Electrotransjection of pp60v-src monoclonal antibody inhibits activation of phospholipase C in platelets. A new mechanism for platelet-activating factor responses.
Antibodies to pp60v-arc and phosphotyrosine were introduced into rabbit platelets using an electropermeabilization technique. The presence of these antibodies inside platelets was detected by flow cytometry. Platelet-activating factor (PAF)-stimulated phospholipase C activity (inositol phosphate production) and aggregation were dramatically inhibited in platelets transjected with either of these antibodies. Incubation of these antibodies with intact cells (i.e. nonpermeabilized) or electrotransjection of several nonspecific antibodies/agents (e.g. goat anti-mouse IgG, mouse serum, human platelet glycoprotein Ib monoclonal antibody, and fetal calf serum) into platelets had no effect on the PAF responses. trpE (another isotype-matched control antibody of pp60v-src) and pp56lck polyclonal antibody (another src-related kinase not present in platelets) also had no effect on PAF-induced aggregation and inositol phosphate production in permeabilized platelets. This indicates that the effect of internalized pp60v-src antibody is direct and specific in platelets. Stimulation of platelets by PAF increased the association and phosphorylation of both pp60c-src (60 kDa) and phospholipase C gamma 1 (145 kDa). This study provides the first evidence in platelets for a direct and specific involvement of pp60c-src in PAF-mediated phospholipase C activation and aggregation response.
Dhar
A
A
Department of Pharmacology, University of Missouri School of Medicine, Columbia 65212.
Shukla
S D
SD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D001792:Blood Platelets / Q000201:enzymology*; D018274:Electroporation; D004789:Enzyme Activation; D066298:In Vitro Techniques; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D015688:Oncogene Protein pp60(v-src) / Q000276:immunology*; D010972:Platelet Activating Factor / Q000502:physiology*; D010974:Platelet Aggregation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology*; D011817:Rabbits; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
D000911:Antibodies, Monoclonal; D010972:Platelet Activating Factor; D011518:Proto-Oncogene Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D015688:Oncogene Protein pp60(v-src); D016392:Proto-Oncogene Proteins pp60(c-src); D010738:Type C Phospholipases