C e l l u l a r A d h e s i o n a n d th e E n d o t h e l i u m : P-Selectin Abdullah Kutlar,
MD
a
, Stephen H. Embury,
MD
b,c,
*
KEYWORDS Sickle cell disease Impaired blood flow in sickle cell disease Sickle red cell adhesion P-selectin Pentosan polysulfate sodium Microvascular occlusion KEY POINTS Impaired blood flow is the ultimate morbid effect of the canonical sequence deoxygenation / sickle hemoglobin polymerization / erythrocyte sickling. Adhesion of sickle red blood cells to the vascular endothelium has been shown to initiate acute vascular occlusion; numerous polymerization-dependent and polymerizationindependent mechanisms contribute to its completion. Effective correction of abnormal blood flow acting through any mechanism will provide therapeutic benefit. Endothelial P-selectin is initiatory and necessary for adhesion of sickle red blood cells to the endothelium. Although several selectin-blocking drugs are in their development phase, an orally active agent is preferable for long-term, prophylactic therapy.
INTRODUCTION
The importance of P-selectin to the pathophysiology of sickle cell disease (SCD) is understood through its effect on blood flow. P-selectin is central to the abnormal blood flow in SCD, and abnormal blood flow is paramount to the morbidity and mortality of the disorder. Although the expression of P-selectin makes important contributions to normal platelet activity, hemostasis, coagulation, and inflammation,1–4 the focus of
Funding Sources: Dr A. Kutlar: Novartis Pharmaceuticals, Inc, Celgene Corporation, Inc, and GlycoMimetics, Inc; Dr S.H. Embury: Consultant for Global Blood Therapeutics, Inc and JUNCTIONRx. Conflict of Interest: Dr A. Kutlar: None; Dr S.H. Embury: Executive of Vanguard Therapeutics, Inc. a Sickle Cell Center, Department of Medicine, Georgia Regents University, 1120 15th Street, Augusta, GA 30912, USA; b Department of Medicine, University of California San Francisco School of Medicine, 505 Parnassus Avenue, San Francisco, CA 94143, USA; c Vanguard Therapeutics, Inc, 108 Eagle Trace Drive, Half Moon Bay, CA 94019-2286, USA * Corresponding author. Vanguard Therapeutics, Inc, 108 Eagle Trace Drive, Half Moon Bay, CA 94019-2286. E-mail address: [email protected] Hematol Oncol Clin N Am 28 (2014) 323–339 http://dx.doi.org/10.1016/j.hoc.2013.11.007 hemonc.theclinics.com 0889-8588/14/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
324
Kutlar & Embury
this review is the detrimental effect of endothelial P-selectin–mediated sickle red cell adhesion on sickle cell blood flow. ABNORMAL BLOOD FLOW IN SICKLE CELL DISEASE
Abnormal blood flow is responsible for most of the morbidity associated with SCD.5–9 Stroke,10,11 acute painful episodes,12–14 splenic sequestration and infarcts, osteonecrosis, acute chest syndrome, and priapism are among the clinical events caused by or significantly contributed to by impaired blood flow. Stroke is an important cause of disability and death15,16; pain crises are major determinants of the quality of life17 and have been associated with increased mortality.18 In this regard, deficient blood flow can be regarded as the principal defect of SCD. Microvascular blood flow has been found to be consistently abnormal in sickle cell patients even when they are not experiencing pain crises, independent of the technique used for measurement (nail-bed microscopy, laser Doppler measurement, or ocular computer-assisted intravital microscopy).19–22 During acute pain crises, additional flow changes are detected using these same methods.20–22 The contribution of numerous polymerization-independent processes to impaired sickle cell blood flow23–33 provides several therapeutic targets for remedying abnormal sickle cell blood flow as alternatives to unraveling the paradigmatic Gordian knot of deoxygenation / sickle hemoglobin (HbS) polymerization / sickle red blood cell (SRBC) rigidification and sickling.34 This understanding suggests that genetic diseases do not necessarily require genetic treatments. The primacy of abnormal blood flow to these complex interrelated pathophysiologies is revealed by the dependence of polymerization-independent mechanisms on ischemia/ reperfusion.35,36 DETERMINANTS OF SICKLE CELL BLOOD FLOW
The flow of sickle cell blood is influenced by classical descriptors of Newtonian fluid flow, as well as by additional determinants that pertain to its non-Newtonian nature, unique properties as sickle cell blood, and exogenous influences to which it is singularly susceptible.32,37 The relative influences of these myriad factors vary in different parts of the circulation and variable conditions affecting those vessels.38 It has been reasoned that blood viscosity is a more important determinant of blood flow in large vessels and that individual SRBC deformability is of greater consequence to microcirculatory flow, and that this accounts for the contrasting clinical effects of RBC transfusion that differ according to the conflicting influences on the two circulations.39–43 In this analysis the importance of SRBC folding to squeeze through small vessels is paramount in the microcirculation where rigidification of SRBC is highly detrimental.9,39,44–47 Most critical to the present discussion, the tight squeeze of flowing cells through the smallest vessels exposes the entire circumference of these cells to endothelial cell adhesion molecules, which renders microcirculatory flow more susceptible than large-vessel flow to the detrimental effects of cell adhesion.30 This concept was illustrated clearly by Hebbel,30 as shown in Fig. 1. IMPORTANCE OF SRBC ADHESION TO BLOOD FLOW
