GPIb and the β1 integrin α2β1

Thrombosis Research, Vol. 81, No. 1, pp. 113-l 19, 1996 Cowright 0 1995 Elsevier Science Ltd P&&d-in the USA. All rights reserved 0049-3848/96 $12.00 + 00

Pergamon 0049-3848(95)00219-7

PLATELET ADHESION AT HIGH WILLEBRAND FACTOR/GPIb

SHEAR AND

RATES: THE ROLES OF VON THE p1 INTEGRIN c@1

Harvey R. Gralnick§*, Wendy S. Kramerg, Laurie P. McKeown§, Leonard Garfinkelt, Amos Pinott, Sybil B. Williams§, and Henry Krutzsch$ $The Hematology Service, Clinical Center, National Institutes of Health, Bethesda, MD 20892, tBio-Technology General (Israel) Ltd., Rehovot, Israel, *Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD (Received 11 August 7995 by Editor D. Triplett; revised/accepted

Abstract

18 October 1995)

We have previously described a monomeric rvWf fragment, Leu504 Lys728 that contains one disulfide bond linking Cys509Cys695. This fragment, VCL, has previously been shown to inhibit vWf-ristocetin, asialo-vWf, and botrocetin-induced vWf binding and aggregation of platelets. VCL inhibited 50% of vWf binding to heparin, but it did not At a high shear force (2600-l inhibit vWf binding to type I collagen. set), VCL inhibited platelet adhesion to the subendothelial surface of human umbilical arteries. The maximum inhibition of platelet adhesion was 83 + 4% at a VCL concentration of 7.6pmol/L. Various monoclonal anti-Very Late Activation antigens (VLA) antibodies were added to the VCL and tested for their ability to enhance the inhibition of platelet adhesion at high shear forces. Of all of the VLA antibodies tested, only the anti-VLA-2 antibody (176D7) inhibited platelet aggregation in the absence of VCL and enhanced the inhibition of platelet adhesion in the presence of VCL. The VLA-2 antibody and VCL together inhibited 96 + 4% of platelet adhesion at high shear forces.

These studies indicate that the primary mechanism(s) of platelet attachment to the subendothelial surface are dependent on GPIb-IX-vWf interactions, and a second mechanism is related to the platelet VLA-2 binding to subendothelial collagen. Understanding the role(s) of inhibition of GPIba binding to vWf and the role of the platelet VLA-2 provides a new strategy of inhibition of thrombosis at high shear forces. Platelet adhesion to vascular subendothelium hemostasis and thrombosis. von Willebrand

is a critical step in the initiation of factor is necessary to stabilize platelets

Key words: thrombosis, VLA-2, GPIb-a Corresponding author: Dr. Harvey R. Gralnick, NIH, Building 10, Room 2C390, Bethesda, MD 20892. 113

9000 Rockville

Pike,

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which are adherent under conditions of high shear stress. von Willebrand factor binds to extracellular matrix proteins and links them to the platelet glycoproteins, e.g., GPIb/IX and GPIIb/IIIa. We have previously synthesized, isolated, and reported a recombinant fragment of von Willebrand factor Leu504-Lys728 with a single disulfide bond linking residues Cys509 and Cys695 (1). This monomeric vWf fragment inhibited the binding of von Willebrand factor to the GPIb-IX complex, and it inhibited platelet adhesion to the subendothelial surface. When this recombinant protein was studied at high shear forces (2600-l set), the IC50 of platelet adhesion was 0.94pM. The maximal inhibition of platelet adhesion was 83% which was observed at a concentration of 7.6pmol/L. In an attempt to further understand the mechanisms(s) of platelet adhesion to the subendothelium under high shear forces, we have studied the effect(s) of various monoclonal antibodies against the VLAs to identify other platelet glycoprotein(s) involved in platelet-adhesion. MATERIALS

AND METHODS

Blood was obtained from healthy medical personnel who had not used aspirin or nonsteroidal anti-inflammatory drugs for at least 10 days. In these studies, platelet adhesion to human umbilical artery subendothelium was performed at a shear force of 2600-lsec, as previously described (2). Briefly, human umbilical arteries were everted and de-endothelialized by exposure to air. Arterial segments wee mounted on a rod in an annular perfusion chamber. Blood entering the chamber flowed through the annular space between the subendothelial space between the subendothelial surface and the chamber wall, thereby exposing the platelets tohe subendothelium. VCL was added to citrate anticoagulated whole blood at concentrations varying from 0.46-8.Opmol/L, In a second group of experiments, we added either anti-VLA-2 (176D7), produced in our laboratory, or anti-VLA4, antiVLA5, or anti-VLA-6 purchased from AMAC, Inc., Westbrook, ME. These monoclonal antibodies were added at concentrations of 5, 10, or 20i.t.g/mL to whole blood containing 7.6pmol/L VCL; platelet adhesion at a shear force of 2600-l set was determined. In other studies the antibodies directed against the VLA’s were individually incubated with whole blood in the absence of VCL and the effect on platelet adhesion was analyzed. All of the antibodies employed were IgG. In some experiments, we compared the anti-VLA-2 Fab fragments to the intact anti-VLA-2 antibody in the inhibition of platelet adhesion. Static platelet adhesion to type I collagen coated microtiter plates was performed as previously described (3). VCL was incubated with platelets at final concentrations of 0.94-7.6pmol/L for 20 minutes prior to their addition to collagen coated wells. We also studied the ability of VCL to inhibit the interaction of von Willebrand factor with collagen (4,5) and heparin (6). Three mg of heparin CLdB Sepharose were equilibrated with water and washed on a glass frit initially with distilled water and then with 0.2mol/L Mes-Tris Buffer, pH 6.0. The heparin-Sepharose was washed with Mes-Tris in a 50 ml conical tube, suspended in Mes-Tris to 25%, and stored at

