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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:611-620.)
© 1996 American Heart Association, Inc.

Articles

Platelet Adhesion to Multimeric and Dimeric von Willebrand Factor and to Collagen Type III Preincubated With von Willebrand Factor

Ya-Ping Wu; Hans H.F.I. van Breugel; Hanneke Lankhof; Robert J. Wise; Robert I. Handin; Philip G. de Groot; Jan J. Sixma

From the Department of Hematology, University Hospital, Utrecht, Netherlands, and the Division of Hematology-Oncology (R.J.W., R.I.H.), Department of Medicine, Brigham and Women's Hospital, Boston, Mass.

Correspondence to Dr J.J. Sixma, Department of Haematology, University Hospital Utrecht, PO Box 85500, 3508 GA Utrecht, Netherlands. E-mail jsixma@lab.azu.nl.

	Abstract

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Abstract As part of a systematic study of platelet interaction with adhesive proteins under flow conditions, we studied platelet adhesion to multimeric and dimeric von Willebrand factor (vWF) coated to glass. vWF-dependent adhesion to collagen type III was studied for comparison. Adhesion to glass-coated vWF and vWF-mediated adhesion to collagen type III were in many respects similar. Both showed no decrease at increasing shear rates and a decline to 50% of maximum with a low-molecular-weight multimeric fraction. Adhesion to glass-coated vWF was partially inhibited by heparin and completely inhibited by prostaglandin I₂ and anti–glycoprotein (GP) Ib and anti–GPIIb-IIIa antibodies. vWF-dependent adhesion to collagen was not inhibited by heparin, was partially inhibited by anti–GPIIb-IIIa, and was completely inhibited by prostaglandin I₂ and anti-GPIb. Recombinant dimeric vWF was made by deletion of the propeptide and expression in Chinese hamster ovary cells. Adhesion was 50% of that with plasma vWF, and larger concentrations of dimeric vWF were required. Adhesion to dimeric vWF was optimal at 1500 s^-1, with a gradual decrease at higher shear rates. We conclude that adhesion to collagen type III is strongly but not completely determined by the adhesive properties of vWF.

Key Words: von Willebrand factor • collagen type III • platelet • adhesion

	Introduction

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Platelet adhesion is the first step of the hemostatic and thrombotic process. It is mediated by a number of ligand-receptor interactions, among which the interaction of vWF with its receptors, the GPIb-IX and GPIIb-IIIa complexes, plays a crucial role, particularly at high shear rates. The role of vWF in platelet adhesion has been established in perfusion studies^{1 2 3 4} ; the vWF present in the subendothelium^{5 6} or secreted from platelets can contribute to adhesion.^{7 8} The essential contribution of vWF to adhesion to collagen and to fibrin(ogen) in flowing blood has also been established.^{9 10} These studies were performed under conditions in which other adhesive molecules besides vWF were present. Only a few studies were performed in which vWF was the only adhesive molecule. Such studies are needed to shed light on the special role of vWF in platelet adhesion. Adhesion studies under static conditions show collaboration between GPIb and GPIIb-IIIa as receptors.¹¹ Studies under flow conditions with radiolabeled platelets adhering to vWF-coated capillaries show a concentration and shear-dependent adhesion that can be increased by prior activation of platelets with ADP or thrombin¹² and indicate the role of GPIb and GPIIb-IIIa.¹³

The multimeric size of vWF is important for its function. vWF multimers of lower molecular weight show less binding to platelets in the presence of ristocetin¹⁴ and less binding to collagen.^{15 16} In contrast to ristocetin-induced platelet aggregation,¹⁷ the support of platelet adhesion to subendothelium by vWF is not dependent on multimeric size. Studies with SpIII, an amino-terminal dimeric proteolytic fragment of vWF, show that this fragment can also support adhesion to collagen.¹⁸

In the present study we examined platelet adhesion to purified vWF from flowing blood and compared its characteristics with those of the adhesion of platelets to collagen type III preincubated with vWF. We paid special attention to the role of the multimeric size of vWF.

	Methods

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Materials
Monoclonal antibody AK2 directed against GPIb and inhibiting ristocetin-induced platelet aggregation¹⁹ was a kind gift from Dr M. Berndt, Baker Institute, Melbourne, Australia. C17, a monoclonal antibody directed against GPIIIa and inhibiting ADP-induced platelet aggregation,²⁰ was a kind gift from Dr A.E.G. von dem Borne, Central Laboratory Blood Transfusion Service, Amsterdam, Netherlands. Human collagen type III from placental origin was obtained from Sigma Chemical Co. Unfractionated heparin (Thromboliquine) was purchased from Organon and LMWH (Fragmin) from Kabi Pharmacia. Recombinant nonsulfated hirudin was from Ciba-Geigy (courtesy of Dr R. Wallis). PPACK was purchased from Bachem Feinchemikalien AG. All reagents used were of the highest purity commercially available.

