Inhibition of tyrosine kinase activity prevents the adhesive and cohesive properties of platelets and the expression of procoagulant activity in response to collagen.
BACKGROUND
Platelet activation leads to signal transduction mechanisms, in which phosphotyrosine proteins play a relevant role.


METHODS
Platelet suspensions were independently activated by collagen and thrombin in the absence and in the presence of two tyrosine kinase inhibitors, tyrphostin 47 and genistein. Samples were processed to visualize morphological changes by electron microscopy, to evaluate changes in cytoskeletal assembly, to analyze modifications in the expression of activation dependent antigens, and the procoagulant activity at the surface level by flow cytometry. Additional experiments applying flow conditions were performed to assess the effect of inhibiting tyrosine phosphorylation on primary platelet adhesion and fibrin formation.


RESULTS
Inhibition of tyrosine phosphorylation blocked shape change and cytoskeletal assembly induced by collagen, and inhibited, though partially, those effects due to thrombin. Both activating agents induced the expression of the intraplatelet antigens CD62P and CD63 at the surface, although only collagen promoted expression of anionic phospholipids. Both tyrphostin 47 and genistein prevented those effects. The extent of platelet adhesion on both collagen-coated and subendothelial surfaces was significantly diminished by the presence of the tyrosine kinase inhibitors assayed. Fibrin formation was also significantly reduced.


CONCLUSIONS
Platelet shape change and secretion during platelet activation depends on tyrosine phosphorylation. In addition, primary adhesion of platelets induces signaling through tyrosine kinases to achieve full spreading, and results in the exposure of a procoagulant surface on platelets.
Diaz-Ricart
Maribel
M
Servicio de Hemoterapia-Hemostasia, Hospital Clínic, IDIBAPS, Universidad de Barcelona, Spain. mdiaz@clinic.ub.es
Palomo
Marta
M
Fuste
Berta
B
Lopez-Vilchez
Irene
I
Carbo
Carla
C
Perez-Pujol
Silvia
S
White
James G
JG
Escolar
Gines
G
D001777:Blood Coagulation / Q000187:drug effects*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects*; D003094:Collagen / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005337:Fibrin / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D005434:Flow Cytometry; D019833:Genistein / Q000494:pharmacology*; D006801:Humans; D008854:Microscopy, Electron; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects; D010973:Platelet Adhesiveness / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D012016:Reference Values; D013499:Surface Properties; D013917:Thrombin / Q000494:pharmacology; D014443:Tyrosine / Q000037:antagonists & inhibitors / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology*
D047428:Protein Kinase Inhibitors; D020032:Tyrphostins; C060152:tyrphostin 47; D014443:Tyrosine; D005337:Fibrin; D003094:Collagen; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D013917:Thrombin