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Platelet activation by fMLP-stimulated polymorphonuclear leukocytes: the activity of cathepsin G is not prevented by
V Evangelista1, G Rajtar, G de Gaetano
1Giulio Bizzozero Laboratory of Platelet and Leukocyte Pharmacology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy.
Abstract:
Human polymorphonuclear leukocytes (PMN) activated by fMLP (in the presence of CaCl2, fibrinogen, and cytochalasin B) were able to induce aggregation, cytoplasmic Ca2+ increase, and thromboxane A2 production in coincubated autologousplatelets. Cell-free supernatants prepared from n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN were able also to induce platelet activation. Antibodies against cathepsin G and different serin protease inhibitors completely suppressed the activity of PMN-derived supernatants, indicating that cathepsin G is the major platelet activator released by PMN in our system. However, antiproteinases only partially affected platelet activation induced by PMN in mixed cell suspensions. Superoxide dismutase and catalase added to the cell suspension did not affect platelet activation nor potentiated serin protease inhibitors, making a role for short-lived oxygen radicals in our experimental system unlikely. Electron microscopic observation of stirred mixed cell suspensions preincubated for 2 minutes at 37 degrees C before stimulation showed a close PMN-platelets contact without any morphologic or biochemical event suggesting platelet activation. Preincubation of the cells without stirring to minimize PMN-platelet interaction before stimulation did not modify subsequent aggregation and platelet cytoplasmic Ca2+ increase in control samples. However, in this condition trypsin inhibitor from soybean completely prevented PMN-induced platelet activation. In samples preincubated without stirring in the presence of the antiproteinase, activated PMN stuck together but platelets preserved their discoid shape and did not appear significantly activated. We propose that membrane-to-membrane contact could create a microenvironment in which cathepsin G, discharged from stimulated PMN on adherent platelets, is protected from antiproteinases.
Insights
Human neutrophils (PMN) activate platelets via cathepsin G. Direct cell contact creates a protected environment for cathepsin G, enhancing platelet activation and aggregation.
Area of Science:
- Hematology
- Immunology
- Cell Biology
Background:
- Human polymorphonuclear leukocytes (PMN) play a role in inflammatory responses.
- Platelet activation is a critical step in hemostasis and thrombosis.
Purpose of the Study:
- To investigate the mechanism by which activated human PMN induce platelet activation.
- To identify the specific molecules released by PMN responsible for platelet activation.
Main Methods:
- Coincubation of human PMN and autologous platelets.
- Stimulation of PMN with fMLP.
- Analysis of platelet aggregation, cytoplasmic Ca2+ increase, and thromboxane A2 production.
- Use of cell-free supernatants from stimulated PMN.
- Inhibition studies using antibodies against cathepsin G, serine protease inhibitors, superoxide dismutase, and catalase.
- Electron microscopy to observe PMN-platelet interactions.
- Preincubation experiments with and without stirring.
Main Results:
- Activated PMN induced platelet aggregation, cytoplasmic Ca2+ increase, and thromboxane A2 production.
- Cell-free supernatants from stimulated PMN also activated platelets.
- Cathepsin G was identified as the major PMN-derived platelet activator.
- Antiproteinases partially inhibited PMN-induced platelet activation in mixed cell suspensions.
- Oxygen radicals were unlikely to be involved.
- Direct PMN-platelet contact, particularly without stirring, was crucial for maximal PMN-induced platelet activation, suggesting a protected microenvironment for cathepsin G.
Conclusions:
- Cathepsin G released from activated PMN is a key mediator of platelet activation.
- Membrane-to-membrane contact between PMN and platelets creates a microenvironment that protects cathepsin G from inhibitors, leading to enhanced platelet activation.