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Thrombin-mast cell interactions. Binding and cell activation
Abstract:
Activation of mouse bone marrow-derived mast cells (BMMC) by thrombin (0.05-0.5 U/million cells) resulted in a concentration-dependent release of histamine, which levelled off by 0.1 U thrombin. Rat peritoneal mast cells (RMC) were not stimulated by thrombin, though in control experiments, both types of mast cells degranulated upon exposure to IgE-antigen. Pretreatment of thrombin with 0.2 mM diisopropylfluorophosphate (DFP), a specific serine protease inhibitor, resulted in 90% loss of thrombin degranulation and coagulant activity. Fluorescently labelled thrombin (FITC-thrombin) specifically bound to the BMMC surface, as measured by fluorescence cytometry. Pre-exposure of the BMMC to 20-fold excess of unlabelled thrombin prior to incubation with FITC-thrombin, prevented the binding of the labelled-thrombin to the cells. Incubation of thrombin with DFP or with antithrombin III (AT-III) resulted in losses of procoagulant and of BMMC degranulatory activities. DFP treatment of FITC-thrombin had no effect on the binding of the labelled enzyme to the cell surface. However, preincubation of the FITC-thrombin with AT-III prevented thrombin binding to the BMMC. Thus, the binding and the catalytic regions of the thrombin molecule are operationally distinct from one another. Kinetic analysis of the BMMC exposed to 0.5 U thrombin revealed a transient rise in intracellular cAMP, which peaked by 15 sec and was not measurable after 1 min. This suggests that differential activation of mast cells can occur at sites of tissue injury.
Insights
Thrombin activates mouse mast cells to release histamine, but not rat mast cells. Thrombin
Area of Science:
- Immunology
- Biochemistry
Background:
- Mast cells play a crucial role in allergic reactions and inflammation.
- Thrombin is a key enzyme in the coagulation cascade with potential roles beyond hemostasis.
Purpose of the Study:
- To investigate the direct effect of thrombin on mast cell activation and histamine release.
- To determine the binding characteristics and functional domains of thrombin involved in mast cell interaction.
Main Methods:
- Activation of mouse bone marrow-derived mast cells (BMMC) and rat peritoneal mast cells (RMC) with varying concentrations of thrombin.
- Measurement of histamine release.
- Use of diisopropylfluorophosphate (DFP) and antithrombin III (AT-III) to inhibit thrombin activity.
- Fluorescently labeled thrombin (FITC-thrombin) for binding studies using fluorescence cytometry.
- Intracellular cyclic AMP (cAMP) measurements.
Main Results:
- Thrombin induced a concentration-dependent histamine release from BMMC, but not RMC.
- DFP inhibited both thrombin's degranulatory and coagulant activities.
- FITC-thrombin specifically bound to BMMC, and this binding was blocked by excess unlabeled thrombin.
- AT-III inhibited thrombin's degranulatory activity and binding to BMMC, while DFP did not affect binding.
- Thrombin induced a transient increase in intracellular cAMP in BMMC.
Conclusions:
- Thrombin directly activates mouse mast cells, leading to histamine release, suggesting a role in inflammatory responses at injury sites.
- The binding and catalytic regions of thrombin are distinct, with AT-III interfering with binding and DFP affecting catalytic activity.
- Differential mast cell activation by thrombin may occur in specific physiological contexts like tissue injury.