Von Willebrand Factor Mediates Platelet Adhesion to Virally Infected Endothelial Cells (Adhesion Molecule/Herpes Simplex Virus)

Proc. Nati. Acad. Sci. USA Vol. 90, pp. 5153-5156, June 1993 Cell Biology

von Willebrand factor mediates platelet adhesion to virally infected endothelial cells (adhesion molecule/herpes simplex virus)

ORLI R. ETINGIN*t, ROY L. SILVERSTEIN*, AND DAVID P. HAJJARt Departments of *Medicine and tPathology, and the Specialized Center of Research in Thrombosis, Cornell University Medical College, New York, NY 10021 Communicated by Bernard L. Horecker, March 8, 1993

ABSTRACT von Willebrand factor is an adhesive glyco- MATERIALS AND METHODS protein critical to normal hemostasis. It is stored in the Weibel-Palade body of endothelial cells and upon release may Culture of Endothelial Cells. Human umbilical vein endo- mediate platelet adhesion. Herpesvirus-infected endothelium is thelial cells (HUVECs) were isolated as described (13). Cells known to be prothrombotic and to support enhanced platelet were routinely grown in RPMI 1640 medium (GIBCO) with adherence. We previously 2%o fetal bovine serum, heparin (100 ,ug/ml; Sigma), and 1% identified P-selectin as a monocyte penicillin/streptomycin solution (Sigma). Cells were con- receptor that is translocated from the Weibel-Palade body to firmed to be HUVECs by immunofluorescent staining with the endothelial cell surface in response to the local generation anti-vWF antiserum. All cells in a single experiment were of thrombin on herpesvirus infected cells. In this study, we obtained from the same umbilical cord. Endothelial cell show that viral injury to vascular endothelial cells induces matrices were prepared as described (14). secretion of von Willebrand factor which mediates enhanced Viruses. HSV-1 (strain F) and adenovirus type 2 (Ad2) platelet adhesion to these cells. were purchased from the American Type Culture Collection. Confluent monolayers of HUVECs were infected with 0.1- Vascular endothelial cells and megakaryocytes synthesize 1.0 plaque-forming unit of virus per cell. After 2 hr of von Willebrand factor (vWF), an adhesive glycoprotein adsorption at 37°C, the virus was removed and the cells were which circulates in plasma (1-3). Activated platelets secrete washed and refed with fresh medium for 24 hr prior to each vWF and express at least two receptors for vWF. Interac- experiment. Control studies were performed on mock- tions ofvWF with platelet glycoprotein lIb/IlIa may regulate infected and Ad2-infected cells. In selected studies, platelet-to-platelet contact and platelet adhesion to subendo- HUVECs were infected with a previously characterized thelial matrix. vWF interaction with platelet glycoprotein lb mutant of HSV-1, NS-1, which does not express herpesviral is instrumental in mediating platelet adhesion to subendothe- glycoprotein C (gC) on the infected endothelial cell surface lial collagen in vessels at high shear rates (4-6). These (11). In these experiments, the number of virus plaque- forming units used was lower due to the toxicity of this processes are critical to hemostasis, since qualitative or mutant virus for endothelial cells (12). quantitative defects ofvWF result in a mild to severe bleeding Platelet Adhesion Assay. Platelets were freshly isolated diathesis. from the blood of normal volunteers by gel filtration. Platelet Endothelial vWF is both secreted constitutively and stored adhesion was measured by using 51Cr-labeled platelets, in intracellular organelles known as Weibel-Palade bodies which were prepared by the addition of 51CrC13 to a suspen- (7)' The stored vWF is predominantly in the form of high sion of platelets (10w per ml) at 22°C for 90 min. After they molecular weight multimers that are biologically potent and were washed in phosphate-buffered saline, labeled platelets are more active in in vitro platelet binding assays than (106 per well) were added to monolayers of cells in 24-well circulating forms. Upon release, this vWF may mediate plates and incubated at 37°C. After 2 hr, culture medium, platelet adhesiveness to the vascular endothelium (8, 9). containing nonadherent platelets, was removed and the cells Herpes simplex virus (HSV)-infected endothelium has were gently rinsed once with culture medium. NaOH (0.2 M) been shown to be prothrombotic and to support enhanced was added to each well for 1 hr to solubilize the cells, and platelet adherence (10). We have previously shown that radioactivity was measured in a 'y counter. In some experi- HSV-infected endothelial cells generate significantly in- ments, antibodies to vWF (American Diagnostica, Green- creased amounts of thrombin (11) and that increased throm- wich, CT), HSV gC (courtesy of Harvey Friedman, Univer- bin generation promotes monocyte adhesion to the surface of sity of Pennsylvania, Philadelphia), or platelet glycoprotein infected endothelial cells. We identified P-selectin as a mono- Ib or Ilb/IIIa (courtesy of Adam Asch, Cornell Medical cyte receptor on these cells, and postulated that the