Figure 1
Figure 1. SFKs are required for localization of activated β2 integrins at sites of PMN-platelet contacts and for αMβ2-dependent PMN-platelet interactions in suspension. (A) PMNs alone or with PFA-fixed or unfixed thrombin (0.25 U/mL)–activated platelets (1:10 ratio) were incubated for 0 to 10 minutes at 37°C in stirring conditions (1000 rpm). At each time point, stirring was stopped and KIM127 (10 μg/mL) was added to the cells for an additional 5 minutes at 37°C. The cells were then fixed in 2% PFA for 30 minutes, washed, and stained with Alexa-Fluor 488–conjugated goat antimouse antibody at 4°C for 30 minutes. Nonspecific fluorescence was determined by incubation of PMNs with Alexa-Fluor 488–conjugated goat antimouse antibody only. Values are reported as percentages of PMNs expressing KIM127-specific binding. (B) PMNs alone or with thrombin-activated, PFA-fixed platelets (C-D) were stirred at 37°C for 3 minutes, at which time 10 μg/mL mAb 327C (B-C, E-F) or KIM127 (D) was added. Confocal microscopy to detect KIM127 and 327C mAb binding and rhodamine-phalloidin was performed as described in “Materials and methods.” (C-D) The actin staining in these images was captured at a lower amplification than in panels B, E, or F in order to allow clear identification of platelets that contain higher concentration of F-actin than do PMNs. PMNs are identified by a white line around the membrane edge and show intense 327C (C) or KIM127 (D) binding in areas with attached platelets. (E-F) PMNs were pretreated with vehicle (E) or PP2 (F) for 5 minutes at RT before incubation with platelets. PP2 treatment dramatically reduces expression of activated β2 integrins at platelet contact sites. (G) PMNs from wild-type or αM−/− mice were stirred in the absence or in the presence of fixed-activated wild-type or P-selectin–deficient platelets. Samples were immediately fixed with an equal volume of 2% PFA (open bars) or, alternatively, 5 mM EGTA was added to mixed cell suspensions 30 seconds before fixation to disrupt P-selectin–mediated bonds (closed bars designated stop EGTA). The formation of mixed platelet–PMN conjugates was analyzed by flow cytometry, as described in “Materials and methods.” The numbers of platelets recruited by 100 PMNs are graphed and the numbers on the top of the bars are the percentage of PMNs with attached platelets. Average results (mean ± SEM) from 6 different experiments for wild-type cells and 4 experiments for αM−/− or P-selectin–deficient cells are presented. *P < .05 versus WT. (H) PMNs from wild-type or double hck−/−fgr−/− or triple hck−/−fgr−/−lyn−/− mice were stirred in the absence or in the presence of activated, fixed wild-type platelets and treated as described in panel F. The numbers of platelets recruited by 100 PMNs are graphed and the numbers on top of the bars are the percentage of PMNs with attached platelets (mean, n = 4). The figure shows average results (mean ± SEM) from 4 different experiments performed with PMNs isolated from pooled bone marrow obtained from 5 wild-type or 5 knock-out mice. *P < .05 versus WT.

SFKs are required for localization of activated β2 integrins at sites of PMN-platelet contacts and for αMβ2-dependent PMN-platelet interactions in suspension. (A) PMNs alone or with PFA-fixed or unfixed thrombin (0.25 U/mL)–activated platelets (1:10 ratio) were incubated for 0 to 10 minutes at 37°C in stirring conditions (1000 rpm). At each time point, stirring was stopped and KIM127 (10 μg/mL) was added to the cells for an additional 5 minutes at 37°C. The cells were then fixed in 2% PFA for 30 minutes, washed, and stained with Alexa-Fluor 488–conjugated goat antimouse antibody at 4°C for 30 minutes. Nonspecific fluorescence was determined by incubation of PMNs with Alexa-Fluor 488–conjugated goat antimouse antibody only. Values are reported as percentages of PMNs expressing KIM127-specific binding. (B) PMNs alone or with thrombin-activated, PFA-fixed platelets (C-D) were stirred at 37°C for 3 minutes, at which time 10 μg/mL mAb 327C (B-C, E-F) or KIM127 (D) was added. Confocal microscopy to detect KIM127 and 327C mAb binding and rhodamine-phalloidin was performed as described in “Materials and methods.” (C-D) The actin staining in these images was captured at a lower amplification than in panels B, E, or F in order to allow clear identification of platelets that contain higher concentration of F-actin than do PMNs. PMNs are identified by a white line around the membrane edge and show intense 327C (C) or KIM127 (D) binding in areas with attached platelets. (E-F) PMNs were pretreated with vehicle (E) or PP2 (F) for 5 minutes at RT before incubation with platelets. PP2 treatment dramatically reduces expression of activated β2 integrins at platelet contact sites. (G) PMNs from wild-type or αM−/− mice were stirred in the absence or in the presence of fixed-activated wild-type or P-selectin–deficient platelets. Samples were immediately fixed with an equal volume of 2% PFA (open bars) or, alternatively, 5 mM EGTA was added to mixed cell suspensions 30 seconds before fixation to disrupt P-selectin–mediated bonds (closed bars designated stop EGTA). The formation of mixed platelet–PMN conjugates was analyzed by flow cytometry, as described in “Materials and methods.” The numbers of platelets recruited by 100 PMNs are graphed and the numbers on the top of the bars are the percentage of PMNs with attached platelets. Average results (mean ± SEM) from 6 different experiments for wild-type cells and 4 experiments for αM−/− or P-selectin–deficient cells are presented. *P < .05 versus WT. (H) PMNs from wild-type or double hck−/−fgr−/− or triple hck−/−fgr−/−lyn−/− mice were stirred in the absence or in the presence of activated, fixed wild-type platelets and treated as described in panel F. The numbers of platelets recruited by 100 PMNs are graphed and the numbers on top of the bars are the percentage of PMNs with attached platelets (mean, n = 4). The figure shows average results (mean ± SEM) from 4 different experiments performed with PMNs isolated from pooled bone marrow obtained from 5 wild-type or 5 knock-out mice. *P < .05 versus WT.

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