Characteristics of vWF
vWF was purified from cryoprecipitates by gel filtration on Sepharose 4B (Pharmacia AB).^{9 21} vWF in the void volume was stored at 4°C and used within 2 weeks after purification. 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–polyacrylamide gel electrophoresis of reduced vWF. In experiments in which the effect of the multimeric composition of vWF was studied, separate 3-mL fractions from the Sepharose 4B gel filtration column were studied for their multimeric composition, ristocetin cofactor activity, and vWF antigen content. These fractions were then used for coating coverslips or binding of vWF to collagen type III.

Determination of the Multimeric Composition of vWF
The multimeric structure of vWF was assayed by using the Pharmacia Phast Gel System (Pharmacia LKB Biotechnology) as described by Lawrie et al.²² Briefly, samples diluted in 10 mmol/L Tris/HCl, 1 mmol/L EDTA, 2% SDS, 8 mol/L urea, and 0.05% bromophenol blue, pH 8.0, were applied to a 1.7% agarose gel (LE, Seakem, FMC Bioproducts) in 0.5 mol/L Tris/HCl, pH 8.8, and 0.1% SDS with a stacking gel consisting of 0.8% agarose (HGT, Seakem) in 0.125 mol/L Tris/HCl, pH 6.8, and 0.1% SDS. After electrophoresis the protein was transferred to a polyvinylidene fluoride membrane (Immobilon P, Millipore) by diffusion blotting for 1 hour at 60°C. The membrane was then blocked with 5% nonfat dry milk protein solution (Protifar, Nutricia) for 1 hour at room temperature. After washing with PBS/T, pH 7.4, the blot was incubated with horseradish peroxidase–conjugated rabbit polyclonal antibodies to human vWF (Dakopatts) diluted 1:1000 in PBS/T for 1 hour at room temperature. After three washes with PBS/T, the membrane was incubated with the substrate solution (25 mg 3,3'-diaminobenzidine tetrahydrochloride [Sigma] in 50 mL PBS with 10 µL 30% H₂O₂). The enzyme reaction was stopped by washing the membrane with distilled water.

vWF-PRO
vWF-PRO was synthesized in Chinese hamster ovary cells transfected with vWF cDNA containing a deleted propeptide-coding region. Construction of the vWF-PRO cDNA and establishment of the Chinese hamster ovary cell line has been described.^{23 24} Serum-free conditioned medium was collected²⁴ and concentrated in dialysis bags covered with Aquacide-3 (CalBiochem). Following concentration and dialysis against 50 mmol/L Tris-HCl (pH 7.6) and 150 mmol/L NaCl, the medium was applied to a Bio-Gel A.5 (Bio-Rad) chromatography column. vWF was collected in the void volume and concentrated by ultrafiltration.

Coating and Spraying of Coverslips
Glass coverslips (Menzel Gläser, 18x18 mm) were cleaned overnight by a chromium trioxide solution and rinsed with distilled water before coating. Coating was performed by incubating the coverslips with 100 µL purified vWF in PBS for 1 hour followed by a 1-hour incubation with 4% human albumin solution in PBS to block further protein binding. After coating, coverslips were kept in PBS until perfusion. The quantity of vWF that was coated to the coverslip was measured with vWF labeled with ¹²⁵I by using lactoperoxidase-glucose-oxidase (Enzymobeads, Bio-Rad) according to the instructions of the manufacturer. The effect of radioactive labeling of vWF was checked by mixing different amounts of labeled and unlabeled protein and keeping the total amount of vWF constant. No differences in coating behavior were observed, indicating that radioactive labeling had not affected the adsorption of vWF (data not shown).

Monomeric collagen type III (acid soluble) was solubilized in 50 mmol/L acetic acid and sprayed with a density of 30 µg/cm² on glass coverslips with a retouching airbrush (model 100, Badger Brush Co).^{25 26} After the spraying procedure the collagen surface was incubated for 1 hour with 1% human albumin in PBS to block further protein binding. The surface was then incubated with vWF (10 µg/mL in PBS) for 1 hour at room temperature and washed three times with PBS. Fibronectin was isolated from human plasma by affinity chromatography on gelatin-Sepharose.⁹ SDS–polyacrylamide gel electrophoresis after reduction showed the characteristic doublet at 220 000 kD. Fibronectin was coated by spraying at a concentration of 20 µg/cm². In some experiments, vWF solubilized in PBS was sprayed in the same way as collagen type III or fibronectin. After the spraying procedure, vWF or fibronectin was blocked with 1% human albumin in PBS by incubation for 1 hour.

Human endothelial cells derived from umbilical veins were isolated and cultured^{27 28} 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.²⁹ The extracellular matrix was washed three times with PBS before use.