receptor College) were added 1 hr prior to the addition of platelets. translocated from the Weibel-Palade bodies to the external Hirudin (Sigma) and PPACK (D-Phe-L-Pro-L-Arg-CH2Cl), plasma membrane in response to the localized generation of which are thrombin inhibitors, were added separately to thrombin on these cells (12). Since vWF and P-selectin designated wells 1 hr before the labeled platelet adhesion colocalize to this intracellular organelle, we hypothesized assay was done. In separate experiments, thrombin genera- that endothelial cells may concomitantly secrete increased tion was measured with the chromogenic substrate S-2268 amounts of vWF in response to HSV infection. In this study, (Kabi Diagnostica, Stockholm) to confim inhibition of we show that subacute injury to vascular endothelial cells thrombin generation in the presence of inhibitors. In specific caused by HSV infection induces secretion of vWF which mediates increased platelet adhesiveness to the endothelial Abbreviations: Ad2, adenovirus type 2; gC, glycoprotein C; HSV, cell. herpes simplex virus; HUVEC, human umbilical vein endothelial cell; PPACK, D-Phe-L-Pro-L-Arg-CH2Cl; vWF, von Wilebrand factor. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Division of payment. This article must therefore be hereby marked "advertisement" Hematology-Oncology, Comell Medical College, 1300 York Ave- in accordance with 18 U.S.C. §1734 solely to indicate this fact. nue, New York, NY 10021. 5153 Downloaded by guest on September 24, 2021 5154 Cell Biology: Etingin et al. Proc. Natl. Acad. Sci. USA 90 (1993) experiments, platelet adhesion to endothelial cell-derived 3.0 extracellular matrices was similarly measured. Finally, vWF antigen levels were measured in conditioned media from 2.5 control and HSV-infected endothelial cells with a vWF ELISA kit (American Diagnostica). A standard curve was E m 2.0 generated with commercial vWF (American Diagnostica). ._ Statistics. Data were examined by analysis of variance. 0 0 1.5 CR0 RESULTS 30 1.0 HSV-Infection Induces vWF Secretion by Cultured HUVEC. U- HSV-infected HUVECs secreted 5-fold more vWF than uninfected cells (Fig. 1). Ad2-infected cells (a DNA virus 0.5 control) secreted vWF at similar levels as control cells (data not shown). vWF secretion required surface expression of HSV gC, since specific murine monoclonal antibodies to gC 0 4 8 12 16 inhibited vWF secretion of HSV-infected cells by >90% 20 24 28 when added at least 1 hr prior to removal of cell conditioned Time (hrs) medium for vWF measurement (Fig. 1). Nonimmune serum FIG. 2. Time course ofvWF release by HSV-infected endothelial treatment did not inhibit vWF secretion of HSV-infected cells. Conditioned medium from HSV-infected (s) and mock- cells. In addition, cells infected with HSV mutants that do not infected (v) HUVECs was collected at intervals 0-24 hr after express gC did not secrete increased amounts of vWF (Fig. infection. Conditioned media were stored at -80°C before vWF 1). HSV-infected cells similarly treated with the thrombin ELISA. Data represent the mean + SD from two experiments each inhibitor PPACK (10 ,uM) did not secrete vWF above the performed in triplicate. level of uninfected cells (Fig. 1). These data suggest that enhanced vWF secretion by HSV-infected HUVECs was Platelet Adherence to HSV-Infected Endothelium Is Medi- dependent on both the local generation of thrombin and the ated by vWF. HSV infection induced a 4-fold increase in surface expression of HSV gC. platelet adhesion as compared with uninfected cells or cells Endothelial cell secretion of vWF significantly increased infected with the non-gC expressing HSV mutant NS-1 (Fig. by 4-6 hr. Release appeared to decrease by 24 hr (Fig. 2). 3). Target-cell viability was from 90-95% as assessed by This time course parallels that of platelet adhesion, where trypan blue exclusion. This increased adhesion was inhibited maximal adhesion is seen 24 hr postinfection, with a signif- 90%o by goat anti-human vWF antibodies, whereas nonim- icant increase in platelet adhesion seen as early as 4-6 hr mune serum did not inhibit adhesion to infected or uninfected postinfection (10). vWF secretion also paralleled the course cells. Noninfected endothelial cells exposed for 30 min to of P-selectin expression that we previously reported (12). thrombin (3 units/ml) supported an 8-fold increase in platelet Specifically, increments in P-selectin and vWF expression adhesion compared with nontreated cells (data not shown). were seen by 4 hr and remained elevated up to 24 hr. Microscopic examination of the cells revealed that the platelets were adherent to the HUVECs and not to exposed 3.0 I extracellular matrix. In addition, extracellular matrices prepared from HSV-infected and uninfected cells did not signif- 2.5 icantly support differential platelet adhesion, further support- 7 ing the hypothesis that the platelets were adhering to endothelial cells and not to the underlying matrix (data not E 2.0 shown). The binding of platelets to HSV-infected cells was

