I-_, b ‘2
~P~TELL!l?FIBRlX%ENIiZPLASW
I, pp. 161-l,i, Pergamon Press, -
I
??
19,j
Inc.
DEF'l33~~~
IXXS AND IX URENIC PATIENTS BEo?JZ AN2 AFTER H3xx)IALk-SIS
Kxns Johnsson Departmantof Elcd CoagulationResearch, Karolinska Sjukhuset, 104 01 Stockholm, &eden.
(Received
3.3.1975;
Accepted
in revisecl form 19.5.1975. H.C. Godal)
by Editor
Platelet fibrinogenin dogs was estimated inrnunologically before and during Dafibrase-induceddefibrinogenationof plasma. The antigen was identifiedby imnunoelectrophoresisand by SDS gel electrophoresisof reduced and non reduced sanples. The concentrationof platelet associated fibrinogenremained unaffected by the Defibrase treatment.The results indicate that neither fibrinogennor fibrin(-ogen)degradationproducts (FDP) are interchangedbetween plasma and the platelet. Uremic patients showed a normal concentrationof platelet fibrinogen. During the course of hdalysis no decrease in platelet fibrinogen content was observed in uremic patients.
IXIRCDUClTCN Fibrinogen in marsralianblcod platelets has been isolated and quantified by several workers (1,2,3,4,5).This intraplatelet-associated fibrinogenprobably related to the intracellulargranulae (61, has in some respects been distinguisablefrom plasma fibrinogen.Ithas been reportedthatithas
a
slower clotting with throsbin, a differentrobility in SDS gel electrophcresis and in agar gel irrmunoelectrophoresis (2,5). me twopro&ins
seemtohaxsz
the samx sedimentatim Coefficients,total amino
acid ccmpositionand Wterminal amino acids (2). SCE investigatorsclaim 161
that
tkey
are
t.k
~~ucts
cf fter plasr?;in vitro of platelet fibrinogen,hmever, &gradation products zere i&ztified. These fitdir.gs indicate,that platelet fibrinogenis not degrackd by plasrkn dluringCefibrase treatmaM,.that platelet fibrinogenis degraded by plasrih in vitro and that ED? not are interchangedbeti~eenplatelets and slsm. In spite of that Cefibrase in vitro was capable to coagulate platelet fibrincgen,'bysplitting of fibrinopeptide-A,platelet fibrinogenremaked quantitatively unaffected during Defibrase treatrrent of the dogs. This discrepancy rnightsuggestthatoertain properties of the platelet fibrinogen
is changed
when it is released from the,platelets.Suciza statemant is in accordancewith a reported enzymatic activity in the platelets ~~hichin a reversiblemnner reducescertain disulfide bonds in the fibrinogenmlecule
(32). In this reduced
form fibrinogenis very difficult to coagulatewith throrrbinenzynes. Previous results have shm
that the administrationof substances capable
of inhibiting the platelet function, including platelet release reaction to defibrinogenisedindividuals, significantlyincrease the bleeding temkncy (31,33).It would, therefore,appear probable that in a plasma defibrinogenatedindividual plateletfibrinogenis of intact hemostatic capacity. Also
importance in the maintenanceof an
in vitro, the platelet fibrinogenseems to
be of certain significance for the platelet behaviour. Solurnand Stomrken (34)have pointed out that fibrinogenserves as an important co-factor for the ADP-rmadiated platelet aggregation.It is hw.?everpossible to aggregate plateletswith ADP even in the abscence of fibrinogen
in plasma in dogs trea-
ted with Defibrase,if the platelets are pre-exposed to thror&in (24).This mightbe a consequenceof a thrmbin-mediated release ofplateletfibrinogen whicfiin tum stistitutes for plasma fibrinogenas a co-factor for ADP, Keenah and Solum (8) and Holma et al (9) have shun that platelet fibrinogen my be released in vitro by thrtiin. In order to investigatewhether there was any release of platelet fibrinogenin viva, this content w=
