Photometric assay of platelet factor 4 with a chromogenic substrate.

Haemostasis 6 : 283-293 (1977)

Photometric Assay of Platelet Factor 4 with a Chromogenic Substrate H. V lN A Z Z E R Blood Coagulation Laboratory, Linz

Key Words. Platelet factor 4 • Factor Xa • Heparin • S-2222 Abstract. A photometric assay procedure for platelet factor 4 is described. The syn thetic oligopeptide benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S-2222) is used as a substrate. By the action of factor Xa,/>-nitroaniline (pNA) is split form the peptide bond. The amount of pNA liberated from S-2222 per minute is in direct relation to the activity of factor Xa. This reaction permits a photometric assay. Addition of heparin to an activation system consisting of plasma, thromboplastin and calcium chloride inhibits development of Xa activity. Since platelet factor 4 neutralizes heparin, its activity can be measured in such a system when all other components are kept at a constant level. Experimental details of the reactions involved and clinical results of the assay in comparison to a clotting method are described.

Introduction

Received: February 2, 1977; in revised form: April 4, 1977; accepted by editor E.F.Liischer: April 28,1977.

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Recent evidence of the role of platelet factor 4 (PF 4) as a possible inducer of increased thrombotic tendency [2, 5, 7] raised the general interest in this particular platelet activity. The present assay methods for PF 4 are either based on the principle of the heparin-thrombin time [4-6, 11] or on the paracoagulation phenomenon induced by PF 4 [5]. All these methods are time-consuming and difficult to perform. Simpler assays [12] cannot be quantitated. For these reasons, an attempt was made to develop a quanti tative method which would meet with the requirements for clinical routine tests.

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Materials Benzol-1 le-Glu-Gly-Arg-/)-nitroanilide (S-2222): KABI AB, Stockholm, Sweden. The contents of one vial was dissolved in 17 ml distilled water to obtain a 2 mM solution. The dissolved substance was stable for 3 months at 4 °C. Buffer: 0.15 m Tris-HCl, pH 8.4. Heparin: Liquemin® (Hoffmann-La Roche, Basle, Switzerland). The stock solution con tained 5,000 units/ml. This was diluted with distilled water to obtain a test concentration of 1 unit/ml. This dilution was prepared daily. Ancrod: Arwin® (Knoll, Ludwigshafen, FRG) at a concentration of 70 units/ml. Thromboplastin reagent: Thromboplastin liquid (Hyland, Costa Mesa, Calif., USA). Calcium chloride: 0.025 m solution in Tris buffer pH 7.35. Thrombin: Topostasin® (Hoffmann-La Roche). The contents of one vial was dissolved in 30 ml distilled water to get a stock solution of 100 N1H units/ml. This solu tion was frozen in 1-ml portions at —36 °C. Collagen: Kollagenreagens Horm (Hormonchemie, Munich, FRG). This reagent was used at a concentration of 10/ig/ml. Epinephrine: Suprarenin® (Hoechst, Frankfurt am Main, FRG). This reagent was diluted to a final concentration of 20 mM. Aprotinin: Trasylol® (Bayer, Leverkusen, FRG). By dilution of 100/il of the original solution with 4.9 ml isotonic saline solution, a concentration of 200 kallikrein inhibitor units/ml was achieved. Defibrinated plasma: By addition of 400 /d ancrod to 10 ml plasma as described previously [9], Plasma was also frozen in 1-ml portions at —36 °C. Homogenized platelets: Platelets in PRP were counted in a phase contrast microscope. PRP was centrifuged at 6,000 g for 30 min to obtain PPP as a source for defibrinated plasma. The platelet button was washed three times by centrifugation with 10 ml saline solution containing 10% 0.15 m sodium citrate. After the last washing process, platelets were brought to a concen tration of 3000,000/ul by addition of isotonic saline solution. Homogenization was achieved by sonication (3 W/cm2, 5 min). Homogenates not used immediately were frozen in 1-ml portions at —36 °C. Centrifuge: UJ III E model (Christ, Osterode, FRG. Coagulometer: MKG 300 (Labtronic, Zurich, Switzerland). Aggregometer: Elvi 840 (Elvi, Milan, Italy). Photo meter: Labtronic S 100 with an automatic cuvette changer S 304 connected to a micro processor ES 23 for automatic enzyme and substrate programs. Cuvettes: 1 cm semimicro cuvettes (glass), minimal filling volume 400 ¡i\.

