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Anticoagulant activity of peptides from the human placenta
~HI;OXBO3I3 RESEXRCH 16; 689-69$ Printed Per$anlon Press L:d.ly;q.
Keczys$aw
in Grear
Britain
UszyAski
(Received 2.4.1979; in revised form 7.8.1979. Accepted by Editor H.C. Godal)
A ocptil e with ecticoagulont activity ~~3s isolated fro2 a human placenta hoaogenate.It was found that this ptptidc prolongs cephalin-kaolin clotting tine noderately and thrombin clotting time of human plssza considersbly.Thc peptide was purified by a three-step procedure; l/ dialysis of the homogenate, 2/ estraction of the peptides fro= the filterable fraction, 3/ separation of the peptide nixturF on 8 Doses column.
In the humn plecents there are several substances which affect the clotting systea and fibrinolysis in vivo. These substances fall into opposing groups: procosgulante vs.anticosgulants 2nd fibrinolysis activators vs. fibrinolysis inhibitors. Tissue throzboplastins are known to be procoagulants in the placental and decidual ti ssues where they dominate the anticoagulant sctivity /3/. Sinilarly,the olasninogen activator inhibitors /uro?-inase inhibitors/ dominate plasminogen activet:3r5 /l/. The presence of an anticoagulant in the human placenta :'I a s first reported by Uszydski and Abildgaard /2/. LQte?, Uszyriski 2nd Godol /3/ found that the sntico3gulant fron the
huEen placenta inhibits extrinsic cosgulgtion by inhibiting the complex consisting of fen+or VII.tissue throzboplsstins, 689
;,; Ja,TZ31
.i“T
‘,.-I ;iY
I
yEyLL,~<-J5
l-The human plscenta homogenstes and the filterable frstion from the homogenates were preoared as I'ollows: placent3s obtained from full-term deliveries ..vere,afterremoval :I‘ the connective tissue,washed in tap rtster and iomediately hcmogenize3 for 5 minutes at 19,17OC _-pa in 3 Xaring blender /Krups 1, F.P?,S./ using 2 31 of C.C1 >i!Tris/FJ,:lbluffer,pZ 7.25, per gram of tlaoue.ifter 30 minuzee' centrifug3tlon it 9,G;sG rpm 111 a cooled centrifuge /type ?3 13, ?JSS Lt.?., zon:or., -2cO:_ England/ the cleer supern3tsnt .*:3spooled 3~: k?pt 3: The filtersble fraction of the pIac?nt5 l?O2C~e?-3t~ ‘.$3f Ohtanned together with other low-zo:ecul.3r components b;; di3lysis of the homogenate against 3 l?-fold volu?e of deionized water in dialysis bags /Union :?rbi:io,,iicago,uS~./3t ~4~: for 48 hours. 2. Preparation of the peptide fraction The filterable fraction of the human placenta homogenate ~32 lyophilized in a vacuum sgstem /sotavspor, Ei. x!rvI, "V _..& S,:litzerland/. The peptides were extrscted from the sediment .~ith A mixture of methanol,butanol,and ethyleneglycocoll /2:2:1/. The pspti.les ::'eredesalted by filtration thro uzh Yepha:ex G-25 gel /5Ccm x 20 cm column/. 3. Measurement of the peptide concentration In .a system of 0.2.21 of the sample + 0.5 ml of the reagent with ninhydrin /25G ml 4 M CI-! lGCE;a x >Fi,G, p;i 5.5, + 7jC ~1 2 methoxyethanol C, H, O7 + 2ag-r' .Inhydrin'+ C.0 g SbC1, x 2H2G ?? 2.5 ml 4 M $H3COOka x 3 X,0, pIi 5.5/ a colour reaction was induced at 100 C for 30 min.-and the extinction ~~39 resd at a wave-length of 546 nm. The peptide concentration ~3s expressed in leucine units calculated by compsring the sample extinctions .tiith the a?andaA curve prep3red from ieucine / 13-g triglycine = 1 leucine un1ii. 4.Fractionatlon of the peptide mixture The peptides dissolved in 1 ml pyridine-acetic buffer at pX 1.96 were placed on a Dowex-50 x S column /17cm x 0.9 cm/ 3nd eluted :vitQ 3 flow rate of SO ml/h,at a temperature of the coThe lumn of 50 C,and a linearly increasing pH /1.96-+5.G/. peptide concentration in the eluate / 3ml portions/ ~39 determined, the buffer was then removed by evaporation in 3 vacuum system snd the effect of the fraction on the clotting system was then investigated.P inelly the number of peptides.in the fraction was determined by fingerprinting.Descending chromatography of the heterogeneous frsctions was carried out on Machery-Nagel paper No. 218 in the following system:pyridine -butanol-concentrated acetic acid-distilled water /l,OO:150:30 :120/. 5. Two-dimensional peptide mapping - fingerprinting The peptides investigated /50 leucine units/ were placed on a gel plate Sil G-25 /&ERCK,Darmstadt,F.R.G./ and electrophoresis was performed in a buffer et pH 6.5 consisting of 1Oml
Vol.