examinedin uremicpatients before and after
hemdialysis. Lindsay (35) has previously shmn that an adhesion of platelets to the thrtiogenic surface of the dialysis filter takes place during extracorporeal circulation,despite a general heparihisation.It also appeared to us prabable that sane platelets do not adhere pemanently to the dialysis rrerrbrane, but are activated in connectionwith the wntact blood-dialysis filter, and thus activated, release part of their contents. Ma were Iunableto &serve any &crease in fibrinogencmaentration in platelets during hemdia-
lysis. This need net necessarilyexcluck a release of platelet fibrinogendluring dialysis as it is still not established-&ere the platelet fibrinogenis formed. It has been speculatedtiether the platelet fibrinogenmay be formed in circulatingplatelets (36) and in that case a release might be overlocked by this procedure. Ms consider that further stties of this aspect are iqortar-k,and we believe that the plasma defibrinogenisedanimal may well be a valuable experiment subject, possibly :tiththe help of labelled precursors. Uremic patients, irrespecti= etiology, have a documentarypathological platelet function and an increasedbleeding tendency (37).Gur results
estab-
lish that the content of platelet fibrinoqenin uremic shjects dces not differ from that of healthy subjects. A leered platelet fibrinogencontent is, therefore,not the caluseof this abnormal platelet faction, and preliminary results have shcklnthat fibrinogenis released from "uraemicplatelets" as well from 'normal platelets"with a cannercial cephalin (lhrombofaxR)(38) whicfiis capable to aggregateplatelets in vitro (39,40).
This workwas supportedby grants fromthe Swedish ~MadicalFksearch Comcil
(No. 19X-520). I express mj sincere gratitude to Dr. Margareta
Bloti&% for valuable discussion and for placing facilities.
1. NACHMAN, R.L. Irmoslologic studies of platelet protein. Blood. 25, 703, 1965. 2. SOLUM,N.O. andUIPACIUK, S. Eovine plateletproteins. III. SoXEproperties of platelet fibrinogen.Tbrorrb.Diath. Haemorlhag. 21, 428, 1969. 3. KARACA, M., NILSSON, I.M. and HELNER, U. Quantitativedeterminationof platelet fibrinogen.J. Lab. Clin. &kd. 77, 485, 1971. 4. KEENAN, I.P. Platelet fibrinogen.I. Quantitatiglusing fibrinogensensitized red cells. Med. Lab. Technol. 29, 71, 1972. 5. CANGULY, P. Isolation and some properties of fibrinogen from human blood platelets. J. Biol. C&m. 247, 1809, 1972. 6. NAcZEiM?B,R.L. and MARCUS, A.J. Immunologicalstudies of proteins associatedwith the s&cellular fractionsof throrrbastenic andafibrinogmc platelets. Brit. J. Haematol. 15, 181, 1968. 7. mLITM:, R-P.,-, T., and WITFELL, B.A. Platelet and plasma fibrinogens are identical gene products. ScienQ. 185, 368, 1974.
,,
9.
.2
i
.
‘-:
)
.
._*
*
’
R., SIZE%, J.J.,b$im,, E.H., and HOTIIG,T. Demonstrationof platelet fibrincgensecretion via the surface connecting system. Thrombosis &search. 3, 347, 1973.
HOLME,
10. CASTALDI, P.A. and CA!ZN,J. Platelet fibrinogen.J. Clin. Pathol. 18, 579, 1965. U., EUME&K, B., 11. EGBERG, N., BLOFIBfiCX, PI.,JOHNSSCN, H., AB-, L., JOHAF+SSC%-, S.A., KcIXNGH, J., S., GijRANS~, DIENER, L., E.XESTRjfjI, McCONAGH, R., NILSSON, S.E., NORDSTR@~, S., OLSSON, P., and WIMAN, B. Clinical and experimentalstties on Reptilase. Thromb. Diath. Paen-orrhag. Suppl. 47, 379, 1971. 12. -CR, 387,
