Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 1996;93:992-999

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sobel, M.
Right arrow Articles by Harris, R. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sobel, M.
Right arrow Articles by Harris, R. B.

(Circulation. 1996;93:992-999.)
© 1996 American Heart Association, Inc.

Articles

Heparins Designed to Specifically Inhibit Platelet Interactions With von Willebrand Factor

Michael Sobel, MD; Karyn E. Bird, PhD, DVM; Ruth Tyler-Cross, PhD; Dalila Marques, BS; Naoto Toma, MD; H. Edward Conrad, PhD; Robert B. Harris, PhD

From the Division of Vascular Surgery (M.S., K.E.B., D.M., N.T.) and Department of Biochemistry and Biophysics (R.T.-C., R.B.H.), Medical College of Virginia, Virginia Commonwealth University (Richmond); H.H. McGuire Veterans Affairs Medical Center (M.S., K.E.B., D.M., N.T., R.T.-C., R.B.H.), Richmond, Va; Department of Biochemistry (H.E.C.), University of Illinois (Urbana); and Glycomed Inc (H.E.C.), Alameda, Calif.

Correspondence to Michael Sobel, MD, Box 980108 MCV Station, Richmond, VA 23298. E-mail sobel@gems.vcu.edu.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Background Platelet interactions with the injured vessel wall may contribute significantly to the early and late failures of many cardiovascular interventions; the adhesive protein von Willebrand factor (vWF) is thought to play an important role. Previously, we demonstrated that heparin interfered with platelet/vWF hemostatic mechanisms by binding to vWF within the protein's domain responsible for binding the platelet vWF receptor, glycoprotein Ib. The purpose of the present study was to develop and refine heparins with greater potency to inhibit platelet/vWF interactions.

Methods and Results Immobilized synthetic peptides based on a known heparin-binding domain of vWF were used to yield novel fractions of standard heparin that demonstrated a sevenfold increase in their ability to inhibit vWF-dependent platelet agglutination and vWF/platelet binding. The high vWF affinity heparin showed enhanced anti–factor Xa activity but comparable activated partial thromboplastin time activity. Chemical modification of a standard heparin by periodate oxidation and borohydride reduction enhanced its ability to inhibit platelet/vWF interactions by threefold, while eliminating more than 90% of its activated partial thromboplastin time and anti–factor Xa activity. Affinity chromatography of the chemically modified heparin yielded a heparin with an eightfold higher inhibitory potency than the original heparin.

Conclusions Subspecies of heparin can be developed with significantly enhanced potency to inhibit vWF/platelet interactions. The vWF-inhibiting property of heparin can be dissociated from its antithrombin-binding activity. Based on a growing understanding of heparin/vWF interactions, combinations of affinity separations and chemical modifications could be designed to yield heparins uniquely suitable for prevention of arterial thrombosis.


Key Words: heparin • platelets • von Willebrand factor • thrombosis • platelet aggregation inhibitors


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
A wide range of injuries to the arterial wall may set in motion a series of cellular and biochemical responses leading to arterial thrombosis. Although the process of arterial thrombosis at sites of vascular injury is not fully understood, platelets and vWF are known to play principal roles during the initial events. Two distinct platelet receptors bind vWF: GPIb and GPIIb/IIIa.1 GPIIb/IIIa, an {alpha}IIßß3 of the integrin superfamily, binds to a number of adhesive proteins containing the cell adhesion sequence RGD, including fibrinogen, fibronectin, and vitronectin.2 GPIb contributes significantly to the adhesive functions of vWF, which dominate the deposition of platelets on exposed subendothelium or foreign surfaces in areas of high shear stress.3 4 5 6 Shear-induced platelet aggregation (as may occur in stenosed coronary arteries) is also vWF dependent.7 8 When platelets accumulate at sites of injury, they initiate a cascade of events that may lead to acute vessel closure or contribute to the late complications of recurrent stenosis resulting from intimal and fibromuscular hyperplasias. Collectively, these are the most common causes for failure of balloon angioplasty and vascular grafts.

Attention has been focused on pharmacological strategies to control the early events of platelet interaction with the injured endothelium. Aspirin and other platelet-inhibiting drugs have shown only limited efficacy in reducing the frequency of acute closure of injured arteries. Thrombin inhibitors and platelet receptor antagonists show promise but possibly at the expense of increased bleeding complications.9 10 In part, this may be due to their global inhibition of the later events in the hemostatic sequence (thrombin action or fibrinogen binding to GPIIb/IIIa). Ruggeri and others11 12 13 suggested that inhibition of the vWF/GPIb interaction might be an effective antithrombotic strategy as it would block the initial events associated with arterial thrombosis. Alternative hemostatic mechanisms (eg, integrin functions), which are optimized for lower shear conditions, would be preserved. Although no antithrombotic drug has achieved the ideal balance between efficacy and safety, recent data have suggested that selective blockade of the GPIb/vWF interaction may be a useful antithrombotic strategy.14 15 16 An ideal drug would limit pathological platelet interactions while still preserving the global hemostatic mechanisms needed to prevent hemorrhage.

