Abstract
LMAN1 and MCFD2 encode the components of a mammalian cargo-receptor that facilitates the ER-to-Golgi transport of coagulation factors V (FV) and VIII (FVIII) for secretion to the plasma. Mutations in LMAN1 or MCFD2 result in a rare bleeding disorder known as combined deficiency of coagulation factors V and VIII (F5F8D), characterized by FV and FVIII levels that are ~10% of normal. No other clinical phenotypes are known in human patients. Lman1 null mice have ~50% of normal FV and FVIII levels and exhibit a partially penetrant, perinatal lethality, suggesting a critical role for yet unknown LMAN1 secretory cargo(s).
To further characterize the function of the LMAN1/MCFD2 complex and identify new cargos, we generated several murine models of F5F8D, including ubiquitous null Lman1 (Lman1-/-) and Mcfd2 (Mcfd2-/-) mice maintained on a C57BL/6J genetic background. Adult Lman1-/- mice were mildly thrombocytopenic, exhibiting a 25% decrease in platelet count relative to wild-type (WT) mice (9.3 x 105 vs. 12.3 x 105 cells/uL, p < 0.004), but no other CBC abnormalities. In contrast, Lman1 heterozygous and Mcfd2-/- mice exhibited normal platelet counts. To further explore the role of LMAN1 in megakaryocyte/platelet development or survival, bone marrow (BM) histology and platelet transmission electron microscopy were performed. Lman1-/-mice had no platelet morphologic abnormalities by light or transmission electron microscopy, as well as normal number and morphology of BM megakaryocytes. Hematopoietic stem cells and megakaryocyte progenitors were indistinguishable between WT and Lman1-/- mice by flow cytometry.
In order to determine whether the thrombocytopenia phenotype results from LMAN1 deficiency specifically in the hematopoietic compartment, mice with tissue-specific knockout of Lman1 in hematopoietic and endothelial cells (Tie2-Cre) were generated. Platelet counts of mice with LMAN1 deficiency restricted to the hematopoietic compartment were indistinguishable from those in WT controls. In contrast, hepatocyte-specific (Alb-Cre) Lman1 deficiency, resulted in significant thrombocytopenia relative to WT controls (p < 0.017), with platelet counts comparable to those observed in ubiquitous Lman1 null mice. Since thrombopoietin (TPO) is a major hepatocyte-derived regulator of platelet synthesis, plasma TPO levels were measured by ELISA in ubiquitous Lman1 and Mcfd2 null mice. Plasma TPO levels in Lman1-/- mice were ~56% lower than those in WT levels (128.7 x 103 vs. 229.5 x 103 pg/mL, p < 0.0025). However, TPO levels were not reduced in Mcfd2-/- mice (p > 0.17). TPO mRNA expression in the liver of Lman1-/-mice was indistinguishable from livers of WT littermate controls.
In conclusion, global LMAN1-deficient and hepatocyte-specific LMAN1 deficient mice exhibit thrombocytopenia, a phenotype not previously reported in F5F8D patients. Lman1-/- mice, but not Mcfd2-/- mice, exhibit low plasma TPO levels, suggesting that TPO may be a novel LMAN1-dependent secretory cargo. These results raise the possibility that F5F8D patients with LMAN1 mutations may have mild thrombocytopenia, previously unappreciated as a result of the small number of F5F8D patients and the wide range of clinically normal platelet counts.
Disclosures
No relevant conflicts of interest to declare.
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