16, No. j/6
.?.ST.ICOhCVLAXT PLXCESTX
PEPTIDE
631
+ 2321 EPO. Tne 31ate pyridine + 1 ml concentrated ,CX,CC\;'r: zvgs then dried and ascending chro=atogrsphy X5s csrried out in 3 !direction perpendicular to thst of the electrophoresis; the buffer consisted of 80 ail butanol + 20-l distilled ,tiater."he plates were dried,sprayed with ninhydrin solution /,J.25,ninhydrin in 95 ml butenol/, and keFt at a teapersture of 50 C for 20 ninutes in order to induce a colour reaction. r The cephalin - kaolin time o. This was determined in the following system: 0.1 ml of the saeple - 0.2 ml of the PTT reagent /Bering+ferke,Uarburg-Lahn, F.R.G./, 1 ininute incubation, + 0.2 ml standard olasza /Beringwerke,Xsrburg-Lahn,F. R.G./, 2 min.incubatioc,+ 0.i ml ,CaCi, 0.05 Y. 7. Antithrornbin activity The activity was measured in the following system: 0.1 ml of the sample + 0.1 ml thrombin /6 units NISI/ produced by 3eringwerke,?Aarburg-Lahn, F.R.G., + 0.1 ~1 standard plgsaa. S. Thromboelastography r q'Kellirre" . __ apparatus ~4s used for the thro3boelastogrsohy which wsz carried out in the following system: iI.2 ml 2% bovine fibrinogen /Beringwerke,Marburg-Lahn, F.R.G./, 0.05 ml veronli buffer at pH 7.75, 0.05 ml of the peptides investigated, snd 0.06 ml thrombin /6 NIH units/. RESULTS The peptide mixture obtained from the filtrable fraction of the human placenta homogenate was separated ir.to four fractions /Fig.l/ the first of which contained one peptide /Fig.2/ .$/hereas the remaining fractions contained fro% 3 to 4.
Figure 1 of the peptide Tixture from the hu,len plscente; colu2l.n 9 ,P linearly incressing ;;F; /1.96-c 5.0/ Zi"?-
Frsctionstion 3oxe.x-50
,J;Pnt iA_._".
/-/
Electrophoreais
/+/
c
3oil
zou
w----
lu 3
JU C
I I
\ \
I \
ct_--.---._
--
__.-___
_
Figure 3 Throaboclastography perforord with difl"srrr.t qusntities of activa p?ptidr /Fraction 1/ in the system /z-r T.Iateriql and Methods/. c - control, u - peFtides In leucine units, a-amplitude of thrmboelasto,zraz. >r ‘ S.E. the elssticitv decreases with an incroaqn i.A peptide concentration /sysYtea .:./ 3~3 my 53 ectireigi~li~lnated /systea: B qnd C/.
Vol.l6,No.jj6
_xXTICOAGCLAXT PLACEXTX
PEPTIDE
693
Not all thr fractions had an equal rff-ct on coeguletion: e/ only Fraction, 1 ~33 found to have a high coegu1er.t nctivitjprolonging both cephelin-kaolin and throrcbin tln?s /Tables I ah.dII/ and r-tarAing clot foraotion /Fig.3/, /b/ the ??p?ides of Fractions 2,3, am? 4 separated by means of descendins chrometogrephg had relatively less antlcoa+ont effect; e length?nine of the respactiv? clotting times m the ccphalin-kaolin test artd thAe throrrbin test was observed where the ??:-:ije concentration was ten tir?as hither than ir:Fractier: 1. It was calculated that fro9 lo of plazer.ta ab3lJt _I,CC'; leucine units of peptides are obtorned of Aich only 1G" /300 leucir,e units/ was found to h&eve a high anticca::_ilar.t activity.
