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Dependence of thromboplastin activity in cell extracts upon salt concentration
THROMBOSIS RESEARCH 46; 737-740, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.
BRIEF
COMMUNICATION
DEPENDENCEOF THROMBOPLASTINACTIVITY IN CELL EXTRACTSUPON SALT CONCENTRATION P. LWNI and R.T. DEAN Cell BiologyResearchGroup, Departmentof Applied Biology, Brunei University,UxbridqeUB8 3PH, U.K. (Received 28.8.1986; Accepted in revised form 12.3.1987 by Editor M.C. Scrutton)
IhmODUCTION Thromboplastinactivityin the one step clottingassay has been traditionally measuredby preparingthe sample (in which activityis to be assessed)in a buffer containing0.9 % (w/v)NaCl (l-4).We show here that the concentraticn cf NaCl in such an assay has a dramaticeffect on the activitydetected,and should be optimisedfor each individualsource (tissueor celullar)of samples. MATERIALSAND METHODS Preparationof cell and tissue extracts.Human monocytesfrom peripheral blood were isolatedfrcm cell separatorresidues (NorthLondon Transfusion Centre) by sedimentationat lg and subsequentcentrifugationthrough Ficoll-Paque(Pharmacia,Uppsala,Sweden),as describedbefore (5). Cells were used after adherencein the absenceof serum, or after subsequentstimulation of adhered cells for 3 hours with 10 pg/ml Lipopolysaccharide (LPS) from E. coli (DifcoLabs., Detroit,U.S.A).After the incubationperiod; the cells were washed 4 times with phosphatebufferedsaline,harvestedin 50 n-MVerona1 buffer pH 7.4 and frozen at -70' C. The frozen cell extractswere homogenized using a syringewith a 25gx5/8"needle.Brain and lungs from Wistar rats were homogenizedin a mechanicallydriven glass-Teflonhcrqenizer. Homogenates were centrifugedfcr 20 min at 3000xgand the supernatantdiscarded.The pelletswere resuspendedin Verona1buffer and frozen at -70' C. The frozen extractswere homogenizedusing a syringewith a 21gxl l/2" needle. Measurementof thrcmboplastinactivity.Thrcmboplastinactivitywas measured in a one step clottingassay. 0.1 ml of cell or tissue extractwas used to measure its ability to shcrtenthe clottingtime of 0.1 ml of human plasma. The reactionwas startedwith the additionof 0.1 ml of 30 r@lCaCl,. Results are expressedin arbitraryunits derived by comparingthe sample clottingtime with those producedby dilutionsof brain standardthron-boplastin (National ReferenceLaboratory,Manchester,U.K.) which is taken to have 100 units/ml. Rabbit brain cephalin (Sigma,Dorset, U.K.) was reconstitutedwith 10 ml of water and 20 ~1 of the suspensionused when specified.Each determinationwas Key wcrds: Thromboplastinassay, human rronocyte, tissue factor. 737
738
THROMBOPLASTIN AND CELL EXTRACTS
Vol. 46, No. 5
water and 20 ~1 of the suspensionused when specified.Each determinationwas performedin duplicate. The activityassayedwas shown to be thromboplastinby the imnunologicaland enzymaticcriteriawe describedpreviously(4). RESULTS Throrcboplastin activityof human n-onocyte extractshas been measured in the presence of increasingconcentrationof NaCl. Using a preparationwith a clottingtime over 30 seconds (samplesB and C, Fig.1) the activityappearsto decreasewith increasingconcentrationsof NaCl; if the sample is concentrated so that the clottingtime is under 30 seconds (sampleA, Fig.11 the presence of salt hardly affects the determinationand can even be stimulatory,but the meausurenient of clottingtimes under 30 second is very inaccurate(Fig 1).
120 -
.> 80 J : m c ._ C .-
60
-
40 -
; :
20O-r
I
0
0.9
I
I
1.8
2.7 NaCl
3.6
I
4.5
I
Thrcmboplastin activityin LPS stimulatedhuman moncqteswas measuredin the presenceof the specifiedfinal concentrations of NaCl (A). The same extract was diluted 5 times (B) and 10 times (C).