Awareness of the potential importance of SRBC adhesivity to microcirculatory blood flow began with the initial reports of abnormal SRBC adhesivity to cultured endothelial cells in comparison with normal RBC.48,49 Evidence for the clinical importance of this
Cell Adhesion to Endothelial P-Selectin
Fig. 1. Conceptual models illustrating the significance of circumferential contact between a red cell and vascular wall. Here, the red cell is conceived as being sufficiently rounded (rendered as a black oval) so that adhesion can represent a single point-of-contact phenomenon. Blood flow is from right to left in A and B, and is perpendicular to the plane of the page in C to E. In large vessels precluding circumferential contact (A), detachment forces countering the adhesive bond are derived from shear flow and a peeling torque. If the vessel is small enough to allow adhesive contacts on opposite sides of the red cell (B), the effect of the peeling torque is lost. Also, because twice as many attachments to endothelium exist in B, the avidity of each can be lower than in A to still allow the red cell to maintain endothelial adhesion. The potential influence of multiple adhesion molecules is not realized as long as the red cell makes contact in a large vessel (C), so adhesion there is most likely to develop via high-affinity mechanisms. However, in a microcirculatory vessel that allows complete (D) or partial (E) circumferential contact, the forces promoting detachment can be countered by development of multiple contacts, even if they individually are of much lower affinity than that required to allow attachment via a single point. In the case of complete circumferential contact (D), the effect of peeling torque is lost as well. This highly simplified model can be greatly complicated by inclusion of a wide variety of parameters relevant to real physiology, but this does not materially change the basic conclusions illustrated here. (From Hebbel RP. Adhesive interactions of sickle erythrocytes with endothelium. J Clin Invest 1997;99:2563, Fig. 2. Ó the American Society for Clinical Investigation; with permission.)
adhesion followed promptly, with the determination that the degree of SRBC adhesivity correlates with vaso-occlusive severity of disease.50 It subsequently was demonstrated that acute vaso-occlusion occurs as a 2-step process initiated by the adhesion of a stickier subset of SRBC and completed by the physical trapping of a more rigid, polymerization-prone subset.51,52 In these studies, human SRBC were separated according to density and fractions studied in an ex vivo rat mesocecum flow system, in which intravital microscopy was used to assess the adherence of SRBC and the pressure resistance units were measured to detect flow obstruction in the microcirculation. Infusion of low-density SRBC resulted in adherence but not obstruction of flow; infusion of high-density SRBC resulted in neither adherence nor obstruction of flow; and sequential infusion of low-density followed by high-density SRBC resulted in both adherence and obstruction of flow. This discovery suggested that the preceding 79 years of sickle cell research had been focused on the second step of vaso-occlusion and established SRBC adhesion as a valid initial target for sickle cell research and therapy. In this regard, therapies that abrogate SRBC adhesion have a major impact on SRBC flow in vitro, ex vivo, in mouse models of SCD, and in patients with SCD.32,53–55 The chronic expression of an SRBC-binding adhesion molecule, P-selectin,56 on the vascular endothelium in sickle cell mouse models57 and human endothelial cells from sickle cell patients58 supports the notion that adhesion-mediated chronic drag on SRBC flow might account for the chronically abnormal microcirculatory blood flow in animal models and patients with SCD.19–22,59 CELLULAR MECHANISMS OF SRBC ADHESION
Based on results from experiments using bone marrow transplantation of sickle cell mouse bone marrow60 to C57BL control mice and aggressive methods of endothelial activation with high doses of tumor necrosis factor (TNF)-a in vivo, it was concluded
325
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Kutlar & Embury
that leukocyte adhesion to the endothelium precedes and mediates SRBC adhesion.61 Follow-on considerations posited that heterocellular aggregates also contribute to abnormalities of blood flow.62 The possible relevance of these adhesion mechanisms to blood rheology notwithstanding, direct adhesion of SRBC to the endothelium independent of leukocytes is well established in the vascular perturbation of SCD,63–67 and argues against the hypothesis that leukocyte adhesion must precede SRBC adhesion. MOLECULAR MECHANISMS OF CELL ADHESION
