Download files
Abstract
Primary Immune thrombocytopenia (ITP) is an autoimmune platelet disorder mediated by antiplatelet antibodies that cause platelet destruction and suppression of platelet production. The latter can be caused by the effect of ITP antibodies on various aspects of megakaryocytic development. Uncovering the effects of these antibodies on megakaryocytes (MKs), would enhance the understanding of the pathogenic mechanisms of ITP and may lead to improvement in treatment options. This thesis proposes that ITP antibodies bind MKs and directly inhibit their proplatelet formation thus impeding platelet production and contributing to the observed thrombocytopenia.
19 ITP sera and purified IgGs were incubated with day 8-9 MKs, derived from cord blood haematopoietic stem cells (CD34+). At various days after treatment, the number of proplatelet-bearing MKs and platelets released in culture as well as the MK total numbers, maturation and apoptosis were assessed. 13 ITP antibodies significantly decreased the number of proplatelet-bearing MKs and this was accompanied by a decline in platelet release. Importantly, this occurred despite normal MK proliferation, differentiation and apoptotic status. In addition, this study shows that thrombopoietin receptor agonists (TPORAs), romiplostim and eltrombopag, reversed the deleterious effects of a proportion of ITP antibodies by restoring the MKs ability to form proplatelets.
This thesis investigated the capacity of ITP antibodies to activate the RhoA/ROCK pathway, which is known to inhibit proplatelet formation. It shows that ITP antibodies activate RhoA in Meg-01 cells. Also, they increased the percentage of cells positive for phosphorylated myosin light chain 2 (pMLC2), one of the downstream events following RhoA activation. Moreover, ITP antibodies increased the percentage of human glycoprotein (GP) IIb/IIIa-CHO cells (an ITP antibody target receptor) exhibiting stress fibers, one of the outcomes of the RhoA/ROCK pathway.
In conclusion, antiplatelet antibodies in ITP impair proplatelet formation by MKs and their ability to release platelets. This effect can be mediated by activation of the RhoA/ROCK pathway. Notably, TPORAs reverse the ITP-IgG inhibitory effects and rescue proplatelet formation.