Perfusion Studies
Perfusion studies were performed in a parallel-plate perfusion chamber with well-defined rheologic characteristics devised to accommodate duplicate protein-coated glass coverslips.³⁰ Whole blood obtained by venipuncture from healthy volunteer donors was anticoagulated with 1:10 vol 110 mmol/L trisodium citrate (citrated blood) or with 1:10 vol 200 U/mL LMWH. Whole blood (15 mL) was 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, fixed in 0.5% glutaraldehyde, dehydrated in methanol, and stained with May-Gruenwald-Giemsa.³¹ Platelet adhesion was evaluated by using light microscopy, and the coverage was measured with an image analyzer (AMS 40-10). Adhesion was expressed as the percentage of the surface covered with platelets.

For the perfusion studies over collagen preincubated with vWF, reconstituted blood was used that was prepared as follows. Platelet-rich plasma was obtained from whole blood by centrifugation (10 minutes at 200g and 20°C). One volume of Krebs-Ringer buffer (in mmol/L: KCl 4, NaCl 107, NaHCO₃ 20, and Na₂SO₄ 2, pH 5.0) containing 19 mmol/L sodium citrate was added to 1 vol platelet-rich plasma (final pH 6). A platelet pellet was obtained by centrifugation (10 minutes at 500g and 20°C). The platelet pellet was resuspended in Krebs-Ringer buffer, pH 6.0, and washed twice by centrifugation (10 minutes at 500g). After the second wash, platelets were resuspended to a platelet count of 190 000/µL in a human albumin solution (4% human albumin in Krebs-Ringer buffer without citrate, pH 7.35, containing 20 U/mL LMWH, 5 mmol/L -D-glucose, and 2.5 mmol/L CaCl₂). Red cells were washed three times with PBS containing 5 mmol/L -D-glucose (2000g at 20°C, twice for 5 minutes and the last time for 15 minutes).⁹ Washed red cells were added in a volume fraction of 40% of total volume 5 minutes before perfusion.

To reach higher shear rates, platelet adhesion was studied by using square glass microcapillaries (Microcell, The Wilmad Glass Co Inc). The glass microcapillaries were coated by adsorption of vWF as described for the coverslips. Perfusions were performed with a single passage, drawing blood from a container through the tube at the appropriate speed by using a syringe and an automated syringe pump (Harvard Apparatus).

The studies with vWF-PRO were performed with a specially devised small perfusion chamber (in mm: width 2, length 18, and height 0.1). Because of the limited availability of vWF-PRO, a template was prepared that allowed spraying of the area of the coverslip that was exposed to the flowing blood. Perfusions were performed with a single passage by using aspiration with a pump-driven syringe as described above. Flow rates varied from 0.1 to 0.8 mL/min during 5 minutes. Platelet adhesion was evaluated by using light microscopy and an image analyzer as described above. Evaluation was in 12 fields perpendicular to the flow rate in the middle of the chamber, 12 fields 3 mm upstream, and 12 fields 3 mm downstream (magnification x1000).

SEM
For SEM, coverslips with platelets fixed for 1 hour with 2% glutaraldehyde at room temperature were postfixed in 1% osmium tetroxide for 1 hour, dehydrated in a graded series of ethanols, and dried by the critical-point procedure with CO₂ as transitional fluid. The samples were sputter coated with a thin layer of gold and viewed in an SEM (Cam Scan S2).

	Results

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Adhesion of Platelets to vWF Coated to Glass
Concentration Dependence
Two methods of coating vWF to glass coverslips were used. For the first method, glass coverslips were coated with vWF by adsorption. The amount of vWF that bound was tested with radiolabeled vWF. Maximum coating was found after 30 minutes. Bound vWF began to level off above 50 µg/mL, but it had not yet reached saturation at 100 µg/mL: a coating of 400 ng/cm² was found at this concentration (Fig 1). The question of whether vWF was removed from glass by perfusion with whole blood was tested separately. No changes in the amount of adsorbed protein were found after perfusion for 5 minutes at shear rates between 300 and 1800 s^-1 (data not shown). Platelet adhesion to adsorbed vWF was subsequently tested and compared with the adhesion to vWF that had been coated to a coverslip by spraying it with a retouching airbrush (Fig 2). Homogeneous coverage was obtained with both methods. Preference was given to adsorption for the subsequent studies, because this is an easier and less time-consuming method. Unless indicated otherwise, a vWF concentration of 10 µg/mL was chosen, since this caused optimal adhesion and deposited 60 ng/cm² vWF on the glass coverslip. Because proteins adsorbed to a surface will denature with time, the effect of time after adsorption on platelet adhesion was studied. Platelet adhesion to a vWF-coated surface started to decrease after 6 hours of storage at room temperature. After 48 hours of storage, platelet adhesion was decreased to 50% of the adhesion value after 2 hours of storage (data not shown). The role of vWF present in the blood was tested by comparing adhesion from whole blood with adhesion from reconstituted blood in which plasma was replaced by a human albumin solution. No difference in adhesion was observed (results not shown).

