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Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses.

Hartwell DW, Mayadas TN, Berger G, Frenette PS, Rayburn H, Hynes RO, Wagner DD - J. Cell Biol. (1998)

Bottom Line: The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells.Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma.Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

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Interaction of resting and activated platelets with HL-60 cells. The interaction between platelets and HL-60 cells was  determined under phase microscopy and the percentages of HL-60 cells bound to two or more platelets are shown. The data represent the mean of three separate experiments.
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Figure 6: Interaction of resting and activated platelets with HL-60 cells. The interaction between platelets and HL-60 cells was determined under phase microscopy and the percentages of HL-60 cells bound to two or more platelets are shown. The data represent the mean of three separate experiments.

Mentions: Previous studies have shown that the binding of activated platelets to neutrophils and monocytes is mediated by P-selectin (Larsen et al., 1989; Hamburger and McEver, 1990). The ability of ΔCT–P-selectin to mediate platelet– leukocyte interactions was assessed by a rosetting assay (Larsen et al., 1989). Resting or thrombin-activated platelets were incubated with HL-60 cells which express functional PSGL-1, the major ligand for P-selectin. The interaction between the two cell types was evaluated by light microscopy. As shown in Fig. 6, few rosetting events were observed between HL-60 cells and resting platelets of either genotype. When activated with thrombin, the ΔCT platelets bound to HL-60 cells to a similar extent as wild-type platelets. Rosetting was eliminated by the presence of EDTA or pretreatment of HL-60 cells with neuraminidase (data not shown).


Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses.

Hartwell DW, Mayadas TN, Berger G, Frenette PS, Rayburn H, Hynes RO, Wagner DD - J. Cell Biol. (1998)

Interaction of resting and activated platelets with HL-60 cells. The interaction between platelets and HL-60 cells was  determined under phase microscopy and the percentages of HL-60 cells bound to two or more platelets are shown. The data represent the mean of three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132959&req=5

Figure 6: Interaction of resting and activated platelets with HL-60 cells. The interaction between platelets and HL-60 cells was determined under phase microscopy and the percentages of HL-60 cells bound to two or more platelets are shown. The data represent the mean of three separate experiments.
Mentions: Previous studies have shown that the binding of activated platelets to neutrophils and monocytes is mediated by P-selectin (Larsen et al., 1989; Hamburger and McEver, 1990). The ability of ΔCT–P-selectin to mediate platelet– leukocyte interactions was assessed by a rosetting assay (Larsen et al., 1989). Resting or thrombin-activated platelets were incubated with HL-60 cells which express functional PSGL-1, the major ligand for P-selectin. The interaction between the two cell types was evaluated by light microscopy. As shown in Fig. 6, few rosetting events were observed between HL-60 cells and resting platelets of either genotype. When activated with thrombin, the ΔCT platelets bound to HL-60 cells to a similar extent as wild-type platelets. Rosetting was eliminated by the presence of EDTA or pretreatment of HL-60 cells with neuraminidase (data not shown).

Bottom Line: The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells.Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma.Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

Show MeSH
Related in: MedlinePlus