M. Purificationof antihe.mophilic globulin. Arkiv Xemi. 12, 1958.
13. BUW!&CX, B. and BLCMEWX, M. Purificationof human and bovine fibrinogen. Arkiv Xemi. 10, 415, 1956. 14. Biologisk styrkebest&minq av trombin. PharmacopeaNordica Editio Suecia. 4, 137, 1969. 15. B.U%It%CX,B. and BL@Il%CK, M. A method for the assay of factor V. Stand. J. Clin. Lab. Invest. 15, 639, 1963. 16. BERGSTR&I,X., BWCK, B., and XLEEN, G. Studies on the plasma fibrinolytic activity in a case of liver cirrhosis. Acta Med. Stand. 168, 291, 1960. 17.MBRSXBY, c., LALBZARI, P., and JOHNSON, A.J. A rapid, simple, sensitive method for masuring fibrinolyticsplit products in hm serum. Proc. Sot. ~xp. Biol. F&d. 131, 871, 1969. 18. KRISTENSON,A.A. A new method for the direct counting of the so-called blood platelets in man. Acta Med. Stand. 57, 301, 1922. 19. LAURBLL, C.E. Quantitativeestimation of proteins by electrophoresisin agarose gel containing antibodies.Anal. Biochem. 15, 45, 1966. 20. SCHEIDEGZER,J.J. Une micro-r&hode de l'imnuno-electrophorese. Intern. Arch. Allergy. 7, 103, 1955. 21. XUDRYX, B., REOTERBY, J., and WCX, B. Adsorption of plasmic fragment D to thrcxrbin modified fibrinogen-Sepharose. Thrombosis Research. 2, 297, 1973. 22. MZONAGH, J., MESSEL, H., MclX%lAGH,R.P., MURANO, G., and -CX, B. mlecular weight analysis of fibrinogen and fibrin chains by an improved sodium dodecyl sulfate gel electrophoresismethod. Biochim. Biophys. Acta. 257, 135, 1972.
23.
?;OSm,
H.L.,
K. , lclIimR,
I., CLXEELD, R-P., EC,? dZR, V.P., SPAUXDIS, and &?SHI, G.D. %easuremnt of fibri2opeptid.e .?. in
YLDEZLMX~,
G.D.,
humn blocd. J. Clin. Invest. 54, 43, 1974. 23 . EGBERG, N. and JOHNSSON, H. Platelet aggregation induced by ADP and
throrrbinin Reptilase defibrinateddcgs. Thro&osis Research. 1, 95, 1972 25. E!L&mCK, B. Personal cm?nmication. 26.
ARNESEN, H. and GODAL, H.C. Studies on the th.rmbin clotting time. The influence of fibrinogendegradationproducts. Scud. J. Haerratol.10, 232, 1973.
2; .
JAMES, H.L., BRADFORD, and GANGVLY, P. C?.xmtitation of human platelet fibrinogen.AHA Conferenceon Throtisis and Hermstasis.Dallas, Tems. Nov. 20-22 1974. PresentationNo. 1084.