5.6
%,
FIGURE 1 Inappropiateconditionslead to drastic underestimationof activityand in some cases, dependingon the sample,the activitycould go completely undetected.In the absence of NaCl, we have been able to measure readily the thron-boplastin activityof freshlyadheredmonocytes, which in the presenceof salt had shown hardly any activity.Moncxyteswere allowed to adhere for 1, 2 and 3 hours in the absenceof serum, and the thrc&oplastin activities (units/q prot. + S.D.; n=3) were determined.In the presenceof NaCl the activitywas undetectedfor the first 2 hours, and 0.58 + 0.1 after 3 hours. In the absenceof NaCl it was 1.1 + 0.1, 2.8 k 0.1 and 6.20 k 0.4 (at 1, 2 and 3 h respectively).If rabbit brain cephalinwas added to the assay of extracts of stimulatedmonocytesthe values rexrained at control levels until a salt concentrationof 0.36 % (W/V),and decreasedonly at higher concentration.The cephalinby itself has no procoagulantactivityin the conditionsused for the
739
THROMBOPLASTIN AND CELL EXTRACTS
Vol. 46, No. 5
assay. Using crude rat lung or brain extractsa similardependenceof activity on salt concentrationis observed:at the concentrationof NaCl routinelyused in the assay (0.3 % (w/v))the activitymeasured is less than maximal (Fig. 2), but lower salt concentrationsgive higher activity.With the British comparativethronboplastinpreparation,which containsa much higher proportionof lipids,differentresultsare obtained (Fig 2); IWaClin the assay is necessaryfor optimalactivity.
w
100 -
.Z A 80> ..I:
The thromboplastin activityof rat brain (0) and lung (A) extracts,and of standard thromboplastin(0) was measuredin the presenceof increasing concentrations of NaCl.
60-
a, .E 40.a E
20O0
0.9
1.8 2.7 3.6 NaCl %,
4.5
5.4
FIGURE 2 In a separateexperimentthe clottingtime of recalcifiedplasma was found to be much inhibitedin the presenceof NaCl (Table1). TABLE 1 Effect of NaCl on plasma clottingtime Clottingtime (minutes) NaCl
(% w/v)
0.18
0.27
0.36
0.45
0.54
0.63
0.72
Plasma I
5.75
6.75
7.00
8.50
9.33
11.45
14.50
Plasma II
7.20
8.50
10.50
11.00
11.50
12.50
15.00
Plasma III
7.00
8.25
10.25
11.20
15.50
17.00
18.00
The clottingtime of plasma from healthydonors was measuredas described,
740
THROMBOPLASTIN AND CELL EXTRACTS
Vol. 46, No. 5
using Verona1 buffer insteadof a tissue factor sample in the presenceof the specifiedconcentrationsof NaCl. DISCUSSION NaCl seems to have a biphasiceffect on the activityof thromboplastin detectedin the one stage clottingassay in agreementwith earlier observationson the conformationof thrcmboplastin(6). The resultsobtained with human monocytesand rat brain and lung extractsindicatethat the effect of NaCl depends on the activityof the preparation.The concentrationof the samples should not be such that the clottingtime is lower than 30 seconds, since the measurementof such a rapid clottingtime is imprecise.NaCl is inhibitoryat concentrationsabove 0.18 % (w/v).The two stage coagulation assay requiresthe additionof phospholipidsfor optimal activity (7). Thus an excess of lipids seems to overcomethe inhibitionby NaCl in the one stage assay, and so the interactionof the apoproteinwith phospholipidsmight be affectedby NaCl (6). The effect of NaCl on the clottingtime of plasma indicatesthat many reactionsinvolvinginteractionof clottingfactorswith phospholipidsare affected.In many cases, particularlywith cell extracts, when the thromboplastinactivitycould be low, it is of great importanceto determinewhether the presenceof NaCl is desirableor whether the additionof lipid is required. ACKNCWLEIXEMENTS This work was supportedby the Medical ResearchCouncil,U.K. REFERENCES 1.
RIVERS, R.P.A.,HATHAWAY,W.E. and WESTON, W.L. The endotoxininduced coagulantactivityof human monocytes.Br. J. Haematol.30, 311-316, 1975.
2. PRYDZ, H. and ALLISON.A.C. Tissue thron-boplastin activityof isolated humen n-onocytes. Thrombos.Haen-ostas. (Stuttg.)39, 582-591,1978. 3. EDWARDS,R.L. and RICKLES,F.R. The role of human T cells and T cell productsfor monocytetissue factor generation.J. of Inmunology.125, 606-609,1980. 4. LMlNI, P. and DEAN R.T. An intracellularpool of the procoagulant thromboplastinin human monocytes.Thrcanbosis Res. 40, 199-205,1985. 5. HVATUM,M. and PRYDZ, H. Studieson tissue throrrboplastin. I Solubilization with sodium deoxycholate.Biochim.et Biophys.ACta 130 92-99, 1966. 6. HOWELL,R.M., REZOAN,R.H. and SEPPES,P.R. Conformationaltransitionsof thromboplastinapoproteinfrom pig brain. FEBS Letters 97, 23-26, 1979. 7. BOLHUIS,P-A., SYIVA-STEENLAND, R-M-R.,TUTUARIMA,J.A., HISCHE, E-A. and VAN DER HELM, H.J. Ccnnparison of the spectrophotcmetric determinationand the two-stagecoagulationassay of tissue factor activity.ThrombosisResearch,27, 429-434,1982.
