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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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Related in: MedlinePlus

Flow cytometry analysis of P-selectin expression on  platelets. Wild-type and ΔCT platelets were stained for membrane P-selectin and analyzed by flow cytometry. In the resting  state, platelets of both genotypes displayed virtually no P-selectin  on their plasma membranes. Thrombin activation induced in  wild-type as well as in mutant platelets a similar increase in mean  fluorescence with ∼90% of P-selectin–positive platelets. Representative histograms are shown. Shaded area, negative control  staining with only the FITC-conjugated goat anti–rabbit IgG.
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Figure 4: Flow cytometry analysis of P-selectin expression on platelets. Wild-type and ΔCT platelets were stained for membrane P-selectin and analyzed by flow cytometry. In the resting state, platelets of both genotypes displayed virtually no P-selectin on their plasma membranes. Thrombin activation induced in wild-type as well as in mutant platelets a similar increase in mean fluorescence with ∼90% of P-selectin–positive platelets. Representative histograms are shown. Shaded area, negative control staining with only the FITC-conjugated goat anti–rabbit IgG.

Mentions: The level of P-selectin expressed on the surfaces of platelets was determined by FACS® analysis. Fig. 4 illustrates typical fluorescence histograms of P-selectin staining of platelets. Very little P-selectin was detected on the surface of ΔCT platelets in the resting state, as was the case for wild-type platelets (Fig. 4). After activation by thrombin, ∼90% of the platelets, from both the mutant and the wild-type animals, expressed P-selectin on their surfaces (Fig. 4). In addition, similar means of fluorescence intensities were observed in platelets of the two genotypes. These results indicate that, similar to wild-type platelets, the resting ΔCT platelets do not express significant amounts of P-selectin on the plasma membrane, and that levels of P-selectin stored in a releasable pool inside the platelets are comparable in the wild-type and ΔCT platelets.


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)

Flow cytometry analysis of P-selectin expression on  platelets. Wild-type and ΔCT platelets were stained for membrane P-selectin and analyzed by flow cytometry. In the resting  state, platelets of both genotypes displayed virtually no P-selectin  on their plasma membranes. Thrombin activation induced in  wild-type as well as in mutant platelets a similar increase in mean  fluorescence with ∼90% of P-selectin–positive platelets. Representative histograms are shown. Shaded area, negative control  staining with only the FITC-conjugated goat anti–rabbit IgG.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Flow cytometry analysis of P-selectin expression on platelets. Wild-type and ΔCT platelets were stained for membrane P-selectin and analyzed by flow cytometry. In the resting state, platelets of both genotypes displayed virtually no P-selectin on their plasma membranes. Thrombin activation induced in wild-type as well as in mutant platelets a similar increase in mean fluorescence with ∼90% of P-selectin–positive platelets. Representative histograms are shown. Shaded area, negative control staining with only the FITC-conjugated goat anti–rabbit IgG.
Mentions: The level of P-selectin expressed on the surfaces of platelets was determined by FACS® analysis. Fig. 4 illustrates typical fluorescence histograms of P-selectin staining of platelets. Very little P-selectin was detected on the surface of ΔCT platelets in the resting state, as was the case for wild-type platelets (Fig. 4). After activation by thrombin, ∼90% of the platelets, from both the mutant and the wild-type animals, expressed P-selectin on their surfaces (Fig. 4). In addition, similar means of fluorescence intensities were observed in platelets of the two genotypes. These results indicate that, similar to wild-type platelets, the resting ΔCT platelets do not express significant amounts of P-selectin on the plasma membrane, and that levels of P-selectin stored in a releasable pool inside the platelets are comparable in the wild-type and ΔCT platelets.

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