Synthetic Peptide From the V3 Loop Consensus Motif With A Potent Anti-HIV Activity Inhibits Ristocetin-Mediated vWF-GPIb Interaction

Peptides, Vol. 18, No. 9, pp. 1289 –1293, 1997 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 1 .00

PII S0196-9781(97)00205-2

Synthetic Peptide From the V3 Loop Consensus Motif With A Potent Anti-HIV Activity Inhibits Ristocetin-Mediated vWF-GPIb Interaction HIROSHI MOHRI,* YUSUKE ASAKURA,* JUN FUKUSHIMA,† SUSUMU KAWAMOTO,† TAKAO OKUBO* AND KENJI OKUDA† *The First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236, Japan †Department of Bacteriology, Yokohama City University School of Medicine, Yokohama 236, Japan Received 3 April 1997; Accepted 23 June 1997 MOHRI, H., Y. ASAKURA, J. FUKUSHIMA, S. KAWAMOTO, T. OKUBO AND K. OKUDA. Synthetic peptide from the V3 loop consensus motif with a potent anti-HIV activity inhibits ristocetin-mediated vWF-GPIb interaction. PEPTIDES 18(9) 1289 –1293, 1997.—The V3 loop consensus motif. Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile (HIV-1 IIIB), inhibits an interaction of HIV with CD4-positive lymphocytes. Recently, both proline-rich peptides and peptides containing proline-glycine loops (b-turns) form a complex with ristocetin dimers. These peptides interact with ristocetin-loaded platelet membrane glycoprotein (GP) Ib and act as inhibitors of von Willebrand factor (vWF)-GPlb interaction by preventing the subsequent formation of ristocetin dimer bridges. The Pro-Gly sequence is also present in the V3 loop consensus motif, Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile (HIV-1 IIIB). In this report, we have evaluated the effect of the HIV-1 IIIB peptide on vWF binding to GPIb. This peptide only inhibited vWF binding to GPIb as well as platelet aggregation in the presence of ristocetin while it had no effect on botrocetin-mediated vWF interaction with platelets. The peptide inhibited a binding of anti-vWF monoclonal antibody (RG-46) to immobilized vWF. Furthermore, ristocetin inhibited the binding of HIV-1 IIIB peptide to immobilized CXC-chemokine receptor-4 (CXCR-4) peptide. These results indicate that ristocetin may prevent HIV infection and would be useful a tool to understand the mechanism of HIV tissue tropism and infection. © 1997 Elsevier Science Inc. HIV-1 IIIB peptide

von Willebrand factor

Glycoprotein Ib

HUMAN immunodeficiency virus type-1 (HIV-1) infection is a poor prognostic and sometimes fetal disease. The third variable domain (V3) of HIV-1 surface envelope glycoprotein gp120 is an important site of HIV tissue tropism and infection (15,26). This region contains the principal neutralization domain of HIV-1 gp120, and anti-V3 antibodies inhibit HIV-induced cell fusion (5). The V3 loop consensus motif, Arg-Gly-Pro-Gly-Arg-Ala-PheVal-Thr-Ile (RGPGRAFVTI;HIV-1 IIIB), has been shown to involve the interaction of HIV with CD4-positive lymphocytes (16,27). Due to the key role of the V3 loop in a process of the HIV-1 infection, this region has been extensively studied for development of vaccinal and/or therapeutical strategies. Although several attempts have been made to inhibit HIV-1 infection by V3-loop-related peptides, controversial results have been reported (15,23). Recently, the receptors for HIV have been studied and a chemokine receptor functioned as a coreceptor for lymphocytetropic HIV-1 strains. The binding of CD4 receptor with gp120 envelope protein is the first step in HIV infection. However, CD4 exposition is not per se sufficient to allow viral entry (18). Recent studies have demonstrated the role that CXCR-4 (the receptor for the stromal cell-derived factor [SDF]-1 chemokine) (2,17) and CC-CKR5 (24) play as coreceptors of syncytium-inducing T-

