Abstract
Thrombocytopenia Cargeeg is a rare autosomal dominant disorder identified in a New Zealand family with low platelet counts. It is caused by a heterozygous mutation (G41S) in the CYCS gene, which encodes for cytochrome c (cyt c). The G41S mutation enhances the ability of human cyt c to trigger caspase activation in vitro without affecting its role in the mitochondrial electron transport chain.
The cellular basis of Thrombocytopenia Cargeeg has yet to be fully characterised. In response to thrombopoietin, haematopoietic stem cells (HSCs) in the bone marrow undergo a complex programme of differentiation into megakaryocytes (MKs). Mature MKs then release platelets into the bloodstream via long, thin intravascular protrusions called proplatelets. Our previous study has suggested that the early platelet production observed using Thrombocytopenia Cargeeg CD34+ cell-derived MKs could be the result of altered megakaryopoiesis. Here, we showed that the early platelet production observed in previous work was caused by Thrombocytopenia Cargeeg HSCs maturing to MKs at an accelerated rate. Mature Thrombocytopenia Cargeeg MKs also extended and formed proplatelets to the same extent as control MKs. Transmission electron microscopy (TEM) analysis of a Thrombocytopenia Cargeeg bone marrow previously revealed the abnormal presence platelet-like structures in the marrow space, suggesting premature platelet release. However, this observation is difficult to reconcile with evidence that blood shear forces are needed to separate platelets from the proplatelet projections that extend from the MK cell body. To address this question, we reconstructed these structures in 3D from serial TEM sections. The 3D reconstruction showed that these structures had the dimension of circulating platelets. Thrombocytopenia Cargeeg circulating platelets have the marginal microtubule coil consistent with effective platelet release via the proplatelet mechanism. The platelet-like structures, in contrast, lack the marginal microtubule coil, suggesting that they are released into the bone marrow space via a mechanism that is independent of proplatelet formation.
The relationship of enhanced caspase-inducing activity of human G41S cyt c to low platelet count phenotype is still unclear. Furthermore, the role of the intrinsic apoptosis pathway in platelet production is still a matter of debate. To further understand the molecular basis of Thrombocytopenia Cargeeg, we attempted to establish the megakaryoblastic SET-2 cell line expressing human G41S cyt c. While validating the suitability of the clonal cells as a human cell-based model of Thrombocytopenia Cargeeg, we found that endogenous cyt c expression was upregulated in high cell density cultures and during phorbol-12-myristate-13-acetate (PMA)-induced MK differentiation. Same observations were also found in PMA-induced macrophage differentiation. The observation that cyt c expression varied with different cell density rendered SET-2 cells unsuitable for studying the role of cyt c-mediated caspase activation in megakaryopoiesis and platelet production.
The functional consequences of G41S mutation of human cyt c on apoptosis induction were also investigated in this thesis. Treatment of Thrombocytopenia Cargeeg and control peripheral blood mononuclear cells with intrinsic apoptosis inducers ABT-199 and ABT-737 showed no difference in caspase-3 activity, indicating that the presence of human G41S cyt c in heterozygous Thrombocytopenia Cargeeg subjects does not alter the induction of apoptosis ex vivo. One hypothesis to explain the basis of enhanced caspase-inducing activity of human G41S cyt c is increased binding affinity to Apaf-1. We have made many attempts to express the WD40 domain of Apaf-1 in E. coli for use in interaction study with human G41S cyt c. However, none of the attempts yielded soluble and correctly folded proteins.
In summary, this thesis has provided further insights into the cellular basis of Thrombocytopenia Cargeeg. This thesis is the first to describe platelet-like structures in the human bone marrow in 3D detail. Upregulation of cyt c expression in high cell density cultures and during PMA-induced differentiation are also novel discoveries. This thesis also provides the first direct evidence that the enhanced caspase-inducing function of human G41S cyt c, at least in its heterozygous form, does not alter the induction of apoptosis ex vivo.