Summary
We have developed a chromogenic assay to measure the phospholipid-related procoagulant
activity (PPA) in whole blood, or platelet-rich plasma. The test is based upon thrombin
formation from prothrombin by prothrombinase and is designed in such a way that procoagulant
lipids are rate limiting for the prothrombinase activity. In the chromogenic test
PPA concentrations equivalent to 0-10 nM phospholipid vesicles containing 75% phosphatidyl
choline (PC) and 25% phosphatidyl serine (PS) can be measured.
The thrombin, which develops during the test, is measured with a chromogenic substrate.
By the action of thrombin on this chromogenic substrate p-nitroaniline is liberated,
which causes an increase in absorbance. Thrombin formed in the assay mixture activates
the present platelets. This causes a linear increase of the velocity of thrombin generation
during the test, i. e. a parabolic increase of product formation. For that reason
the thrombin generation in time is characterized by two parameters, the basal PPA
(PPA-B) of the original mixture and the increase in PPA due to platelet activation
(PPA-A). To determine these figures the absorbency-data were fitted to parabolas.
In most cases the contribution of PPA-A to the total amount of formed thrombin becomes
considerable already after 30 s.
Preliminary tests show that PPA-B activity in whole blood or platelet-rich plasma
of patients with a thrombotic disorder is significantly higher than the activity of
a control group of the same age.