The process of leukocyte adhesion to the endothelium during the inflammation is understood in great detail4 and serves as a model for deciphering SRBC adhesion to endothelial cells. Pioneering studies disclosed that during inflammation leukocyte adhesion uses a cascade of adhesion molecules, triggered by initial contact with selectins and completed by firm adhesion to molecules of other types.68–70 Among the selectins, P-selectin has been found to be initiatory in the adhesion cascade,68 predominant over the other selectins,71 and essential for a complete inflammatory response.72 Downstream molecules that mediate firm adhesion and transendothelial migration of leukocytes are members of the integrin family and immunoglobulin superfamily.4,73 Those molecules have considerable overlap in their function, which makes it unlikely that absence or inhibition of any one of these would abrogate their aggregate function. These considerations define P-selectin as the initiatory linchpin molecule for leukocyte adhesion to the vascular endothelium. The selectin family of cytoadhesion molecules consist of P-selectin, E-selectin, and L-selectin, each of which mediates cytoadhesion through Ca21-dependent recognition of specific cell surface carbohydrates.74,75 P-selectin requires fucose and sialic acid in its recognition determinant; its glycan ligand, PSGL-1, has sialyl Lewis X (sLeX) in close proximity to sulfated tyrosine near the amino terminus of the ligand.74–77 Adhesion by any of the selectins requires hydrodynamic shear stress and is nonexistent in the absence of flow.78,79 Expression of P-selectin on the surface of platelets and endothelial cells requires their activation.74,80 The prompt translocation of P-selectin from a storage granules within platelets to the cell surface is induced by the rapid-acting secretagogue thrombin81; thrombin, histamine, complement components, oxygen radicals, phorbol esters, calcium ionophores, hypoxia, hypoxia/reoxygenation, and heme induce the rapid translocation of P-selectin from the Weibel-Palade body storage granules in endothelial cells to the surface of those cells.74,80,82–86 Constitutive transcription of P-selectin within endothelial cells supplies Weibel-Palade bodies with the molecule, and induction of more rapid transcription74,82 may overload the Weibel-Palade bodies, resulting in chronic surface expression of P-selectin.87 Initial reviews of the therapeutic potential of blocking SRBC adhesion did not include a molecular cascade mechanism,88,89 as selectins had not been tested for SRBC adhesivity. Subsequent studies established that endothelial P-selectin mediates abnormal static adhesion and rolling adhesion of SRBC in vitro, as shown in Fig. 2,56,90 and that this SRBC adhesion to endothelial P-selectin can be prevented in vitro using approved P-selectin–blocking therapeutic agents.90,91 Induction of endothelial P-selectin expression with an N-terminal peptide of protease-activated receptor-1, which selectively activates mouse endothelial cells but not mouse platelets,92 triggered prompt SRBC adhesion and acute stoppage of blood flow in the microcirculation of sickle cell chimeric mice.66 These in vivo adhesion studies were rigorously controlled by use of a combination of leukocyte adhesion–blocking
Cell Adhesion to Endothelial P-Selectin
Fig. 2. Effect of P-selectin antibodies on the adherence of nonsickle and sickle erythrocytes to human umbilical vein endothelial cells (HUVEC) treated with or without thrombin. The data shown indicate the static adherence of red blood cells (RBC) to HUVEC that were treated with thrombin or medium alone and then exposed to medium with or without P-selectin–blocking antibody 9E1. The 100% adherence level is the mean number of nonsickle (AA) RBC/field adherent to untreated HUVEC. SS, sickle erythrocytes. The reduction of erythrocyte adherence to untreated or thrombin-treated HUVEC is shown in the presence (hatched bars) or absence (open bars) of monoclonal antibody (mAb) 9E1. The data are mean percent adherence from 12 replicate experiments. Significant inhibition of adherence (P
MD
a
, Stephen H. Embury,
MD
b,c,
*
KEYWORDS Sickle cell disease Impaired blood flow in sickle cell disease Sickle red cell adhesion P-selectin Pentosan polysulfate sodium Microvascular occlusion KEY POINTS Impaired blood flow is the ultimate morbid effect of the canonical sequence deoxygenation / sickle hemoglobin polymerization / erythrocyte sickling. Adhesion of sickle red blood cells to the vascular endothelium has been shown to initiate acute vascular occlusion; numerous polymerization-dependent and polymerizationindependent mechanisms contribute to its completion. Effective correction of abnormal blood flow acting through any mechanism will provide therapeutic benefit. Endothelial P-selectin is initiatory and necessary for adhesion of sickle red blood cells to the endothelium. Although several selectin-blocking drugs are in their development phase, an orally active agent is preferable for long-term, prophylactic therapy.
