF on the surface is still abundant (see below).

The clinical indication for which VCL will probably be most useful is to prevent adhesion and thrombus formation on an atherosclerotic lesion during and after percutaneous transluminal coronary angioplasty. It was therefore relevant to study adhesion to an atherosclerotic plaque. In a previous study,30 we found that thrombus formation on atherosclerotic intima was strongly increased; this thrombus formation was dependent on vWF. In the present study, we found that a concentration of 5 µmol/L VCL inhibited aggregate formation at 2250 s-1, a shear rate that may occur in a stenosed coronary artery. The decrease in aggregate size rather than an absence of aggregates is in agreement with the effect that was observed with suboptimal concentrations of a monoclonal antibody directed against the GPIb-binding domain of vWF30 (G.H. van Z., personal observation, 1995).

The inhibition of adhesion to ECM in the present study attained a maximum of 60%, which is less than we and others attained previously with antibodies directed against vWF.37 53 One explanation may be that a monoclonal antibody that blocks a functional site on vWF may be more effective than a fragment that has to compete with a highly repetitive ligand such as the multimeric vWF.

VCL has been shown to inhibit binding of vWF to GPIb on the platelets in the presence of ristocetin or botrocetin.24 This suggests that VCL inhibits platelet adhesion via interference with this interaction. The studies with 125I-labeled VCL (Fig 4Up) showed a concentration-dependent binding to platelets that was much enhanced by ristocetin. This binding in the presence of ristocetin was inhibited by vWF and an anti-GPIb monoclonal antibody. Binding in the absence of ristocetin was not blocked by vWF and only weakly by anti-GPIb (Fig 4Up), which argues in favor of a qualitatively different binding. Our data, together with those of Gralnick et al,24 who showed inhibition of vWF binding in the presence of ristocetin, suggest that VCL works by competing with vWF for interaction with GPIb.

We have also studied an alternative explanation. VCL contains a collagen-binding site, and we wondered whether VCL might compete with vWF in its binding to collagen and whether this might contribute to its inhibitory action. This appeared not to be the case. vWF competed only weakly with VCL, which conversely did not compete at all with vWF at 500-fold molar excess (Table 2Up), thus excluding the possibility that VCL might act by blocking vWF binding to collagen.

The most likely scenario is that VCL inhibits platelet adhesion when vWF is present as a secondary adhesive molecule (eg, in adhesion to collagen, fibronectin, or fibrinogen) but not when vWF is directly immobilized because of stoichiometric reasons. Concentrations of VCL in greater orders of magnitude may be required in the latter case because many more vWF molecules are present at the surface.

Comparison of our data with those of Sugimoto et al,54 who studied a similar recombinant fragment, shows the importance of apparently relatively minor changes in the composition of the fragment. The fragment reported by Sugimoto et al contained seven cysteine residues, and because of that it formed dimers and tended to be insoluble unless it was first reduced and alkylated. This was not the case for VCL, which contains only the two cysteines that form the disulfide loop between 509 and 695. The fragment described by Sugimoto, called R12986, was recently used in adhesion studies in oscillatory flow on the extracellular matrix of bovine corneal endothelial cells.55 Notwithstanding large differences in technique, results similar to those with VCL were observed. R12986 inhibited platelet adhesion in the same range as VCL. The data of Gralnick et al24 and those of Dardik et al,55 combined with our detailed studies reported in this article, suggest that GPIb-binding fragments of vWF may be of value as inhibitors of adhesion in situations in which vWF is involved as a second adhesive molecule.


*    Selected Abbreviations and Acronyms
 
ECM = endothelial cell matrix
ELISA = enzyme-linked immunosorbent assay
FC = final concentration
GP = glycoprotein
HBS = HEPES-buffered saline
LMWH = low-molecular-weight heparin
PBS = phosphate-buffered saline
SDS-PAGE = SDS–polyacrylamide gel electrophoresis
TBS = Tris-buffered saline
UFH = unfractionated heparin
vWF = von Willebrand factor


*    Acknowledgments
 
This study was supported by grants from the Netherlands Foundation of Medical Research (900-526-067) and the Netherlands Heart Foundation (88-066).

Received June 6, 1995; accepted August 31, 1995.


*    References
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*References
 

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