In pursuit of this goal, we observed previously that pharmaceutical heparin significantly impaired vWF-dependent platelet hemostatic mechanisms in vitro and in vivo in humans.17 Further experiments showed that this selective platelet-inhibitory effect was exerted by heparin binding to a portion of the vWF domain responsible for binding GPIb. The effect was not mediated by heparin binding to the platelet or ristocetin. vWF is a multivalent protein: the binding domain for platelet GPIb (its principal platelet receptor) overlaps with an important heparin-binding domain.18 Heparin bound to vWF blocks the protein's binding to GPIb, most likely through steric hindrance or induced conformational changes. We localized the responsible heparin-binding domain of vWF to a 23-residue sequence, Y565-A587, located within the GPIb-binding region.19 Synthetic peptides based on this domain specifically bind heparin with as high affinity as the native protein itself19 and are effective affinity ligands for purifying subfractions of heparin that display high affinity for vWF.20 21

We report the results of two strategies for developing such heparins: refinement of high vWF affinity heparin species by affinity chromatography and chemical modification. Using vWF-based synthetic peptides, we devised large-scale affinity matrices to extract from heterogeneous pharmaceutical heparin species with especially high affinity for vWF. We also showed that periodate oxidation (with or without affinity chromatography) enhanced heparin's affinity for vWF. In the present report, we describe these methods and the biological and biophysical properties of these novel heparin species.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Reagents
For the study, we purchased ristocetin (BioData Co), unfractionated porcine mucosal heparin (170 to 180 USP U/mg; molecular mass, {approx}14 000 to 15 000 D; Celsus Co); and sodium [125I]iodide (Amersham Co). Synthetic peptides were prepared with standardized solid-phase methods, and their purity was confirmed as previously described.19 21 22 The chemically modified heparin Astenose was generously provided by Glycomed Inc (Alameda, Calif).

HPLC and Heparin Assays
A Shimadzu-6A system equipped with a UV detector and recorder (SPD-6A and C-R4A, Shimadzu) was used for HPLC. The molecular masses of heparins were estimated with GPC-HPLC on serial SynChroPak 30/100 analytic columns run at 1 to 1.5 mL/min and equilibrated in 0.2 mol/L PBS, pH 7.4. Unfractionated heparin and heparin oligosaccharide standards of known molecular weight and mass were used for calibration. Heparin mass was determined with a modified carbazole assay.23 The anti–factor Xa activity of the heparins was determined with a chromogenic substrate assay described by Teien and Lie24 and adapted to microtiter plates with reagents supplied by Kabi Pharmacia. aPTT values were determined with a semiautomated method using the Organon Teknika automated aPTT reagent. The aPTT activities of different heparins were compared according to the concentration of heparin that prolonged the baseline aPTT by twofold. The aPTT activity of experimental heparins was then expressed as a percentage of the activity of the standard Celsus heparin (defined as 100%).

Affinity Chromatography
By theoretical design and empirical testing, novel immobilization chemistries were developed to couple the synthetic vWF peptides to Sepharose beads through a spacer arm without compromising essential cationic residues while enabling maximal specific binding between peptide and heparin. Initial experiments were performed with a heterobifunctional cross-linking reagent to conjugate the peptide to Sepharose through a pendant sulfhydryl group, as previously described.20 For economies of cost and scale, this original method was modified to prepare a large bed volume of affinity matrix.

Sepharose CL-6B was first activated with 1,1'-carbonyldiimidazole in anhydrous dioxane solution as described25 and then substituted with 1,6-diaminohexane to a level of 23 µmol/mL settled bed volume (0.285 µmol -NH2 group equivalents/mg of acetone-dried Sepharose). The resulting pendant amino group was derivatized with iodoacetic acid (Aldrich; fivefold molar excess over calculated amino groups) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Aldrich; 10-fold molar relative to the number of amino groups) to affect condensation. The coupling reaction was done in water, pH 4.8 (adjusted with 0.1 N HCl), overnight at room temperature. The reaction was monitored with a ninhydrin assay of a weighed aliquot of acetone-dried gel, and after more than 90% of amino groups were derivatized, the gel was washed extensively with water until the pH of the effluent was neutral. Underivatized amino groups are then covalently modified with a 10-fold molar excess of acetic anhyride in aqueous solution after a 3-hour reaction at room temperature.

The activated and substituted Sepharose gel now bears an iodoacetyl functional group and can be stored at 4°C in water solution (containing 0.2% wt/vol sodium azide) for at least 8 months without apparent loss of functional group substitution. In three different preparations, iodoacetic acid was substituted at {approx}14 µmol/mL of settled bed volume.

Next, the peptide (extended at the carboxyl terminus20 by the sequence ß-Ala-Cys) was coupled to the substituted Sepharose in 50 mmol/L sodium borate per 1 mmol/L EDTA buffer, pH 8.5 (overnight, room temperature); the incorporation of peptide was monitored by the disappearance of reaction with 5,5'-dithiobis-2-nitrobenzoic acid (Ellmans reagent), and the absolute concentration of peptide on each gel was determined by quantitative amino acid analysis of a portion of acetone-dried, weighed substituted Sepharose.

It was previously shown20 that optimal binding of heparin took place at a substitution level of peptide of {approx}1.5 mmol/L. Thus, the desired level of peptide was controlled with an excess of Sepharose with a fixed amount of peptide. Nonderivatized iodoacetyl functional groups were blocked with 2-mercaptoethanol.

Two vWF peptides with confirmed high affinity for heparin were prepared and used as affinity ligands: Y565-A587, the originally described full-length sequence of the heparin-binding domain, and K569-I580, a truncated version that includes the core binding sequence. In previous work, we demonstrated that the full-length and core sequence peptides possess comparable heparin-binding properties.20 Affinity gels were prepared and substituted with 1.6 mmol/L K569-I580 vWF peptide (75 mL) and 1.4 mmol/L Y565-Y586 vWF peptide (70 mL). For chromatography, the affinity gels are equilibrated in 50 mmol/L Tris buffer, pH 7.5. The starting material for fractionation was standard porcine mucosal heparin (Celsus) in equilibrating buffer at 3 g/20 mL. After elution of the nonbinding (breakthrough) fraction of heparin, the vWF peptide columns were washed exhaustively, and then the bound fraction was eluted sequentially with 1 and 2 mol/L NaCl in equilibrating buffer. In initial experiments, the equilibrating buffer was prepared at low ionic strength (0.1 mol/L NaCl), and a step of 0.5 mol/L NaCl was included. For later production purposes, the buffer was prepared in 0.5 mol/L NaCl, and steps of 1 and 2 mol/L alone were collected.

