title: RGD-ligand mimetic antagonists of integrin alphaIIbbeta3 paradoxically enhance GPVI-induced human platelet activation.
abstract: BACKGROUND
  The integrin alpha(IIb)beta(3) is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of alpha(IIb)beta(3), for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD-ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported.
  
  
  OBJECTIVE
  To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation.
  
  
  METHODS
  We assessed the effect of lotrafiban, and other alpha(IIb)beta(3) antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro-coagulant activity) and dense granule release.
  
  
  RESULTS
  In all cases, no effect of alpha(IIb)beta(3) antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)-associated FcR gamma-chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y(12) antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response.
  
  
  CONCLUSIONS
  These data provide a mechanism for enhancement of platelet activity by alpha(IIb)beta(3) inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.
authors.affiliation: Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol, UK.
authors.forename: M L
authors.initials: ML
authors.lastname: Jones
authors.forename: M T
authors.initials: MT
authors.lastname: Harper
authors.forename: E W
authors.initials: EW
authors.lastname: Aitken
authors.forename: C M
authors.initials: CM
authors.lastname: Williams
authors.forename: A W
authors.initials: AW
authors.lastname: Poole
mesh_terms: D001569:Benzodiazepines / Q000494:pharmacology; D032701:Biomimetics; D001777:Blood Coagulation / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D004789:Enzyme Activation; D006801:Humans; D007142:Immunoglobulin gamma-Chains / Q000097:blood; D047908:Intracellular Signaling Peptides and Proteins / Q000097:blood; D008024:Ligands; D009842:Oligopeptides / Q000097:blood*; D010718:Phosphatidylserines / Q000097:blood; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000009:adverse effects / Q000494:pharmacology*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000097:blood; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000097:blood; D058925:Receptors, Purinergic P2Y12; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism; D000072377:Syk Kinase; D013997:Time Factors; D000077466:Tirofiban; D014443:Tyrosine / Q000031:analogs & derivatives / Q000097:blood / Q000494:pharmacology; D019061:src-Family Kinases / Q000097:blood
chemical_list: D007142:Immunoglobulin gamma-Chains; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D009842:Oligopeptides; C548576:P2RY12 protein, human; D010718:Phosphatidylserines; D010880:Piperidines; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; C098341:platelet membrane glycoprotein VI; D001569:Benzodiazepines; D014443:Tyrosine; C047981:arginyl-glycyl-aspartic acid; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D019061:src-Family Kinases; D000077466:Tirofiban; C412702:lotrafiban