Glycoprotein (GP) Ib-IX-transfected Cells Roll on a von Willebrand Factor Matrix under Flow

Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbα cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbα blocking antibody (AK2) and was not observed with CHO cells expressing GPIbβ and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20–200 s−1), GPIb-IX was able to support cell rolling at both venous (150 s−1) and arterial (1500–10,500 s−1) shear rates. Studies with a mutant GPIbα receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000–6000 s−1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbα and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress. Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbα cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbα blocking antibody (AK2) and was not observed with CHO cells expressing GPIbβ and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20–200 s−1), GPIb-IX was able to support cell rolling at both venous (150 s−1) and arterial (1500–10,500 s−1) shear rates. Studies with a mutant GPIbα receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000–6000 s−1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbα and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress. Cell/cell and cell/matrix adhesive interactions are fundamental to a broad range of physiological processes, including inflammation, immunity, and hemostasis. The ability of circulating cells, such as leukocytes and platelets, to adhere to the vessel wall requires specialized adhesion mechanisms capable of withstanding the mechanical challenge of flowing blood. The mechanism by which leukocytes adhere to the vascular endothelium is the paradigm for cell adhesion under flow. This is a multistep process in which the initial interaction of these cells with the endothelium involves selectin-mediated leukocyte rolling. This interaction serves to reduce the velocity of cell movement over the endothelium and increases the probability of forming irreversible stationary adhesion contacts between leukocyte β2-integrins and ICAM-1 (intercellularadhesion molecule 1) on the endothelial cell surface (1Lawrence M.B. Springer T.A. Cell. 1991; 65: 859-873Abstract Full Text PDF PubMed Scopus (1882) Google Scholar, 2Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6400) Google Scholar). A similar multistep adhesion mechanism has recently been proposed to explain stable platelet adhesion to areas of vessel wall injury under conditions of high shear stress, such as those encountered in the microvasculature or in stenotic arteries (3Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar, 4Ruggeri Z.M. J. Clin. Invest. 1997; 99: 559-564Crossref PubMed Scopus (192) Google Scholar). Platelet tethering to the injured vessel wall under conditions of rapid blood flow is critically dependent on the interaction between subendothelial-bound vWf 1The abbreviations used are: vWf, von Willebrand factor; BvWf, bovine von Willebrand factor; HvWf, human von Willebrand factor; GP, glycoprotein; ABP, actin-binding protein; CHO, Chinese hamster ovary; mAb, monoclonal antibody; PBS, phosphate-buffered saline. and the GPIb-V-IX complex on the platelet surface. This adhesive interaction is characterized by a rapid receptor/ligand on/off rate that supports reversible adhesion contacts. Similar to leukocyte adhesion under flow, the vWf/GPIb-V-IX interaction is critical for platelet tethering as a prerequisite for integrin-mediated cell arrest (3Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar, 5Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar). A major difference between selectins and GPIb-V-IX is the shear forces under which they must operate in vivo to support cell adhesion. For example, adhesion contacts mediated by selectins are relatively sensitive to disruption by increasing shear, with detachment of leukocytes occurring at shear stresses below 7 dyne/cm2(∼200 s−1) (1Lawrence M.B. Springer T.A. Cell. 1991; 65: 859-873Abstract Full Text PDF PubMed Scopus (1882) Google Scholar). Platelets, on the other hand, adhere to vessel walls at shear stresses of up to 60 dyne/cm2(∼1600 s−1) under normal physiological conditions and can also form stable adhesion contacts at pathological levels of shear of up to 300 dyne/cm2 (∼8000 s−1) (6Kroll M.H. Hellums D. McIntire L.V. Schafer A.I. Moake J.L. Blood. 1996; 88: 1525-1541Crossref PubMed Google Scholar). The ability of platelets to form stationary adhesion contacts under conditions of high shear is dependent on several factors. First, bond formation between vWf and GPIb must occur with sufficient rapidity to enable the capture of platelets from flowing blood. Second, the vWf/GPIb interaction must have sufficient tensile strength to resist the detaching effects of high shear. Third, integrin engagement of the adhesive surface must occur rapidly to retain platelets at the site of vessel wall injury. Despite its fundamental importance, the unique biomechanical characteristics of the vWf/GPIb interaction enabling platelet tethering under high shear remain poorly understood. The high tensile strength of the vWf/GPIb interaction is likely to reflect the formation of a large number of active bonds between vWf and GPIb, a process favored by the high GPIb density on the platelet surface and the multivalency of vWf. In this regard, GPV may play an important role, as it has been postulated to cross-link two GPIb molecules on the cell surface, thereby facilitating multivalent interactions. Anchorage of GPIb to the membrane skeleton may also be important for regulating the vWf/GPIb interaction by regulating the receptor distribution on the cell surface. Previous studies examining the role of the cytoplasmic tail of GPIbα in regulating receptor function have yielded conflicting results. Cunningham et al. (7Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) have reported normal binding of vWf to mutant GPIbα receptors with C-terminal deletions of up to 52 amino acids. In et 1997; PubMed Scopus Google Scholar) have recently reported that the of as as C-terminal amino from GPIbα has a on the ability of the receptor to vWf. The for this is but may reflect between the two Studies of the GPIb-V-IX complex have demonstrated that of GPIb is to actin-binding protein in vivo J.E.B. J. Clin. Invest. 