title: Thrombin-induced expression of endothelial P-selectin and intercellular adhesion molecule-1: a mechanism for stabilizing neutrophil adhesion.
abstract: Thrombin-induced expression of endothelial adhesivity toward neutrophils (PMN) was studied using human umbilical vein endothelial cells (HUVEC). HUVEC were challenged with human alpha-thrombin for varying durations up to 120 min, after which the cells were fixed with 1% paraformaldehyde and 51Cr-labeled human PMN were added to determine PMN adhesion. Endothelial adhesivity increased within 15 min after alpha-thrombin exposure, and the response persisted up to 120 min. Expression of endothelial adhesion proteins, P-selectin (GMP-140, PADGEM, CD62), and intercellular adhesion molecule-1 (ICAM-1; CD54) on the endothelial surface was quantitated by increase in the specific binding of anti-P-selectin mAb G1 and anti-ICAM-1 mAb RR1/1 labeled with 125I. P-selectin expression was maximal at 5-15 min alpha-thrombin exposure and decayed to basal levels within 90 min. In contrast, ICAM-1 activity increased at 30 min and remained elevated for 120 min after alpha-thrombin challenge. The initial endothelial adhesivity was dependent on P-selectin expression since PMN adhesion occurring within the first 30 min after alpha-thrombin challenge was inhibited by mAb G1. The later prolonged PMN adhesion was ICAM-1 dependent since this response was inhibited by mAb RR1/1 and to the same degree by the anti-CD18 mAb IB4. Anti-ELAM-1 mAb BB11 had no effect on adhesion of PMN to the alpha-thrombin-challenged cells. The initial P-selectin expression and PMN adhesion responses were reproduced by the 14-amino peptide (SFLLRNPNDKYEPF) (thrombin-receptor activity peptide; TRP-14) which comprised the NH2 terminus created by thrombin's proteolytic action on its receptors. However, TRP-14-induced PMN adhesion was transient, and TRP-14 did not cause ICAM-1 expression. The ICAM-1-dependent PMN adhesion mediated by alpha-thrombin was protein synthesis independent since ICAM-1 expression and PMN adhesion were not inhibited by cycloheximide pretreatment of HUVEC. Moreover, Northern blot analysis indicated absence of ICAM-1 mRNA signal up to 180 min after alpha-thrombin challenge. In conclusion, thrombin-induced endothelial adhesivity involves early- and late-phase responses. The initial reversible PMN adhesion is mediated by rapid P-selectin expression via TRP-14 generation. Thrombin-induced PMN adhesion is stabilized by a protein synthesis-independent upregulation of the constitutive ICAM-1 activity which enables the interaction of ICAM-1 with the CD18 beta 2 integrin on PMN.
authors.affiliation: Department of Physiology, Albany Medical College, New York 12208.
authors.forename: Y
authors.initials: Y
authors.lastname: Sugama
authors.forename: C
authors.initials: C
authors.lastname: Tiruppathi
authors.forename: K
authors.initials: K
authors.lastname: offakidevi
authors.forename: T T
authors.initials: TT
authors.lastname: Andersen
authors.forename: J W
authors.initials: JW
authors.lastname: Fenton
authors.forename: A B
authors.initials: AB
authors.lastname: Malik
mesh_terms: D000595:Amino Acid Sequence; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000096:biosynthesis*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000502:physiology*; D019007:P-Selectin; D010446:Peptide Fragments / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D015815:Cell Adhesion Molecules; D019007:P-Selectin; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D018799:Intercellular Adhesion Molecule-1; D013917:Thrombin