Methods Platelet count [3]. Assay of thrombin activity: A reference curve was obtained by clotting pooled donor plasma with serial dilutions of the thrombin stock solution. Thrombin activities which developed during tests were measured in N1H units per milliliter plasma from this reference curve. Coagulation assay of PF 4 [11]. Assay methods with S-2222 as described below. These assays were carried out at 37 °C and at a wavelength of 405 nm. The final reaction volumes in the cuvettes were kept constant at 500/d.

Results


In a preliminary series o f experiments, the development of factor Xa and of thrombin was examined. For this purpose an activation mixture of de-

Assay of Platelet Factor 4 with S-2222

285

(fig-1). According to these results, the kinetics o f the development of factor Xa and of thrombin in the reaction mixture were slightly different. Whilst a maximum of factor Xa was reached 1 min after addition o f calcium chloride, thrombin activity at that time was still comparatively low. After 2 min, the thrombin peak was reached while factor Xa was already decreasing. When the incubation time was extended further, there was a rapid decrease of both thrombin and factor Xa, apparently due to inactivation by antithrombin III (fig. 1). From these results, an incubation time of 1 min most favorable for assays of factor Xa since more than 97 % o f the amidolytic reaction was actually caused by this activity (table II). A regression curve obtained by


fibrinated plasma, thromboplastin and calcium chloride in equal parts was used. Thrombin activities were assayed by the clotting method described above. Factor Xa was measured by its amidolytic activity on the substrate S-2222. A possible interference of thrombin with the amidolytic reaction between factor Xa and S-2222 was tested in the following way: Total amidolytic activity of the plasma activation mixture was measured. In parallel series of tests, aprotinin was pipetted into the plasma mixture im mediately prior to the addition of S-2222. Thereby, factor Xa was inactivated, and the remaining amidolytic activity reflected the action of thrombin on the substrate. Furthermore, the stock solution of thrombin was diluted to obtain a series of concentrations equal to the thrombin activities found in the plasma activation mixture after various lengths of incubation time with calcium chloride. Since factor Xa is generally present in commercial throm bin preparations, the amidolytic activities of these thrombin solutions were also tested in parallel experiments with and without addition of aprotonin. The presence of thromboplastin and of calcium chloride in the assay series for the reaction between commercial thrombin and S-2222 was in fact un necessary. These reagents were nevertheless added in order to obtain com parable experimental conditions. The technical details of these experiments are listed in table I, the results are given in table II. The difference in optical density per minute (OD/min) caused by throm bin was negligible compared to the intensity of amidolysis of S-2222 by factor Xa. Since thrombin as well as factor Xa are inactivated by antithrom bin III when plasma is present in the reaction mixture, an optimal incubation time had to be established. This was achieved by examination of the kinetics of activation and inactivation of both enzymes. Therefore, assays of throm bin and of factor Xa in the activation mixture were carried out at 1- to 3-min intervals between 0 and 10 min after addition of calcium chloride

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Table I. Activation mixtures for assays of the influence of factor Xa and of thrombin on the liberation of pNA from the chromogenic substrate (all quantities in /d) Cuvette No.

Plasma, defibrinated Thromboplastin Saline Thrombin 0.025 m CaCl2 Incubation time, sec. Aprotinin S-2222 (1 part) in buffer (4 parts)

1

2

50 50 100

50 50 50

-

-

50 0 250

50 600 50 250

4

3 _

_

50 100 50 50

50 50 50 50

-

-

50 250

250

Table 11. Average difference in optical density per minute (n = 10) Incubation, min

0 1 2 3 5 7 10

Thrombin, units

Cuvette No. 1

2

0.000 0.158 0.094 0.073 0.051 0.033 0.017

0.000 0.004 0.010 0.007 0.006 0.003 0.002

0 18 63 47 33 20 11

Cuvette No. 3

4

0.000 0.008 0.027 0.021 0.013 0.009 0.005

0.000 0.002 0.012 0.008 0.006 0.002 0.002

Contents of cuvettes: 1: plasma activation mixture; 2: factor Xa inhibited by apro tinin; 3: commercial thrombin, concentration indicated; 4: factor Xa in commercial thrombin inhibited by aprotinin.


serial dilutions o f pooled defibrinated plasma followed a pattern of a first order reaction (fig. 2). When factor X was kept at a constant level, addition of heparin to the activation mixture (50 /A heparin and 50 ¡A saline instead o f 100 ¡A saline) resulted in a diminution o f factor Xa activity, and in consequence also in a diminution of the increase in OD/min. Variations o f the heparin concen trations between 0.20 and 2.0 units/ml allowed the drawing of a regression

287

Assay of Platelet Factor 4 with S-2222 100 -,

8060-

£ §

40 -

x

z

/\r£./. Factor Xa and thrombin after exogenous activation of defibrinated plasma. Kinetics of the formation of both enzymes and of their inactivation by antithrombin III present in plasma.