Ristocetin

lymphotropic (9) and non-syncytium-inducing macrophage-tropic (7) HIV strains, respectively. Previous work has demonstrated that proline-rich peptides containing proline-glycine loops (b-turns) form a complex with ristocetin dimers (14). Moreover, the ristocetin-dimer-promoted stabilization of von Willebrand factor (vWF) on platelet membrane glycoprotein (GP) Ib was abolished by low concentrations of poly (Pro-Gly-Pro). The proline-rich peptides flanking the vWF A1 loop, Cys474-Pro488 and Leu694-Pro708 (21), inhibited vWF binding by competitively interfering with the formation of vWF-GPIb complex rather than by complexation of free ristocetin dimers (4). However, in a flow chamber with blood circulating at high shear forces these peptides failed to prevent the binding of collagenassociated vWF to GPIb, suggesting that these peptides could not be part of binding sites for GPIb (13). Moreover the Pro-Gly sequence found in the N-terminal domain of GPIb (22). These results indicate that the peptides containing the Pro-Gly sequence could be active in inhibition of ristocetin-mediated vWF binding to GPIb. In this report we have studied the effect of the HIV-1 IIIB peptide on ristocetin-mediated vWF binding to GPIb. This peptide inhibited ristocetin-mediated vWF binding to GPIb probably by complex formation with ristocetin. Furthermore, ristocetin by itself

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inhibited the binding of the HIV-1 IIIB peptide to immobilized CXCR-4 peptide. It may suggest that ristocetin is a good and useful tool to understand the mechanism of HIV tissue tropism and infection.

TABLE 1 INHIBITION OF VWF-GPIB INTERACTION BY SYNTHETIC PEPTIDES IC50 (mM) Peptide Sequence

METHOD

vWF was a kind gift from Dr. Zaverio M. Ruggeri at the Scripps Research Institute (La Jolla, CA). Botrocetin from the crude Bothrops jararaca venom was a gift from Dr. Yoshihiro Fujimura (Nara Medical School, Nara, Japan). Ristocetin was purchased from Sigma (St Louis, MO).

HIV-1 IIIB Substituted peptide Substituted peptide Substituted peptide Substituted peptide Substituted peptide

A B C D E

RGPGRAFVTI RGRRRAFVTI RGEERAFVTI RGPPRAFVTI RGGGRAFVTI RGGPRAFVTI

Ristocetin

Botrocetin

18 6 3.8 .1000 .1000 35 6 7.6 .1000 66 6 6.8

.1000 .1000 .1000 .1000 .1000 .1000

Synthetic Peptides Peptides were synthesized on a peptide synthesizer at TANA Bio-Systems LC (Houston, TX). A peptide, which was comprised of Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile (RGPGRAFVTI), interfered the interaction of HIV-1 with CD4-positive lymphocytes (16,27). Three peptides, corresponding to residues 474 – 488, 694 – 708, and 514 –542 of vWF, were also synthesized at TANA Bio-System LC. These three peptides inhibited ristocetin-mediated vWF binding to GPIb (4,21). A CXCR-4 peptide, a receptor for HIV-1 and HIV-2 gp120 (2), was also synthesized using an automated model 430A peptide-synthesizer (Applied Biosystems, Foster City, CA). A sequence of this peptide was as follows: SDNYTEEMGSGDYDSMKEPCFREE. Lyophilized crude peptides were purified by reverse-phase HPLC on a C18 column, using elution gradient of 0 – 80% acetonitrile with 0.1% trifluoracetic acid. The purity and composition of peptides were verified by HPLC analysis of hydrolysates prepared by treating peptides under nitrogen with 6 N HCl containing 0.1% phenol at 160°C. Antibodies The anti-vWF monoclonal antibodies, RG-46, 52K-2, and 52K-8 were prepared and characterized as described in detail elsewhere (11,12). These antibodies inhibit the interaction of vWF with GPIb and were also gifts of Dr. Zaverio Ruggeri. Polyclonal anti-HIV-1 IIIB peptide antibody (AG3) was raised by injecting coupled peptide into New Zealand White rabbits in multiple subcutaneous sites. The first injection consisted of 1 mg of peptidecarrier complex in complete Freund’s adjuvant. Booster injections consisted of the same amount of peptide in incomplete Freund’s adjuvant given at weekly intervals in the same manner. Animals were bled 2 weeks after the last injection and every 2 weeks thereafter. IgGs were purified by affinity chromatography on protein A-Sepharose CL-6B as previously described (8). Protein Labeling Purified proteins were labeled with carrier-free 125I (Amersham Corp.) using IODO-GEN (Pierce Chemical Co., Rockford, IL) according to the method described elsewhere (10). Unbound reagent was removed by gel filtration on a 0.5 3 20 cm column of Sepharose G-25 equilibrated with TBS (20 mM Tris-HCl, 0.15 M NaCl, pH 7.3). Binding Studies Binding of 125I-vWF to GPIb was measured in the presence of 1.0 mg/ml ristocetin or 10 mg/ml botrocetin using formalin-fixed platelets at a final count of 1 3 108/ml as described elsewhere (19). In brief, binding was measured after incubation for 30 min at 22°C without agitation. Bound ligand was separated from free ligand by centrifugation. Binding was expressed as a percentage of that

