title: Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.
abstract: BACKGROUND
  After activation, platelets adhere to neutrophils via P-selectin and beta2-integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown.
  
  
  RESULTS
  Whole blood was sheared at approximately 100 s(-1). The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (approximately 30%) and neutrophil aggregation (approximately 70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation.
  
  
  CONCLUSIONS
  The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.
authors.affiliation: Cox Laboratory for Biomedical Engineering, Institute of Biosciences and Bioengineering, Rice University, Houston, Tex, USA.
authors.forename: K
authors.initials: K
authors.lastname: Konstantopoulos
authors.forename: S
authors.initials: S
authors.lastname: Neelamegham
authors.forename: A R
authors.initials: AR
authors.lastname: Burns
authors.forename: E
authors.initials: E
authors.lastname: Hentzen
authors.forename: G S
authors.initials: GS
authors.lastname: Kansas
authors.forename: K R
authors.initials: KR
authors.lastname: Snapp
authors.forename: E L
authors.initials: EL
authors.lastname: Berg
authors.forename: J D
authors.initials: JD
authors.lastname: Hellums
authors.forename: C W
authors.initials: CW
authors.lastname: Smith
authors.forename: L V
authors.initials: LV
authors.lastname: McIntire
authors.forename: S I
authors.initials: SI
authors.lastname: Simon
mesh_terms: D000077284:Abciximab; D000328:Adult; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000737:chemistry / Q000166:cytology* / Q000648:ultrastructure; D018821:CD18 Antigens / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002449:Cell Aggregation / Q000187:drug effects / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D016177:Macrophage-1 Antigen / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron; D008875:Middle Aged; D009504:Neutrophils / Q000737:chemistry / Q000166:cytology* / Q000648:ultrastructure; D019007:P-Selectin / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology; D013314:Stress, Mechanical; D014680:Veins
chemical_list: D000911:Antibodies, Monoclonal; D018821:CD18 Antigens; D002412:Cations; D002614:Chelating Agents; D007140:Immunoglobulin Fab Fragments; D016177:Macrophage-1 Antigen; D019007:P-Selectin; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D004492:Edetic Acid; D016285:Iloprost; D000077284:Abciximab