title: Development of a perfusion chamber assay to study in real time the kinetics of thrombosis and the antithrombotic characteristics of antiplatelet drugs.
abstract: BACKGROUND
  Arterial thrombosis triggered by vascular injury is a balance between thrombus growth and thrombus fragmentation (dethrombosis). Unbalance towards thrombus growth can lead to vascular occlusion, downstream ischemia and tissue damage.Here we describe the development of a simple methodology that allows for continuous real time monitoring and quantification of both processes during perfusion of human blood under arterial shear rate conditions. Using this methodology, we have studied the effects of antiplatelet agents targeting COX-1 (aspirin), P2Y12 (2-MeSAMP, clopidogrel), GP IIb-IIIa (eptifibatide) and their combinations on the kinetics of thrombosis over time.
  
  
  RESULTS
  Untreated samples of blood perfused over type III collagen at arterial rates of shear promoted the growth of stable thrombi. Modulation by eptifibatide affected thrombus growth, while that mediated by 2-MeSAMP and aspirin affected thrombus stability. Using this technique, we confirmed the primacy of continuous signaling by the ADP autocrine loop acting on P2Y12 in the maintenance of thrombus stability. Analysis of the kinetics of thrombosis revealed that continuous and prolonged analysis of thrombosis is required to capture the role of platelet signaling pathways in their entirety. Furthermore, studies evaluating the thrombotic profiles of 20 healthy volunteers treated with aspirin, clopidogrel or their combination indicated that while three individuals did not benefits from either aspirin or clopidogrel treatments, all individuals displayed marked destabilization profiles when treated with the combination regimen.
  
  
  CONCLUSIONS
  These results show the utility of a simple perfusion chamber technology to assess in real time the activity of antiplatelet drugs and their combinations. It offers the opportunity to perform pharmacodynamic monitoring of arterial thrombosis in clinical trials and to investigate novel strategies directed at inhibiting thrombus stability in the management of cardiovascular disease.
authors.affiliation: Portola Pharmaceuticals Inc, 270 E, Grand Avenue, Ste 22, South San Francisco, CA 94080, USA. gstephens@portola.com.
authors.forename: Gillian
authors.initials: G
authors.lastname: Stephens
authors.forename: Ming
authors.initials: M
authors.lastname: He
authors.forename: Connie
authors.initials: C
authors.lastname: Wong
authors.forename: Marzena
authors.initials: M
authors.lastname: Jurek
authors.forename: Hans-Christian
authors.initials: HC
authors.lastname: Luedemann
authors.forename: Golnaz
authors.initials: G
authors.lastname: Shapurian
authors.forename: Kevin
authors.initials: K
authors.lastname: Munnelly
authors.forename: Craig
authors.initials: C
authors.lastname: Muir
authors.forename: Pamela B
authors.initials: PB
authors.lastname: Conley
authors.forename: David R
authors.initials: DR
authors.lastname: Phillips
authors.forename: Patrick
authors.initials: P
authors.lastname: Andre