The elution profile was monitored at 206 nm, and the appropriate fractions were pooled and desalted by exhaustive dialysis against multiple changes of deionized, glass-distilled water (1000 D molecular mass cutoff dialysis membrane; Fisher). The heparins were recovered with lyophilization, and the heparin content was determined with the carbazole method.

Chemical Derivatization of Heparin
Porcine mucosal heparin was chemically modified by a proprietary process of periodate oxidation and sodium borohydride reduction in which polymer chain length of the polysaccharide is relatively preserved.26 27 By this process, the 1,2 ring diols are converted to the corresponding open chain alcohols, selectively disrupting the structure of the antithrombin-binding pentasaccharide domain. This heparin, Astenose (Glycomed Inc), has an average molecular mass of 11 000 D and ATIII-binding activity of {approx}5 to 7 anti–factor Xa U/mg.

Platelet Aggregometry
The ability of heparins to inhibit vWF-dependent platelet aggregation was quantified by the measurement of ristocetin-induced agglutination of fixed platelets. In earlier work, we demonstrated that the inhibitory effects of heparin were the same with the use of either fresh or fixed platelets in plasma, so formaldehyde-fixed platelets were prepared and used as previously described.17 20 Briefly, fixed platelets were suspended in 0.15 mol/L Tris-buffered saline at a final concentration of 200 000/µL. For each batch of platelets, a standard amount of normal pooled, citrated plasma (the source of vWF) was chosen based on titrations to the lowest concentration needed to achieve maximal agglutination to ristocetin. Platelets were preincubated with plasma and heparin or buffer control for 10 minutes at 37°C. Agglutination in response to ristocetin (1 mg/mL final concentration) was measured with a Scienco aggregometer in standardized aggregation units. Inhibition by heparins was expressed as a reduction in the percent of the maximal agglutination in response to ristocetin alone. To compare the potency of the different heparins, the concentration at which there was 80% inhibition of agglutination (IC80) was interpolated. This level was chosen to provide practical comparisons that would be relevant to their clinical application as potential antithrombotic drugs, where a high level of inhibition would be desirable. Studies were performed in quadruplicate or more, and the mean and SEM values are reported.

Platelet/vWF Radioligand Binding Assay
The previously described assay17 was modified for use in specialized 96-well microtiter plates (Millipore Corp). Human vWF was purified from cryoprecipitate and labeled with [125I]iodide.17 125I-vWF was added to each well at a final concentration of 1.0x10-10 mol/L ({approx}50 000 counts per well). Washed human platelets were added to a final concentration of 200 000/µL, along with doubling dilutions of each heparin (or buffer control), beginning at the highest concentration of 3.33x10-5 mol/L. Each heparin dilution was run in duplicate. The plate was incubated at 37°C for 15 minutes. Ristocetin was then added to a final concentration of 0.25 mg/mL, and the plate was again incubated for 15 minutes at 37°C. Platelet-bound vWF was separated from free by vacuum filtration across the hydrophilic 0.22-µm-filter bottoms of each well. The wells were washed with vWF assay buffer (0.05 mol/L Tris-HCl, 0.15 mol/L NaCl, 0.1% ovalbumin, pH 7.5); the membranes were dried and punched out; and the radioactivity of each was measured with a gamma counter (LKB-Wallac 1282 CompuGamma). Total binding was assessed in the absence of heparin. Nonspecific binding (typically {approx}7% of total counts) was estimated by incubation of the platelets in the presence of 125I-vWF without ristocetin. Alternative methods of estimating nonspecific binding (including high doses of heparin and excess unlabeled vWF) yielded similar results. The data presented represent specific binding, which was calculated by subtraction.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
down arrowReferences
 
Affinity Chromatography Yields
Peptide/Sepharose matrices were prepared containing the full-length heparin-binding domain of vWF (Y565-A587) and containing the core sequence (K569-I580) at densities of 1.4 and 1.6 mmol/L, respectively. The heparin fractions that eluted from these two peptide matrices were indistinguishable by all the parameters detailed below and are therefore summarized together. Sepharose matrices that were identically derivatized and prepared, but without conjugated peptides, did not bind heparin (data not shown). Approximately 8% of the heparin mass eluted in 1.0 mol/L NaCl, and 2% eluted in 2.0 mol/L NaCl. When an equivalent quantity of high-affinity refined heparin was reapplied to the same column, more than 85% was bound, and reeluted at high ionic strength. Fig 1Down compares the yields of reapplied high-affinity material with that of the standard unfractionated heparin. Approximately 27% of the loaded Astenose eluted at high NaCl concentrations (1.0 and 2.0 mol/L together) from the same peptide/affinity matrix.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 1. Two different heparins were independently applied to the peptide affinity matrix, and the total quantities that did not bind and that bound and eluted at high ionic strength (1.0 and 2.0 mol/L NaCl together) were measured. For standard unfractionated heparin, only 10% bound. When heparin that had been previously fractionated as high affinity was reapplied, more than 85% bound again. Because of losses during the process, the values of bound and unbound total slightly less than 100%.