View larger version (11K): [in this window] [in a new window]   Figure 1. Line graph shows effect of 125I-labeled vWF concentration (conc.) on adsorbed amount of protein (1-hour incubation at room temperature). Data are mean±SEM; n=6.

View larger version (17K): [in this window] [in a new window]   Figure 2. Line graphs show platelet coverage on (top) adsorbed and (bottom) sprayed vWF at different concentrations. Data are mean±SEM of three separate experiments, each performed in quadruplicate, with blood from different donors. Perfusions were performed for 5 minutes at 1000 s-1.

Time Dependence
The adhesion of platelets to vWF was studied at 1, 3, 5, 10, and 15 minutes at shear rates of 300, 1000, and 1500 s^-1 (Fig 3, top). Fast initial platelet adhesion was found in the first minute followed by a more or less linear phase between 1 and 5 minutes. The increase in adhesion gradually declined between 5 and 15 minutes. This pattern of time dependence differed from that of platelet adhesion to fibronectin, collagen type III at 300 s^-1 and 1500 s^-1, and collagen type IV, which showed a linear phase in the first 10 minutes but corresponded closely to that observed on ECM (Fig 3, bottom).

View larger version (21K): [in this window] [in a new window]   Figure 3. Line graphs show time dependence of platelet adhesion to vWF. Top, Perfusions were performed for the indicated times at shear rates of 300 (), 1000 (), and 1600 () s-1. Data are mean±SEM of three separate perfusions, each performed in quadruplicate, with blood from different donors. Bottom, Time dependence of adhesion at 1000 s-1 to collagen type III at 300 () and 1500 () s-1, ECM at 1500 s-1 (), fibronectin at 300 s-1 (), and collagen type IV at 300 () and 1500 () s-1. Data are mean±SEM of three separate experiments, each performed in quadruplicate.

Shear Rate Dependence
vWF is regarded as specifically suited for adhesion interactions at high shear rates. The perfusion system that is used in most studies, the parallel-plate perfusion system of Sakariassen et al,³⁰ is not suited for perfusions at very high shear rates (>2600 s^-1). We therefore used square glass microcapillaries through which blood was aspirated from a container at 37°C with a pump-driven syringe. Comparison studies with the parallel-plate perfusion system were first performed. These studies showed no essential difference between platelet adhesion in a single-passage system and a system with repeated recirculation of blood (results not shown). The shear rate dependence of platelet adhesion over a large range of shear rates is shown in Fig 4. Adhesion to vWF did not decrease at higher shear rates (up to 4000 s^-1), in contrast to adhesion to fibronectin, laminin, and thrombospondin, which show optimal adhesion at 300, 800, and 1500 s^-1, respectively.^{32 33 34}

View larger version (8K): [in this window] [in a new window]   Figure 4. Line graph shows shear rate dependence of platelet adhesion to multimeric vWF (), ECM (), and collagen type III preincubated with vWF (). Blood was aspirated for 5 minutes from a container in which it was kept at 37°C at the indicated shear rate. Data are mean±SEM of three separate experiments, each performed in quadruplicate, with blood from different donors.

Several studies that used platelet-rich plasma or platelets in vWF-containing buffer have shown that platelets aggregate under high shear rate conditions in a plate-and-cone viscometer.^{35 36} This aggregation is dependent on the GPIb-vWF interaction, which at high shear stress may activate platelets.^{36 37 38} We tried to discern whether this mechanism occurs in whole blood during flow at high shear rates. Adhered platelets showed dendritic and spread forms (Fig 5). There was more pronounced spreading of platelets adhering at the highest shear rate (4000 s^-1; Fig 5c) than at lower shear rates of 300 (Fig 5a) and 1000 (Fig 5b) s^-1, but no or only small aggregates were observed, apart from single dendritic cells lying on top of thin, fully spread platelets. We also studied adhesion at 4000 s^-1 at 1 and 3 minutes to ascertain that no aggregates had been present that were subsequently swept away. No aggregates were observed at these earlier times. We also studied platelet adhesion to vWF at 4000 s^-1, using hirudin-anticoagulated blood, to see whether aggregate formation might occur under these conditions. Again, no aggregates were observed.

View larger version (146K): [in this window] [in a new window]   Figure 5. SEM of platelets adhering to vWF at (a) 300, (b) 1000, and (c) 4000 s-1 and to dimeric vWF at (d) 1000 s-1. All perfusions were for 5 minutes (magnification x1000; bar=10 µm).