The present study is a continuation of investigaticr.5 3i: coagulant inhibitors if, the human placenta /2,3,(,/. It S:rc TC-W been shown that the filtrable fraction of a placenta hczz:cnate contains a peptide with a high acticoegulcnt oc?ivit;. which CSUSPC ceF~.e'_i_~.-~:solin ti,l.: 8 mr'erate len$.Ceclng Of
anticoagulant activi!; :k;lch ;;rolonga TeptiFies with an cephalin-kaolin tine snd throzbin clottins tlae occur in human placenta homogenates. REFERENCES l.USZyNSKI,!J. and U~ZY~~S%~-FOLE~~ISK~.,R.Rlasminogen activgtor and urokinase inhibitor in zyometriun,placenta, and amiotic fluid. As.;. Obstet.Cynec., z,lC!41,1g69. 2.uSzYiiSicI,1vI. and hBILDGAXR~,U. Separation and chsrscterisation Of two fitirinolytic inhibitors froc lunar! pl5ceKltB. Thrombos.!Iiathes.S,qeaorrh.,2'j,580,1971. --3.USZYNSKI,M. and GCIZ\L,F;.Z.P.ninhibitor of fgctor human placenta. Scz+nd.;.Haemat., 3, 392,1971.
VII
from
J.USZYNSKI,M. Blood coagulation and fibrinolytic system activator inhibitors in the human placenta and zyOmetriUm. /In Folish/.Roczn.A.M.~ia~ystok,16,159,1971. 5.USZyNSKI,M. Peptides with anti-urokinase activity from human placenta. / In preparation/ 6.NILSSON,I.M.,ASTEDT,B.,XEDNER,U. and BEREZIN,2: IntraUterine death and circulating anticoagulant /"antlthromboplastin"/. Acts Med.Scand., 197, 153, 1975.
Keczys$aw
in Grear
Britain
UszyAski
(Received 2.4.1979; in revised form 7.8.1979. Accepted by Editor H.C. Godal)
A ocptil e with ecticoagulont activity ~~3s isolated fro2 a human placenta hoaogenate.It was found that this ptptidc prolongs cephalin-kaolin clotting tine noderately and thrombin clotting time of human plssza considersbly.Thc peptide was purified by a three-step procedure; l/ dialysis of the homogenate, 2/ estraction of the peptides fro= the filterable fraction, 3/ separation of the peptide nixturF on 8 Doses column.
In the humn plecents there are several substances which affect the clotting systea and fibrinolysis in vivo. These substances fall into opposing groups: procosgulante vs.anticosgulants 2nd fibrinolysis activators vs. fibrinolysis inhibitors. Tissue throzboplastins are known to be procoagulants in the placental and decidual ti ssues where they dominate the anticoagulant sctivity /3/. Sinilarly,the olasninogen activator inhibitors /uro?-inase inhibitors/ dominate plasminogen activet:3r5 /l/. The presence of an anticoagulant in the human placenta :'I a s first reported by Uszydski and Abildgaard /2/. LQte?, Uszyriski 2nd Godol /3/ found that the sntico3gulant fron the
huEen placenta inhibits extrinsic cosgulgtion by inhibiting the complex consisting of fen+or VII.tissue throzboplsstins, 689
;,; Ja,TZ31
.i“T
‘,.-I ;iY
I
yEyLL,~<-J5
l-The human plscenta homogenstes and the filterable frstion from the homogenates were preoared as I'ollows: placent3s obtained from full-term deliveries ..vere,afterremoval :I‘ the connective tissue,washed in tap rtster and iomediately hcmogenize3 for 5 minutes at 19,17OC _-pa in 3 Xaring blender /Krups 1, F.P?,S./ using 2 31 of C.C1 >i!Tris/FJ,:lbluffer,pZ 7.25, per gram of tlaoue.ifter 30 minuzee' centrifug3tlon it 9,G;sG rpm 111 a cooled centrifuge /type ?3 13, ?JSS Lt.?., zon:or., -2cO:_ England/ the cleer supern3tsnt .*:3spooled 3~: k?pt 3: The filtersble fraction of the pIac?nt5 l?O2C~e?-3t~ ‘.$3f Ohtanned together with other low-zo:ecul.3r components b;; di3lysis of the homogenate against 3 l?-fold volu?e of deionized water in dialysis bags /Union :?rbi:io,,iicago,uS~./3t ~4~: for 48 hours. 