Characterization of the Heparins
Molecular Mass
When standard heparin was fractionated on the peptide matrices, the portions that eluted at different ionic strengths were characterized by GPC-HPLC (Fig 2Down). The TableDown and Fig 2Down compare the average peak molecular masses of the fractions and the unfractionated starting heparin. There is a clear trend toward increasing molecular mass with increasing ionic strength of elution. The heparin fraction that did not bind to the affinity gel was also analyzed. Fig 3Down demonstrates that this "depleted" or breakthrough fraction shows the loss of a species of specific molecular mass range corresponding to the peak of the high-affinity fraction. However, in this breakthrough fraction, there clearly remains a heparin species with higher molecular mass than the fraction eluting at high ionic strength. In separate experiments, Astenose showed a modest shift in molecular mass, from 11 000 to 12 500 D (2 mol/L fraction).



View larger version (13K):
[in this window]
[in a new window]
  		Figure 2. GPC-HPLC analysis of the molecular mass distribution of heparin fractions (see "Methods" for details). Compared are the standard unfractionated porcine heparin starting material (Unfx Heparin) and fractions eluting from a stepwise gradient of increasing ionic strength (1.0 and 2.0 mol/L NaCl). The passage of time from sample injection is shown on the abscissa from left to right, and the elution profile was monitored at 210 nm.


View this table:
[in this window]
[in a new window]
  		Table 1. Physicochemical Properties of the Heparin Species



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. A composite of GPC-HPLC profiles of the standard heparin starting material (Unfx. Heparin), the heparin eluting at high NaCl (2.0 mol/L NaCl), and the heparin fraction that did not bind to the affinity column (Depleted Heparin). See Fig 2Up and "Methods" for details.

Anticoagulant Activity
The anti–factor Xa activity and conventional anticoagulant activity (aPTT) of the affinity fractionated and chemically modified heparins are presented in the TableUp and Fig 4Down. Although the heparins with high affinity for vWF also showed enhancement of their anti–factor Xa activity, this was not reflected in their global anticoagulant activity as measured by the aPTT. Binding to the peptide matrix did not appear to be related to anticoagulant activity, as the nonbinding and low-affinity fractions also had normal aPTT activity. The process of chemical modification reduced anti–factor Xa activity and aPTT activity by 96% and 93%, respectively.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 4. Conventional anticoagulant activity of representative heparins, as measured by aPTT assay. Control heparin is the standard, unfractionated mucosal heparin; breakthrough effluent is the fraction that did not bind the peptide affinity matrix. vWF heparin is a high-affinity fraction eluted at 2.0 mol/L NaCl, and 0.5 mol/L is a preceding fraction. Astenose is the chemically modified heparin. Comparisons were made based on the relative concentration of heparin required to prolong the aPTT to twice the control value. Data points represent the mean and SEM of two or three independent assays.

Heparin Inhibition of vWF/Platelet Interactions
Affinity-Separated Heparins
The fractions from each of five affinity separation experiments of standard heparin were independently tested for their ability to inhibit vWF-dependent platelet agglutination. These assay results are presented in Fig 5Down. The nonbinding or breakthrough effluent shows distinctly lower inhibitory potency (IC80 >20 µmol/L) compared with the unfractionated starting heparin (IC80=7.6 µmol/L). The 0.5 mol/L NaCl eluate was not appreciably different from the original heparin, whereas the heparins eluting at 1 and 2 mol/L NaCl show significantly enhanced inhibitory potency. The TableUp summarizes the calculated IC80 values of each fraction.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. An assessment of the ability of different heparin fractions to inhibit vWF-dependent platelet agglutination induced by ristocetin. Standard porcine mucosal heparin was subjected to affinity chromatography on vWF/peptide matrix. The heparin fraction that did not bind at all (Breakthrough effluent), and the fractions eluting at 0.5, 1.0, and 2.0 mol/L NaCl were tested. The heparin fractions from five separate chromatographic experiments were independently tested in quadruplicate, and mean and SEM values of each fraction type are presented. The IC80 (the point of 20% maximal agglutination) is used for comparison between heparins.

Chemically Modified Heparins
The original heparin used for periodate oxidation/borohydride reduction was of a different commercial source than the standard heparin (Celsus) used for simple affinity fractionation. Therefore, to determine the effects of chemical modification, independent of affinity fractionation, this source heparin was also tested for its ability to inhibit vWF-dependent platelet agglutination (Fig 6Down). The starting material (a pharmaceutical-grade porcine heparin of molecular mass {approx}14,000 D obtained commercially by Glycomed Inc) was slightly less potent at inhibiting vWF-dependent platelet agglutination than the standard heparin used for the preceding fractionations (Celsus and Sigma). Fig 6Down shows that the chemical modification alone significantly enhanced the inhibitory potency of the source heparin despite some loss in polymer chain length (molecular mass {approx}11 000 D).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 6. Measurement of the ability of different heparin fractions to inhibit vWF-dependent platelet agglutination induced by ristocetin (see "Methods" for details). A commercial porcine mucosal heparin (Astenose Source Heparin) was subjected to periodate oxidation and reduction to yield Astenose, which was then affinity-fractionated and analyzed as in Fig 5Up. The breakthrough fraction (not shown) had a profile similar to the 0.5 mol/L eluate. The heparin fractions from two separate chromatographic experiments were independently tested in quadruplicate, and mean and SEM values of each fraction type are presented.

Fig 6Up also shows the results of subsequent affinity fractionation of this periodate-oxidized, borohydride-reduced heparin under identical conditions as above. Affinity fractionation further enhanced the vWF-inhibiting potency of the oxidized heparin. Although the 1 mol/L eluate was no different from the starting material, the 2 mol/L eluate clearly showed enhanced activity, comparable in potency to the 2 mol/L elution fraction of standard heparin in Fig 5Up. The 0.5 mol/L elution of Astenose showed reduced potency to inhibit platelet agglutination, more closely resembling the breakthrough or nonbinding fraction derived from standard heparin. The behavior of the breakthrough fraction of Astenose (not shown) was the same as its 0.5 mol/L elution. The characteristics of Astenose and its fractions are summarized in the TableUp.

