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Article Figures & Data

Figures

  • Fig. 1.

    Expression of chemokine receptor mRNA in platelets.

    PCR analysis was performed using primers against the chemokine receptors CCR1-9, CXCR1-5, CX3CR1, and XCR1 with GAPDH as a housekeeping probe.

  • Fig. 2.

    Surface expression of CCR1, CCR3, CCR5, CXCR1, CXCR3, and CXCR4 on platelets assessed by fluorescence-activated cell sorter analysis.

    Surface expression of GPVI, a collagen receptor, is shown for comparison. Platelets were labeled with 10 μg/mL antireceptor antibody (black line) or an isotype control antibody (or preimmune polyclonal) (gray filled), then labeled with an fluorescein isothiocyanate (FITC) conjugate. Washed platelets were analyzed by flow cytometry and accumulated events were gated against the isotype control. The experiment shown is representative of 3 similar experiments.

  • Fig. 3.

    Expression of CCR1 and CCR4 in platelets assessed by immunoprecipitation and immunoblotting using antipeptide polyclonal antibodies.

    Left panel, detection with anti-CCR1 polyclonal antibodies; 1, platelet lysate; 2, immunoprecipitate from platelets of donor A with anti-CCR1 polyclonal antibodies; 3, immunoprecipitate from platelets of donor B with anti-CCR1 polyclonal antibodies; 4, immunoprecipitate from monocytes with anti-CCR1 polyclonal antibodies. Right panel, detection with anti-CCR4 polyclonal antibodies; 1, platelet lysate; 2, immunoprecipitate from platelets of donor A with anti-CCR4 polyclonal antibodies; 3, immunoprecipitate from platelets of donor B with anti-CCR4 polyclonal antibodies; 4, immunoprecipitate from CCR4 transfected cells with anti-CCR4 polyclonal antibodies.

  • Fig. 4.

    Platelet cytoplasmic Ca++ response to various chemokines measured using fura-2 fluorescence.

    Washed platelets (108/mL) were loaded with fura-2 and then activated with 100 nmol/L chemokine. When the signal had returned to baseline or stabilized (generally at 2 minutes) either a second dose of the same chemokine or of another one was added. Partial mutual desensitization of the Ca++ signal response by chemokines using different receptors was observed but was almost complete when the same chemokine was used twice or when chemokines with the same receptor were used. The results shown are typical of at least 3 similar experiments.

  • Fig. 5.

    Titration of platelet cytoplasmic Ca++response to increasing amounts of chemokines measured using fura-2 fluorescence.

    Washed platelets (108/mL) loaded with fura-2 were activated with various concentrations of chemokines. (A) TARC: plot 1, 10 nmol/L; plot 2, 20 nmol/L; and plot 3, 50 nmol/L. (B) Eotaxin: plot 1, 10 nmol/L; plot 2, 20 nmol/L; and plot 3, 50 nmol/L. (C) MIP-1α: plot 1, 10 nmol/L; plot 2, 20 nmol/L; and plot 3, 30 nmol/L. The results shown are typical of at least 3 similar experiments.

  • Fig. 6.

    Platelet cytoplasmic Ca++ response to various agonists measured using fura-2 fluorescence.

    Several agonists such as ADP (10 μmol/L), TRAP (1 μmol/L), and thrombin (0.05 U/mL) caused a partial reduction in a second chemokine-induced signal or vice-versa. The ADP receptor inhibitor, AR-C66096 (100 nmol/L), also caused a partial but not complete inhibition of Ca++ signaling by chemokines and the other agonists. The signals were lower but not cross-desensitized in the presence of apyrase (5 U/mL, not shown). The results shown are typical of at least 3 similar experiments.

  • Fig. 7.

    Platelet cytoplasmic Ca++ response to eotaxin and TARC alone and in the presence of a blocking antibody directed against CCR3, measured using fura-2 fluorescence.

    (A) Plot 1: washed platelets (108/mL) loaded with fura-2 were activated with eotaxin (10 nmol/L) (a), followed by TARC (10 nmol/L) (b) and thrombin (0.1U/mL) (c). Plot 2: eotaxin (10 nmol/L) (d) and TARC (10 nmol/L) were added together. The Ca++ signal is equivalent to that of 0.1 U/mL thrombin in plot 1. (B) Washed platelets (108/mL) loaded with fura-2 were incubated with Fab fragments of anti-CD32 antibodies and then treated with anti-CCR3 antibodies (a) that cause the inhibition of Ca++ signaling by eotaxin (b) but not TARC (c). The results shown are typical of at least 3 similar experiments.

  • Fig. 8.

    Aggregation response of platelets to chemokines alone and in presence of ADP receptor inhibitor AR-C66096 or ADPase.

    In the upper left series, washed platelets (5 × 108/mL) were treated with 100 nmol/L TARC, RANTES, SDF-1, or MDC, respectively. In the upper right series, washed platelets (5 × 108/mL) were treated with a mixture of 100 nmol/L each TARC plus SDF-1 or RANTES plus TARC, respectively. No signal addition was seen when the same doses were given successively (Figure4). In the lower 2 aggregation profiles, washed platelets were treated with 100 nmol/L TARC in the presence of 100 nmol/L ADP receptor inhibitor (AR-C66096) or (bottom profile) with 100 nmol/L RANTES in the presence of 5 μmol/L apyrase. The results shown here are representative of at least 3 similar experiments.

  • Fig. 9.

    Aggregation response of platelets to chemokines alone and in combination as well as in the presence of heparin, low-molecular-weight heparin (Fragmin), heparinase, or chondroitinases.

    Upper series of aggregation profiles shows washed platelets activated with 100 nmol/L TARC, from left, control, in the presence of 1 U/mL, 5 U/mL, and 20 U/mL heparin, respectively. Middle series shows washed platelets activated with 100 nmol/L TARC, from left, in the presence of 1 U/mL, 5 U/mL, and 20 U/mL Fragmin, respectively. Lower series shows washed platelets activated with 100 nmol/L TARC, from left, control, after incubation for 30 minutes at 37°C with 5 U/mL heparinases I and III, 5 U/mL chondoitinases ABC, and 5 U/mL heparitinase I and III plus 5 U/mL chondoitinase ABC, respectively. These results are typical of at least 3 similar experiments.

  • Fig. 10.

    Time dependence of tyrosine phosphorylation in proteins from platelets activated by RANTES, TARC, or ADP and immunoprecipitation of PLCγ2.

    (A) Washed platelets (5 × 108) were activated with 100 nmol/L RANTES, TARC, or 10 μmol/L ADP, respectively. Aliquots were removed at times indicated and dissolved in SDS containing inhibitors. After separation by SDS-PAGE and transfer to PDVF membranes the proteins were incubated with the antiphosphotyrosine antibody 4G10 before detection by chemiluminescence. Proteins with increased tyrosine phosphorylation are indicated by arrows on the left and identified phosphoproteins are indicated on the right. The results shown here are typical of at least 3 experiments performed with these doses and chemokines but analogous experiments with other chemokines interacting with platelets also gave similar results. (B) Aliquots of 108 platelets from points on a time range activated with TARC (50 nmol/L) solubilized in RIPA buffer were immunoprecipitated with antibodies against PLCγ2. After SDS-PAGE and Western blotting, the proteins were detected with 4G10 antiphosphotyrosine antibody. The membranes were stripped and treated with anti-PLCγ2antibodies.