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Microparticle generation during in vitro platelet activation by anti-CD9 murine monoclonal antibodies
1First Department of Internal Medicine, Kansai Medical University, Osaka, Japan.
Abstract:
We used flow cytometry and two anti-CD9 murine monoclonal antibodies (NNKY1-19, MALL13) to investigate the glycoprotein composition and the potential functions of microparticles (MP) released by platelets exposed to these antibodies in vitro. NNKY1-19 produced aggregation with characteristics similar to those noted in previous reports. The action of MALL13 on platelets in platelet-rich plasma (PRP), however, differs from that of other anti-CD9 antibodies. The normal fluctuation in the MALL13-induced change in optical density disappeared when complement was present. MALL13-induced effect for platelet in PRP was not inhibited by preincubation with monoclonal anti-GPIIb/IIIa antibody, but was inhibited in washed platelets (WP). Furthermore, following MALL13 stimulation in PRP platelets, the amount of buffer LDH markedly increased and electron microscopy findings showed vacuoles appearing inside the platelets. These results suggest that MALL13 has at least two effects on platelets that differ for PRP platelets and WP. The number of MP released was increased by the addition of anti-CD9 antibodies. MP surfaces were found to be rich in CD9 protein. MALL13 stimulation lead to a significant increase in the binding of C1q and C3 to platelets and caused the production of MP to occur more rapidly than it did the exposure of fibrinogen binding sites in the presence of complement. The analysis of the relationship of MP to anti-CD9 monoclonal antibody may be useful in the investigation of the relationship between platelet function and coagulation regulation.
Insights
Two anti-CD9 antibodies were studied for their effects on platelets and microparticle release. MALL13 demonstrated unique platelet interactions and increased microparticle production, suggesting roles in platelet function and coagulation.
Area of Science:
- Immunology
- Hematology
- Cell Biology
Background:
- Platelets play a crucial role in hemostasis and thrombosis.
- Microparticles (MPs) are released from activated platelets and contribute to various physiological and pathological processes.
- CD9 is a cell surface glycoprotein found on platelets, implicated in platelet activation and aggregation.
Purpose of the Study:
- To investigate the effects of two anti-CD9 murine monoclonal antibodies (NNKY1-19 and MALL13) on platelet function and microparticle release.
- To characterize the glycoprotein composition and functions of antibody-induced platelet-derived microparticles.
- To elucidate the distinct mechanisms of action of MALL13 on platelets in different conditions.
Main Methods:
- Flow cytometry was employed to analyze platelet responses and microparticle formation.
- Two anti-CD9 monoclonal antibodies, NNKY1-19 and MALL13, were used to stimulate platelets in vitro.
- Platelet-rich plasma (PRP) and washed platelets (WP) were utilized to assess antibody effects under varying conditions.
- Electron microscopy was used to examine ultrastructural changes in platelets.
- Complement components (C1q, C3) binding and fibrinogen binding were measured.
Main Results:
- NNKY1-19 induced platelet aggregation, consistent with previous findings.
- MALL13 exhibited distinct effects on platelets in PRP compared to WP, including complement-dependent optical density changes and inhibition by anti-GPIIb/IIIa in WP but not PRP.
- MALL13 stimulation led to increased lactate dehydrogenase (LDH) release and vacuole formation within platelets in PRP.
- Both antibodies increased the number of released MPs, which were rich in CD9 protein.
- MALL13 significantly enhanced C1q and C3 binding to platelets and accelerated MP production in the presence of complement.
Conclusions:
- MALL13 exerts distinct, dual effects on platelets depending on the presence of plasma components.
- Anti-CD9 antibodies, particularly MALL13, promote the release of CD9-rich microparticles.
- MALL13's interaction with platelets, complement, and subsequent MP generation may link platelet function to coagulation regulation.