Inhibition of Platelet/vWF Binding
The same products of affinity fractionation of standard heparin were also tested for their ability to inhibit the binding of 125I-vWF to platelets. This assay system is {approx}10-fold more sensitive to the inhibitory effects of heparin because of the much lower concentrations of vWF used. Fig 7Down summarizes the typical results. The 1 and 2 mol/L eluates fractionated from unmodified heparin again showed a nearly identical pattern of significantly enhanced inhibitory potency compared with the starting heparin. The breakthrough fraction that did not bind to the peptide affinity column was an extremely poor inhibitor of vWF binding to platelets, as it was in the agglutination assay. Astenose and its 2 mol/L chromatographic product also showed results comparable to their behavior in the agglutination assay.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 7. Heparin inhibition of 125I-vWF binding to platelets, as described in "Methods." Standard and chemically modified heparins were affinity-fractionated on vWF/peptide matrix. Samples from four separate chromatographic experiments were independently assayed two to four times for their ability to block the binding of vWF to platelet GPIb. The data points represent the mean and SEM values. Control heparin and its elution products correspond to materials tested in Fig 5Up; Astenose and its high-affinity product correspond to Fig 6Up.

The possibility that differences in molecular mass were responsible for the enhanced biological activity of the fractionated heparins was assessed by testing fractions of heparin that had been selected solely according to molecular mass. Moieties of peak molecular masses of 19 000 and 36 000 D (a generous gift from Per Ostergaard, Novo Nordisk, Denmark) were thus tested for their ability to inhibit vWF binding to fixed platelets in the same assay system. Fig 8Down compares their potency with that of the 1.0 mol/L (molecular mass {approx}16 000 D) and 2.0 mol/L (molecular mass {approx}19 000 D) affinity-purified fractions previously studied. The data show that size for size, the affinity-fractionated heparins had significantly higher potency to inhibit vWF/platelet binding. Even a 36 000-D size-selected fraction had less potency than the 19 000-D 2.0 mol/L affinity fraction.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 8. Heparin inhibition of 125I-vWF binding to platelets: a comparison of fractions selected by molecular mass versus affinity for vWF peptide. The high vWF affinity heparin fractions derived from standard heparin (1.0 mol/L, molecular mass {approx}16 000 D; 2.0 mol/L, molecular mass {approx}19 000 D) are compared with subfractions of standard heparin separated exclusively according to molecular mass (peak molecular mass, 36 000 and 19 000 D).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
Because heparin is such a structurally heterogeneous substance, it is not surprising that its diverse biological properties might be separable through chemical modification or affinity methods. For example, the ability of heparin to inhibit smooth muscle proliferation can be dissociated from its binding affinity for ATIII.28 29 The hypothesis that the novel ability of heparins to inhibit vWF/platelet interactions might be a discrete and dissociable property of the heparin led us to pursue the present study. The goal was to determine whether a subspecies of pharmaceutical heparin could be practically refined or modified to enhance its potency to inhibit platelet/vWF interactions.

We found that affinity fractionation with vWF-based peptides yields a subspecies of heparin with unique characteristics. Fractionation selects a subgroup of heparins with higher molecular mass but does not simply select the largest heparins present in the heterogeneous starting material. The high vWF affinity heparins show at least a sevenfold enhancement of potency with respect to inhibiting platelet/vWF interactions as measured by platelet aggregometry and vWF/platelet radioligand binding assay. Chemical modification of unfractionated heparin by periodate oxidation and reduction also enhances the vWF affinity by at least threefold, while destroying most of its ATIII-binding affinity. Despite some reduction in molecular mass, this chemical modification amplifies its affinity for vWF. Through a combination of chemical modification and chromatographic refinement, an approximately eightfold enhancement of vWF-inhibiting activity was obtained.

Just as other biological activities of heparin can be dissociated, we have shown that the vWF-binding and ATIII-binding properties of heparin can be separated. This is most dramatically seen in the chemically modified heparin, in which the critical antithrombin-binding pentasaccharide structure is disrupted.30 This modified heparin has negligible anti–factor Xa activity, very reduced aPTT activity, yet a threefold enhancement of vWF inhibitory activity. This dissociation of anticoagulant and vWF-binding properties of heparin is further supported by the recent studies of Baruch et al.31 They observed that separation of heparins according to their affinity for ATIII did not alter their affinities for vWF: the pentasaccharide sequence responsible for binding ATIII was not essential for binding vWF. They saw a molecular mass dependency for heparin's affinity for vWF, as we did. And although they suggested that molecular mass primarily governs heparin's affinity for vWF, some of their chemically modified heparin fractions possessed vWF affinity comparable to that of other heparins nearly twice their molecular mass.31

Several lines of evidence support the concept that there is some structural specificity to heparin's vWF affinity. First, when the high-affinity refined material is reapplied to the same affinity matrix, it behaves like a discrete species. More than 85% of it is bound on the second pass. Second, although affinity fractionation selected a generally higher molecular mass moiety of heparin, the process did not select the largest heparin species. Size-equivalent heparins that were prepared solely according to their molecular mass were not as biologically active as those selected for their affinity for vWF peptides. Finally, chemical modification of heparin can enhance its vWF inhibitory activity even while reducing its molecular mass. This especially suggests that there is some structural specificity to this biological activity, independent of polysaccharide chain length.

