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.
References
1
Bevers EM,
Comfurius P,
Van Rijn JLML,
Hemker HC,
Zwaal RFA.
Generation of prothrombin-converting activity and the exposure of phos-phatidylserine at the outer surface of platelets. Eur J Biochem 1982; 122: 429-436
5
Van Dieijen G,
Tans G,
Rosing J,
Hemker HC.
The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem 1981; 256: 3433-3442
6
Mertens K,
Bertina RM.
The contribution of Ca2+ and phospholipids to the activation of human blood-coagulation factor X by activated factor IX. Biochem J 1984; 223: 607-615
7
Van Dieijen G,
Van Rijn JLML,
Govers-Riemslag JWP,
Hemker HC,
Rosing J.
Assembly of the intrinsic factor X activating complex: Interactions between factor IXa, factor VIIIa and phospholipid. Thromb Haemost 1985; 53: 396-400
8
Esmon CT,
Owen WG,
Jackson CM.
A plausible mechanism for prothrombin activation by factor Xa, factor Va, phospholipid and calcium ions. J Biol Chem 1974; 249: 8045-8057
9
Rosing J,
Tans G,
Govers-Riemslag JWP,
Zwaal RFA,
Hemker HC.
The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-283
10
Van Rijn JLML,
Govers-Riemslag JWP,
Zwaal RFA,
Rosing J.
Kinetic studies of prothrombin activation: Effect of factor Va and phospholipids on the formation of the enzyme substrate complex. Biochemistry 1984; 23: 4557-4563
11
Krishnaswamy S,
Jones KC,
Mann KG.
Prothrombinase complex assembly. Kinetic mechanism of enzyme assembly on phospholipid vesicles. J Biol Chem 1988; 263: 3823-3834
12
Van Rijn J,
Rosing J,
Van Dieijen G.
Activity of human blood platelets in prothrombin and in factor X activation induced by ionophore A23187. Eur J Biochem 1983; 133: 1-10
13
Jesty J,
Beltrami E,
Willems G.
Mathematical analysis of a proteolytic positive-feedback loop: Dependence of lag time and enzyme yields on the initial conditions and kinetic parameters. Biochemistry 1993; 32: 6266-6274
14
Bevers EM,
Tilly RH,
Senden JMG,
Comfurius P,
Zwaal RFA.
Exposure of endogenous phosphatidylserine at the outer surface of stimulated platelets is reversed by restoration of amino-phospholipid translocase activity. Biochemistry 1989; 28: 2382-2387
15
Comfurius P,
Senden JM,
Tilly RH,
Schroit AJ,
Bevers EM,
Zwaal RFA.
Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase. Biochim Biophys Acta 1990; 1026 (02) 153-160
19
Owen WG,
Esmon CT,
Jackson CM.
The conversion of prothrombin to thrombin. I. Characterization of the reaction products formed during the activation of bovine prothrombin. J Biol Chem 1974; 249: 594-605
20
Wagenvoord RJ,
Hendrix H,
Soria C,
Hemker HC.
Localization of the inhibitory site(s) of pentosan polysulphate in blood coagulation. Thromb Haemost 1988; 60: 220-225
21
Lindhout T,
Govers-Riemslag JWP,
Van de Waart P,
Hemker HC,
Rosing J.
Factor Va-Factor Xa interaction. Effects of phospholipid vesicles of varying composition. Biochemistry 1982; 21: 5494-5502
24
van Rijn JLML,
Govers-Riemslag JWP,
Zwaal RFA,
Rosing J.
Kinetic studies of prothrombin activation: Effect of factor Va and phospholipids on the formation of the enzyme substrate complex. Biochemistry 1984; 23: 4557-4563
27
Chesney CM,
Pifer DD,
Colman RW.
Subcellular localization and secretion of factor V from human platelets. Proc Natl Acad Sci USA 1981; 78: 5180-5184
28
Rosing J,
Van Rijn JLML,
Bevers EM,
Van Dieijen G,
Comfurius P,
Zwaal RFA.
The role of activated human platelets in prothrombin and factor X activation. Blood 1965; 319-332