Multimeric Composition
Unlike ristocetin-induced platelet aggregation,¹⁷ vWF-dependent platelet adhesion to subendothelium is not influenced by the multimeric size of vWF. In the present study we considered the effect of multimeric size on platelet adhesion to purified vWF. Different 3-mL fractions from gel filtration purification of vWF were collected separately. Coverslips were adsorbed with these various samples at 10 µg/mL as described above, and platelet adhesion at 1000 s^-1 was estimated. Fig 6 (top) presents a curve in which the percent surface coverage is plotted against the column fraction. There was a gradual decrease in platelet adhesion with lower molecular weights. The multimeric distribution present in the various fractions is also shown (Fig 6, bottom). To exclude the possibility of different adsorption of various multimers leading to differences in adhesion, we also coated glass coverslips by spraying the different multimeric fractions onto the coverslips. A similar decrease in adhesion with decreasing multimeric size was observed (data not shown).

View larger version (149K): [in this window] [in a new window]   Figure 6. Top, Line graph shows platelet coverage on vWF adsorbed to glass () or collagen type III () for different fractions obtained by gel filtration over a Sepharose 6B column. Data are mean±SEM of three similar experiments, each performed in quadruplicate, with blood from different donors. Data were normalized by taking the adhesion with fraction 1 as 100%. The absolute adhesion with fraction 1 varied between 20% and 30% on collagen type III incubated with vWF and between 50% and 60% on vWF coated to glass. Adhesion to collagen type III in the absence of preincubated vWF was negligible. Bottom, Representative picture of multimeric distribution of vWF multimers in the fractions obtained from the Sepharose 6B gel filtration column. PL indicates plasma.

To obtain further insight into the role of the multimeric structure of vWF, we studied adhesion to dimeric vWF. The vWF-PRO molecule forms dimers but fails to multimerize because it is synthesized in the absence of the vWF propeptide.^{23 39} We first performed a concentration dependence study by spraying vWF-PRO on a coverslip (Fig 7, top). Higher concentrations of vWF-PRO than of plasma vWF were required (Fig 2) to obtain optimal adhesion, and adhesion to vWF-PRO was lower. A time curve of adhesion to vWF-PRO shows the same initial fast adhesion as that observed with plasma vWF (data not shown; see Fig 3). Adhering platelets were less spread than on multimeric vWF and tended to form lateral aggregates (Fig 5d). After 10 minutes definite aggregate formation had occurred. Since a special perfusion procedure had been used to minimize the consumption of recombinant vWF-PRO, we also studied adhesion to plasma vWF by using this system. No aggregate formation was observed, and platelet adhesion was similar to that which was observed with the other perfusion systems. The adhesion to vWF-PRO showed an increase, with the shear rate up to 1500 s^-1 (Fig 7, bottom). A gradual decrease occurred at higher shear rates, in contrast to that which was observed with plasma vWF.

View larger version (18K): [in this window] [in a new window]   Figure 7. Line graphs. Top, Platelet coverage on sprayed vWF-PRO at different concentrations. Perfusions were for 5 minutes at 1000 s-1 with a specially devised small perfusion chamber. Data are the mean of three separate runs per concentration. Bottom, Shear rate dependence of platelet adhesion to vWF-PRO. Conditions are as described in Fig 4.

Modulation of Adhesion to vWF
Divalent cations. The effect of divalent cations on the adhesion to vWF was tested by comparing perfusions performed with citrated blood and blood anticoagulated with a mixture of hirudin 20 U/mL and PPACK 30 nmol/L. Similar values were found in two separate duplicate experiments, indicating that divalent cations at plasma concentrations are not required for adhesion to vWF. vWF-dependent adhesion to the subendothelium of umbilical arteries is inhibited by treatment of vWF with EDTA.⁴⁰ In the present study vWF was adsorbed to glass and then treated with EDTA. Treatment with EDTA gave only a weak inhibition of adhesion (Table 1).

View this table: [in this window] [in a new window]   Table 1. Effect of Pretreatment With EDTA on Adhesion to vWF

Heparin. Adhesion of platelets to GPIb-binding fragments of vWF is inhibited by heparin.⁴¹ We studied whether heparin had any effect on adhesion to vWF in two different ways (Table 2). In one set of experiments unfractionated heparin or LMWH was added in different concentrations to citrated blood, and adhesion from these samples was compared. Heparin addition yielded a concentration-dependent inhibition of platelet adhesion. LMWH had less effect. In a second set of experiments, adsorbed vWF was preincubated with unfractionated heparin or LMWH; this preincubation was followed by perfusion studies in which citrated blood was circulated over the incubated surfaces. Preincubation of vWF with heparin caused a somewhat stronger inhibiting effect than addition of heparin to blood (51.8% of control versus 62.3% at 5 U/mL). Addition of LMWH had less effect (Table 2).