To date, the heparins we have studied are still relatively large polymers, despite their narrower biological profiles. We do not yet know whether the domains of heparin that bind vWF and interfere with platelet/vWF binding are as structurally unique as the antithrombin-binding pentasaccharide, nor do we understand clearly how the chemical modification alters the resulting biological activity of the glycosaminoglycan. This emphasizes the importance of our ongoing work to identify discrete vWF-binding domains of heparin. It is possible that the structural features of heparin that direct binding to vWF represent a more broadly conserved strategy for protein binding. The same features responsible for binding vWF may regulate heparin's interactions with a number of proteins outside the serpin superfamily. For example, in related studies of heparins that bind directly to platelets, we have identified structurally unique oligosaccharides (derived by periodate oxidation) that dictate heparin's specific binding to platelet membranes.32

Clinical Relevance
vWF/platelet interactions play an important role in the events of thrombosis at sites of vascular injury. Especially under high shear conditions, vWF-GPIb binding is important for the initial platelet/platelet and platelet/surface interactions.5 33 Ikeda et al13 demonstrated a biphasic aggregation response to low and high shear rates. Total blockade of all ligand/GPIIb/IIIa interactions suppresses platelet aggregation at both low and high shear rates, but specific inhibition of vWF adhesive function (either via selective blockade of the vWF/GPIIb/IIIa or vWF/GPIb interaction) only suppresses aggregation at high shear rate. These findings point to a promising method for pharmacological prevention of thrombosis in stenosed, injured arteries: selective inhibition of the platelet responses mediated by GPIb and vWF. This strategy could potentially avoid the global suppression of platelet reactivity and hemorrhagic diathesis associated with GPIIb/IIIa blockade.10

Currently, much debate centers around the relative roles played by thrombin,34 35 GPIb,36 37 GPIIb/IIIa,38 39 and adhesive proteins in the genesis of platelet-dominated thrombosis. With appropriate models, all of these constituents can be shown to contribute significantly. We have attempted to specifically isolate the interaction of vWF with platelet GPIb by using fixed platelets and ristocetin as a cofactor. These methods have proved to be a valid surrogate for the native interactions of vWF with GPIb,40 41 42 to the exclusion of vWF/GPIIb/IIIa interactions or thrombin effects.6 43 Only continued studies of more complex models of thrombosis will determine whether strategies to inhibit vWF/GPIb interactions hold significant therapeutic potential. Nevertheless, antibodies to GPIb,36 peptide antagonists,12 and anionic compounds that bind vWF16 44 already show promise. Related studies have documented that heparin can also reduce platelet deposition on injured arteries,4 45 even more effectively than specific thrombin inhibitors in some models.15 We are evaluating the efficacy of the current heparins in more complex, clinically relevant models of thrombosis.

It is not clear why the high vWF affinity fractions derived from standard heparin showed enhanced affinity for ATIII but normal aPTT activity. For low-molecular-mass heparins the converse is true: higher anti–factor Xa activity versus aPTT activity. These current observations are favorable from a clinical therapeutic point of view, suggesting that an enhanced antiplatelet effect can be achieved without the liabilities of an increased anticoagulant effect. The limiting factor for capitalizing on this antiplatelet property of heparin has been the intense anticoagulation that would accompany the required high doses. This current report raises the possibility that a combination of chemical and affinity-based processes could yield heparins with unique and extremely focused antithrombotic activities, for example, a heparin that could prevent platelet-mediated thrombosis (where vWF was an important mediator) but that did not significantly impair plasma coagulation. Conversely, based on our growing understanding of what dictates heparin/vWF interactions, a heparin could be devised with potent conventional anticoagulant activity that has minimal effects on platelet function.


*    Selected Abbreviations and Acronyms
 
aPTT = activated partial thromboplastin time
ATIII = antithrombin III
GPC = gel permeation chromatography
GPIb = glycoprotein Ib
GPIIb/IIIa = complex of glycoproteins IIb and IIIa
HPLC = high-performance liquid chromatography
vWF = von Willebrand factor


*    Acknowledgments
 
This work was funded in part by grants from the National Institutes of Health (RO1-HL-39903, Dr Sobel), the Veterans Administration Research Service (Dr Sobel), Commonwealth Biotechnologies Inc (Dr Harris), and Glycomed Inc (Drs Sobel and Harris). Preliminary data from this work were previously published in abstract form (Circulation. 1992;86[suppl I]:I-869). We are grateful to Dr Per Ostergaard for his generous sharing of heparin materials.


*    Footnotes
 
Dr Conrad was an employee of Glycomed Inc. during the early phases of this work. He is no longer employed with Glycomed and retains no financial interest in the company. Likewise, Glycomed Inc. supported the initial work with an unrestricted grant, which has concluded.

Received June 9, 1995; revision received October 5, 1995; accepted October 10, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