Substitution of the sequence of proline-glycine residues to argininearginine, to aspartic acid-aspartic acid, to proline-proline, to glycineglycine or to glycine-proline.

measured in a control mixture in the absence of inhibitor, after subtracting the nonspecific binding. Nonspecific binding was evaluated by adding a 50-fold excess of unlabeled ligand. The concentration of a competing substance required to inhibit specific binding by 50% (IC50) was then calculated from dose-response curves in which the percentage of residual binding was plotted against the logarithm of competing ligand concentration. Platelet Aggregation Studies Platelet aggregation studies were conducted in siliconized cuvettes as described previously (20), using a NNK platelet aggregation tracer (Nikkou Bioscience, Tokyo, Japan) at 37°C with stirring at 1,000 rpm. Synthetic peptides at various concentrations were added to platelet-rich plasma. ADP (final concentration; 5 3 1026 M), collagen (10 mg/ml), ristocetin (1.2 mg/ml), or botrocetin (20 mg/ml) was then added. Aggregation was measured indirectly as the increase in light transmission through the mixture. ELISA Assay Effects of peptides on antibody binding to immobilized vWF were evaluated. These studies were conducted using enzymelinked immunoassay (ELISA) as described elsewhere (3). Polystyrene microtiter wells were coated with vWF at a concentration of 2 mg/ml. For measurement of inhibition of antibody binding to vWF, purified IgG of selected anti-vWF antibodies at a concentration sufficient to give an optical density reading of 1.0 –1.5 in the ELISA assay was incubated with peptides at various concentrations for 2 h at 22°C. The mixtures were then added to the wells containing immobilized vWF, and residual antibody binding was measured. The binding of antibody preincubated with peptides was expressed as percentage of that measured in a control mixture containing Tris-buffered saline instead of peptide. Effect of ristocetin on binding of the HIV-1 III B peptide to immobilized CXCR-4 peptide (100 mg/ml) was also evaluated. For measurement of inhibition of ristocetin on this binding, the HIV-1 III B peptide at a concentration sufficient to give an optical density reading of 1.0 –1.5 in the ELISA assay was incubated with ristocetin at various concentrations for one hour at 22°C. The mixtures were added to the microtiter wells coated with CXCR-4 peptide. Residual binding of the HIV-1 III B peptide was detected by an addition of the polyclonal antibody against HIV-1 III B peptide (designated as AG3). Then peroxidase-coupled goat anti-rabbit IgG was added and the intensity of the color developed in 30 min

HIV-1 IIIB INHIBITS VWF-GPIB INTERACTION

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FIG. 2. Inhibitory effect of synthetic peptides on monoclonal antibody binding to immobilized vWF. Polystyrene microtiter wells were coated with purified intact vWF (2 mg/ml), and purified IgG from the monoclonal antibodies were used at a concentration sufficient to give an optical density between 1.0 –1.5 in ELISA assay (RG-46, 6 mg/ml; 52K-2, 16 mg/ml; 52K-8, 18 mg/ml). Antibodies (F, RG-46; E, 52K-2; ‘, 52K-8) were incubated with indicated concentrations of peptide for 2 h at 22°C before addition to the wells. Thus, a decrease in optical density indicates interaction of the peptide with given monoclonal antibody in solution.

