Combined deficiency of coagulation factors V and VIII (F5F8D) is a human autosomal recessive bleeding disorder caused by mutations in LMAN1 or MCFD2, which encode the components of the only known cargo-receptor complex in the mammalian secretory pathway. The LMAN1/MCFD2 complex facilitates secretion of coagulation factors V (FV) and VIII (FVIII) to the plasma. F5F8D patients exhibit FV and FVIII levels that are ~10-15% of normal. The relationship between Lman1 expression levels and the corresponding secretion of LMAN1-dependent cargos has not been characterized, and additional functions of LMAN1 remain to be determined. We previously described Lman1 mutant mice carrying a gene-trap insertion in intron 10 (Lman1gt1). Lman1gt1/gt1 mice have ~50% of normal FV and FVIII activity levels, and are therefore less severely affected than human F5F8D patients. This difference could be explained by low levels of residual LMAN1 expression, or by differences in FV and FVIII secretion between mice and humans. Lman1gt1/gt1 mice also exhibit a partially penetrant, perinatal lethality that is restricted to certain mouse genetic strains. We generated a second, independent LMAN1-deficient mouse with no Lman1 gene expression (Lman1-/-), as well as hypomorphic LMAN1-deficient mice (Lman1cgt/cgt) that retain ~7% of wildtype Lman1 expression levels on a C57BL/6J genetic background. Plasma FV and FVIII levels in Lman1-/- and Lman1cgt/cgtmice were measured by functional coagulation assays and compared to wildtype (WT) and Lman1gt1/gt1 mice. A dose-response relationship was observed, with Lman1-/- mice exhibiting FV and FVIII levels that are ~50% of normal (compared to ~10-15% in human patients), while hypomorphic Lman1cgt/cgtmice demonstrate an intermediate reduction in FV and FVIII (~70% of WT levels). A similar result was demonstrated for alpha-1 antitrypsin, another known LMAN1-dependent cargo. Taken together with the presence of two null LMAN1 alleles in nearly all reported LMAN1 F5F8D patients, these results suggest that hypomorphic LMAN1 mutations in humans may also result in milder reductions in plasma FV and FVIII that may have been missed clinically, though still contributing to FV and FVIII level variation and to bleeding susceptibility.

The partially penetrant, strain-specific, perinatal lethality previously reported in Lman1gt1/gt1 mice was also confirmed in mice homozygous for the independently generated Lman1- allele on a pure C57BL/6J genetic background (p < 0.007), thereby ruling out a passenger gene effect as the cause of the originally reported Lman1gt1/gt1 lethality. In contrast, Lman1cgt/cgt mice on the same background are viable and observed in the expected Mendelian ratios (p > 0.19). Lman1-/- lethality was not observed on a mixed C57BL/6J and 129S1/SvImJ background (p > 0.79). F5F8D is not associated with fetal loss in humans, nor would fetal loss be expected from moderate deficiency of FV and FVIII. These results thus suggest a critical role for additional, as yet unknown, LMAN1 cargo(s).

Finally, adult Lman1-/- mice exhibit a mild thrombocytopenia compared to WT controls (9.3 x 105 vs. 12.3 x 105 cells/uL, p < 0.004). In contrast, Lman1+/-, Lman1cgt/cgt, and Mcfd2-/- mice all exhibit normal platelet counts indistinguishable from WT controls. No Lman1-/- platelet morphologic abnormalities are evident by standard or transmission electron microscopy, with normal appearance of megakaryocyte number and morphology on bone marrow examination. Although thrombocytopenia has not been noted previously in human F5F8D patients, these results suggest a novel role for an LMAN1-dependent secretory cargo in thrombopoiesis.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.