  1. Ruggeri ZM, Zimmerman TS. von Willebrand factor and von Willebrand disease. Blood. 1987;70:895-904. [Abstract/Free Full Text]
  2. Hawiger J. Adhesive interactions of blood cells and the vascular wall. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and Thrombosis. Philadelphia, Pa: JB Lippincott Co; 1994:762-796.
  3. Moroose R, Hoyer LW. von Willebrand factor and platelet function. Annu Rev Med. 1986;37:157-163. [Medline] [Order article via Infotrieve]
  4. Badimon L, Badimon JJ, Rand J, Turitto VT, Fuster V. Platelet deposition on von Willebrand factor-deficient vessels: extracorporeal perfusion studies in swine with von Willebrand's disease using native and heparinized blood. J Lab Clin Med. 1987;110:634-647. [Medline] [Order article via Infotrieve]
  5. Moake JL. Insolubilized von Willebrand factor and the initial events in hemostasis. J Lab Clin Med. 1989;114:1-3. [Medline] [Order article via Infotrieve]
  6. Olson JD, Zaleski A, Hermann D, Flood PA. Adhesion of platelets to purified solid-phase von Willebrand factor: effects of wall shear rate, ADP, thrombin, and ristocetin. J Lab Clin Med. 1989;114:6-18. [Medline] [Order article via Infotrieve]
  7. Moake JL, Turner NA, Stathopoulos NA, Nolasco L, Hellums JD. Shear-induced platelet aggregation can be mediated by vWF released from platelets, as well as by exogenous large or unusually large vWF multimers, requires adenosine diphosphate, and is resistant to aspirin. Blood. 1988;71:1366-1374. [Abstract/Free Full Text]
  8. Alevriadou BR, Moake JL, Turner NA, Ruggeri ZM, Folie BJ, Phillips MD, Schreiber AB, Hrinda ME, McIntire LV. Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets. Blood. 1993;81:1263-1276. [Abstract/Free Full Text]
  9. Antman EM, for the TIMI 9A Investigators. Hirudin in acute myocardial infarction: safety report from the Thrombolysis and Thrombin Inhibition in Myocardial Infarction (TIMI) 9A Trial. Circulation. 1994;90:1624-1630. [Abstract/Free Full Text]
  10. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty: the EPIC Investigation. N Engl J Med. 1994;330:956-961. [Abstract/Free Full Text]
  11. Ruggeri ZM. New insights into the mechanisms of platelet adhesion and aggregation. Semin Hematol. 1994;31:229-239. [Medline] [Order article via Infotrieve]
  12. Ruggeri ZM. von Willebrand factor as a target for antithrombotic intervention. Circulation. 1992;86(suppl III):III-26-III-29.
  13. Ikeda Y, Handa M, Kawano K, Kamata T, Murata M, Araki Y, Anbo H, Kawai Y, Watanabe K, Itagaki I, Sakai K, Ruggeri ZM. The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J Clin Invest. 1991;87:1234-1240.
  14. Yao S-K, Ober JC, Garfinkel LI, Hagay Y, Ezov N, Ferguson JJ, Anderson HV, Panet A, Gorecki M, Buja LM, Willerson JT. Blockade of platelet membrane glycoprotein Ib receptors delays intracoronary thrombogenesis, enhances thrombolysis, and delays coronary artery reocclusion in dogs. Circulation. 1994;89:2822-2828. [Abstract/Free Full Text]
  15. Lawrence JB, Bodenheimer SL. Thrombin inhibition is not responsible for the inhibition of platelet adherence produced by unfractionated heparin. Blood. 1991;78S:217a.
  16. Girma JP, Fressinaud E, Christophe O, Rouault C, Obert B, Takahashi Y, Meyer D. Aurin tricarboxylic acid inhibits platelet adhesion to collagen by binding to the 509-695 disulphide loop of von Willebrand factor and competing with glycoprotein Ib. Thromb Haemost. 1992;68:707-713. [Medline] [Order article via Infotrieve]
  17. Sobel M, McNeill PM, Carlson P, Kermode JC, Adelman B, Conroy R, Marques D. Heparin inhibition of von Willebrand factor in vitro and in vivo. J Clin Invest. 1991;87:1787-1793.
  18. Meyer D, Girma J-P. von Willebrand factor: structure and function. Thromb Haemost. 1993;70:99-104. [Medline] [Order article via Infotrieve]
  19. Sobel M, Soler DF, Kermode JC, Harris RB. Localization and characterization of a heparin binding domain peptide of human von Willebrand factor. J Biol Chem. 1992;267:8857-8862. [Abstract/Free Full Text]
  20. Tyler-Cross R, Sobel M, Marques D, Soler DF, Harris RB. Heparin-von Willebrand factor binding as assessed by isothermal titration calorimetry and by affinity fractionation of heparins using synthetic peptides. Arch Biochem Biophys. 1993;306:528-533. [Medline] [Order article via Infotrieve]
  21. Tyler-Cross R, Sobel M, Marques D, Harris RB. Heparin binding domain peptides of antithrombin III: analysis by isothermal titration calorimetry and circular dichroism spectroscopy. Protein Sci. 1994;3:620-627. [Abstract]
  22. Soler DF, Sobel M, Harris RB. Design and synthesis of a helix heparin-binding peptide. Biochemistry. 1992;31:5010-5016. [Medline] [Order article via Infotrieve]
  23. Blumenkrantz N, Asboe-Hansen G. New method for quantitative determination of uronic acids. Anal Biochem. 1973;54:484-489. [Medline] [Order article via Infotrieve]
  24. Teien AN, Lie M. Evaluation of an amidolytic heparin assay method: increased sensitivity by adding purified antithrombin III. Thromb Res. 1977;10:399-410. [Medline] [Order article via Infotrieve]
  25. Bethell GS, Ayers JS, Hancock WS, Hearn MT. A novel method of activation of cross-linked agaroses with 1,1'-carbonyldiimidazole which gives a matrix for affinity chromatography devoid of additional charged groups. J Biol Chem. 1979;254:2572-2574. [Abstract/Free Full Text]
  26. Patent No. 5250519. Non-anticoagulant heparin derivatives. Assignee 226565, Glycomed Inc, California, 1993.
  27. Patent No. 5280016. Non-anticoagulant heparin derivatives. Assignee 226565, Glycomed Inc, California, 1994.