View this table: [in this window] [in a new window]   Table 2. Effect of Unfractionated Heparin and LMWH on Platelet Adhesion to vWF

Effect of prostacyclin. Prostacyclin was added to whole blood 5 minutes before recirculation of the blood through the perfusion system. A concentration-dependent inhibition of platelet adhesion was observed, with half-maximal adhesion at 0.5 ng/mL (Fig 8).

View larger version (10K): [in this window] [in a new window]   Figure 8. Dose-response curve of the effect of prostacyclin on adhesion to vWF () and collagen type III preincubated with vWF (). Data are the means of four coverslips and are representative of two similar experiments.

Platelet Receptors for Adhesion to vWF
Platelets have two receptors for vWF, the GPIb-IX complex and the GPIIb-IIIa complex.⁴² We have studied the involvement of these two receptors in adhesion to vWF in flow by using monoclonal antibodies to both receptors. Powerful inhibition was found with anti-GPIb as well as with anti–GPIIb-IIIa antibodies (Fig 9), indicating that both receptors are involved in adhesion to vWF.

View larger version (22K): [in this window] [in a new window]   Figure 9. Dose-response curves of the effect of (top) anti-GPIb monoclonal antibody AK2 and (bottom) anti–GPIIb-IIIa monoclonal antibody C17 on adhesion to vWF () and collagen type III preincubated with vWF (). Data are the means of four coverslips and are representative of three similar experiments.

Role of vWF in Platelet Adhesion to Collagen Type III
Because adhesion of blood platelets to collagen in flow is mediated by vWF,⁹ we also studied the role of various variables of vWF-mediated adhesion to collagen type III. Adhesion showed time dependence, with a more or less linear increase in adhesion over 15 minutes at 300 s^-1 and a somewhat more rapid increase in the first minute at 1500 s^-1, which was less rapid than the results found for vWF at all shear rates (Fig 3). The effect of the shear rate on platelet adhesion is shown in Fig 4. Platelet adhesion increased slowly between 1000 and 4000 s ^-1, similar to that for vWF alone. Fig 4 also shows the shear rate dependence of adhesion to ECM, which is also vWF dependent. This adhesion follows a similar pattern as adhesion to vWF alone or vWF-mediated adhesion to collagen type III. As in the case of adhesion to vWF alone, preincubation of vWF with EDTA had no effect (data not shown). Platelet adhesion was also not significantly affected by the addition of 5 U/mL unfractionated heparin to citrated blood (results not shown). The role of the multimeric size of vWF was studied by preincubating collagen type III with various fractions obtained from the gel filtration purification of vWF (Fig 6), similar to those used for the study of the influence of multimeric structure on platelet adhesion to vWF described above. The effect of multimeric size tended to be less gradual than that for vWF alone. There was no effect in the two higher molecular weight fractions and a relatively steep decline in the two lower molecular weight fractions. The effect of prostacyclin on vWF-dependent adhesion to collagen type III is shown in Fig 8. A degree of inhibition by prostacyclin similar to that occurring when vWF alone was the adhesive molecule was found. The role of the platelet receptors GPIb and GPIIb-IIIa was studied by using monoclonal antibodies (Fig 9). Anti-GPIb gave complete inhibition of adhesion. Anti–GPIIb-IIIa also inhibited adhesion, but more antibody was required, and the dose-response curve was flatter than for vWF alone.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The crucial role of vWF in adhesion to the vessel wall at high shear rates is well established. vWF is present in subendothelium deposited by endothelial cells^{43 44} and binds to the subendothelium from the circulation.⁴ The actual binding protein(s) has not been completely identified, but collagen type VI is a strong candidate.^{45 46} Platelets also adhere to connective tissue in deeper layers of the vessel wall and to the connective tissue of atherosclerotic tissue.⁴⁷ This process is mediated by binding of vWF from the circulation to collagen types I and III, which mediate platelet adhesion at high shear rates.⁹ This interaction is essentially different from the binding of vWF to subendothelium.⁴⁸ We studied the properties of vWF as a single adhesive molecule to shed more light on the question of how far properties of vWF can affect overall adhesion to the vessel wall. We also studied the mediation of platelet adhesion to collagen by adsorbed vWF. The comparison between the two conditions may involve two different variables: adsorption of vWF to collagen type III may lead to differences in the function of vWF, and the interaction of platelets with collagen as ligand together with the interaction with vWF may lead to differences in adhesion. Unfortunately, it is not possible to distinguish between these two mechanisms when differences occur, but we found that both processes showed considerable similarity: a gradual increase in adhesion between shear rates of 1000 and 4000 s^-1 and similar inhibition by anti-GPIb and prostacyclin. Both were affected by the multimeric size, but adhesion to vWF itself was somewhat more sensitive. Heparin inhibited adhesion to vWF but had no effect on adhesion to collagen type III; anti–GPIIb-IIIa inhibited adhesion to vWF completely but adhesion to collagen type III only partly. The initial rate of adhesion was higher on vWF than on collagen type III. We conclude that adhesion to collagen type III is strongly but not completely determined by the adhesive properties of vWF. As mentioned above, the differences may be due to differences in properties of vWF when adsorbed to collagen type III compared with glass and/or differences in platelet adhesion caused by the presence of a second ligand interacting with its platelet receptors.

