title: Preincubation in the Prothrombinase-induced Clotting Time test (PiCT) is necessary for in vitro evaluation of fondaparinux and to be avoided for the reversible, direct factor Xa inhibitor, rivaroxaban.
abstract: BACKGROUND
  There is no clear consensus about tests in vitro that are suitable for evaluating various factor Xa inhibitors. The availability of reversible and irreversible inhibitors further complicates the application of available assays.
  
  
  METHODS
  We evaluated the suitability of the prothrombinase-induced clotting test (PiCT) for fondaparinux and rivaroxaban, as representatives for irreversible and reversible inhibition of factor Xa, with specific attention to preincubation times prior to re-calcification, in the context of automate program limitations.
  
  
  RESULTS
  We demonstrate that the PiCT assay requires a preincubation step to allow inhibitory activity by fondaparinux. Without this step, inhibition in the test is minimal and lacking sufficient dynamic range. In contrast, to measure the reversible inhibition by rivaroxaban, we found any preincubation introduced an artifact in inhibition as exemplified by a biphasic pattern and only the test without a preincubation phase gave informative results.
  
  
  CONCLUSIONS
  It is concluded that PiCT in its format with two steps is suitable for fondaparinux evaluation, while its format without preincubation (the one-addition format) is suitable for reversible inhibitors such as rivaroxaban. Unfortunately, both types of inhibitors cannot be compared in vitro in a single assay format.
authors.affiliation: Centre for Human Drug Research, Leiden, The Netherlands. kluft@euronet.nl
authors.forename: C
authors.initials: C
authors.lastname: Kluft
authors.forename: P
authors.initials: P
authors.lastname: Meijer
authors.forename: R
authors.initials: R
authors.lastname: Kret
authors.forename: J
authors.initials: J
authors.lastname: Burggraaf
mesh_terms: D000925:Anticoagulants / Q000737:chemistry*; D001681:Biological Assay / Q000592:standards*; D001777:Blood Coagulation; D065427:Factor Xa Inhibitors; D000077425:Fondaparinux; D006801:Humans; D007700:Kinetics; D009025:Morpholines / Q000737:chemistry*; D010314:Partial Thromboplastin Time / Q000592:standards*; D011134:Polysaccharides / Q000737:chemistry*; D000069552:Rivaroxaban; D013876:Thiophenes / Q000737:chemistry*; D013925:Thromboplastin / Q000737:chemistry; D013997:Time Factors
chemical_list: D000925:Anticoagulants; D065427:Factor Xa Inhibitors; D009025:Morpholines; D011134:Polysaccharides; D013876:Thiophenes; D013925:Thromboplastin; D000069552:Rivaroxaban; D000077425:Fondaparinux