I---------------- 1---------------- 1----------------1

0.25

0.50

1.0

2.0

Fig. 2. Calibration curves for factor Xa and for heparin activity.


Heparin, units

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Plasma

Thrombo Saline plastin

_

_

50 50

50 50 50

200 100 100 50

Platelets

50 50 50

CaCl2 Incu Substrate bation, + buffer sec _

50 50 50

60 60 60 60

250 250 250 250

OD/min

1 SD

0.000 0.003 0.158 0.162

0.000 0.002 0.014 0.017

curve for heparin which proved that the reaction was linear within the con centrations indicated (fig. 2). In the following series, a possible influence of homogenized platelets on the amidolytic reaction and on the activation of factor X by thromboplastin and calcium was examined. The data of this experiment are presented in table III. From the results of these experiments, the conclusion could be drawn that the chromogenic substrate was neither split by platelet enzymes nor by a mixture of platelets with thromboplastin and calcium chloride. Further more, addition of platelets to the plasmatic activation mixture had no influence on the formation of factor Xa. Addition of homogenized platelets to an activation mixture containing heparin however, resulted in partial neutralization of heparin activity. When heparin was kept at a constant level of 1 unit/ml, the amount of heparin neutralization could be brought into relation with platelet antiheparin activity. When the difference in OD/min obtained by an activation mixture containing heparin but no platelets was subtracted as a blank from OD/min achieved by serial dilutions o f homogenized platelets with saline, a calibra tion curve for PF 4 could be made (fig. 3). An activity o f 100% PF 4 was assumed when pooled homogenized pla telets from 10 healthy donors were tested at a concentration o f 3 x 105 plalets//d. Instead of a relative percentage, an expression of PF 4 in heparin units neutralized by this factor was also possible. This could be read from the inactivation curve of factor Xa by heparin (fig. 2). When activities of PF 4 were simultaneously established as a percentage and as the amount of heparin neutralizing power, 100 % PF 4 was equal to neutralization of 0.028


Table III. Influence of homogenized platelets on exogenous activation of factor Xa (n = 10; quantities in/d)


i—

i— i--------1—

20 30 PF 4, %

i— i— i— m

50

289

------------ 1--------- 1

100

200

units of heparin by 1.5 x 107 platelets. This figure could, however, only be applied when heparin from the same source was used. For this reason, the relative percentage of PF 4 appeared to be more reliable. After completion of the experimental part of this assay, the following procedure was used in all further tests. At a reaction temperature o f 37 °C in a 1-cm semimicro cuvette were mixed (/d): plasma, defibrinated, 50; heparin (1 unit/ml), 50; platelets, homogenized (3 x 105//(d), 50; after 120 sec, thromboplastin, 50; 0.025 m calcium chloride, 50; after 60 sec, S-2222 (1 part) in buffer (4 parts), 250. OD was measured at 405 nm at 15 and 75 sec after addition of the substrate. The blank was obtained by substitution of platelets by saline. The result was subtracted from the total difference in OD. This test was compared with an assay o f PF 4 which is based on the he parin-thrombin clotting time [11]. The results are listed in table IV. According to the results, there was no significant difference between the methods applied. This was observed in PF 4 levels within normal range as well as in diminished levels o f PF 4. Since an idiopathic diminution of PF 4 is rare, diminished levels for comparison of the methods were obtained from pa tients admitted to the intensive care department for hemorrhagic shock after severe accidents. A minor variation of the photometric method also permitted an assay of PF 4 release from platelets after stirring of PRP with aggregating substances. For this purpose, patient PRP was adjusted ot a concentration of 3 x 10s platelets/jul by dilution with patient PPP. In a platelet aggregometer, 400 /d PRP was stirred with 100 fx\ epinephrine and with 100 [A collagen, respec tively.


