TRAP induces more intense tyrosine phosphorylation than thrombin with differential ultrastructural features.
We have analyzed modifications on platelet ultrastructural morphology, cytoskeletal assembly, and tyrosine phosphorylation developing in platelets activated by both thrombin and the thrombin receptor-activating peptide (TRAP). Washed platelets exposed to various concentrations of thrombin or TRAP, for different periods, were: fixed and examined by electron microscopy, or lysed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under similar activating conditions, thrombin and TRAP induced different sequences of activation causing distinctive morphological and biochemical changes. Platelets exposed to thrombin showed centralized organelles encircled by constricted microtubule coils and granules secreting their contents through narrow channels of the open canalicular system. In contrast, activation by TRAP induced swelling of the open canalicular system with organelles remaining randomly dispersed and microtubules peripherally distributed. Compared to thrombin activation, TRAP induced higher rates of actin polymerization; increased association of actin-binding protein, myosin, and alpha-actinin; and higher association of tyrosine-phosphorylated proteins with the insoluble cytoskeletal fraction. Secretion of intragranule substances, measured as expression of P-selectin and lysosomal integral membrane protein at the surface level, were similar for both agonists at equivalent concentrations. Our biochemical observations indicate that TRAP causes more intense changes in signaling through tyrosine phosphorylation of proteins associated with the cytoskeletal fraction than thrombin. However, as derived from ultrastructural observations, TRAP seems to be less efficient in triggering cytoskeletal assembly and internal contraction in an organized manner in contrast with the natural protease.
Fusté
Berta
B
Servicio de Hemoterapia y Hemostasia, Hospital Clínic, Institut d'Investigacions Biomèdiques Agustí Pi Sunyer, Barcelona, Spain. agalan@clinic.ub.es
Díaz-Ricart
Maribel
M
Jensen
Morten Krogh
MK
Ordinas
Antonio
A
Escolar
Ginés
G
White
James G
JG
D000185:Actinin / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000648:ultrastructure*; D003599:Cytoskeleton / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D010974:Platelet Aggregation; D011506:Proteins / Q000494:pharmacology*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism*
D008840:Microfilament Proteins; D011506:Proteins; D018179:Receptors, Thrombin; D000185:Actinin; D014443:Tyrosine; D013917:Thrombin; D009218:Myosins