FIG. 1. Effect of the HIV-1 IIIB peptide on platelet aggregation in the presence of ristocetin or botrocetin. Synthetic peptides at various concentrations were added to platelet-rich plasma with platelet count of 3 3 108/ml. Ristocetin (1.2 mg/ml) or botrocetin (20 mg/ml) was then added.

after addition of the peroxidase substrate O-phenylenediamine was evaluated by measuring the absorbance at 492 nm. Protein Concentrations Protein concentrations were measured by Bradford’s method with bovine serum albumin (BSA) as the standard (6). RESULTS

Binding of vWF to GPIb occurred in the presence of modulators such as ristocetin and botrocetin in vitro. The effect of the HIV-1 III B peptide to inhibit the binding of 125I-vWF to GPIb was evaluated. Interestingly, this peptide inhibited ristocetin-mediated vWF binding to GPIb in a dose-dependent manner with IC50 of 18 6 3.8 mM while it failed to block botrocetin-mediated vWF binding to GPIb. Previous studies suggested that the Pro-Gly sequence was crucial for inhibition of ristocetin-mediated vWF binding to GPIb (4,14). Then we synthesized five substituted

peptides. Substitution of the sequence of two residues Pro-Gly in the HIV-1 III B peptide to Pro-Pro or Gly-Pro also led to inhibit ristocetin-mediated vWF binding to GPIb while the substitution to Asp-Asp, to Arg-Arg or Gly-Gly led to complete loss (Table 1). The HIV-1 III B peptide was inactive in fibrinogen binding to thrombin-stimulated platelets (data not shown). Platelet aggregation studies also supported these binding studies. The HIV-1 III B peptide inhibited ristocetin-induced platelet agglutination in a dose-dependent manner with IC50 of 90 6 13 mM. However, the peptide was inactive in botrocetin-induced platelet aggregation. The peptide was also inactive in ADP-, collagen-, and epinephrine-induced platelet aggregations (Fig. 1). Anti-vWF monoclonal antibodies (RG-46, 52K-2 and 52K-8) are well-known to inhibit vWF binding to GPIb in the presence of ristocetin. RG-46 recognizes sequence corresponding to residues 474 – 488 of vWF and 52K-2 and 52K-8 recognize sequence corresponding to residues 694 –708 (11,12,21). The inhibitory effect of preincubation with the HIV-1 III B peptide on anti-vWF antibody binding to immobilized vWF was evaluated. The peptide only inhibited the binding of RG-46 monoclonal antibody to immobilized vWF while it did not inhibit the interactions with 52K-2 and 52K-8 to immobilized vWF (Fig. 2). Peptides with a sequence of proline-glycine form a complex with ristocetin dimers (14). As the HIV-1 III B peptide inhibited ristocetin-mediated vWF binding to GPIb, this peptide probably bound to ristocetin. To confirm this possibility, we evaluated the effect of ristocetin on the binding of HIV-1 III B peptide to immobilized CXCR-4 peptide. At first we studied a reactivity of the anti-HIV-1 III B peptide polyclonal antibody (AG3) to immobilized HIV-1 III B peptide. This antibody (AG3) bound to immobilized HIV-1 III B peptide while anti-vWF monoclonal antibody (52k-2) did not (Fig. 3A). These results suggested that this antibody specifically recognized the HIV-1 III B peptide. Then we studied the effect of ristocetin on the binding of this peptide to immobilized CXCR-4, HIV coreceptor. Indeed ristocetin inhibited the binding of the HIV-1 III B peptide to immobilized CXCR-4 peptide at optimal concentrations between 0.8 –2 mg/ml (Fig. 3B).

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MOHRI ET AL. TABLE 2 AMINO ACID SEQUENCES Peptide Sequence

HIV-1 III B (318–327) vWF (514–542) vWF (474–488) vWF (694–708)

RGPGRAFVTI DLVFLLDGSSRLSEAEFEVLKAFVVDM CQEPGGLVVPPTDAPV LCDLAPEAPPPTLPPD

It should be noted that the Pro-Gly sequence is only found in HIV-1 III B and vWF (474 – 488).

FIG. 3. A) Binding of the peptide-specific antibody to immobilized HIV-1 IIIB peptide. Polystyrene microtiter wells were coated with a solution of the HIV-1 IIIB peptide at a concentration of 100 mg/ml. The polyclonal anti-peptide antibody (AG3) was added to the wells in increasing concentrations, indicated on the abscissa, and its binding to the immobilized peptide was measured with the peroxidase-coupled goat anti-rabbit IgG. B) Effect of ristocetin on the binding of HIV-1 IIIB peptide to immobilized CXCR-4 peptide. The HIV-1 IIIB peptide was used at a concentration of 100 mg/ml and incubated with ristocetin at a concentrations shown on the abscissa. After incubation for one hour at 22°C, the mixtures were added to microtiter wells coated with CXCR-4 peptide and the ELISA assay was performed.

