The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1β and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element–binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli.

The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are dependent on the interaction of the leukocyte integrin Mac-1 (α M β 2 , CD11b/CD18) and the platelet counter receptor glycoprotein (GP) Ibα. Previous studies have established a central role for the I domain, a stretch of ∼200 amino acids within the α M subunit, in the binding of GP Ibα. This study was undertaken to establish the molecular basis of GP Ibα recognition by α M β 2 . The P 201 –K 217 sequence, which spans an exposed loop and amphipathic α4 helix in the three-dimensional structure of the α M I domain, was identified as the binding site for GP Ibα. Mutant cell lines in which the α M I domain segments P 201 –G 207 and R 208 –K 217 were switched to the homologous, but non-GP Ibα binding, α L domain segments failed to support adhesion to GP Ibα. Mutation of amino acid residues within P 201 –K 217 , H 210 –A 212 , T 213 –I 215 , and R 216 –K 217 resulted in the loss of the binding function of the recombinant α M I domains to GP Ibα. Synthetic peptides duplicating the P 201 –K 217 , but not scrambled versions, directly bound GP Ibα and inhibited α M β 2 -dependent adhesion to GP Ibα and adherent platelets. Finally, grafting critical amino acids within the P 201 –K 217 sequence onto α L , converted α L β 2 into a GP Ibα binding integrin. Thus, the P 201 –K 217 sequence within the α M I domain is necessary and sufficient for GP Ibα binding. These observations provide a molecular target for disrupting leukocyte–platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.

The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are both dependent on the interaction of the leukocyte integrin, Mac-1, and a heretofore unknown platelet counterreceptor. Here, we identify the platelet counterreceptor as glycoprotein (GP) Ibα, a component of the GP Ib-IX-V complex, the platelet von Willebrand factor (vWf) receptor. THP-1 monocytic cells and transfected cells that express Mac-1 adhered to GP Ibα–coated wells. Inhibition studies with monoclonal antibodies or receptor ligands showed that the interaction involves the Mac-1 I domain (homologous to the vWf A1 domain), and the GP Ibα leucine-rich repeat and COOH-terminal flanking regions. The specificity of the interaction was confirmed by the finding that neutrophils from wild-type mice, but not from Mac-1–deficient mice, bound to purified GP Ibα and to adherent platelets, the latter adhesion being inhibited by pretreatment of the platelets with mocarhagin, a protease that specifically cleaves GP Ibα. Finally, immobilized GP Ibα supported the rolling and firm adhesion of THP-1 cells under conditions of flow. These observations provide a molecular target for disrupting leukocyte–platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.