76: PubMed Scopus Google Scholar). This to play an important role in maintaining the normal of platelets and in of the receptor complex the of the membrane 1997; PubMed Scopus Google Scholar). studies of the cytoplasmic tail of GPIbα have demonstrated that the interaction between GPIbα and is essential for the receptor complex to the membrane skeleton (7Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). binding and antibody studies have demonstrated that the of the GPIbα cytoplasmic tail and with Fox J.E.B. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). studies have an important role for in this interaction in vivo (7Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google the of amino between and in regulating the interaction in to be In an to the structural domains of the GPIb-V-IX complex involved in regulating cell we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbα cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined the ability of these receptors to support cell adhesion under flow Our studies demonstrate that the GPIb-IX complex is in mediating cell rolling at both venous and arterial levels of shear in the of other platelet adhesion receptors. GPV not to cell rolling or the strength of cell to a vWf Studies of GPIbα have also demonstrated that the cytoplasmic tail of GPIbα not the initial tethering or rolling of CHO cells on vWf at low shear stress, but plays an important role in maintaining cell to the matrix under high shear. The monoclonal and from was from The of the The other from we have J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1997; PubMed Scopus Google Scholar). CHO cells in a of and with bovine CHO cells with a of which the for and These a from and the as B. Fox J.E.B. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). The from was to to the and cells under CHO cells with the GPIb-IX and and under the with and for their surface of GPIbα by and a for their level of GPIbα and GPIX by flow a flow CHO cells expressing GPIb-IX with GPV and a from to CHO cells for of GPV by cell and to two of cell to to the CHO cells with the GPIbα in by the was two to to et al. PubMed Scopus Google and cells by with or of GPIbα in and the was and for and by CHO in and for at which the was on The cells and for on with in two the cells and with of protein for at the with or an protein for on and from the by in for by and to with for the an with bovine vWf human vWf or at and with bovine at for and cells with with PBS, in to a concentration of and to the for at For a of cells was with the anti-GPIbα to to cells and the with cells an as by et al. PubMed Scopus Google Scholar). with a of with the concentration of or at with PBS, and with bovine or human at for In the of CHO cells the at a shear rate of for and the of the cell the was with at the shear stress, and was on for for the shear was to and shear rate the on the was for In studies examining cell tethering to vWf, cells the at shear rates from to 300 for the the and the number of was and the velocity of rolling was as cells in and the was The was a and the rolling velocity was expressed in For capture and the was used was to the of PubMed Scopus Google Scholar). the protein with bovine as a and human vWf from by to the of and J. Clin. Invest. PubMed Scopus Google Scholar). was from as by and E. Full Text PDF Scopus Google Scholar). demonstrated to be by of of and the for is that GPIb-IX expressed on the surface of cells is to human vWf in the presence of such as or (7Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, B. Fox J.E.B. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). these a to the interaction in their effects on vWf or GPIb-IX not those that occur in involves bovine vWf, which GPIb-IX in the of J.L. Full Text PDF PubMed Scopus Google M.B. Blood. 76: PubMed Google Scholar). In we examined the ability of to support adhesion of CHO conditions but not or to This adhesion was to the interaction in that it not occur on a matrix and was by the anti-GPIbα with CHO cells not form stable adhesion contacts with a human vWf matrix in the of a (7Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). To the effects of shear on the we in flow-based as under In initial cells to the matrix for to the of flow. shear (150 s−1) to cells their adhesion from stationary to rolling adhesion of the CHO cells contrast demonstrated a of the cells, of cell rolling. The of cells to at a cells to their rolling cell with the matrix to the of flow not to irreversible cell adhesion or rolling of cells in the presence of not the rolling a major role for CHO cell in this of cells or the vWf matrix to a broad range of shear rates s−1) not to in the interaction between cells and For example, of the matrix to high shear not cell tethering or rolling. Furthermore, the matrix for at shear stresses yielded results to on cells to at a similar velocity over