A chromogenic test to determine the procoagulant phospholipids in platelet-rich plasma and whole blood.
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.
Wagenvoord
R J
RJ
Department of Biochemistry, Faculty II, University of Limburg, Maastricht The Netherlands.
Hendrix
H H
HH
Kai
H
H
Hemker
H C
HC
D016477:Artifacts; D001769:Blood; D001777:Blood Coagulation; D002863:Chromogenic Compounds; D003124:Colorimetry; D004151:Dipeptides; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D010743:Phospholipids / Q000097:blood* / Q000737:chemistry; D010865:Pilot Projects; D010949:Plasma; D015539:Platelet Activation; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000096:biosynthesis*; D013925:Thromboplastin / Q000378:metabolism*; D013927:Thrombosis / Q000097:blood
D002863:Chromogenic Compounds; D004151:Dipeptides; D006454:Hemoglobins; D010743:Phospholipids; C021129:S 2238; D013925:Thromboplastin; D013917:Thrombin