Fig. 3. Calibration curve for platelet factor 4.

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Table IV. Percentage of platelet factor 4 in healthy individuals and in hemorrhagic shock; comparison of two methods Patients

n

Method

Significance(t-test)

S-2222

Donors, pooled Healthy Shock

10 50 11

thrombin-heparin time

average

1 SD

range

average

1 SD

range

100 101.4 59.8

5.1 13.2 12.1

89-109 77-121 39- 81

100 97.3 60.4

8.7 18.8 13.3

87-112 68-135 44-77

n.s. n.s. n.s.

Table V. Release of PF 4 by stirring of PRP with collagen (C) and epinephrine (E), respectively; comparison of two methods (figures are percentages of total patient PF 4)

Healthy Aspirin

n

20 20 10 10

Releasing substance

C E C E

Method S-2222

thrombin-heparin time

Significance (t-test)

average

1 SD

range

average

1 SD

range

37.8 54.2 8.4 11.2

11.4 12.3 3.6 4.5

23-54 39-71 3-13 5-16

34.4 56.0 7.1 9.8

13.6 15.3 4.2 4.2

20-56 n.s. 33-74 n.s. 0-12 n.s. 4-14 n.s.

Aggregation was simultaneously recorded, and a maximum o f aggrega tion was reached after 3-5 min at a stirring speed of 1,500 rpm and at 37 °C. Defibrination was obtained by addition o f 20 (i 1 ancrod when aggregation was complete. Retraction o f the fibrin clot was greatly facilitated when the stirrer was left in action. Platelets and aggregates were simultaneously re moved by incorporation into the clot. 320 ¿ul saline was added to the stirred and defibrinated PRP. Thereby, a 50 % dilution o f the defibrinated plasma was obtained since the quantity o f the aggregating substance had also to be considered as a diluent. Defibrinated platelet-poor patient plasma was pro vided by simultaneous stirring of 400 /d PPP with 20 [A ancrod. Tests were carried out in the following way (volumes in /¿l):


Patients


blank PPP, defibrinated PRP, stirred, defibrinated Heparin Saline After 120 sec thromboplastin Platelets Calcium chloride After 60 sec S-2222 (1 part) in buffer (4 parts)

total PF 4

291

PF 4 release _

50

50

-

-

50 50

50 -

—

50

50

50

50 50 50

250

250

250

-

100 50

—

50

The release was expressed as a percentage of total PF 4 o f homogenized patient platelets. Tests were carried out in 20 healthy individuals and in 10 patients who regularly received 1,500 mg aspirin per day. The results were again compared with the clotting method for PF 4 mentioned above. Details are presented in table V. Also in this series o f tests, there was no significant difference between the results o f the amidolytic method and the clotting assay.

Assays of coagulation activities with the amidolytic substrate S-2222 were previously described. Methods for factor X [1] and for heparin [8] are presently in use. However, in both of these an endpoint reaction was applied. The sensitivity of the substrate to factor Xa and as a consequence also to heparin was demonstrated by these methods. It was therefore obvious that an assay o f PF 4 should be possible by a similar technique. The method described is an indirect reaction since factor Xa is used as an indicator. Its diminution by heparin, and the amount of heparin neutralization by PF 4, eventually allowed the evaluation o f PF 4 activity. In order to get comparable results, all participants in the reaction other than PF 4 had to be kept at a constant level. Since the exogenous pathway o f Xa activation was used, variations of factors VII and X would have influenced the formation of Xa. Furthermore, variations in the concentration of antithrombin III and/or heparin would have been reflected in the rate o f Xa inactivation. The use o f pooled defibrinated donor plasma frozen in small portions was assumed to be a simple and