the surface of the and the rolling velocity of cells not to over a These are with the that cell rolling is to the reversible characteristics of the vWf/GPIb interaction to of molecules from the cell surface or the examined the of several independent including shear receptor density, and matrix on the velocity of cell rolling. In rolling under binding conditions as under demonstrated in increases in shear rate in a in the velocity of cell from at up to at similar conditions, with surface of GPIb-IX an in rolling velocity A concentration also a on CHO cell rolling. demonstrated in increasing the concentration of from to in a in the rate of cell rolling at To human vWf also support rolling of CHO cells, cells as under with the bovine cells able to and on a matrix in the of The ability of cells to was by the matrix concentration and wall shear a low concentration cells tethered poorly to the matrix at shear rates at a high matrix concentration cells tethered at low shear rates The bovine protein was at cell tethering at both low and high matrix as with the human cell tethering was at relatively low shear rates s−1). Similar to our with BvWf, the velocity of cell rolling on was dependent on the shear rate the velocity of cell rolling on a human matrix was that observed with the bovine rolling velocity of cells on human or bovine velocity was as under The are from in which cells on matrix at shear rate in a velocity was as under The are from in which cells on matrix at shear rate demonstrated in at a high matrix concentration of cells to at shear rates as high as shear in a in the number of cells, at the shear rate s−1), of cells able to adhere and on the This with the from which of cells at a shear rate of at and at the cells with on their to detachment by high shear forces not with the rolling the of cell detachment was by the matrix concentration and receptor density not of GPV on cell detachment from bovine vWf. and cells from and over a range of shear rates s−1). The shear rate was 60 to a of shear the was and the number of cells was that cells the as cells at a cell concentration of To a role for GPV in regulating the rolling we cells with GPV as under demonstrated in high levels of GPV expressed on the surface of cells by cell and cells expressed levels of expressing GPV to in a similar to cells expressing GPIb-IX alone. difference was in the velocity of cell rolling between and cells at both low (150 s−1) and high s−1) shear rates and examined over a broad range of matrix not The of GPV on cell detachment by increasing shear was also demonstrated in the presence of GPV on the rate of cell detachment at matrix and and shear rates s−1). In to receptor density and matrix concentration regulating the strength of adhesion of cells to vWf, we the that the association of GPIbα with may also play an important role in regulating to detachment by high shear For these we a GPIbα cytoplasmic tail mutant that the major binding domain for and expressed this mutant on the surface of CHO cells as under To the this has on the ability of GPIbα to with ABP, we studies as under The results of these are in In cells, and and of and In cells, and and but to was that the complex GPIbα with amino is to with In on but cells not with an for These results demonstrate that the mutant in contrast to the is to ABP, an important role for the domain of the GPIbα cytoplasmic tail in binding to To the of this on the ability of GPIb to with vWf, we adhesion of and cells on a These cell for levels of GPIb-IX surface and as demonstrated in was difference in the ability of the mutant or receptor to mediate cell tethering at a wall shear rate of or of the rolling velocity and of adhesion at and also difference between and at a shear rate of cells cells and to be from the such that at a shear rate of of cells to the matrix with of To the ability of the mutant receptor to support cell adhesion under high shear conditions, we flow studies on a this it was to examine in adhesive characteristics of a number of and cells at shear rates. with the studies on human vWf, was difference in the ability of the mutant and receptors to support cell adhesion under conditions or tethering under low shear Similar to the human at high shear rates s−1), cells cells and from the adhesive surface, such that at of cells tethered to the matrix with of cells of the interaction in regulating cell adhesion under flow on bovine vWf. Adhesion as under and the number of was over the number of cells under and low shear (150 s−1) conditions was similar for both and of the major domain of GPIbα the rolling velocity of CHO cells at high shear rates. or cells to adhere to at The shear rate was and rolling cells for 60 at shear rate The rolling velocity of cells for cell was and the are the from of the domain of GPIbα the of cells to detachment by high shear In the as cells to increasing shear rates at and the number of cells at the of was that and cells the and that the was for both cell The in which the two cell was between of the of the vWf/GPIb interaction has been from studies such as and to binding of vWf to GPIb-V-IX under low shear these have been in the important domains on both vWf and GPIb, they not the binding conditions by platelets in in which shear plays an important role in the vWf/GPIb The in this a of the structural domains of the GPIb-V-IX complex involved in regulating cell adhesion under flow. CHO cells with partial (GPIb-IX) or complete receptor we have demonstrated that GPIb-IX is both and sufficient to mediate cell tethering and rolling on a vWf matrix under flow. Furthermore, this