BRIEF
COMMUNICATION
DEPENDENCEOF THROMBOPLASTINACTIVITY IN CELL EXTRACTSUPON SALT CONCENTRATION P. LWNI and R.T. DEAN Cell BiologyResearchGroup, Departmentof Applied Biology, Brunei University,UxbridqeUB8 3PH, U.K. (Received 28.8.1986; Accepted in revised form 12.3.1987 by Editor M.C. Scrutton)
IhmODUCTION Thromboplastinactivityin the one step clottingassay has been traditionally measuredby preparingthe sample (in which activityis to be assessed)in a buffer containing0.9 % (w/v)NaCl (l-4).We show here that the concentraticn cf NaCl in such an assay has a dramaticeffect on the activitydetected,and should be optimisedfor each individualsource (tissueor celullar)of samples. MATERIALSAND METHODS Preparationof cell and tissue extracts.Human monocytesfrom peripheral blood were isolatedfrcm cell separatorresidues (NorthLondon Transfusion Centre) by sedimentationat lg and subsequentcentrifugationthrough Ficoll-Paque(Pharmacia,Uppsala,Sweden),as describedbefore (5). Cells were used after adherencein the absenceof serum, or after subsequentstimulation of adhered cells for 3 hours with 10 pg/ml Lipopolysaccharide (LPS) from E. coli (DifcoLabs., Detroit,U.S.A).After the incubationperiod; the cells were washed 4 times with phosphatebufferedsaline,harvestedin 50 n-MVerona1 buffer pH 7.4 and frozen at -70' C. The frozen cell extractswere homogenized using a syringewith a 25gx5/8"needle.Brain and lungs from Wistar rats were homogenizedin a mechanicallydriven glass-Teflonhcrqenizer. Homogenates were centrifugedfcr 20 min at 3000xgand the supernatantdiscarded.The pelletswere resuspendedin Verona1buffer and frozen at -70' C. The frozen extractswere homogenizedusing a syringewith a 21gxl l/2" needle. Measurementof thrcmboplastinactivity.Thrcmboplastinactivitywas measured in a one step clottingassay. 0.1 ml of cell or tissue extractwas used to measure its ability to shcrtenthe clottingtime of 0.1 ml of human plasma. The reactionwas startedwith the additionof 0.1 ml of 30 r@lCaCl,. Results are expressedin arbitraryunits derived by comparingthe sample clottingtime with those producedby dilutionsof brain standardthron-boplastin (National ReferenceLaboratory,Manchester,U.K.) which is taken to have 100 units/ml. Rabbit brain cephalin (Sigma,Dorset, U.K.) was reconstitutedwith 10 ml of water and 20 ~1 of the suspensionused when specified.Each determinationwas Key wcrds: Thromboplastinassay, human rronocyte, tissue factor. 737
738
THROMBOPLASTIN AND CELL EXTRACTS
Vol. 46, No. 5
water and 20 ~1 of the suspensionused when specified.Each determinationwas performedin duplicate. The activityassayedwas shown to be thromboplastinby the imnunologicaland enzymaticcriteriawe describedpreviously(4). RESULTS Throrcboplastin activityof human n-onocyte extractshas been measured in the presence of increasingconcentrationof NaCl. Using a preparationwith a clottingtime over 30 seconds (samplesB and C, Fig.1) the activityappearsto decreasewith increasingconcentrationsof NaCl; if the sample is concentrated so that the clottingtime is under 30 seconds (sampleA, Fig.11 the presence of salt hardly affects the determinationand can even be stimulatory,but the meausurenient of clottingtimes under 30 second is very inaccurate(Fig 1).
120 -
.> 80 J : m c ._ C .-
60
-
40 -
; :
20O-r
I
0
0.9
I
I
1.8
2.7 NaCl
3.6
I
4.5
I
Thrcmboplastin activityin LPS stimulatedhuman moncqteswas measuredin the presenceof the specifiedfinal concentrations of NaCl (A). The same extract was diluted 5 times (B) and 10 times (C).