15 I I0 11 1.5

U- 0 X 12 - 1.0 - go

0.5 - 0 - i [ 9 0.ci rL 1KC = Control HSV HSV HSV HSV HSV 6 -

+ + + (gC-) 3- a-gc ctrl IgG PPACK i FIG. 1. vWF release by HSV-infected endothelial cells. HUVECs 0 I _-----I. were cultured to confluency in 24-well plates (Costar). Cells were Ctrl HSV NS-1 Ctrl HSV CtrI HSV infected with HSV-1 or with the gC-negative (gC-) HSV mutant, (gC-) NS-1. Sixteen to 24 hr after infection, antibodies to HSV gC (a-gC, a-vWF Ctrl IgG 1:500), or control nonimmune serum IgG (ctrl, 1:500), or PPACK (10 ,uM) was added separately to specific wells. Cells were incubated for FIG. 3. Platelet adhesion to HSV-infected endothelial cells. 1-2 hr after antibody addition, and then the conditioned medium was HUVECs were mock-infected (control, black bars) or were infected removed and frozen at -80°C until the vWF assay was performed. with HSV-1 (open bars) or the gC-negative HSV mutant NS-1 vWF was measured by ELISA (American Diagnostica) in which a (hatched bar). Cells were preincubated with polyclonal goat anti- standard curve was constructed by using pooled human plasma. One human vWF or control antiserum (Ctrl IgG) for 30 min. Radiolabeled unit of activity corresponds to the amount of vWF in 1 ml of plasma. platelets were added and after 2 hr of incubation, the percentage of Data represent the mean ± SD from three experiments each per- adherent platelets was measured. Data represent the mean t SD formed in quadruplicate. Control cells (black bar) were mock-infected. from three experiments each performed in quadruplicate. Downloaded by guest on September 24, 2021 Cell Biology: Etingin et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5155 most likely mediated by platelet glycoprotein Ib-vWF interaction since polyclonal antibodies to glycoprotein Ib com- pletely inhibited adherence of platelets to virally infected cells (Figs. 4 and 5). Furthermore, antibodies to platelet glycoprotein MIb/IIIa or IV did not significantly inhibit platelet adhesion to HSV-infected endothelium, demonstrating specificity (Fig. 4). Taken together, our findings suggest that platelet adhesion to infected endothelial cells was dependent upon expression of vWF, which in turn was mediated by microenvironmental generation of thrombin.