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4°C. A working solution was made by removing an aliquot and washing 3 times with 0.5mol/L Tris-O.lOmol/L NaCl, pH 7.35 with a final working suspension of 1.5%. The total volume in the assay was 25OpL. Two hundred PL of the labeled ligand were mixed with 25pL of l%BSA and 25pL of Heparin-Sepharose diluted to 1.5%. This was mixed every 5 minutes during the 45 minutes incubation. Then 2OOpL of the mixture was layered on 3OOuL of 10% LarcollB and centrifuged at 9,000g for 4 minutes. The supernatant was aspirated, the tip of the tube was clipped and counted. Non-specific binding was determined with heparin sodium salt. RESULTS VCL at a concentration of 7.6vmol/L inhibited 83 + 4% of platelet adhesion to the subendothelium at high shear forces. When the various anti-VLA antibodies, at 5, 10, or 20pg/mL were added to VCL, they did not significantly change the platelet adhesion except in the presence of the anti-VLA-2 antibody which at 5 to 20 pg/mL inhibited platelet adhesion to collagen 19-22% (nl 8). When the anti-VLA-2 was added to 7.6pmol/L VCL at 10 pg/mL, it resulted in the total inhibition of platelet adhesion 97 53% (n=3) (Figure 1).

I

2.5

I

5.0 Peptide, FM

I

7.5

FIG. 1. The effects of the rvWf fragment VCL on the platelet adhesion to deendothelialized umbilical artery segments at shear rates of 2600-l sec.

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0

Antibody Alone

5

Antibody + 7.4 FM VCL

Antibody Concentration

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@g/ml)

FIG. 2 At 5pg/mL, the anti-a2PI antibody inhibited platelet adhesion 81+27% and at lOpg/mL 79236%. 7.4pM VCL added to Spg/mL of the antibody inhibited platelet adhesion 90 t 4%, and lOpg/mL of the antibody inhibited adhesion 9723%.

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Similar results were obtained when the Fab fragment of anti-VLA-2 was employed (data not shown). In contrast, the anti-VLA-5 (a5pl) antibody, which recognizes a fibronectin receptor, did not affect inhibition of platelet adhesion in the presence of antibody recognizes a platelet laminin VCL 83+2% (n=2). The anti-VLA-6 (a6PI) receptor. The addition of anti-VLA-6 to VCL did not affect inhibition of platelet adhesion, 84 + 5% (n=2). VCL inhibited platelet adhesion to collagen in a static system in a concentration The results were similar to those obtained from experiments dependent manner. with adhesion to subendothelium at a high shear rate in that 7.6pmol/L VCL inhibited platelet adhesion to collagen 81% f 10% (n=ll). The VCL did not inhibit vWf binding to collagen, but it did inhibit 50 ~fi 6% (n=S) of vWf binding to heparin in a static system. DISCUSSION We have previously described the characteristics of the rvWf fragment VCL, Leu504-Lys728 (1). This fragment inhibited vWf binding to the GPIb-IX complex, inhibited platelet adhesion to subendothelium at high shear forces, and inhibited platelet adhesion to collagen in a static system. In contrast, we found that the VCL did not inhibit von Willebrand factor binding to collagen and only inhibited 50% of von Willebrand factor binding to heparin. VCL maximally inhibited platelet adhesion to the umbilical artery subendothelium at a concentration of 7.6lmol/L. At this concentration, the inhibition of platelet adhesion was 83% + 4 of the total platelets. Cruz et al had initially identified a collagen binding site in the Al domain of vWf (7). A recent report by Cruz et al indicates that a collagen binding site is not present in the Al domain of von Willebrand factor but most likely is present in the A3 domain. This seems to explain the inability of VCL to inhibit von Willebrand factor binding to collagen at high shear forces (8, 9). Hemler and coworkers have defined the VLA-2 receptor on platelets (10). Sealman et al have utilized the antibody 176D7 to demonstrate that platelet adhesion to collagen is mediated by the a2P1 integrin (11, 12). We considered whether the inability of VCL to inhibit von Willebrand factor binding to collagen resulted in the partial retention of approximately 20% of platelet adhesion in the presence of VCL. We investigated this by performing platelet adhesion studies at high shear rates in the presence of a variety of monoclonal antibodies directed against the bl integrin. We found that of the anti-VLA antibodies tested, only one effectively augmented the VCL inhibition of platelet adhesion. This antibody, an anti-VLA-2 produced in our laboratory, when added to 7.6pM VCL, inhibited platelet adhesion by 97 + 3% (n=S). Other VLA antibodies which included anti-VLA-4, anti-VLA-5 and anti-VLA-6, did not effect platelet adhesion in the absence of VCL. The second heparin binding site present in

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residues l-298 of mature vWf was not inhibited by VCL or the anti-VLA-2 This could explain why only 50% of heparin binding was affected by VCL.

antibody.