Adsorption of radiolabeled vWF to glass coverslips was linear with a concentration below 50 µg/mL. This agrees well with data showing saturation near concentrations of 200 µg/mL when glass capillaries are incubated with vWF (Table 1). The amount of vWF that bound also accords with reported data.¹² Platelet adhesion to the coated coverslips reached a maximum, however, before the surface was saturated with vWF. Maximum adhesion was reached at 10 µg/mL, which corresponded to a surface binding of 60 ng/cm². No increase in adhesion was seen at higher surface concentrations. The data obtained with adsorbed vWF were compared with those obtained with vWF sprayed on the surface. This technique is the preferred one for many adhesive proteins. It has as a major advantage in that the amount of protein present on the surface is precisely known. A maximum adhesion equivalent to that obtained with adsorbed vWF was found at 3 µg/cm². Since 60 ng/cm² of adsorbed vWF gave optimal adhesion, only 2% of sprayed material appears to be reactive. Whether this is due to denaturation resulting from the spraying or the air drying is not clear. What is evident, however, is that the denaturation is an all-or-nothing phenomenon in view of the excellent adhesion to the sprayed material. The time-dependent adhesion of platelets to vWF shows a fast initial attachment that is not seen with other adhesive proteins. Why adhesion levels off after this first minute is not clear. This cannot be due to coating of the surface with plasma proteins because the vWF has been blocked with a human albumin solution. Nor can it be due to saturation of the surface with platelets, because this tends to occur at surface coverages between 60% and 80% and is common to all adhesive proteins. The fast initial attachment was also observed for ECM and to a lesser extent for collagen type III at 1500 s^-1, which is in agreement with the hypothesis that the vWF-GPIb interaction is responsible for the rapid initial attachment of platelets to subendothelium.⁴⁹

The shear rate dependence of the adhesion to vWF shown here gives a slight but continuous increase in adhesion, with a shear rate of up to 4000 s^-1. This is not identical to previous data in the capillary system that show a leveling off above 2000 s^-1,¹² but more experimental points at high shear rates were studied in the present investigation. The main point of the effect of shear is that adhesion to vWF and vWF-dependent adhesion to ECM and collagen types I and III are the only situations that show no decrease at high shear rates. This suggests that there is a difference in resistance to shear stress between adhesion to vWF and vWF-dependent adhesion to collagen or ECM on the one hand and adhesion to other adhesive molecules on the other.

Platelets aggregate under high shear stress (>30 dynes/cm²=3 Pa), such as that attained in a plate-and-cone viscometer in dependence of vWF.^{35 36} This interaction is mediated by GPIb and leads to activation of platelets followed by exposure of GPIIb-IIIa and interaction of vWF with this receptor.^{37 38} The interaction is particularly mediated by high-molecular-weight vWF multimers.³⁵ We wondered whether this would also occur in whole blood. The shear stress values that are attained in the microcapillary system at a shear rate of 4000 s^-1 are on the order of 16 Pa when one assumes the viscosity of blood to be 4 mPaxs. This is probably not correct in small tubes, where there is a plasma layer near the vessel wall due to the Fahraeus-Lindqvist effect⁵⁰ in which platelets are concentrated. If one calculates the shear stress with a plasma viscosity of 1.1 mPaxs, a shear stress of 4.4 Pa is obtained, which is still in the range found to give platelet aggregation in the plate-and-cone viscometer. It is thus surprising that we did not observe aggregates in our studies, as a possible activating effect of the vWF-GPIb interaction should be further enhanced by the platelet spreading on the surface, which also causes platelet activation. High shear stress may have a different effect on vWF-mediated platelet-platelet interaction than on platelet-substrate interaction.