DISCUSSION HSV infection of cultured endothelial cells is known to promote a prothrombotic endothelial cell phenotype by vir- tue of increased tissue-factor and thrombin generation, decreased prostacyclin production, and the adhesion of circulating platelets, monocytes, and neutrophils (10, 15, 16). Thrombin may act in an autocrine fashion by promoting localized cell-cell adhesion by expressing new adhesion molecules (12). In this study, we demonstrate that increased vWF expression by HSV-infected endothelial cells promotes platelet adhesion to these cells. Our data further implicate the autocrine role of thrombin in this system, since the mechanism for vWF expression is a thrombin-inducible one. In our model put forth earlier, the Weibel-Palade body, a source of preformed vWF, translocates to the endothelial cell membrane when stimulated by thrombin, resulting in expression of P-selectin and monocyte adhesion. We now extend this hypothesis to include simultaneous vWF secretion by HSV- FIG. 5. Glycoprotein lb mediates platelet adhesion to HSV- infected cells, with resultant increased platelet adhesion. In infected cells. Endothelial cells grown in 24-well plates were infected cells with acute herpesvirus infection, protein synthesis and with HSV. Platelet adherence was measured 18 hr after infection. (a mRNA transcription are down-regulated because the virus and b) Platelet adhesion to control and HSV-infected cells, respec- degrades total RNA transcripts in the cell (17). However, tively. (c) Platelet adherence to HSV-infected cells in the presence HSV infection can nevertheless promote novel expression of of anti-glycoprotein lb. (x 140.) adhesion molecules on the endothelial surface by promoting The concept of adhesion molecules mediating pathogen- translocation of the endothelial storage-granule membrane vWF- contents to the surface of these cells. This is also consistent induced host-cell injury is not derived solely from with generalized HSV-induced cellular actions, since it does platelet interaction on HSV-infected endothelium. Rickett- not require de novo protein synthesis of either P-selectin or sial infection of cultured cells similarly induces vWF release vWF. which is thought to participate in the pathogenesis of Rocky Mountain spotted fever (18). Bacterial endotoxin has also 12 - been shown to enhance vWF release from endothelial cells, suggesting a common pathway for pathogen-mediated throm- 0 bocytopenia (19). Hostetter and coworkers (20) have described an integrin analogue on Candida albicans as a pos- < 9 - ,;Vg sible mediator of yeast-endothelial adhesion. This yeast 0 surface protein is structurally and antigenically homologous to the a subunit of the leukocyte adhesion molecule Mac-i 6 - found on neutrophils and monocytes. Mac-1, a receptor for C complement component C3bi, also has a functional homolog 0 m on HSV-infected cells-namely, gC, which can bind and activate coagulation factor X, leading to thrombin generation X0 3 - and monocyte adhesion on infected cells (12). In this study, [' we have shown that platelet adhesion to virally injured O3- endothelial cells is mediated by surface expression of vWF, 0- which depends on thrombin generation on these cells. Visser I et ctrl HSV HSV HSV HSV gc(-) gc(-) al. (10) postulated that decreased prostacyclin synthesis + + + + may account in part for increased platelet adherence to a-gplb a-gp a-gplV a-gplb HSV-infected endothelial cells. However, indomethacin Ilb/lila treatment did not abolish the observed increase in platelet adhesion, suggesting that otherfactors, such as vWF, may be FIG. 4. Platelet glycoprotein lb mediates platelet adhesion to involved. HSV-infected endothelial cells. HUVECs were mock-infected (black Platelet adhesion to the endothelium is an early event in bar) or were infected with HSV-1 or the gC-negative mutant NS-1. HSV-infected cells were preincubated for 30 min with polyclonal some animal models of inflammation and atherosclerosis. antibodies to platelet glycoprotein Ib, Ilb/IlIa, or IV (1:500). Platelet Intravascular coagulation which occurs clinically in sepsis adherence was measured as described earlier. Data represent the and thrombotic thrombocytopenic purpura (TTP) is also mean ± SD from three experiments each performed in quadruplicate. characterized by diffuse platelet-endothelial adhesion. The Downloaded by guest on September 24, 2021 5156 Cell Biology: Etingin et al. Proc. Natl. Acad. Sci. USA 90 (1993) role of large vWF multimers in mediating such platelet- 7. Wagner, D. D., Olmstead, J. B. & Marder, V. J. (1982) J. Cell endothelial adhesion in TTP has been postulated by Moake Biol. 95, 355-360. et al. (21). In vitro, large vWF multimers have been shown to 8. Wagner, D. D. & Marder, V. J. (1983) J. Biol. Chem. 258, mediate increased adhesion of sickle erythrocytes to endo- 2065-2067. thelial cells (22). 9. Sporn, L. A., Marder, V. J. & Wagner, D. D. (1986) Cell 46, Platelet adhesion may also play an important role in tumor 185-190. metastasis. In animal models, thrombocytopenia and im- 10. Visser, M. R., Tracy, P. B., Vercellotti, G. M., Goodman, paired platelet function have been shown to correlate with J. L., White, J. G. & Jacob, H. S. (1988) Proc. Natl. Acad. Sci. reduced tumor metastasis. vWF, platelet glycoprotein Ilb/ USA 85, 8227-8230. IlIa, and fibronectin have all been implicated in vitro in 11. Etingin, 0. R., Silverstein, R. L., Friedman, H. M. & HajiJar, platelet-tumor cell interactions and D. P. (1990) Cell 61, 657-662. metastasis (23). 12. Etingin, 0. R., Silverstein, R. L. & Hajiar, D. P. (1991) Proc. In summary, we propose that HSV-infected endothelium is Natl. Acad. Sci. USA 88, 7200-7203. prothrombotic, in part due to expression of adhesion mole- 13. Jaffe, E. A., Nachman, R. L., Becker, C. G. & Minick, C. R. cules for circulating platelets. One mechanism for such (1973) J. Clin. Invest. 52, 2745-2756. expression may be virus-induced translocation of a pre- 14. Knudsen, B. S., Harpel, P. C. & Nachman, R. L. (1987) J. formed pool of these molecules to the endothelial cell sur- Clin. Invest. 80, 1082-1089. face. This type of pathophysiologic mechanism may also be 15. MacGregor, R. R., Friedman, H. M., Macarak, E. J. & Ke- involved in virus-mediated atherogenesis and virus-induced falides, N. A. (1980) J. Clin. Invest. 65, 1469-1471. thrombocytopenia. 16. Key, N. S., Vercellotti, G. M., Winkelmann, J. C., Moldow, C. F., Goodman, J. L., Esmon, N. L., Esmon, C. T. & Jacob, We acknowledge the outstanding technical assistance of Barbara H. S. (1990) Proc. Natl. Acad. Sci. USA 87, 7095-7099. Summers and Brendan Bottari. This work was supported by Grants 17. 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