Discussion

VlNAZZER

safe way to meet with the requirement of identical concentrations in different samples of factors VII, X, and antithrombin III. Defibrination of plasma was an unavoidable step since formation of a fibrin clot in the reaction mix ture would have interfered with photometry. The Hyland preparation of thromboplastin was chosen because of its stability. Thromboplastin always caused a certain amount of turbidity in the reaction mixture. This had, however, no influence on the accuracy of the kinetic test since the intensity of turbidity remained stable. An alternative pathway o f Xa activation by Russell viper venom was also tried. These experiments were, however, unsuccessful since phospholipids present in platelets greatly enhanced the formation o f factor Xa. Thereby, two variables were present in the test system, and an accurate assay was impossible. The influence o f the presence of thrombin in the reaction mixture was thoroughly examined. Though S-2222 is known to be mainly sensitive to factor Xa, it is also split to some extent by thrombin. It was, however, demonstrated that an influence o f thrombin could only be found when the incubation time o f the plasmatic activation mixture was extended until a maximum of thrombin activity was reached. In order to investigate this influence on the result of the assay of PF 4, the incubation time was extended to 2 min. At this point, thrombin activity was highest whilst Xa activity was already beyond its peak. Even in these experiments antiheparin activity was measurable. Though the slope of the calibration curve obtained in these tests was considerably decreased, the reaction was still linear. This pheno menon was explained by the fact that both thrombin and factor Xa undergo rapid inactivation when heparin and antithrombin III are present, and that inactivation of both enzymes is diminished when the heparin activity is partially neutralized. The steeper slope of the regression curve in a system containing high factor Xa and low thrombin activity was, however, pre ferable since the evaluation of the results was more accurate. A possible influence of platelet enzymes on the amidolytic reaction was checked but was found to be absent. There was also no influence o f platelets on the formation o f factor Xa. This was checked though such an influence was improbable even from the theoretic point of view. Phospholipids from platelets cannot be expected to enhance the exogenous activation of coagu lation when tissue thromboplastin is present in abundance whilst factor V, which is also present in platelets, does not participate in the activation of factor Xa. To overcome some of the difficulties described, the assay was initially tried in a system consisting of purified factor Xa, purified anti thrombin III and heparin. This was, however, found to be o f disadvantage


292


293

because of the poor stability of factor Xa after solution o f the lyophilized substance, and of slight changes in activity of different vials of the same batch. The amidolytic method described was partly automated by the use of a cuvette changer and o f a microprocessor. Full automation would be just a minor technical step. From the present experience, this method appears to be suitable for routine tests of PF 4.

References

Prof. Dr. H elmut Vinazzer , Blood Coagulation Laboratory, A-4020 Linz (Austria)


1 A urell, L.; O lausson, A., and C laeson, G.: Determination of factor Xa activity by means of chromogenic substrates based on the primary structure of prothrombin. Thromb. Diath. haemorrh. 34: 591-592 (1975). 2 Chmielewski, J. and F arbiszewski, R.: Platelet factor 4 release during human platelet aggregation in diabetic patients. Thromb. Diath. haemorrh. 24: 203-205 (1970). 3 D erlath, S .: Die direkte phasenoptische Thrombozyten- und Retikulozytenzählung. Ärztl. Forsch. 10: 552-555 (1956). 4 D eutsch , E. and K ain , W.: Studies on platelet factor 4; in J ohnson et al. Blood pla telets, pp. 337-345 (Little, Brown, Boston 1961). 5 F arbiszewski, R.; N iewiarowski, S.; W orowski, K., and L ipinski, B.: Release of platelet factor 4 in vivo during intravascular coagulation and in thrombotic states. Thromb. Diath.haemorrh. 19: 578-583 (1968). 6 H arada, K. and Z ucker , M.B.: Simultaneous development of platelet factor 4 activity and release of 14C-serotonin. Thromb. Diath.haemorrh. 25: 41-46 (1971). 7 L üscher, E.F. and K äser-G lanzmann , R.: Platelet heparin neutralizing factor. Thromb. Diath. haemorrh. 33: 66-72 (1975). 8 T eien, A .N .; L ie, M., and A bildgaard, U.: Assay of heparin in plasma using a chromogenic substrate for activated factor X. Thromb. Res. 8: 413-416 (1976). 9 Vinazzer , H.: Photometric assay of antithrombin III with a chromogenic substrate. Haemostasis 4: 101-109 (1975). 10 Vinazzer , H. and Loew , D.: On the mechanism of platelet dysfunction induced by acetylsalicylic acid (unpublished). 11 Vinazzer , H.; P ütter, J., and Loew , D .: Influence of intravenously administered acetylsalicylic acid on platelet functions. Haemostasis 4: 12-22 (1975). 12 W amhoff, D . und Schmechel, H.: Beeinflussung der Blutstillung durch kombinierte Verabreichung von Azetylsalizylsäure und Heparin. Dt. Gesundh. Wes. 28: 2389-2391 (1973).

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