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Thrombopoietin in Patients With Congenital Thrombocytopenia and Absent Radii: Elevated Serum Levels, Normal Receptor Expression, But Defective Reactivity to Thrombopoietin

Matthias Ballmaier, Harald Schulze, Gabriele Strauβ, Klara Cherkaoui, Nicole Wittner, Stefanie Lynen, Susanne Wolters, Jakob Bogenberger and Karl Welte

Article Figures & Data

Figures

  • Fig. 1.

    Expression of c-Mpl on platelets from TAR patients. (A) c-Mpl expression on the surface of platelets as measured by flow cytometry (see Materials and Methods). Gray, isotype control; black, anti c-Mpl. (B) Detection of c-Mpl in platelet lysates after immunoprecipitation and Western-blot analysis with anti-c-Mpl antibody M1 (chemoluminescence detection, see Materials and Methods). Lane 1, healthy donor; lane 2, TAR patient no. 1.

  • Fig. 2.

    Costimulation of platelets with rhTPO: TAR patient versus healthy control. Platelets of TAR patient no. 1 (A and C) and of a healthy control (B and D) were stimulated with either ADP (50 μmol/L; A and B) or TRAP (5 μmol/l; C and D) with or without preincubation with rhTPO (20 ng/mL). Flow cytometric analysis of platelet activation was performed using a MoAb against CD62P. CD62P expression of unstimulated platelets as well as the isotype control (dotted line) are shown in each histogram.

  • Fig. 3.

    Induction of tyrosine phosphorylation of platelet proteins after stimulation with rhTPO. Platelets (patient no. 5) were preincubated with different concentrations of rhTPO before lysis and Western-blot analysis with HRP-conjugated antiphosphotyrosine antibody (chemoluminescence detection) (see Materials and Methods)

Tables

  • Table 1.

    TAR Patients

    Patient No.SexAbsent RadiiRequirement of Platelet TransfusionsAgeWBC (μL−1)Hgb (g/dL)Plt (μL−1)Tests Performed*
    1F++2 yr 6 mo8,10011.757,0001, 2, 3, 4, 5
    3 yr 7 mo6,70012.187,0003, 6
    2F++1 yr 11 mo15,0008.628,0001, 3, 4, 5
    2 yr 9 moNDND31,0001, 2, 3, 4, 6
    3M++6 wk10,8008.025,0001, 5
    10 mo11,90013.672,0001, 2, 3, 4, 6
    4F+6 dNDNDND7
    6 mo27,0008.710,0001, 2, 3, 5
    5 F++6 yr 11 mo12,00010.712,0001, 2, 6
    7 yr 8 mo8,4008.412,0001, 2, 3, 4, 5
    • Abbreviations: WBC, white blood count; Hgb, Hemoglobin; Plt, platelets; ND, not determined.

    • * Code for the different examinations: 1, TPO serum level; 2, IL-11, IL-6, LIF level; 3, c-Mpl (flow cytometry); 4, c-Mpl (Western-blotting); 5, TPO reactivity (flow cytometry); 6, TPO reactivity (Tyrosine phosphorylation); 7, CFU-mega.

    • This patient has been described in an earlier abstract.6

  • Table 2.

    Levels of TPO, IL-6, IL-11, and LIF in Sera of TAR Patients

    Patient No.AgePlatelet CountTPO (pg/mL)IL-6 (pg/mL)IL-11 (pg/mL)LIF (pg/mL)
    12 yr 6 mo57,000>1,000<8<30<30
    21 yr 11 mo28,000<100ndndnd
    2 yr 9 mo31,000>1,000<8380<30
    36 wk25,000>1,000ndndnd
    10 mo72,000500-1,000<8490<30
    46 mo10,000500-1,000<8<300* <30
    56 yr 11 mo12,000500-1,000<8660<30
    7 yr 8 mo34,000100-500 <8330<30
    Normal range<100 (n = 36)<8 <30 <30 (n = 50)
    • Abbreviation: nd, not determined.

    • * Because of the little volume of the serum sample of patient 4 we had to dilute the serum for IL-11 determination.

    • Information provided by the manufacturer of the ELISA system.

  • Table 3.

    CFU Assay (Day 14)

    Patient No. 4Normal Values3-150
    CFU-GMBFU-ECFU-MegaMedian (range)
    CFU-GMBFU-ECFU-Mega
    Control0000 (0-3)0 (0-2)0 (0)
    TPO3-151 2000 (0-1)0 (0-3)11 (4-40)
    G-CSF,† GM-CSF,† IL-3,† SCF,† EPO3-151 428507nd3-152 125 (68-240)180 (91-431)nd3-152
    • Colony numbers are based upon 105 BM-MNC. Data represent the mean of duplicates.

    • F3-150 Obtained in our laboratory from 12 healthy individuals, using the same experimental conditions.

    • F3-151 10 ng/mL each, except for EPO (1 U/mL).

    • F3-152 Counting Mega-colonies was not possible in dishes with “maximum stimulation” because of the high numbers of colonies.

  • Table 4.

    TPO Synergism in Platelet Activation

    Patient No.123456
    ADPTPO + ADPEmbedded Image TRAPTPO + TRAPEmbedded Image
    126.823.30.8748.645.40.93
    243.945.31.0368.565.60.96
    324.924.00.9623.919.90.83
    49.810.91.116.88.61.26
    527.328.71.0529.631.41.06
    TAR (mean ± SD; n = 5)27 ± 1226 ± 121.00 ± 0.0936 ± 2434 ± 221.01 ± 0.16
    Healthy control (mean ± SD; n = 10)25 ± 1138 ± 101.61 ± 0.2839 ± 2550 ± 281.43 ± 0.33
    • Platelets of TAR patients and of 10 healthy controls were stimulated with either ADP (50 μmol/L, columns 1-3) or TRAP (5 μmol/L, columns 4-6) with (columns 2 and 5) or without (columns 1 and 4) preincubation with rhTPO (20 ng/mL) and analyzed for CD62P expression by flow cytometry. The percentage of CD62P-positive cells after stimulation with or without rhTPO preincubation, as well as the ratio between these data (columns 3 and 6), are shown. Values are the percentage of CD62P+ cells in all columns except 3 and 6.

    • Abbreviation: SD, standard deviation.