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Human leukocyte elastase and cathepsin G inactivate factor VII by limited proteolysis
T Anderssen1, H Halvorsen, S P Bajaj
1Institute of Medical Biology, University of Tromsø, Norway.
Abstract:
The effect of supernatant from phorbol myristate acetate (PMA) stimulated human polymorphonuclear granulocytes (PMN) on human factor VII was studied in vitro. The supernatant caused a rapid loss in factor VII coagulant activity by the action of human leukocyte elastase (HLE) and cathepsin G in the supernatant, as demonstrated by the use of specific inhibitors of the two serine proteases, respectively. Preincubation of the supernatant with the elastase inhibitor and the cathepsin G inhibitor preserved 80% and 25% of the clotting activity, respectively. Calcium protected factor VII completely from the supernatant mediated inactivation. Cathepsin G and HLE purified from PMN each destroyed the coagulant activity of factor VII when added to a non-plasma system. There were, however, no effect on factor VII activity when cathepsin G was added to plasma. Polyacrylamide gel electrophoresis in the presence of SDS indicated that HLE and cathepsin G cleaved the zymogen in the same manner, producing (a) peptide(s) of low molecular mass and a single large product of 48 kDa. Preincubation of factor VII with calcium ions inhibited the proteolytic action of HLE and cathepsin G. It is suggested that HLE and cathepsin G from activated granulocytes may be partly responsible for the loss in factor VII activity that is observed during sepsis.
Insights
Human leukocyte elastase (HLE) and cathepsin G from activated granulocytes rapidly inactivate factor VII. Calcium ions protect factor VII from this degradation, suggesting a role in sepsis-related factor VII loss.
Area of Science:
- Biochemistry
- Hematology
- Immunology
Background:
- Human factor VII is crucial for blood coagulation.
- Activated polymorphonuclear granulocytes (PMN) release proteases that can affect coagulation factors.
- The impact of PMN-derived proteases on factor VII activity is not fully understood.
Purpose of the Study:
- To investigate the effect of supernatant from phorbol myristate acetate (PMA)-stimulated human PMN on human factor VII coagulant activity.
- To identify the specific proteases responsible for factor VII inactivation.
- To explore the protective role of calcium ions against protease-mediated factor VII degradation.
Main Methods:
- In vitro study using supernatant from PMA-stimulated human PMN.
- Assay of factor VII coagulant activity.
- Use of specific inhibitors for human leukocyte elastase (HLE) and cathepsin G.
- Protease activity assays with purified HLE and cathepsin G.
- Polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) to analyze protein cleavage.
- Investigation of calcium ion effects on factor VII stability.
Main Results:
- Supernatant from stimulated PMN caused rapid loss of factor VII coagulant activity.
- HLE and cathepsin G were identified as the primary proteases responsible for factor VII inactivation.
- Specific inhibitors for HLE and cathepsin G partially preserved factor VII activity (80% and 25%, respectively).
- Calcium ions completely protected factor VII from inactivation by PMN supernatant.
- Purified HLE and cathepsin G degraded factor VII in a non-plasma system, with similar cleavage patterns observed via SDS-PAGE.
- Cathepsin G did not affect factor VII activity in plasma.
- Calcium ions inhibited the proteolytic activity of HLE and cathepsin G against factor VII.
Conclusions:
- HLE and cathepsin G released from activated human PMN can rapidly inactivate human factor VII in vitro.
- Calcium ions play a significant protective role against protease-mediated factor VII degradation.
- These findings suggest that HLE and cathepsin G may contribute to the observed reduction in factor VII activity during sepsis.