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Sustained thrombocytopenia in mice: serial studies of megakaryocytes and platelets
P E Stenberg1, J Levin, L Corash
1Department of Laboratory Medicine, University of California School of Medicine, San Francisco.
Abstract:
We studied thrombopoiesis in mice after the experimental induction of sustained, immune thrombocytopenia with platelet antiserum (PAS). Utilizing light and electron microscopy and a digital image analyzer to determine platelet sectional areas, we examined platelets and megakaryocytes (MK) after 120 h of sustained, severe thrombocytopenia (120CT) and during recovery from thrombocytopenia at 48 h (48R), 72 h (72R), and 120 h (120R) after cessation of administration of PAS. Mean platelet volume (MPV), determined by electrical impedance, also was measured at each time point. Platelets at 120CT (platelet count less than 50,000/microliter), 48R (platelet count 100-200,000/microliter), and 72R (platelet count approximately 1 x 10(6)/microliter) were significantly larger in sectional area than control platelets and contained increased profiles of endoplasmic reticulum and Golgi cisternae, a lower concentration of surface-connected canalicular system, and occasional membrane complexes. The largest median platelet sectional area was detected at 48R and was the largest median value observed in response to either chronic or acute thrombocytopenia. At 120R, most platelets were normal in size and cytoplasmic appearance, although some large cells remained present in the circulation. MPV paralleled the morphometric changes in platelet sectional area. MK were increased in number at 120CT, 48R, 72R, and 120R. In addition, at least half of the MK examined at 48R contained small areas of cytoplasm, devoid of organelles, that were interspersed between larger areas of organelle-filled, undemarcated cytoplasm. The modal bone marrow megakaryocyte ploidy class, determined using two-color fluorescence-activated flow cytometry, shifted from 16N to 32N in response to sustained thrombocytopenia. In contrast, during recovery and development of rebound thrombocytosis, the relative frequency of 8N megakaryocytes was significantly increased. Because there was no consistent correlation between megakaryocyte cytoplasmic characteristics and platelet morphology, these data support the hypothesis that platelet formation is not determined by compartmentalization of MK cytoplasm into platelet areas as MK mature in the bone marrow, but involves a rearrangement of MK cytoplasm immediately prior to platelet release.
Insights
This study reveals that during thrombocytopenia recovery, platelets enlarge and megakaryocytes (MKs) change ploidy. Platelet formation involves cytoplasmic rearrangement before release, not pre-compartmentalization in MKs.
Area of Science:
- Hematology
- Cell Biology
- Thrombopoiesis
Background:
- Immune thrombocytopenia is a condition characterized by low platelet counts.
- Understanding thrombopoiesis, the process of platelet formation, is crucial for managing bleeding disorders.
Purpose of the Study:
- To investigate the morphological and ploidy changes in platelets and megakaryocytes (MKs) during recovery from experimentally induced immune thrombocytopenia in mice.
- To explore the relationship between MK cytoplasmic characteristics and platelet morphology during thrombopoiesis.
Main Methods:
- Induction of sustained immune thrombocytopenia in mice using platelet antiserum (PAS).
- Analysis of platelet and MK morphology using light and electron microscopy and digital image analysis.
- Measurement of mean platelet volume (MPV) by electrical impedance.
- Determination of MK ploidy distribution using two-color fluorescence-activated flow cytometry.
Main Results:
- Platelets were significantly larger in sectional area and had altered cytoplasmic content during thrombocytopenia and early recovery.
- Mean platelet volume (MPV) paralleled the observed changes in platelet size.
- Megakaryocyte (MK) number increased, and their ploidy distribution shifted from 16N to 32N during thrombocytopenia, with a subsequent increase in 8N MKs during recovery.
- No consistent correlation was found between MK cytoplasmic features and platelet morphology.
Conclusions:
- Platelet size and morphology change significantly in response to thrombocytopenia and during recovery.
- Megakaryocyte (MK) ploidy dynamics are altered by sustained thrombocytopenia and rebound thrombocytosis.
- Platelet formation likely involves cytoplasmic rearrangement in MKs immediately preceding platelet release, rather than pre-determined compartmentalization.