2. Preparation of the peptide fraction The filterable fraction of the human placenta homogenate ~32 lyophilized in a vacuum sgstem /sotavspor, Ei. x!rvI, "V _..& S,:litzerland/. The peptides were extrscted from the sediment .~ith A mixture of methanol,butanol,and ethyleneglycocoll /2:2:1/. The pspti.les ::'eredesalted by filtration thro uzh Yepha:ex G-25 gel /5Ccm x 20 cm column/. 3. Measurement of the peptide concentration In .a system of 0.2.21 of the sample + 0.5 ml of the reagent with ninhydrin /25G ml 4 M CI-! lGCE;a x >Fi,G, p;i 5.5, + 7jC ~1 2 methoxyethanol C, H, O7 + 2ag-r' .Inhydrin'+ C.0 g SbC1, x 2H2G ?? 2.5 ml 4 M $H3COOka x 3 X,0, pIi 5.5/ a colour reaction was induced at 100 C for 30 min.-and the extinction ~~39 resd at a wave-length of 546 nm. The peptide concentration ~3s expressed in leucine units calculated by compsring the sample extinctions .tiith the a?andaA curve prep3red from ieucine / 13-g triglycine = 1 leucine un1ii. 4.Fractionatlon of the peptide mixture The peptides dissolved in 1 ml pyridine-acetic buffer at pX 1.96 were placed on a Dowex-50 x S column /17cm x 0.9 cm/ 3nd eluted :vitQ 3 flow rate of SO ml/h,at a temperature of the coThe lumn of 50 C,and a linearly increasing pH /1.96-+5.G/. peptide concentration in the eluate / 3ml portions/ ~39 determined, the buffer was then removed by evaporation in 3 vacuum system snd the effect of the fraction on the clotting system was then investigated.P inelly the number of peptides.in the fraction was determined by fingerprinting.Descending chromatography of the heterogeneous frsctions was carried out on Machery-Nagel paper No. 218 in the following system:pyridine -butanol-concentrated acetic acid-distilled water /l,OO:150:30 :120/. 5. Two-dimensional peptide mapping - fingerprinting The peptides investigated /50 leucine units/ were placed on a gel plate Sil G-25 /&ERCK,Darmstadt,F.R.G./ and electrophoresis was performed in a buffer et pH 6.5 consisting of 1Oml
Vol.
16, No. j/6
.?.ST.ICOhCVLAXT PLXCESTX
PEPTIDE
631
+ 2321 EPO. Tne 31ate pyridine + 1 ml concentrated ,CX,CC\;'r: zvgs then dried and ascending chro=atogrsphy X5s csrried out in 3 !direction perpendicular to thst of the electrophoresis; the buffer consisted of 80 ail butanol + 20-l distilled ,tiater."he plates were dried,sprayed with ninhydrin solution /,J.25,ninhydrin in 95 ml butenol/, and keFt at a teapersture of 50 C for 20 ninutes in order to induce a colour reaction. r The cephalin - kaolin time o. This was determined in the following system: 0.1 ml of the saeple - 0.2 ml of the PTT reagent /Bering+ferke,Uarburg-Lahn, F.R.G./, 1 ininute incubation, + 0.2 ml standard olasza /Beringwerke,Xsrburg-Lahn,F. R.G./, 2 min.incubatioc,+ 0.i ml ,CaCi, 0.05 Y. 7. Antithrornbin activity The activity was measured in the following system: 0.1 ml of the sample + 0.1 ml thrombin /6 units NISI/ produced by 3eringwerke,?Aarburg-Lahn, F.R.G., + 0.1 ~1 standard plgsaa. S. Thromboelastography r q'Kellirre" . __ apparatus ~4s used for the thro3boelastogrsohy which wsz carried out in the following system: iI.2 ml 2% bovine fibrinogen /Beringwerke,Marburg-Lahn, F.R.G./, 0.05 ml veronli buffer at pH 7.75, 0.05 ml of the peptides investigated, snd 0.06 ml thrombin /6 NIH units/. RESULTS The peptide mixture obtained from the filtrable fraction of the human placenta homogenate was separated ir.to four fractions /Fig.l/ the first of which contained one peptide /Fig.2/ .$/hereas the remaining fractions contained fro% 3 to 4.