28. MILLS, D. and KARPATKIN,S. Heterogenityof human fibrinogen:Possible relation to proteolysisby throbin and plasmin as studied by SDS polyacrylamidegel electrophoresis.Biochem. Biophys. Res. Cornnun. 40, 206, 1970. 29. BI.C@aCK, B. Studies on the action of thrombic enzymas on bovine fibrinogen as masured by N-terminal analysis. Arkiv Ksmi. 12, 321, 1958. 30. BLQME%CK, B. and LAURENT, T.C. N-terminal and light-scatteringstudies on fibrinogenand its transformationto fibrin. Arkiv Kemi. 12, 137, 1958 3i. OLSSON, P. and JOHNSSON, H. Interferenceof acetyl salicylic acid heparin and fibrinogendegradationproducts in haemstasis of Reptilase defibrinogenated dogs. Thrombosis Research. 1, 135, 1972. W., mI%REN, A., KOWALSKA-LXH, B. 32. BUXlE&X, B., BT0'+@%X,M., F=INER, andOLaVSON, G. Enzymatic reduction ofdisulfide-bonds in fibrincqenby the thioredoxinsystem I. Identificationof reduced bonds and studies on redoxidationprocess. 'Ihrorrbosis Research. 4, 55, 1974. 33. JOHNSSON, H. and NIKUSSON, P.M. 'Iheeffect of n-c&rate doses of chlor pmmzine on the haermstasisin dogs defibrinogenatedwith Defibrase. 'Ihrmrbceis Research. 4, 229, 1974. 34. SOLIJM,N.O. and s]co~RKEN, H. Influenceof fibrinogenon the aggregation of washed human blood platelets induced by adanosin di@-msphate,thmxrbin, collagen and adrenaline.Stand. J. Clin. Lab. Invest:17.Suppl. 84, 170, 1965. 35. LINDSAY, Rd., PRENTICE, C.R.M., E’EFGEON, D., and BURJDT, J.A. Reduction of thxmtxts formatianon dialyser nenbranes by aspirin and R.A. 233. Lancet. 3, 1287, 1972. 36. F0SIEK, O., WEGRZXNCWICZ,Z., SAWICKI, Z. and KOPEC, M. Fibrinogensynthese in Blutplattden. Folia Haematol. (Laipz.192, 553, 1969. 37. CASTALDI, P.A., F0ZENBERG,M-C., and STEWXU', J.H. The bleeding disorder of umemia. Lancet. 2, 66, 1966.
33. JOmSSP: , ii.To be publis:hed. J. and VEA’IsT~~, >I. Platelet aggregation5~ 39. IX Gc7ET2?0, G., VERGER:, throhofax(R). Studies on the mzhanism of action. Experimntia. 29, 1136, 1973.
platelet 40. DE CLEIMC, F., KUCERS, M., VEIWLElN, J. and DE GAETAPiO,G. Hm aggregationby thrcxrbofax. An electron-microscopicstudy of the sequence of events. Stand. J. Haematol. 12, 93, 1974.
~P~TELL!l?FIBRlX%ENIiZPLASW
I, pp. 161-l,i, Pergamon Press, -
I
??
19,j
Inc.
DEF'l33~~~
IXXS AND IX URENIC PATIENTS BEo?JZ AN2 AFTER H3xx)IALk-SIS
Kxns Johnsson Departmantof Elcd CoagulationResearch, Karolinska Sjukhuset, 104 01 Stockholm, &eden.
(Received
3.3.1975;
Accepted
in revisecl form 19.5.1975. H.C. Godal)
by Editor
Platelet fibrinogenin dogs was estimated inrnunologically before and during Dafibrase-induceddefibrinogenationof plasma. The antigen was identifiedby imnunoelectrophoresisand by SDS gel electrophoresisof reduced and non reduced sanples. The concentrationof platelet associated fibrinogenremained unaffected by the Defibrase treatment.The results indicate that neither fibrinogennor fibrin(-ogen)degradationproducts (FDP) are interchangedbetween plasma and the platelet. Uremic patients showed a normal concentrationof platelet fibrinogen. During the course of hdalysis no decrease in platelet fibrinogen content was observed in uremic patients.
