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A survey of surface hemorheological experiments on the inhibition of fibrinogenin formation employing surface layers of fibrinogen systems with heparins and other substances. A contribution on antithrombogenic action
A.L. Laboratory
of
Copley
Biorheoloa-, Brooklyn,
(Received
and
R,G.
King
Polytechnic Institute 7 1207 , i_;‘s& 8-S
3-2.1984; Accepted in revised by Editor S.E. Laskerf
fom
of
Ye:; York?
2.4.1984
\
JiORDS:
Fibrinogen, fibrinogenin, surface bemorheology, antithrombogenic action, heparins, chondroitins, hyalilro-, nate, de_utrans 237
period The main ob.jec Give o f this survey, m-2-6e Q~ST .an exre=ded of more +ihan tez years (l-3), is to compare the surface effects of different DfeFafations of heparin of varying a=ticoagulzxt and antithrom3otic actions. These heparins were added to sollutions of PT! human fibrinogen and hemorheological properties highly ourif?.,_ of the surface layers formed on these solutions were measured for comparison. The properties investigated xere rigidity, viscosity and elasticity. The other objective :r;as to study these surface properties of fibrinogen systems containing substances of knows and The paraunknown antithrombotic activity other than heparins. meters for testing antithrombotic activity estra vivum, r;hich are This is dealt ?
f-03?
There have been several pathways suggested by different groups of investigators for the action of heparin. They concern the treatment of experimental thrombosis in animals and the treatment of thrombosis in patients, in cases where there was no significant A satisfactory anticoagulant action but an antithrombotic effect, explanation has thus far not been given by different investigators for the incongruity between antithrombotic and anticoagulant actions of certain agents in thrombosis therapy. significance as Our extra vivum findings are of particular to be applicable to the prevention and treatment of they appear This is based on Copley's concept of thromthrombotic conditions. of fibrinogen molebogenesis due to the aggregation and gelation without the oarticipation forming so-called l'fibrinogenin", cules, i The inhibition of fibrinogenin formation by Cerof thrombin (7,8): tain heparin preparations and other substancesis considered to be an indicator of their antithrombogenic activity, whichis disc*ussed.
Highly clottability)
purified human prepared by
and bovine the method
fibrinogen of BlombZck
(97-100 per cent and Blomb'ick (9)
HzFL)hepafin prepafations h-ere sac12red I”rom different 5oLfrces. r;ere manuThe com>mercial sodium heparins employed in this survey _&bbott, Chicago? 1ll.(USX); companies: factured by the following X'.J, jUS_?i); Laboratojres Choay, Paris (France); Organon, Orange, _a Xich. (USA.). and Upjohn, Kalamazoo, Kort'hridge , Cal.(USX) Riker, were supplied by L.B. Jaques, Univeroreparations Some of these L Saskatooa (Canada). sity of Saskatchewan,
:iere obtained Tram Xon-commercial preparations of heparin Fractionated heparin from U, investigators as follo:m 29,000 and dextran sulfate from S.E. Laslker, several
We were informed that heparin fractions Tjere prepared by alcofractionation (lo), depolymerization with heparinase followed by affinity to antithrombin by fractionation (11) and fractionation Derivatives were also prepared by acid X-desulfation III (72). Dextran sulfate was obtained from Schwarz-Mann Labora(13,!4). tories, Orangeberg, NY (USA). Sodium hyaluronate was secured from Miles Laboratories, Elkhart, Ind. (USA) and depolymerized hyaluronate x-as made according to Harris et al (I?).
hOl
The blood samples, obtained from the jugular veins of dogs, drawn into evacuated collecting tubes containing sodium hepaof the plasma, rin. For the preparation the blood xas centrifuged at 3000 r-pm for 15 min.
were
A Keissenberg F&eogoniometer modified for surface hemorheologistudies (h-6) was used for measurements of surface rigidity viscosity ( ls) and surface elasticity (G,). The ifi, surface volume of sample require 1 for each measurement was 4 ml. All measurements were made at 37 2 0.5OC. cal
It should be noted that detergents should not be used to clean parts with which the test material comes in contact. The meaSUfiIlg geometry device should preferably be rinsed at least three times in physiologic saline to remove residual fibrinogen and other components of the test sample.