We have reported that chelation of calcium with either EDTA or EGTA causes a change in function of vWF in supporting adhesion to subendothelium but not in ristocetin-induced aggregation,⁴⁰ but we could not reproduce that finding in the current studies. Incubation of adsorbed vWF with chelating agents had almost no effect on its function. We also checked whether preincubation of vWF in solution with EDTA followed by adsorption from this solution would give a loss in function (data not shown), but this had no effect. We have no explanation for this discrepancy. One possibility that requires further study is that calcium is involved in binding of vWF to subendothelium, a mechanism that involves a substrate other than collagen types I and III.^{45 48}

When we studied the effect of divalent cations we found an effect of heparin that caused a decrease in platelet adhesion to vWF. Further studies showed that preincubation of adsorbed vWF with heparin caused this inhibition. The addition of unfractionated heparin to citrated blood also had this inhibitory effect but to a lesser extent. These studies suggest that heparin from blood binds to vWF and causes an inhibition of interaction with platelets. The existence of such a mechanism is supported by studies that localize the heparin-binding domain of vWF between residues 565 and 587 of the A1 repeat,⁵¹ which is responsible for GPIb binding as well as for all heparin binding⁵² and is in agreement with studies⁵³ that show that heparin inhibits binding of vWF to platelets in the presence of ristocetin or botrocetin and platelet agglutination caused by bovine vWF or asialo-vWF. The lesser inhibitory effect of LMWH may be due to lower binding affinity but may also be due to a less pronounced steric effect caused by its shorter mean chain length. Why heparin does not have this inhibitory effect on platelet adhesion to collagen type III or to ECM (Y.-P.W., J.J.S., P.G. de G., unpublished data, 1993) is unclear. It could be explained if one assumes that heparin interferes with the interaction with GPIIb-IIIa, which is less important on collagen and ECM. However, the heparin-binding site on vWF is not localized near the RGD sequence on residues 1744 through 1747, which is responsible for the interaction with GPIIb-IIIa.

The effect of the multimeric size differed when adhesion to adsorbed vWF or preincubation of collagen type III was studied. Adhesion to adsorbed vWF showed a gradual decrease with a decreasing molecular weight, whereas adhesion to collagen showed a more abrupt decrease in the two lowest fractions. This last effect may be due to the fact that platelet vWF released by collagen may participate in adhesion to collagen^{7 8} and thus make it less sensitive to the composition of exogenous vWF. Carboxy-terminal disulfide-bonded dimers of vWF were generated by expressing vWF-PRO in Chinese hamster ovary cells. Higher surface concentrations of this vWF were required for adhesion, which was less extensive and had notably less spreading than plasma vWF. The initial fast adhesion that is characteristic of vWF was also seen with the dimeric form, but adhesion was less resistant to shear stress, although considerable adhesion was still present at 4000 s^-1. These data indicated that the multimeric structure of vWF has a definite function in platelet adhesion, with 50% reduction with the lowest molecular weight multimers. The adhesion found with vWF-PRO in the current study and with a proteolytic dimeric fragment³⁰ contradicts the absence of adhesion found with vWF type 2A. The low molecular weight of the multimers is thought to be caused by proteolytic cleavage of vWF in at least half the cases,⁵⁴ but these multimers are not functional. This discrepancy requires further study.

A curious phenomenon was the presence of aggregates occurring on vWF-PRO but not on plasma vWF after >10 minutes of perfusion at 1000 s^-1. The studies were performed with the single-pass system, which excludes platelet activation due to recirculation. The shear stress was also less than the 3 Pa required to cause aggregation with high-molecular-weight multimers in the plate-and-cone viscometer discussed above. We have no explanation for this unusual result.

We studied the participation of GPIb and GPIIb-IIIa in adhesion to vWF by using monoclonal antibodies. Both antibodies gave pronounced inhibition (Fig 9). Anti–GPIIb-IIIa was less effective on vWF-dependent adhesion to collagen type III, and the dose-response curve was flatter. Further studies are required to define their precise roles. One such study, which uses mutants of vWF with defective function, is available.⁵⁵ A role for both GPIb and GPIIb-IIIa in adhesion to vWF is in agreement with data from Savage et al¹¹ in a static system and Danton et al¹³ in a flow system.

Adhesion was inhibited by prostacyclin at equivalent concentrations (Fig 8), whether vWF was directly coated to glass or adsorbed by preincubation to collagen type III. The effect of prostacyclin was probably not on the availability of GPIIb-IIIa as the receptor for ligands because studies with anti–GPIIb-IIIa showed differences between vWF alone and vWF-dependent adhesion to collagen type III. Prostacyclin is a relatively poor inhibitor of adhesion to the vessel wall and a better inhibitor of platelet aggregation.⁵⁶ The current data show that this may not be true when a more limited number of adhesive molecules are involved in adhesion.

	Selected Abbreviations and Acronyms

 

ECM = endothelial cell matrix GP = glycoprotein LMWH = low-molecular-weight heparin PBS = phosphate-buffered saline PBS/T = phosphate-buffered saline/0.1% Tween 20 PPACK = D–Phe-Pro-Arg chloromethyl ketone SEM = scanning electron microscopy vWF = von Willebrand factor vWF-PRO = recombinant dimeric von Willebrand factor

Received June 20, 1995; accepted January 5, 1996.

	References

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