IXIRCDUClTCN Fibrinogen in marsralianblcod platelets has been isolated and quantified by several workers (1,2,3,4,5).This intraplatelet-associated fibrinogenprobably related to the intracellulargranulae (61, has in some respects been distinguisablefrom plasma fibrinogen.Ithas been reportedthatithas
a
slower clotting with throsbin, a differentrobility in SDS gel electrophcresis and in agar gel irrmunoelectrophoresis (2,5). me twopro&ins
seemtohaxsz
the samx sedimentatim Coefficients,total amino
acid ccmpositionand Wterminal amino acids (2). SCE investigatorsclaim 161
that
tkey
are
t.k
~~ucts
cf fter plasr?;in vitro of platelet fibrinogen,hmever, &gradation products zere i&ztified. These fitdir.gs indicate,that platelet fibrinogenis not degrackd by plasrkn dluringCefibrase treatmaM,.that platelet fibrinogenis degraded by plasrih in vitro and that ED? not are interchangedbeti~eenplatelets and slsm. In spite of that Cefibrase in vitro was capable to coagulate platelet fibrincgen,'bysplitting of fibrinopeptide-A,platelet fibrinogenremaked quantitatively unaffected during Defibrase treatrrent of the dogs. This discrepancy rnightsuggestthatoertain properties of the platelet fibrinogen
is changed
when it is released from the,platelets.Suciza statemant is in accordancewith a reported enzymatic activity in the platelets ~~hichin a reversiblemnner reducescertain disulfide bonds in the fibrinogenmlecule
(32). In this reduced
form fibrinogenis very difficult to coagulatewith throrrbinenzynes. Previous results have shm
that the administrationof substances capable
of inhibiting the platelet function, including platelet release reaction to defibrinogenisedindividuals, significantlyincrease the bleeding temkncy (31,33).It would, therefore,appear probable that in a plasma defibrinogenatedindividual plateletfibrinogenis of intact hemostatic capacity. Also
importance in the maintenanceof an
in vitro, the platelet fibrinogenseems to
be of certain significance for the platelet behaviour. Solurnand Stomrken (34)have pointed out that fibrinogenserves as an important co-factor for the ADP-rmadiated platelet aggregation.It is hw.?everpossible to aggregate plateletswith ADP even in the abscence of fibrinogen
in plasma in dogs trea-
ted with Defibrase,if the platelets are pre-exposed to thror&in (24).This mightbe a consequenceof a thrmbin-mediated release ofplateletfibrinogen whicfiin tum stistitutes for plasma fibrinogenas a co-factor for ADP, Keenah and Solum (8) and Holma et al (9) have shun that platelet fibrinogen my be released in vitro by thrtiin. In order to investigatewhether there was any release of platelet fibrinogenin viva, this content w=
examinedin uremicpatients before and after
hemdialysis. Lindsay (35) has previously shmn that an adhesion of platelets to the thrtiogenic surface of the dialysis filter takes place during extracorporeal circulation,despite a general heparihisation.It also appeared to us prabable that sane platelets do not adhere pemanently to the dialysis rrerrbrane, but are activated in connectionwith the wntact blood-dialysis filter, and thus activated, release part of their contents. Ma were Iunableto &serve any &crease in fibrinogencmaentration in platelets during hemdia-
lysis. This need net necessarilyexcluck a release of platelet fibrinogendluring dialysis as it is still not established-&ere the platelet fibrinogenis formed. It has been speculatedtiether the platelet fibrinogenmay be formed in circulatingplatelets (36) and in that case a release might be overlocked by this procedure. Ms consider that further stties of this aspect are iqortar-k,and we believe that the plasma defibrinogenisedanimal may well be a valuable experiment subject, possibly :tiththe help of labelled precursors. Uremic patients, irrespecti= etiology, have a documentarypathological platelet function and an increasedbleeding tendency (37).Gur results
estab-
lish that the content of platelet fibrinoqenin uremic shjects dces not differ from that of healthy subjects. A leered platelet fibrinogencontent is, therefore,not the caluseof this abnormal platelet faction, and preliminary results have shcklnthat fibrinogenis released from "uraemicplatelets" as well from 'normal platelets"with a cannercial cephalin (lhrombofaxR)(38) whicfiis capable to aggregateplatelets in vitro (39,40).
This workwas supportedby grants fromthe Swedish ~MadicalFksearch Comcil
(No. 19X-520). I express mj sincere gratitude to Dr. Margareta
Bloti&% for valuable discussion and for placing facilities.
