Thrombocytopenia and erythrocytosis in mice with a mutation in the gene encoding the hemoglobin β minor chain

Abstract

Diverse mutations in the genes encoding hemoglobin (Hb) have been characterized in human disease. We describe here a mutation in the mouse Hbb-b2 gene, denoted Plt12, that precisely mimics the human hemoglobin Hotel Dieu variant. The mutation results in increased affinity of Hb for oxygen and Plt12 mutant mice exhibited reduced partial pressure of O(2) in the blood, accompanied by erythrocytosis characterized by elevated erythropoietin levels and splenomegaly with excess erythropoiesis. Most homozygous Hbb-b2(Plt12/Plt12) mice succumbed to early lethality associated with emphysema, cardiac abnormalities, and liver degeneration. Survivors displayed a marked thrombocytopenia without significant deficiencies in the numbers of megakaryocytes or megakaryocyte progenitor cells. The lifespan of platelets in the circulation of Hbb-b2(Plt12/Plt12) mice was normal, and splenectomy did not correct the thrombocytopenia, suggesting that increased sequestration was unlikely to be a major contributor. These data, together with the observation that megakaryocytes in Hbb-b2(Plt12/Plt12) mice appeared smaller and deficient in cytoplasm, support a model in which hypoxia causes thrombocytopenia as a consequence of an inability of megakaryocytes, once formed, to properly mature and produce sufficient platelets. The Plt12 mouse is a model of high O(2)-affinity hemoglobinopathy and provides insights into hematopoiesis under conditions of chronic hypoxia.

Fig. 1.

Plt12, an ENU-induced mutation that causes semidominant erythrocytosis and thrombocytopenia. (A) The low platelet count observed in the Plt12 founder G₁ mouse was transferred to second generation (G2) offspring consistent with a heritable, dominantly acting mutation. (B) Red blood cell and platelet numbers in a cohort of offspring from affected Plt12 parents revealing a proportion of animals with both exacerbated thrombocytopenia and marked erythrocytosis. (C) Erythema, particularly evident in the ears, in Plt12 mice. The control mouse (Left) had a platelet count of 1060 × 10⁹/L and a red cell count of 11.49 × 10¹²/L. The affected mouse (Right) had a platelet count of 353 × 10⁹/L and a red cell count of 16.44 × 10¹²/L.

Fig. 2.

Disrupted tissue morphology in Hbb-b2^Plt12/Plt12 mice. Micrographs of hematoxylin/eosin-stained histological sections of representative tissue sections from Hbb-b2^Plt12/Plt12 (Right) and wild-type Hbb-b2^+/+ mice (Left). Fatty degeneration in liver (A), lung emphysema (B), and expanded red pulp in spleen (C) can be observed in Hbb-b2^Plt12/Plt12 mice compared with their healthy littermate controls.

Fig. 3.

Hbb-b2^Plt12 leads to production of a high O₂-affinity variant of Hb. (A) Comparison of the partial pressure of oxygen (pO₂), partial pressure of carbon dioxide (pCO₂), oxygen saturation (SaO₂), and p50 (partial pressure of oxygen at which hemoglobin is 50% saturated) in arterial blood samples from Hbb-b2^Plt12/Plt12 mice and Hbb-b2^+/+ littermates. (B) Alkaline cellulose acetate and acidic citrate agar electrophoretic analysis of blood indicating the expected excess of αβ^maj over αβ^min in wild-type mice, with dominance of the αβ^min variant in Hbb-b2^Plt12/Plt12 mice. Mean values are indicated with error bars denoting 1 SD. *P < 0.05 for comparison of data from Hbb-b2^Plt12/Plt12 mice with that of Hbb-b2^+/+ controls.

Fig. 4.

Erythrocytosis in Hbb-b2^Plt12/Plt12 mice. (A) Similar kinetics of loss of labeled red blood cells from the circulation (Left) but increased emergence of unlabeled cells following pulse labeling (Right) in Hbb-b2^Plt12/Plt12 mice compared with wild type, indicative of normal cellular half life but increased rate of erythrocyte production. (B) Elevated serum erythropoietin (EPO) concentration in Hbb-b2^Plt12/Plt12 mice of 1.5–12 mo of age. Mean values are indicated with error bars denoting 1 SD. *P < 0.05 for comparison of data from Hbb-b2^Plt12/Plt12 mice with that of Hbb-b2^+/+ controls.

Fig. 5.

Thrombocytopenia in Hbb-b2^Plt12/Plt12 mice. (A) Histological sections of spleens showing von Willebrand factor-stained megakaryocytes at similar numbers but of smaller size and with scant cytoplasm in Hbb-b2^Plt12/Plt12 mice compared with wild type. (B) Comparable half-life of platelets in the circulation of Hbb-b2^Plt12/Plt12 and wild-type mice. (C) Numbers of circulating platelets before surgery and 28 and 85 d after splenectomy showing persistent thrombocytopenia in Hbb-b2^Plt12/Plt12 mice. Mean values are indicated with error bars denoting 1 SD.

Fig. 6.

Increased cell cycling of hematopoietic stem cells in Hbb-b2^Plt12/Plt12 mice. (A) Equivalent numbers of LSK, long-term (LT) hematopoietic stem cells (HSC), short-term (ST) HSC, and multipotent progenitor (MPP) cells (Left) and granulocyte-macrophage progenitor (GMP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), and megakaryocyte progenitor (MkP) cells (Right) in the bone marrow of Hbb-b2^Plt12/Plt12 mice. (B) Increased numbers of cycling (G₁) LSK and progenitor cells in Hbb-b2^Plt12/Plt12 mice. Cells were enumerated by flow cytometry based on expression of distinct cell surface markers as outline in SI Materials and Methods. Mean values are indicated with error bars denoting 1 SD. *P < 0.05 for comparison of data from Hbb-b2^Plt12/Plt12 mice with that of Hbb-b2^+/+ controls.