Mega counts with an abnormal colony morphology. In these colonies cells were smaller and the number of eetls per colony was mueh higher than in normal CFU-Mega derived colonies. In addition to the patient we tested earlier,⁶ we were ahle to test the presence of CFU-Mega in the bone marrow From orily one TAR patient, a newborn baby-gin whose bone marrow was aspirated for diagnostic purposes.

We could not detect any megakaryocyte colony growth after stimulation with TPO, indicating that megakaryoeyte devel opment is markedly irnpaired. Ilowever, more patients liave tu be tested to malte a final statement conceming megakaryo eytic colony growth in vitro. In contrast, the numbers of nonmegakaryocytic colonies (CFU-GM and BFU-E) stimulated by other growth factors (G-CSF GM-CSF IL-3 SCF

and EPO) were increased. This is in aeeordance with reports of episodes of leukocytosis and leukemoid reaetions with white cell counts greater than 35,000/µL and a left shift of myeloid eells in infants with TAR syndrome.^2~3 These reie tions eould be caused by elevated leveis of other hematopoi efle growth faetors. In fact, we found elevated levels of IL 11 in at least three out of five patients affected with the TAR syndrome. IL- 11 is a cytokine that is reported tu promote the growth of myeloid and erythroid precursors. ³⁴ However, elevation of this cytokine also could have occurred indepen dent of thrombocytopenia seen in these patients.

Platelet funetions were reported tu be normal in patients with TAR syndrome,³ although few patients had ahnormal platelet aggregation and storage pool defects. ^8,9 Beeause of the high blood volume required in testing platelet aggrega tion horn thrombocytopenic patients. we only measured the expression of the activation-dependent antigen CD62P on platelets after stimulation with ADP or TRAP. Under these experimental conditions. we found normal platelet activation in all TAR patients tested.

We examined the in vitro reactivity of platelets tu TPO. Although TPO by itself does not cause an inerease in a- granule seeretion as assayed by CD62P expression. it upregulates the response tu ADP or thrombin receptor ago nists.^27~35~36 However, TPO by itseif causes signifleant tyro sine phosphorylations in a number of different proteins in platelets.^21~35~37 In contrast, platelets from TAR patients showed no or very little reaetivity tu TPO. We found no synergism of TPO tu the platelet aetivators ADP and TRAP. Furthermore there was no (3 of 4) or very wealt (1 of 4) additional tyrosine phosphorylation of platelet proteins after stimulation with TPO. From this defeetive reactivity of plate lets in response tu TPO we assume a defeet of all cells of the megakaryocytopoiesis ineluding megakatyocyte progenitor cells. This defeetive response might be the cause ofthrombo

cytopenia in the TAR syndrome. Signal transduction path ways downstream of c-Mpl should be essentially similar in platelets and their progenitors in the bone marrow. Regardless of whether the signal transduction in platelets is a simple remnant horn the megakaryocytes or whether there is a phys iological role of TPO in platelet activation. Miyakawa et a1²¹'³⁷ showed phosphorylation of the signal transduction molecules Jak2, Shc, Stat3. and Stat5 in platelets in response to TPO. These data correspond tu those obtained with cell (ines normally expressing or engineered tu express c Mpl.22.23 38. We could show that c-Mpl is expressed on the surface of platelets horn TAR patients in similar amounts as in healthy controls. In addition the molecular weight of the receptor corresponds tu that of normal donors. However. puint muta tions in c-Mpl of TAR patients cannot be excluded as a cause for the defeet in signal transduction. This seenarirt resembles very much the situation in congenital neutropenia patients in which we could show elevated G-CSF serum levels and normal G-CSF receptor expression but defeetive response tu G-CSF in vitro and in vivo.³⁹ Nevertheless, cnn genital neutropenia patients responded weIl tu pharmacologi cal dosages of G-CSF with an inerease of neutrophils associ ated with a reduetion of bacterial infeetions. suggesting that treatment of TAR patients with rhTPO might help tu inerease platelet numbers and improve elinical problems associated with thrombocytopenia.

ACKNOWLEDGMENT

We thank the foHowing physirians for providing us wuh samp(es from their TAR patients: Dr H. Habenicht (Hamburg. Gennany). Dr H. Jakobi, Dr H.-P. Haastert (Celle, Germany). Dr R. Burghard (Memmingen, Germany), Dr H. Schmidt. Dr U. Richter (Detmold. Gennany), and Dr E. Koscielnak (Stuttgart, Germany). In addition we are grateful tu Dr Michael Martin, Department of Molecular Phannaco(ogy. MHH. for performing the IL-6 and IL-1 1 ELISAs and Dr Muhan Huang for perfonning the LIF ELISA.

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