title: New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.
abstract: Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation.
  
  
  
  Background Protein S plays an important role in the down-regulation of coagulation as cofactor for activated protein C (APC) and tissue factor pathway inhibitor (TFPI). Aim To develop functional assays to quantify the APC- and TFPI-cofactor activities of protein S in plasma. Methods APC- and TFPI-cofactor activities of protein S in plasma were measured using calibrated automated thrombography in protein S-depleted plasma supplemented with a small amount of sample plasma either in the presence of anti-TFPI antibodies and APC (APC-cofactor activity) or at excess full-length TFPI without APC (TFPI-cofactor activity). Total and free protein S levels in plasma were measured by ELISAs. Results Average APC-cofactor activities of protein S were 113%, 108% and 89% in plasma from normal individuals (n = 15), FV Leiden heterozygotes (n = 14) and FV Leiden homozygotes (n = 7), respectively, whereas the average APC-cofactor activity of protein S in plasma from heterozygous protein S-deficient individuals (n = 21) was significantly lower (55%). Similar trends were observed for the TFPI-cofactor activity of protein S, with averages of 109%, 115% and 124% in plasma from individuals with normal protein S levels and different FV Leiden genotypes, and 64% in plasma from protein S-deficient patients. APC-cofactor activities of protein S correlated significantly with free and total protein S antigen levels, whereas TFPI-cofactor activities correlated less with protein S antigen levels. Conclusion We have developed functional protein S assays that measure both the APC- and TFPI-cofactor activities of protein S in plasma, which are hardly if at all affected by the FV Leiden mutation.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands.
authors.forename: N A
authors.initials: NA
authors.lastname: Alshaikh
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Rosing
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands.
authors.forename: M C L G D
authors.initials: MCLGD
authors.lastname: Thomassen
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Castoldi
authors.affiliation: Thrombotic and Haemorrhagic Unit, Department of Medicine (DIMED), 5th Chair of Internal Medicine, University of Padua Medical School, Padua, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Simioni
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands.
authors.forename: T M
authors.initials: TM
authors.lastname: Hackeng
mesh_terms: D020016:Activated Protein C Resistance / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D001777:Blood Coagulation; D001780:Blood Coagulation Tests / Q000379:methods*; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay; D005165:Factor V / Q000235:genetics; D006801:Humans; D008074:Lipoproteins / Q000097:blood*; D017354:Point Mutation; D011237:Predictive Value of Tests; D011486:Protein C / Q000378:metabolism*; D017293:Protein S / Q000235:genetics / Q000378:metabolism*; D018455:Protein S Deficiency / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D013917:Thrombin / Q000378:metabolism*
chemical_list: D008074:Lipoproteins; D011486:Protein C; D017293:Protein S; C095381:factor V Leiden; C051928:lipoprotein-associated coagulation inhibitor; D005165:Factor V; D013917:Thrombin
keywords: activated protein C resistance; blood coagulation; factor V Leiden; protein C; protein S; tissue factor pathway inhibitor