Our previous results with VCL combined with our present studies suggest that the primary mechanism of platelet attachment to the subendothelial surface is dependent on the GPIb-IX-vWf interaction(s) (1). This accounts for approximately 80% of platelet adhesion at high shear forces; however, this leaves a secondary site which appears to be responsible for approximately 20% of platelet adhesion at high shear forces. The residual platelet adhesion can be inhibited by the addition of the a2B1 monoclonal antibody which binds to the platelet collagen receptor and inhibits subendothelial-collagen-platelet binding. This results in almost total inhibition of platelet adhesion at high shear forces. Our data agree with other investigators who have indicated that the collagen binding domain is not present in the Al domain of vWf, and only one of the two heparin binding sites are present within the Al domain. In our studies, the VCL inhibited the Al domain binding sites which resulted in 50% of heparin binding. Platelet interaction with collagen, which accounts for approximately 20-30% of platelet adhesion at high shear forces, is dependent on an intact platelet VLA-2 receptor. Inhibition of platelet adhesion and aggregation to subendothelial surfaces is an important aspect of antithrombotic therapy. In strategies to inhibit platelet adhesion to damaged vascular surfaces and thrombus formation in areas of high shear force, multiple antithrombotic agents may be necessary to prevent further vascular compromise and ensuing thrombotic occlusion(s). Acknowledgment The authors DeGraff.

gratefully

acknowledge

the excellent

secretarial

assistance

of Leslie

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ROLE IN PLATELET ADHESION

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carboxyterminal peptides inhibit platelet adherence to arterial subendothelium at high wail shear rates. J Clin Invest 6 1715-1722,199O. 4. PARETI, F.I., NIIYA, K., MCPHERSON, J.M., and RUGGERI, Z.M. Isolation and characterization of two domains of human von Willebrand factor that interact with fibrillar collagen types I and III. J Biol Chem 262,13835-13841,1987. 5. MOHRI, H., YOSHIOKA, A., ZIMMERMAN, T.S., and RUGGERI, Z.M. Isolation of the von Willebrand factor domain interacting with platelet glycoprotein Ib, heparin, and collagen and characterization of its three distinct functional sites. J Biol Chem 264,17361-17365,1989. 6. FUJIMURA, Y., TITANI, K., HOLLAND, L.Z., ROBERTS, J.R., KOSTEL, I’., RUGGERI, Z.M., and ZIMMERMAN, T.S. A heparin-binding domain of human von Willebrand factor. J Biol Chem 262, 1734-1739,1987. 7. CRUZ, M.A., PETERSON, E., TURCI, S.M., and HANDIN, R.I. Functional analysis of a recombinant glycoprotein Iba polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein Ib-IX complex and to collagen. J Biol Chem 267,1303-1309. 1992. 8. CRUZ, M.A., LEE, J., WISE, R.J., and HANDIN, R.I. Characterization of recombinant von Willebrand factor Al and A3 domain proteins demonstrates that the binding sites for collagen and platelet glycoprotein Ib/IX are located in separate domains. Thrombo Haemost @ 1574,1993. Abstract. 9. CRUZ, M.A., HUABING, Y., LEE, J.R., WISE, R.J., and HANDIN, R.I. Interaction of the von Willebrand Factor (vWf) with collagen. J Biol Chem 270, 10822-10827, 1995. 10. HEMLER, M.E., CROUSE, C., TAKADA, Y., and SONNENBERG, A. Multiple very late antigen (VLA) heterodimers on platelets. J Biol Chem 263, 7660-7665, 1988. 11. SAELMAN, U.M., HORTON, L.F., BARNES, M.J., GRALNICK, H.R., HESE, K-M., NIEUWENHUIS, H.K., DE GROOT, P.G., and SIXMA, J.J. Platelet adhesion to cyanogen-bromide fragments of collagen al (I) under flow conditions. Blood 82. 3029-3033,1993. 12. SAELMAN, U.M., NIEUWENI-IUIS, K., HESE, K.M., DEGROOT, P.G., HEIJEN, H.F.G., SAGE, E.H., WILLIAMS, S., MCKEOWN, L., GRALNICK, H.R., and SIXMA, J.J. Platelet adhesion to collagen types I through VIII under conditions of stasis and flow is mediated by GPIa/IIa (a2J31-integrin). Blood & 1244-1250,1994. 13. FRET-IO, L.J., FOWLER, W.E., MCCASLIN, D.R., ERICKSON, HP., and MCKEE, P.A. Substructure of human von Willebrand factor: proteoiysis by Van and characterization of two functional domains. J Biol Chem 261, 15679-15689, 1986.