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Effect of thrombin on maturing human megakaryocytes
E M Cramer1, J M Massé, J P Caen
1INSERM U. 348 and Institut des Vaisseaux et du Sang, Hôpital Lariboisière, Paris, France.
Abstract:
Thrombin causes platelet activation and secretion. In some nucleated cells, it is mitogenic. In this study, we have investigated how human megakaryocytes (MKs) respond to this agonist and whether the response depends on the maturation stage. MKs were cultured from bone marrow precursors in liquid culture in the presence of normal plasma. To determine whether thrombin can activate MKs, 14-day MK cultures were incubated with thrombin for 5 minutes, and cells were studied by electron microscopy, either by standard techniques or after embedding in glycol-methacrylate for immunoelectron microscopy. Ultrastructural examination of thrombin-treated MKs revealed dramatic morphological changes reminiscent of those found in platelets, including shape change and organelle centralization that involved immature as well as mature cells. MKs were also able to secrete alpha-granule proteins in the dilated cisternae of the demarcation membrane system, as shown by immunogold staining for thrombospondin and glycoprotein Ib. These changes were rapid (less than 5 minutes) but despite them, MKs remained viable for more than 24 hours. To determine whether thrombin has a mitogenic activity, it was added to the culture of MKs from day 3 to day 10 of culture at concentrations varying from 0.1 to 10 U/ml. Cells were subsequently studied by a double staining technique using flow cytometry to determine MK number and ploidy. No changes were observed in these two parameters, showing that thrombin is not mitogenic for MKs at the concentrations used. In conclusion, this study confirms for human MKs previous observations made about guinea pig MKs (Fedorko et al, Lab Invest 1977, 36:32). In addition, it demonstrates that immature MKs are able to respond to thrombin and that more mature cells can secrete alpha-granule proteins into the demarcation membrane system, which is in continuity with the extracellular space. This phenomenon may have implications for pathological states such as myelofibrosis formation and for megakaryopoiesis autocrine regulation.
Insights
Thrombin rapidly activates human megakaryocytes (MKs), inducing shape changes and alpha-granule secretion in both immature and mature cells. However, thrombin does not stimulate MK proliferation, indicating it is not mitogenic for these cells.
Area of Science:
- Hematology
- Cell Biology
- Biochemistry
Background:
- Thrombin is known to activate platelets and can be mitogenic in some nucleated cells.
- Human megakaryocytes (MKs) are precursors to platelets, and their response to agonists is crucial for hemostasis.
- Understanding MK responses to thrombin is important for insights into megakaryopoiesis and related pathologies.
Purpose of the Study:
- To investigate the response of human megakaryocytes (MKs) to thrombin.
- To determine if thrombin's effect on MKs is dependent on their maturation stage.
- To assess the mitogenic potential of thrombin on human MKs.
Main Methods:
- Human MKs were cultured from bone marrow precursors.
- Thrombin activation was studied using electron microscopy and immunoelectron microscopy.
- Mitogenic activity was assessed by flow cytometry to measure MK number and ploidy after thrombin exposure.
Main Results:
- Thrombin induced rapid morphological changes in MKs, including shape change and organelle centralization, in both immature and mature cells.
- MKs secreted alpha-granule proteins (thrombospondin, glycoprotein Ib) into the demarcation membrane system upon thrombin stimulation.
- Thrombin did not alter MK number or ploidy, indicating no mitogenic effect at the tested concentrations.
- MKs remained viable for over 24 hours after thrombin treatment.
Conclusions:
- Human megakaryocytes (MKs) respond rapidly to thrombin with activation and secretion, regardless of maturation stage.
- Mature MKs secrete alpha-granule proteins into the demarcation membrane system, which connects to the extracellular space.
- Thrombin is not mitogenic for human MKs, but its secretory effects may influence pathological conditions like myelofibrosis and autocrine regulation of megakaryopoiesis.