receptor/ligand interaction has sufficient tensile strength to cell adhesion at both venous and arterial levels of shear independent of other platelet adhesion receptors. The presence of GPV not the of cell the velocity of cell or the of the cells to detachment by high shear. Studies with a cytoplasmic tail mutant of GPIbα demonstrated that the interaction between GPIbα and is not essential for cell tethering or rolling under low shear conditions, but is important for enabling cells to remain to a vWf matrix under high shear. Our studies examining the of receptor density and matrix concentration on the ability of cells to with vWf have the of the multivalent vWf/GPIb interaction in regulating cell rolling and the strength of the adhesive To is on the that the number of active bonds between vWf and A protein is which with GPIb and has been postulated to cross-link GPIb molecules on the surface of platelets von J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) and on CHO cells J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we for a role for GPV in regulating the vWf/GPIb interaction under flow. of and cells over a range of shear rates and matrix to a role for GPV in regulating the of cell tethering at low shear, velocity of or the of cells to detachment by increasing shear. A the of this McIntire L.V. Blood. PubMed Google Scholar) has reported similar results with to the role of GPV in regulating cell adhesion on a human vWf matrix under flow examining cells expressing high levels of the a important role for GPV in regulating the vWf/GPIb interaction in cells expressing low levels of the physiological of such to be as such low levels of receptor are to be in normal our studies have not an important role for GPV in cell it that GPV may the tethering of platelets under high shear. studies platelets in GPV be to this Our studies with cells have a critical role for the of the GPIbα cytoplasmic independent of in mediating association of the receptor complex with and the membrane These are with that with high in Fox J.E.B. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). is of that also to be for GPIbα binding to in These on the that of the C-terminal amino of GPIb its interaction with and the membrane a mutant its ability to The between and in mediating GPIbα binding to is an important for is that the major site for is in an between these domains and that disruption of of this site the is also that these binding sites on or that they with other in the protein to the also the that the of the GPIbα tail plays an important role in regulating the is that of amino from the tail not it to be that the tail amino for and amino for not the interaction in Our studies suggest for the a important role for the interaction in enabling platelets to remain to a vWf matrix at high shear rates. is important to that the association of GPIb not to the ability of this receptor to vWf under Fox J.E.B. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) or low shear Furthermore, our studies the of examining the vWf/GPIb interaction over a range of shear conditions and the to with a similar level of receptor to of between and mutant receptors. For example, the of rolling studies on cells at a shear rate of have demonstrated a difference in the number of cells to human vWf and their rolling with the cytoplasmic tail regulating the of the vWf/GPIb interaction under flow. by studies over a range of shear conditions, we able to that in of cells and rolling are to a ability of the mutant receptors to the detaching effects of high shear, a ability of the mutant receptors to vWf. The in this is a of the of the vWf/GPIb This is for examining cell tethering under low shear conditions, the velocity of cell rolling at both venous and arterial levels of shear, and the tensile strength of the vWf/GPIb a of the is the to examine cell tethering under high shear conditions, of the major of the vWf/GPIb This may in reflect the large of CHO cells with platelets, platelets also poorly to a vWf matrix with platelets in the of blood cells in wall interactions PubMed Scopus Google Scholar). A of cells on a human vWf matrix is that these cells are sensitive to the detaching effects of high shear with This difference is likely to reflect in receptor density and cell The receptor density on the CHO cell surface is likely to be at of that on the and the on CHO cells with a of be that by a cell at the wall of a flow the is the wall shear Scopus Google Scholar). have been able to this by studies on a bovine vWf this adhesive cells at low shear, and are to the detaching effects of high shear. These reflect of the such as an association a and tensile bond is also that these reflect the formation of a number of active bonds between GPIbα and with this our studies cells with receptor number and matrix demonstrated that increasing the number of bonds rolling and the ability of cells to high shear. A for be to the between GPIb and the of the vWf/GPIb interaction under high shear. we difference in cell tethering or rolling velocity at low shear, it is that of GPIb to the membrane skeleton may the on/off rate of the vWf/GPIb interaction under high shear, a we have not been able to with our is also that the between GPIb and the tensile strength of the the of the GPIb receptors on the cell surface, thereby the of bond formation under high or results in of the receptor complex from the cell These are under and are critical in the role of the interaction in cell tethering under high shear. for the GPIb-IX receptor and for the cell also for monoclonal and for for the and and for