Figure 1 of the peptide Tixture from the hu,len plscente; colu2l.n 9 ,P linearly incressing ;;F; /1.96-c 5.0/ Zi"?-
Frsctionstion 3oxe.x-50
,J;Pnt iA_._".
/-/
Electrophoreais
/+/
c
3oil
zou
w----
lu 3
JU C
I I
\ \
I \
ct_--.---._
--
__.-___
_
Figure 3 Throaboclastography perforord with difl"srrr.t qusntities of activa p?ptidr /Fraction 1/ in the system /z-r T.Iateriql and Methods/. c - control, u - peFtides In leucine units, a-amplitude of thrmboelasto,zraz. >r ‘ S.E. the elssticitv decreases with an incroaqn i.A peptide concentration /sysYtea .:./ 3~3 my 53 ectireigi~li~lnated /systea: B qnd C/.
Vol.l6,No.jj6
_xXTICOAGCLAXT PLACEXTX
PEPTIDE
693
Not all thr fractions had an equal rff-ct on coeguletion: e/ only Fraction, 1 ~33 found to have a high coegu1er.t nctivitjprolonging both cephelin-kaolin and throrcbin tln?s /Tables I ah.dII/ and r-tarAing clot foraotion /Fig.3/, /b/ the ??p?ides of Fractions 2,3, am? 4 separated by means of descendins chrometogrephg had relatively less antlcoa+ont effect; e length?nine of the respactiv? clotting times m the ccphalin-kaolin test artd thAe throrrbin test was observed where the ??:-:ije concentration was ten tir?as hither than ir:Fractier: 1. It was calculated that fro9 lo of plazer.ta ab3lJt _I,CC'; leucine units of peptides are obtorned of Aich only 1G" /300 leucir,e units/ was found to h&eve a high anticca::_ilar.t activity.
The present study is a continuation of investigaticr.5 3i: coagulant inhibitors if, the human placenta /2,3,(,/. It S:rc TC-W been shown that the filtrable fraction of a placenta hczz:cnate contains a peptide with a high acticoegulcnt oc?ivit;. which CSUSPC ceF~.e'_i_~.-~:solin ti,l.: 8 mr'erate len$.Ceclng Of
anticoagulant activi!; :k;lch ;;rolonga TeptiFies with an cephalin-kaolin tine snd throzbin clottins tlae occur in human placenta homogenates. REFERENCES l.USZyNSKI,!J. and U~ZY~~S%~-FOLE~~ISK~.,R.Rlasminogen activgtor and urokinase inhibitor in zyometriun,placenta, and amiotic fluid. As.;. Obstet.Cynec., z,lC!41,1g69. 2.uSzYiiSicI,1vI. and hBILDGAXR~,U. Separation and chsrscterisation Of two fitirinolytic inhibitors froc lunar! pl5ceKltB. Thrombos.!Iiathes.S,qeaorrh.,2'j,580,1971. --3.USZYNSKI,M. and GCIZ\L,F;.Z.P.ninhibitor of fgctor human placenta. Scz+nd.;.Haemat., 3, 392,1971.
VII
from
J.USZYNSKI,M. Blood coagulation and fibrinolytic system activator inhibitors in the human placenta and zyOmetriUm. /In Folish/.Roczn.A.M.~ia~ystok,16,159,1971. 5.USZyNSKI,M. Peptides with anti-urokinase activity from human placenta. / In preparation/ 6.NILSSON,I.M.,ASTEDT,B.,XEDNER,U. and BEREZIN,2: IntraUterine death and circulating anticoagulant /"antlthromboplastin"/. Acts Med.Scand., 197, 153, 1975.