DISCUSSION

In this study, we have studied the effect of synthetic peptide derived from V3 of HIV-1 surface envelope glycoprotein gp120 on the interaction of adhesive proteins with platelets and found that this peptide was active in inhibiting ristocetin-mediated vWF binding to GPIb. This peptide also inhibited the binding of anti-vWF monoclonal antibody, RG-46, to immobilized vWF. Furthermore, ristocetin inhibited the interaction of the HIV-1 III B peptide with CXCR-4, the receptor for the stromal cell-derived factor [SDF]-1 chemokine. The GPIb-binding domain of vWF has been located in a tryptic fragment of vWF extending from Val449 to Lys728 (11). Studies using synthetic peptides have shown more precise regions of vWF that interfere with the interaction of vWF with GPIb. Two discontinuous sequences of vWF (residues 474 – 488 and 694 –708) are apparently necessary for GPIb-binding in the presence of ristocetin (21). However, a recent study suggested that these peptides were not the parts of the binding sites for GPIb (13). Moreover, these

peptides interacted with ristocetin (monomer)-loaded GPIb (14) and acted as inhibitors by preventing the subsequent formation of ristocetin dimer bridges on proline-rich b-turns (25). A study using site-directed mutagenesis showed the evidence that mutation of the sequence of three consecutive proline residues Pro702-Pro704 to Asp702-Asp704 or Arg702-Arg704 led to a complete loss of ristocetin-mediated vWF binding to GPIb (1). Recent study has shown the importance of Pro-Gly or Pro-Pro sequence for preventing the interaction of vWF to GPIb in the presence of ristocetin (14). In this aspect, the Pro-Gly sequence found in the HIV-1 III B peptide is likely a candidate to bind to ristocetin. Indeed this peptide inhibited only ristocetin-mediated vWF binding to GPIb while it was inactive in inhibiting botrocetin-mediated vWF binding to GPIb. To confirm the importance of the Pro-Gly or Pro-Pro sequence, we synthesized the substituted peptides of this sequence. Substitution of the sequence of two residues Pro-Gly in the HIV-1 III B peptide to Pro-Pro or Gly-Pro also led to inhibit ristocetin-mediated vWF binding to GPIb while the substitution to Asp-Asp, to Arg-Arg or Gly-Gly led to complete loss. These results support the evidence that the Pro-Gly or Pro-Pro sequence was crucial for the inhibition of ristocetinmediated vWF binding to GPIb. Furthermore the HIV-1 III B peptide also has a capacity to inhibit ristocetin-mediated vWF binding to GPIB by its binding to ristocetin. This peptide only had a capacity to neutralize binding of monoclonal antibody (RG-46) to immobilized vWF. This monoclonal antibody recognized a sequence 474–488 of vWF and inhibited the interaction of ristocetin mediated vWF binding to GPIb (12,21). Interestingly the Pro-Gly sequence is also found in the residues 474–488 of vWF. Other sequences of vWF, corresponding to residues 694–708 and 514–542, which inhibit ristocetin-mediated vWF binding to GPIb do not contain the Pro-Gly sequence (Table 2). The Pro-Gly in the HIV-1 III B peptide probably binds to ristocetin. Then interest has focused on the effect of ristocetin on the binding of the HIV-1 IIIB peptide to a HIV coreceptor such as CXCR-4. Recent studies indicate that CXCR-4 (the receptor for the stromal cell-derived factor [SDF]-1 chemokine) (2,17) and CC-CKR5 (24) are important and could act as coreceptors of syncytium-inducing T-lymphotropic (9) and non-syncytium-inducing macrophage-tropic (7) HIV strains, respectively. These coreceptors are important to allow viral entry. Ristocetin inhibited the binding of the HIV-1 IIIB peptide to immobilized CXCR-4 peptide at the optimal concentrations of 0.8 –2.0 mg/ml. To our knowledge, this is the first report that ristocetin interferes with the interaction of V3 of HIV-1 surface envelope gp120 to CXCR-4. As the binding of CD4 receptor with gp120 envelope protein is the first step in HIV infection, ristocetin might prevent HIV infection and would be a good tool to understand the mechanism of HIV tissue tropism and infection.

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