  28. Guyton JR, Rosenberg RD, Clowes AW, Karnovsky MJ. Inhibition of rat arterial smooth muscle cell proliferation by heparin: in vivo studies with anticoagulant and nonanticoagulant heparin. Circ Res. 1980;46:625-634. [Free Full Text]
  29. Wright TC Jr, Castellot JJ Jr, Petitou M, Lormeau J-C, Choay J, Karnovsky MJ. Structural determinants of heparin's growth inhibitory activity: interdependence of oligosaccharide size and charge. J Biol Chem. 1989;264:1534-1542. [Abstract/Free Full Text]
  30. Casu B. Methods of structural analysis. In: Lane DA, Lindahl U, eds. Heparin, Chemical and Biological Properties: Clinical Applications. Boca Raton, Fla: CRC Press; 1989:25-49.
  31. Baruch D, Ajzenberg N, Denis C, Legendre P, Lormeau J-C, Meyer D. Binding of heparin fractions to von Willebrand factor: effect of molecular mass and affinity for antithrombin III. Thromb Haemost. 1994;71:141-146. [Medline] [Order article via Infotrieve]
  32. Suda Y, Marques D, Kermode JC, Kusumoto S, Sobel M. Structural characterization of heparin's binding domain for human platelets. Thromb Res. 1993;69:501-508. [Medline] [Order article via Infotrieve]
  33. Lawrence JB, Gralnick HR. Monocolonal antibodies to the glycoproteins involved in adhesive protein binding: effects on platelets spreading and ultrastructure on human arterial subendothelium. J Lab Clin Med. 1987;109:495-503. [Medline] [Order article via Infotrieve]
  34. Heras M, Chesebro JH, Webster MW, Mruk JS, Grill DE, Penny WJ, Bowie EJ, Badimon L, Fuster V. Hirudin, heparin, and placebo during deep arterial injury in the pig: the in vivo role of thrombin in platelet-mediated thrombosis. Circulation. 1990;82:1476-1484. [Abstract/Free Full Text]
  35. Harker LA. Platelets and vascular thrombosis. N Engl J Med. 1994;330:1006-1007. [Free Full Text]
  36. Miller JL, Thiam-Cisse M, Drouet LO. Reduction in thrombus formation by PG-1 F(ab')2, an anti-guinea pig platelet glycoprotein Ib monoclonal antibody. Arterioscler Thromb. 1991;11:1231-1236. [Abstract/Free Full Text]
  37. Roth GJ. Developing relationships: arterial platelet adhesion, glycoprotein Ib, and leucine-rich glycoproteins. Blood. 1991;77:5-19. [Free Full Text]
  38. Kohmura C, Gold HK, Yasuda T, Holt R, Nedelman MA, Guerrero JL, Weisman HF, Collen D. A chimeric murine/human antibody Fab fragment directed against the platelet GPIIb/IIIa receptor enhances and sustains arterial thrombolysis with recombinant tissue-type plasminogen activator in baboons. Arterioscler Thromb. 1993;13:1837-1842. [Abstract]
  39. Weiss HJ, Hawiger J, Ruggeri ZM, Turitto VT, Thiagarajan P, Hoffmann T. Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J Clin Invest. 1989;83:288-297.
  40. Coller BS, Peerschke EI, Scudder LE, Sullivan CA. Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor. Blood. 1983;61:99-110. [Abstract/Free Full Text]
  41. Moake JL, Olson JD, Troll JH Jr, Weinger RS, Peterson DM, Cimo PL. Interaction of platelets, von Willebrand factor, and ristocetin during platelet agglutination. J Lab Clin Med. 1980;96:168-184. [Medline] [Order article via Infotrieve]
  42. Allain JP, Cooper JA, Wagner RH, Brinkhaus KM. Platelets fixed with paraformaldehyde: a new reagent assay of von Willebrand factor and platelet aggregating factor. J Lab Clin Med. 1975;25:318-321.
  43. Hardisty RM. Molecular mechanism of platelet adhesion. Adv Exp Med Biol. 1985;192:411-418. [Medline] [Order article via Infotrieve]
  44. Phillips MD, Moake JL, Nolasco L, Turner N. Aurin tricarboxylic acid: a novel inhibitor of the association of von Willebrand factor and platelets. Blood. 1988;72:1898-1903. [Abstract/Free Full Text]
  45. Adelman B, Stemerman MB, Handin RI. Interaction of platelets and fibrin with injured rabbit aortic neointima: effect of prostaglandin I2 and heparin. Arteriosclerosis. 1983;3:141-148.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CirculationHome page
A. O. Spiel, J. C. Gilbert, and B. Jilma
Von Willebrand Factor in Cardiovascular Disease: Focus on Acute Coronary Syndromes
Circulation, March 18, 2008; 117(11): 1449 - 1459.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. Perrault, N. Ajzenberg, P. Legendre, G. Rastegar-Lari, D. Meyer, J. A. Lopez, and D. Baruch
Modulation by Heparin of the Interaction of the A1 Domain of von Willebrand Factor With Glycoprotein Ib
Blood, December 15, 1999; 94(12): 4186 - 4194.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
E. M. Antman and R. Handin
Low-Molecular-Weight Heparins : An Intriguing New Twist With Profound Implications
Circulation, July 28, 1998; 98(4): 287 - 289.
[Full Text] [PDF]

Home page
 CirculationHome page
G. Montalescot, F. Philippe, A. Ankri, E. Vicaut, E. Bearez, J. E. Poulard, D. Carrie, D. Flammang, A. Dutoit, A. Carayon, et al.
Early Increase of von Willebrand Factor Predicts Adverse Outcome in Unstable Coronary Artery Disease : Beneficial Effects of Enoxaparin
Circulation, July 28, 1998; 98(4): 294 - 299.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
L. F. Poletti, K. E. Bird, D. Marques, R. B. Harris, Y. Suda, and M. Sobel
Structural Aspects of Heparin Responsible for Interactions With von Willebrand Factor
Arterioscler. Thromb. Vasc. Biol., May 1, 1997; 17(5): 925 - 931.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sobel, M.
Right arrow Articles by Harris, R. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sobel, M.
Right arrow Articles by Harris, R. B.

Circulation Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 1996 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.