Participation of protein kinases in complement C5b-9-induced shedding of platelet plasma membrane vesicles.
The formation of membrane microparticles through vesiculation of the platelet plasma membrane is known to provide catalytic surface for several enzyme complexes of the coagulation system, and to underlie the procoagulant responses elicited with platelet activation. This induced shedding of vesicles from the plasma membrane is most prominent when platelets are activated by the terminal complement proteins, C5b-9, by a Ca2+ ionophore, or by the combination of thrombin plus collagen. Although shown to require elevated [Ca2+], the cellular events that initiate plasma membrane evagination and fusion to form the shed vesicles remain unresolved. To gain additional insight into the cellular events that regulate membrane microparticle formation, we have examined how this process is influenced by the activity of cellular protein kinases. Cytoplasmic [Ca2+] of gel-filtered platelets was increased by membrane assembly of the terminal complement proteins C5b-9 in the presence of selective inhibitors of protein kinase or phosphatase reactions, and resulting microparticle formation was quantitated by fluorescence-gated flow cytometry. Pre-equilibration of the phosphatase inhibitor vanadate into the platelet cytosol increased microparticle formation by as much as 40%, suggesting that vesiculation of the platelet plasma membrane is influenced by the state of phosphorylation of a cellular constituent. By contrast to the stimulatory effects of vanadate, microparticle formation was partially inhibited in platelets treated with the protein kinase inhibitor sphingosine, the myosin light chain kinase inhibitor ML-7, the calmodulin-antagonist W-7, and under conditions of elevated cytosolic concentration of cyclic adenosine monophosphate. These results indicate that complement-induced platelet microparticle formation is influenced by one or more protein kinase(s) as well as by calmodulin, and suggest a role for the platelet myosin light chain kinase or another Ca(2+)-pluscalmodulin-regulated membrane component.
Wiedmer
T
T
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Sims
P J
PJ
D001792:Blood Platelets / Q000502:physiology* / Q000648:ultrastructure; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D015938:Complement Membrane Attack Complex / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D009219:Myosin-Light-Chain Kinase / Q000502:physiology; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D013110:Sphingosine / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D002147:Calmodulin; D015938:Complement Membrane Attack Complex; D014638:Vanadates; D000242:Cyclic AMP; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D013110:Sphingosine; D002118:Calcium