INTRODUCTION
The importance of P-selectin to the pathophysiology of sickle cell disease (SCD) is understood through its effect on blood flow. P-selectin is central to the abnormal blood flow in SCD, and abnormal blood flow is paramount to the morbidity and mortality of the disorder. Although the expression of P-selectin makes important contributions to normal platelet activity, hemostasis, coagulation, and inflammation,1–4 the focus of
Funding Sources: Dr A. Kutlar: Novartis Pharmaceuticals, Inc, Celgene Corporation, Inc, and GlycoMimetics, Inc; Dr S.H. Embury: Consultant for Global Blood Therapeutics, Inc and JUNCTIONRx. Conflict of Interest: Dr A. Kutlar: None; Dr S.H. Embury: Executive of Vanguard Therapeutics, Inc. a Sickle Cell Center, Department of Medicine, Georgia Regents University, 1120 15th Street, Augusta, GA 30912, USA; b Department of Medicine, University of California San Francisco School of Medicine, 505 Parnassus Avenue, San Francisco, CA 94143, USA; c Vanguard Therapeutics, Inc, 108 Eagle Trace Drive, Half Moon Bay, CA 94019-2286, USA * Corresponding author. Vanguard Therapeutics, Inc, 108 Eagle Trace Drive, Half Moon Bay, CA 94019-2286. E-mail address: [email protected] Hematol Oncol Clin N Am 28 (2014) 323–339 http://dx.doi.org/10.1016/j.hoc.2013.11.007 hemonc.theclinics.com 0889-8588/14/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
324
Kutlar & Embury
this review is the detrimental effect of endothelial P-selectin–mediated sickle red cell adhesion on sickle cell blood flow. ABNORMAL BLOOD FLOW IN SICKLE CELL DISEASE
Abnormal blood flow is responsible for most of the morbidity associated with SCD.5–9 Stroke,10,11 acute painful episodes,12–14 splenic sequestration and infarcts, osteonecrosis, acute chest syndrome, and priapism are among the clinical events caused by or significantly contributed to by impaired blood flow. Stroke is an important cause of disability and death15,16; pain crises are major determinants of the quality of life17 and have been associated with increased mortality.18 In this regard, deficient blood flow can be regarded as the principal defect of SCD. Microvascular blood flow has been found to be consistently abnormal in sickle cell patients even when they are not experiencing pain crises, independent of the technique used for measurement (nail-bed microscopy, laser Doppler measurement, or ocular computer-assisted intravital microscopy).19–22 During acute pain crises, additional flow changes are detected using these same methods.20–22 The contribution of numerous polymerization-independent processes to impaired sickle cell blood flow23–33 provides several therapeutic targets for remedying abnormal sickle cell blood flow as alternatives to unraveling the paradigmatic Gordian knot of deoxygenation / sickle hemoglobin (HbS) polymerization / sickle red blood cell (SRBC) rigidification and sickling.34 This understanding suggests that genetic diseases do not necessarily require genetic treatments. The primacy of abnormal blood flow to these complex interrelated pathophysiologies is revealed by the dependence of polymerization-independent mechanisms on ischemia/ reperfusion.35,36 DETERMINANTS OF SICKLE CELL BLOOD FLOW
The flow of sickle cell blood is influenced by classical descriptors of Newtonian fluid flow, as well as by additional determinants that pertain to its non-Newtonian nature, unique properties as sickle cell blood, and exogenous influences to which it is singularly susceptible.32,37 The relative influences of these myriad factors vary in different parts of the circulation and variable conditions affecting those vessels.38 It has been reasoned that blood viscosity is a more important determinant of blood flow in large vessels and that individual SRBC deformability is of greater consequence to microcirculatory flow, and that this accounts for the contrasting clinical effects of RBC transfusion that differ according to the conflicting influences on the two circulations.39–43 In this analysis the importance of SRBC folding to squeeze through small vessels is paramount in the microcirculation where rigidification of SRBC is highly detrimental.9,39,44–47 Most critical to the present discussion, the tight squeeze of flowing cells through the smallest vessels exposes the entire circumference of these cells to endothelial cell adhesion molecules, which renders microcirculatory flow more susceptible than large-vessel flow to the detrimental effects of cell adhesion.30 This concept was illustrated clearly by Hebbel,30 as shown in Fig. 1. IMPORTANCE OF SRBC ADHESION TO BLOOD FLOW
