Initial rolling of circulating neutrophils on a blood vessel wall prior to adhesion and transmigration to damaged tissue is dependent upon P-selectin expressed on endothelial cells and its specific neutrophil receptor, the P-selectin glycoprotein ligand-1 (PSGL-1). Pretreatment of neutrophils, HL60 cells, or a recombinant fucosylated soluble form of PSGL-1 (sPSGL-1.T7) with the cobra venom metalloproteinase, mocarhagin, completely abolished binding to purified P-selectin in a timedependent and EDTA-and diisopropyl fluorophosphateinhibitable manner consistent with mocarhagin selectively cleaving PSGL-1. A polyclonal antibody against the N-terminal peptide Gln-1-Glu-15 of mature PSGL-1 immunoprecipitated sPSGL-1.T7 but not sPSGL-1.T7 treated with mocarhagin, indicating that the mocarhagin cleavage site was near the N terminus. A single mocarhagin cleavage site between Tyr-10 and Asp-11 of mature PSGL-1 was determined by N-terminal sequencing of mocarhagin fragments of sPSGL-1.T7 and is within a highly negatively charged amino acid sequence 1-QATEYEYLDY2DFLPETEPPE, containing three tyrosine residues that are consensus sulfation sites. Consistent with a functional role of this region of PSGL-1 in binding P-selectin, an affinity-purified polyclonal antibody against residues Gln-1-Glu-15 of PSGL-1 strongly inhibited P-selectin binding to neutrophils, whereas an antibody against residues Asp-9 -Arg-23 was noninhibitory. These combined data strongly suggest that the Nterminal anionic/sulfated tyrosine motif of PSGL-1 as well as downstream sialylated carbohydrate is essential for binding of P-selectin by neutrophils.In response to inflammatory stimuli, neutrophils in the adjacent vasculature initially roll on the blood vessel wall, then stick, and finally transmigrate to the site of insult (1). The initial rolling event involves a class of adhesion proteins termed selectins (P-, E-, and L-selectin), which mediate the interaction between leukocytes and endothelial cells by recognizing specific carbohydrate counterstructures, including sialyl-Lewis x (2-4). P-selectin binds to ϳ10,000 -20,000 copies of a single class of binding site on neutrophils and HL60 cells (4,5). Studies in a number of laboratories have identified a 220 -240-kDa, disulfide-linked homodimeric protein, which appears to specifically bind P-selectin (6, 7). This protein is probably identical to P-selectin glycoprotein ligand-1 (PSGL-1) 1 (8). PSGL-1 is a 220-kDa, disulfide-linked homodimeric sialomucin, which, when expressed in COS cells with the appropriate fucosyltransferase, binds P-selectin in a similar calcium-dependent manner to the receptor on neutrophils. PSGL-1 has a signal peptide sequence of 17 amino acids followed by a 24-amino acid PACE propeptide sequence (8). The mature N terminus of PSGL-1 contains an unusual stretch of 20 amino acids, which is rich in negatively charged aspartate and glutamate residues and which contains three tyrosine residues that meet the consensus sequence for O-sulfation by Golgi sulfotransferase(s) (9). At least on...

In the inflammatory response, leukocyte rolling before adhesion and transmigration through the blood vessel wall is mediated by specific cell surface adhesion receptors. Neutrophil rolling involves the interaction of P-selectin expressed on activated endothelium and its counter-receptor on neutrophils, P-selectin glycoprotein ligand-1 (PSGL-1). Here, it is reported that P-selectin binding to neutrophils is lost under conditions that cause the release of proteinases from neutrophil primary granules. Treatment of neutrophils with the purified neutrophil granule proteinases, cathepsin G and elastase, rapidly abolished their capacity to bind P-selectin. This inactivation corresponded to loss of the N-terminal domain of PSGL-1, as assessed by Western blot analysis. A loss of intact PSGL-1 protein from the surfaces of neutrophils after the induction of degranulation was also detected by Western blot analysis. Cathepsin G initially cleaved near the PSGL-1 N-terminus, whereas neutrophil elastase predominantly cleaved at a more C-terminal site within the protein mucin core. Consistent with this, cathepsin G cleaved a synthetic peptide based on the PSGL-1 N-terminus between Tyr-7/Leu-8. Under conditions producing neutrophil degranulation in incubations containing mixtures of platelets and neutrophils, the loss of PSGL-1, but not P-selectin, from plateletneutrophil lysates was detected. Cathepsin G-or neutrophil elastase-mediated PSGL-1 proteolysis may constitute a potential autocrine mechanism for downregulation of neutrophil adhesion to P-selectin. IntroductionRecruitment of neutrophils to inflammatory sites is initiated by rolling of circulating cells on activated endothelium before firmer adhesion and subsequent transmigration. [1][2][3][4] Neutrophil rolling is mediated predominantly by expression of adhesive receptors of the selectin family on activated endothelial cells. 5,6 P-selectin is stored on Weibel-Palade body membranes and rapidly expressed on the surface of activated endothelial cells; E-selectin expression is induced in response to inflammatory stimuli. The counter-receptor for P-selectin on neutrophils is P-selectin glycoprotein ligand-1 (PSGL-1), an approximately 220-kd glycoprotein homodimer. The mature protein consists of an extracellular N-terminal anionic sequence containing 1 to 3 sulfated tyrosine residues (Gln-1-Glu-20), a sialomucin domain, a transmembrane domain, and a cytoplasmic tail. 7,8 Several lines of evidence have shown that the sulfated N-terminal sequence of PSGL-1 is critical for P-selectin binding. We found that a cobra venom metalloproteinase, mocarhagin, abolished P-selectin binding to neutrophils by selectively cleaving P-selectin between Tyr-10 and Asp-11. 9 Further, P-selectin binding to recombinant PSGL-1 was dependent on coexpression with tyrosylsulfate sulfotransferase, whereas blocking sulfation of the recombinant receptor inhibited ligand binding. [10][11][12] After initial rolling, tight adhesion of arrested neutrophils is regulated by other adhesion receptors, in particul...

Binding of the adhesive glycoprotein, von Willebrand factor (vWf), to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates platelet adhesion and aggregation at high shear stress in hemostasis and thrombosis. In this study, the GP Ib-IX-V binding site within the vWf A1 domain was analyzed using a panel of murine monoclonal antibodies raised against a 39/34-kd vWf fragment (Leu-480/Val-481–Gly-718) encompassing the A1 domain. One antibody, 6G1, strongly inhibited ristocetin-dependent vWf binding to platelets, but had no effect on botrocetin- or jaracetin-dependent binding, or asialo-vWf–dependent platelet aggregation. The 6G1 epitope was mapped to Glu-700–Asp-709, confirming the importance of this region for modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf A1 domain, because it enhanced binding of the 39/34-kd fragment to platelets. In contrast, 5D2 and CR1 completely inhibited asialo-vWf–induced platelet aggregation and ristocetin-induced vWf binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and jaracetin-induced vWf binding to platelets and binding of vWf to botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were conformationally dependent, but not congruent. Other antibodies mapped to epitopes within the A1 domain (CR2 and CR15, Leu-494–Leu-512; CR2, Phe-536–Ala-554; CR3, Arg-578–Glu-596; CR11 and CR15, Ala-564–Ser-582) were not functional, identifying regions of the vWf A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The combined results provide evidence that the proline-rich sequence Glu-700–Asp-709 constitutes a regulatory site for ristocetin, and that ristocetin and botrocetin induce, at least in part, separate receptor-recognition sites on vWf. (Blood. 2000;95:164-172)