title: Synergistic effect of signaling from receptors of soluble platelet agonists and outside-in signaling in formation of a stable fibrinogen-integrin αIIbβ3-actin cytoskeleton complex.
abstract: BACKGROUND
  Thrombus formation in the injured vessel wall is a highly complex process involving various blood-born components that go through specific temporal and spatial changes as observed by intravital videomicroscopy. Platelets bind transiently to the developing thrombus and may either become stably incorporated into or disengage from the thrombus. The aim of the present study was to reveal the processes involved in the formation of a stable thrombus.
  
  
  METHODS
  Platelet-rich plasma and washed platelets were studied by the aggregometer. The aggregate stability was challenged by eptifibatide. Platelet Triton-insoluble fraction was prepared and the actin and αIIb content in the cytoskeleton was analyzed by western blot.
  
  
  RESULTS
  Maximal actin polymerization is achieved 1min after platelet activation while maximal αIIbβ3-actin cytoskeleton association requires 5 to 10min of activation and fibrinogen-mediated platelet-to-platelet bridging. Thus, actin polymerization is dependent on platelet activation and requires neither αIIbβ3 integrin occupation nor platelet aggregation. Formation of a stable aggregate requires platelet activation for more than 1min, complete increase in actin cytoskeleton fraction and partial association of αIIbβ3 with the actin cytoskeleton. However, direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation. Thus, stable αIIbβ3-fibrinogen interaction, representing stable aggregate, is achieved after more than 1min agonist activation, involving inside-out and outside-in signaling but not after direct integrin activation, involving only outside-in signaling.
  
  
  CONCLUSIONS
  Formation of a stable fibrinogen-αIIbβ3-actin cytoskeleton complex is the result of the combined effect of platelet stimulation by soluble agonists, activation of αIIbβ3, fibrinogen binding and platelet-to-platelet bridging.
authors.affiliation: Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Department of Pathophysiology, I. M. Sechenov First Moscow State Medical University, Moscow, Russia.
authors.forename: Ivan
authors.initials: I
authors.lastname: Budnik
authors.affiliation: National Hemophilia Center, Sheba Medical Center, Tel-Hashomer, Israel.
authors.forename: Boris
authors.initials: B
authors.lastname: Shenkman
authors.affiliation: Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Electronic address: eyeres@post.tau.ac.il.
authors.forename: Naphtali
authors.initials: N
authors.lastname: Savion
mesh_terms: D000199:Actins / Q000737:chemistry*; D000293:Adolescent; D000328:Adult; D000368:Aged; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D005260:Female; D005340:Fibrinogen / Q000737:chemistry*; D006801:Humans; D008297:Male; D018715:Microscopy, Video; D008875:Middle Aged; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D053657:Platelet-Rich Plasma / Q000378:metabolism; D015398:Signal Transduction; D055815:Young Adult
chemical_list: D000199:Actins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen
keywords: actin cytoskeleton; fibrinogen–integrin αIIbβ3 binding; platelet aggregation