Awareness of the potential importance of SRBC adhesivity to microcirculatory blood flow began with the initial reports of abnormal SRBC adhesivity to cultured endothelial cells in comparison with normal RBC.48,49 Evidence for the clinical importance of this
Cell Adhesion to Endothelial P-Selectin
Fig. 1. Conceptual models illustrating the significance of circumferential contact between a red cell and vascular wall. Here, the red cell is conceived as being sufficiently rounded (rendered as a black oval) so that adhesion can represent a single point-of-contact phenomenon. Blood flow is from right to left in A and B, and is perpendicular to the plane of the page in C to E. In large vessels precluding circumferential contact (A), detachment forces countering the adhesive bond are derived from shear flow and a peeling torque. If the vessel is small enough to allow adhesive contacts on opposite sides of the red cell (B), the effect of the peeling torque is lost. Also, because twice as many attachments to endothelium exist in B, the avidity of each can be lower than in A to still allow the red cell to maintain endothelial adhesion. The potential influence of multiple adhesion molecules is not realized as long as the red cell makes contact in a large vessel (C), so adhesion there is most likely to develop via high-affinity mechanisms. However, in a microcirculatory vessel that allows complete (D) or partial (E) circumferential contact, the forces promoting detachment can be countered by development of multiple contacts, even if they individually are of much lower affinity than that required to allow attachment via a single point. In the case of complete circumferential contact (D), the effect of peeling torque is lost as well. This highly simplified model can be greatly complicated by inclusion of a wide variety of parameters relevant to real physiology, but this does not materially change the basic conclusions illustrated here. (From Hebbel RP. Adhesive interactions of sickle erythrocytes with endothelium. J Clin Invest 1997;99:2563, Fig. 2. Ó the American Society for Clinical Investigation; with permission.)
adhesion followed promptly, with the determination that the degree of SRBC adhesivity correlates with vaso-occlusive severity of disease.50 It subsequently was demonstrated that acute vaso-occlusion occurs as a 2-step process initiated by the adhesion of a stickier subset of SRBC and completed by the physical trapping of a more rigid, polymerization-prone subset.51,52 In these studies, human SRBC were separated according to density and fractions studied in an ex vivo rat mesocecum flow system, in which intravital microscopy was used to assess the adherence of SRBC and the pressure resistance units were measured to detect flow obstruction in the microcirculation. Infusion of low-density SRBC resulted in adherence but not obstruction of flow; infusion of high-density SRBC resulted in neither adherence nor obstruction of flow; and sequential infusion of low-density followed by high-density SRBC resulted in both adherence and obstruction of flow. This discovery suggested that the preceding 79 years of sickle cell research had been focused on the second step of vaso-occlusion and established SRBC adhesion as a valid initial target for sickle cell research and therapy. In this regard, therapies that abrogate SRBC adhesion have a major impact on SRBC flow in vitro, ex vivo, in mouse models of SCD, and in patients with SCD.32,53–55 The chronic expression of an SRBC-binding adhesion molecule, P-selectin,56 on the vascular endothelium in sickle cell mouse models57 and human endothelial cells from sickle cell patients58 supports the notion that adhesion-mediated chronic drag on SRBC flow might account for the chronically abnormal microcirculatory blood flow in animal models and patients with SCD.19–22,59 CELLULAR MECHANISMS OF SRBC ADHESION
Based on results from experiments using bone marrow transplantation of sickle cell mouse bone marrow60 to C57BL control mice and aggressive methods of endothelial activation with high doses of tumor necrosis factor (TNF)-a in vivo, it was concluded
325
326
Kutlar & Embury