1. NACHMAN, R.L. Irmoslologic studies of platelet protein. Blood. 25, 703, 1965. 2. SOLUM,N.O. andUIPACIUK, S. Eovine plateletproteins. III. SoXEproperties of platelet fibrinogen.Tbrorrb.Diath. Haemorlhag. 21, 428, 1969. 3. KARACA, M., NILSSON, I.M. and HELNER, U. Quantitativedeterminationof platelet fibrinogen.J. Lab. Clin. &kd. 77, 485, 1971. 4. KEENAN, I.P. Platelet fibrinogen.I. Quantitatiglusing fibrinogensensitized red cells. Med. Lab. Technol. 29, 71, 1972. 5. CANGULY, P. Isolation and some properties of fibrinogen from human blood platelets. J. Biol. C&m. 247, 1809, 1972. 6. NAcZEiM?B,R.L. and MARCUS, A.J. Immunologicalstudies of proteins associatedwith the s&cellular fractionsof throrrbastenic andafibrinogmc platelets. Brit. J. Haematol. 15, 181, 1968. 7. mLITM:, R-P.,-, T., and WITFELL, B.A. Platelet and plasma fibrinogens are identical gene products. ScienQ. 185, 368, 1974.
,,
9.
.2
i
.
‘-:
)
.
._*
*
’
R., SIZE%, J.J.,b$im,, E.H., and HOTIIG,T. Demonstrationof platelet fibrincgensecretion via the surface connecting system. Thrombosis &search. 3, 347, 1973.
HOLME,
10. CASTALDI, P.A. and CA!ZN,J. Platelet fibrinogen.J. Clin. Pathol. 18, 579, 1965. U., EUME&K, B., 11. EGBERG, N., BLOFIBfiCX, PI.,JOHNSSCN, H., AB-, L., JOHAF+SSC%-, S.A., KcIXNGH, J., S., GijRANS~, DIENER, L., E.XESTRjfjI, McCONAGH, R., NILSSON, S.E., NORDSTR@~, S., OLSSON, P., and WIMAN, B. Clinical and experimentalstties on Reptilase. Thromb. Diath. Paen-orrhag. Suppl. 47, 379, 1971. 12. -CR, 387,
M. Purificationof antihe.mophilic globulin. Arkiv Xemi. 12, 1958.
13. BUW!&CX, B. and BLCMEWX, M. Purificationof human and bovine fibrinogen. Arkiv Xemi. 10, 415, 1956. 14. Biologisk styrkebest&minq av trombin. PharmacopeaNordica Editio Suecia. 4, 137, 1969. 15. B.U%It%CX,B. and BL@Il%CK, M. A method for the assay of factor V. Stand. J. Clin. Lab. Invest. 15, 639, 1963. 16. BERGSTR&I,X., BWCK, B., and XLEEN, G. Studies on the plasma fibrinolytic activity in a case of liver cirrhosis. Acta Med. Stand. 168, 291, 1960. 17.MBRSXBY, c., LALBZARI, P., and JOHNSON, A.J. A rapid, simple, sensitive method for masuring fibrinolyticsplit products in hm serum. Proc. Sot. ~xp. Biol. F&d. 131, 871, 1969. 18. KRISTENSON,A.A. A new method for the direct counting of the so-called blood platelets in man. Acta Med. Stand. 57, 301, 1922. 19. LAURBLL, C.E. Quantitativeestimation of proteins by electrophoresisin agarose gel containing antibodies.Anal. Biochem. 15, 45, 1966. 20. SCHEIDEGZER,J.J. Une micro-r&hode de l'imnuno-electrophorese. Intern. Arch. Allergy. 7, 103, 1955. 21. XUDRYX, B., REOTERBY, J., and WCX, B. Adsorption of plasmic fragment D to thrcxrbin modified fibrinogen-Sepharose. Thrombosis Research. 2, 297, 1973. 22. MZONAGH, J., MESSEL, H., MclX%lAGH,R.P., MURANO, G., and -CX, B. mlecular weight analysis of fibrinogen and fibrin chains by an improved sodium dodecyl sulfate gel electrophoresismethod. Biochim. Biophys. Acta. 257, 135, 1972.
23.
