Thrombin generation-based assays to measure the activity of the TFPI-protein S pathway in plasma from normal and protein S-deficient individuals.
BACKGROUND
Protein S acts as a cofactor for full-length tissue factor pathway inhibitor (TFPI) in the downregulation of thrombin formation.


OBJECTIVE
To develop a functional test to measure the activity of the TFPI-protein S system in plasma.


METHODS
Using calibrated automated thrombography, we quantified the activity of the TFPI-protein S system in plasma by measuring thrombin generation in the absence and presence of neutralizing antibodies against protein S or TFPI. Moreover, we designed an enzyme-linked immunosorbent assay (ELISA) to determine the level of full-length TFPI in plasma. The performance of these assays was examined in plasma from 85 normal individuals and from 35 members of protein S-deficient families.


RESULTS
The ratio of thrombin peaks determined in the absence and presence of anti-protein S antibodies (protein S ratio = 0.5 in normal plasma) is a measure of the TFPI cofactor activity of protein S, whereas the ratio of thrombin peaks determined in the absence and presence of anti-TFPI antibodies (TFPI ratio = 0.25 in normal plasma) is a measure of the overall activity of the TFPI-protein S system. Protein S and TFPI ratios were elevated in protein S-deficient individuals, indicating an impairment of the TFPI-protein S system. Both ratios correlated well with full-length TFPI levels, which were significantly lower in protein S-deficient patients than in normal family members.


CONCLUSIONS
Functional assays for the TFPI-protein S system and an ELISA for full-length TFPI were developed. These assays show that the activity of the TFPI-protein S anticoagulant pathway is impaired in individuals with congenital protein S deficiency.
Maurissen
L F A
LF
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands.
Castoldi
E
E
Simioni
P
P
Rosing
J
J
Hackeng
T M
TM
D000595:Amino Acid Sequence; D057134:Antibodies, Neutralizing; D057205:Automation, Laboratory; D015415:Biomarkers / Q000097:blood; D001780:Blood Coagulation Tests / Q000592:standards; D002138:Calibration; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D005260:Female; D006801:Humans; D008074:Lipoproteins / Q000097:blood* / Q000276:immunology; D008297:Male; D008969:Molecular Sequence Data; D011237:Predictive Value of Tests; D017293:Protein S / Q000276:immunology / Q000378:metabolism*; D018455:Protein S Deficiency / Q000097:blood / Q000175:diagnosis*; D015203:Reproducibility of Results; D013917:Thrombin / Q000378:metabolism*
D057134:Antibodies, Neutralizing; D015415:Biomarkers; D008074:Lipoproteins; D017293:Protein S; C051928:lipoprotein-associated coagulation inhibitor; D013917:Thrombin