that leukocyte adhesion to the endothelium precedes and mediates SRBC adhesion.61 Follow-on considerations posited that heterocellular aggregates also contribute to abnormalities of blood flow.62 The possible relevance of these adhesion mechanisms to blood rheology notwithstanding, direct adhesion of SRBC to the endothelium independent of leukocytes is well established in the vascular perturbation of SCD,63–67 and argues against the hypothesis that leukocyte adhesion must precede SRBC adhesion. MOLECULAR MECHANISMS OF CELL ADHESION
The process of leukocyte adhesion to the endothelium during the inflammation is understood in great detail4 and serves as a model for deciphering SRBC adhesion to endothelial cells. Pioneering studies disclosed that during inflammation leukocyte adhesion uses a cascade of adhesion molecules, triggered by initial contact with selectins and completed by firm adhesion to molecules of other types.68–70 Among the selectins, P-selectin has been found to be initiatory in the adhesion cascade,68 predominant over the other selectins,71 and essential for a complete inflammatory response.72 Downstream molecules that mediate firm adhesion and transendothelial migration of leukocytes are members of the integrin family and immunoglobulin superfamily.4,73 Those molecules have considerable overlap in their function, which makes it unlikely that absence or inhibition of any one of these would abrogate their aggregate function. These considerations define P-selectin as the initiatory linchpin molecule for leukocyte adhesion to the vascular endothelium. The selectin family of cytoadhesion molecules consist of P-selectin, E-selectin, and L-selectin, each of which mediates cytoadhesion through Ca21-dependent recognition of specific cell surface carbohydrates.74,75 P-selectin requires fucose and sialic acid in its recognition determinant; its glycan ligand, PSGL-1, has sialyl Lewis X (sLeX) in close proximity to sulfated tyrosine near the amino terminus of the ligand.74–77 Adhesion by any of the selectins requires hydrodynamic shear stress and is nonexistent in the absence of flow.78,79 Expression of P-selectin on the surface of platelets and endothelial cells requires their activation.74,80 The prompt translocation of P-selectin from a storage granules within platelets to the cell surface is induced by the rapid-acting secretagogue thrombin81; thrombin, histamine, complement components, oxygen radicals, phorbol esters, calcium ionophores, hypoxia, hypoxia/reoxygenation, and heme induce the rapid translocation of P-selectin from the Weibel-Palade body storage granules in endothelial cells to the surface of those cells.74,80,82–86 Constitutive transcription of P-selectin within endothelial cells supplies Weibel-Palade bodies with the molecule, and induction of more rapid transcription74,82 may overload the Weibel-Palade bodies, resulting in chronic surface expression of P-selectin.87 Initial reviews of the therapeutic potential of blocking SRBC adhesion did not include a molecular cascade mechanism,88,89 as selectins had not been tested for SRBC adhesivity. Subsequent studies established that endothelial P-selectin mediates abnormal static adhesion and rolling adhesion of SRBC in vitro, as shown in Fig. 2,56,90 and that this SRBC adhesion to endothelial P-selectin can be prevented in vitro using approved P-selectin–blocking therapeutic agents.90,91 Induction of endothelial P-selectin expression with an N-terminal peptide of protease-activated receptor-1, which selectively activates mouse endothelial cells but not mouse platelets,92 triggered prompt SRBC adhesion and acute stoppage of blood flow in the microcirculation of sickle cell chimeric mice.66 These in vivo adhesion studies were rigorously controlled by use of a combination of leukocyte adhesion–blocking
Cell Adhesion to Endothelial P-Selectin
Fig. 2. Effect of P-selectin antibodies on the adherence of nonsickle and sickle erythrocytes to human umbilical vein endothelial cells (HUVEC) treated with or without thrombin. The data shown indicate the static adherence of red blood cells (RBC) to HUVEC that were treated with thrombin or medium alone and then exposed to medium with or without P-selectin–blocking antibody 9E1. The 100% adherence level is the mean number of nonsickle (AA) RBC/field adherent to untreated HUVEC. SS, sickle erythrocytes. The reduction of erythrocyte adherence to untreated or thrombin-treated HUVEC is shown in the presence (hatched bars) or absence (open bars) of monoclonal antibody (mAb) 9E1. The data are mean percent adherence from 12 replicate experiments. Significant inhibition of adherence (P