5.6
%,
FIGURE 1 Inappropiateconditionslead to drastic underestimationof activityand in some cases, dependingon the sample,the activitycould go completely undetected.In the absence of NaCl, we have been able to measure readily the thron-boplastin activityof freshlyadheredmonocytes, which in the presenceof salt had shown hardly any activity.Moncxyteswere allowed to adhere for 1, 2 and 3 hours in the absenceof serum, and the thrc&oplastin activities (units/q prot. + S.D.; n=3) were determined.In the presenceof NaCl the activitywas undetectedfor the first 2 hours, and 0.58 + 0.1 after 3 hours. In the absenceof NaCl it was 1.1 + 0.1, 2.8 k 0.1 and 6.20 k 0.4 (at 1, 2 and 3 h respectively).If rabbit brain cephalinwas added to the assay of extracts of stimulatedmonocytesthe values rexrained at control levels until a salt concentrationof 0.36 % (W/V),and decreasedonly at higher concentration.The cephalinby itself has no procoagulantactivityin the conditionsused for the
739
THROMBOPLASTIN AND CELL EXTRACTS
Vol. 46, No. 5
assay. Using crude rat lung or brain extractsa similardependenceof activity on salt concentrationis observed:at the concentrationof NaCl routinelyused in the assay (0.3 % (w/v))the activitymeasured is less than maximal (Fig. 2), but lower salt concentrationsgive higher activity.With the British comparativethronboplastinpreparation,which containsa much higher proportionof lipids,differentresultsare obtained (Fig 2); IWaClin the assay is necessaryfor optimalactivity.
w
100 -
.Z A 80> ..I:
The thromboplastin activityof rat brain (0) and lung (A) extracts,and of standard thromboplastin(0) was measuredin the presenceof increasing concentrations of NaCl.
60-
a, .E 40.a E
20O0
0.9
1.8 2.7 3.6 NaCl %,
4.5
5.4
FIGURE 2 In a separateexperimentthe clottingtime of recalcifiedplasma was found to be much inhibitedin the presenceof NaCl (Table1). TABLE 1 Effect of NaCl on plasma clottingtime Clottingtime (minutes) NaCl
(% w/v)
0.18
0.27
0.36
0.45
0.54
0.63
0.72
Plasma I
5.75
6.75
7.00
8.50
9.33
11.45
14.50
Plasma II
7.20
8.50
10.50
11.00
11.50
12.50
15.00
Plasma III
7.00
8.25
10.25
11.20
15.50
17.00
18.00
The clottingtime of plasma from healthydonors was measuredas described,
740
THROMBOPLASTIN AND CELL EXTRACTS
Vol. 46, No. 5
using Verona1 buffer insteadof a tissue factor sample in the presenceof the specifiedconcentrationsof NaCl. DISCUSSION NaCl seems to have a biphasiceffect on the activityof thromboplastin detectedin the one stage clottingassay in agreementwith earlier observationson the conformationof thrcmboplastin(6). The resultsobtained with human monocytesand rat brain and lung extractsindicatethat the effect of NaCl depends on the activityof the preparation.The concentrationof the samples should not be such that the clottingtime is lower than 30 seconds, since the measurementof such a rapid clottingtime is imprecise.NaCl is inhibitoryat concentrationsabove 0.18 % (w/v).The two stage coagulation assay requiresthe additionof phospholipidsfor optimal activity (7). Thus an excess of lipids seems to overcomethe inhibitionby NaCl in the one stage assay, and so the interactionof the apoproteinwith phospholipidsmight be affectedby NaCl (6). The effect of NaCl on the clottingtime of plasma indicatesthat many reactionsinvolvinginteractionof clottingfactorswith phospholipidsare affected.In many cases, particularlywith cell extracts, when the thromboplastinactivitycould be low, it is of great importanceto determinewhether the presenceof NaCl is desirableor whether the additionof lipid is required. ACKNCWLEIXEMENTS This work was supportedby the Medical ResearchCouncil,U.K. REFERENCES 1.
RIVERS, R.P.A.,HATHAWAY,W.E. and WESTON, W.L. The endotoxininduced coagulantactivityof human monocytes.Br. J. Haematol.30, 311-316, 1975.
2. PRYDZ, H. and ALLISON.A.C. Tissue thron-boplastin activityof isolated humen n-onocytes. Thrombos.Haen-ostas. (Stuttg.)39, 582-591,1978. 3. EDWARDS,R.L. and RICKLES,F.R. The role of human T cells and T cell productsfor monocytetissue factor generation.J. of Inmunology.125, 606-609,1980. 4. LMlNI, P. and DEAN R.T. An intracellularpool of the procoagulant thromboplastinin human monocytes.Thrcanbosis Res. 40, 199-205,1985. 5. HVATUM,M. and PRYDZ, H. Studieson tissue throrrboplastin. I Solubilization with sodium deoxycholate.Biochim.et Biophys.ACta 130 92-99, 1966. 6. HOWELL,R.M., REZOAN,R.H. and SEPPES,P.R. Conformationaltransitionsof thromboplastinapoproteinfrom pig brain. FEBS Letters 97, 23-26, 1979. 7. BOLHUIS,P-A., SYIVA-STEENLAND, R-M-R.,TUTUARIMA,J.A., HISCHE, E-A. and VAN DER HELM, H.J. Ccnnparison of the spectrophotcmetric determinationand the two-stagecoagulationassay of tissue factor activity.ThrombosisResearch,27, 429-434,1982.