?;OSm,
H.L.,
K. , lclIimR,
I., CLXEELD, R-P., EC,? dZR, V.P., SPAUXDIS, and &?SHI, G.D. %easuremnt of fibri2opeptid.e .?. in
YLDEZLMX~,
G.D.,
humn blocd. J. Clin. Invest. 54, 43, 1974. 23 . EGBERG, N. and JOHNSSON, H. Platelet aggregation induced by ADP and
throrrbinin Reptilase defibrinateddcgs. Thro&osis Research. 1, 95, 1972 25. E!L&mCK, B. Personal cm?nmication. 26.
ARNESEN, H. and GODAL, H.C. Studies on the th.rmbin clotting time. The influence of fibrinogendegradationproducts. Scud. J. Haerratol.10, 232, 1973.
2; .
JAMES, H.L., BRADFORD, and GANGVLY, P. C?.xmtitation of human platelet fibrinogen.AHA Conferenceon Throtisis and Hermstasis.Dallas, Tems. Nov. 20-22 1974. PresentationNo. 1084.
28. MILLS, D. and KARPATKIN,S. Heterogenityof human fibrinogen:Possible relation to proteolysisby throbin and plasmin as studied by SDS polyacrylamidegel electrophoresis.Biochem. Biophys. Res. Cornnun. 40, 206, 1970. 29. BI.C@aCK, B. Studies on the action of thrombic enzymas on bovine fibrinogen as masured by N-terminal analysis. Arkiv Ksmi. 12, 321, 1958. 30. BLQME%CK, B. and LAURENT, T.C. N-terminal and light-scatteringstudies on fibrinogenand its transformationto fibrin. Arkiv Kemi. 12, 137, 1958 3i. OLSSON, P. and JOHNSSON, H. Interferenceof acetyl salicylic acid heparin and fibrinogendegradationproducts in haemstasis of Reptilase defibrinogenated dogs. Thrombosis Research. 1, 135, 1972. W., mI%REN, A., KOWALSKA-LXH, B. 32. BUXlE&X, B., BT0'+@%X,M., F=INER, andOLaVSON, G. Enzymatic reduction ofdisulfide-bonds in fibrincqenby the thioredoxinsystem I. Identificationof reduced bonds and studies on redoxidationprocess. 'Ihrorrbosis Research. 4, 55, 1974. 33. JOHNSSON, H. and NIKUSSON, P.M. 'Iheeffect of n-c&rate doses of chlor pmmzine on the haermstasisin dogs defibrinogenatedwith Defibrase. 'Ihrmrbceis Research. 4, 229, 1974. 34. SOLIJM,N.O. and s]co~RKEN, H. Influenceof fibrinogenon the aggregation of washed human blood platelets induced by adanosin di@-msphate,thmxrbin, collagen and adrenaline.Stand. J. Clin. Lab. Invest:17.Suppl. 84, 170, 1965. 35. LINDSAY, Rd., PRENTICE, C.R.M., E’EFGEON, D., and BURJDT, J.A. Reduction of thxmtxts formatianon dialyser nenbranes by aspirin and R.A. 233. Lancet. 3, 1287, 1972. 36. F0SIEK, O., WEGRZXNCWICZ,Z., SAWICKI, Z. and KOPEC, M. Fibrinogensynthese in Blutplattden. Folia Haematol. (Laipz.192, 553, 1969. 37. CASTALDI, P.A., F0ZENBERG,M-C., and STEWXU', J.H. The bleeding disorder of umemia. Lancet. 2, 66, 1966.
33. JOmSSP: , ii.To be publis:hed. J. and VEA’IsT~~, >I. Platelet aggregation5~ 39. IX Gc7ET2?0, G., VERGER:, throhofax(R). Studies on the mzhanism of action. Experimntia. 29, 1136, 1973.
platelet 40. DE CLEIMC, F., KUCERS, M., VEIWLElN, J. and DE GAETAPiO,G. Hm aggregationby thrcxrbofax. An electron-microscopicstudy of the sequence of events. Stand. J. Haematol. 12, 93, 1974.