any
RESULTS I\r'o surface layers alone were tested as
could be control.
detected
when
heparin
solutions
1 shows the viscous and elastic moduli of surface layers Fig. of 0.4 per cent fibrinogen solutions, to xhich two pfeparatiOnS of sodium heparin from two commercial sources, A, B, in 0.2 per cent concentrations, were added. Both preparations did not change the values of surface viscosity (q's) and surface elasticity (GS) from those of the control.
Ir; Fig. are ShO%v?l of CO which 0.2 heparin were viscosixy and
2 the surface viscosit:; and surface elasticixv values surface layers of 0.4 per cent fibrinogen soi;tions per cent high and low molecular weight fractions of added. Tkese particular hepariDs did not affect the elasticity of the fibrinogen surface layers.
;9
-
q.__a.
+
:cO__~___~--p~,~-a u yz _ .% z T f. z ; 5 : 15. -! -*.;-'9 -z r t:0. -2. r2 _-
a-
FIG.
i
z= z,
;; 1 -'I& iz ! z2 j sn I-. ,C’
2
Surface
r
_.
.- __~ ._~ ,(y :’ .I __~__.. i‘qfC:t’d:Y ,+r91z
viscosity and surversus freface elasticity silency of ssrface layers of t-J solution as * &$ fibrizogen control f comzared with O.k$ fibrinogen to krhich 0.2$ low XV heparin fraction and 0.2$ high M< fraction have beer, added.
the other hand, shoris thar 0.2 -,er cent of a difFig. 3a,on reduces?ts lox molecular weight (LX'!!) heparin fraction. ferent CA) The LAX% heparin was prepared by Lasand Gs by about 60 per cent. ker (11,16). Fig.3b demonstrates that a 0.1 per cent; heparin fraction, which has a high affinity for antithrombin, ioxered 'q's and Gs -by while another heparin fraction which had a loi< about 90 per cent, affinity for antithrombin showed no change in either 7's or Gs. The heparins, shohn in Fig. li,were received from Vlf Lindahl. Fig. and high heparins
3~ demonstrates zhat different heparin fractions of 1074 The affinity for antithrombin did noz weakes:Q', and Gs. shown in this Figure were also received from Elf Lizdahl,
rii~h high affinity for Fig. 3d shows that a heparin preparation layers of 0.4 the elastic modulus of surface antithrombin reduces However, fibrinogen solution by abour. JO per cent. per cent bovine viscosity moduli did not change significantly.
FIGS .
3a,3b,3c,3d
elasticity viscosity and surface Comparisons between the surface , of 0.476 fibrinogen solution as control, and 0.4$ fibrinogen to which Fig. 321~ 0,2$ of a iow Mk heparin fraction (A) and a 0.2$ (3) have been added; Fig. 3b, fraction of another low MW beparin n.l$ of a heparin h;ith low affinity for antithrombin, and O.l$ Of a heparin with high affinity for antithrombin have been added; Fig. 3c, 0.2$ of heparin with low affinity for antithrzmbin, and affinity for antithrombin have been 0.27/o of a heuarin with high Pig. jd, 0.1% of a heparin preparation of high affinity added; for antithrombin has been added. Figs. ha and kb are data curves of the surface viscosir;y ar_d elaszicity, respectively of 0.4 per cent fibrinogen as control, to which (a) 0.1 per cent sodium heparin, (b) 0.1 per cent calcium heparin and (c), as control, SmM CaC12 were added. Both the calcium he?arin and the calcium ions increased both T's and Gs by almost 50 per cent.
viscosity (Fig. ha) and surface elasxicity (Fig. 45) Surface versus frequency of O.tig fibrinogen solution as 32.ontrol I comsolutions of 0.576 fibrinogen fo which O.l$ pared -with three O,l$ calcium heparin and jmX C-a?+, respectivesodium heparin, 17~. have been added.
i _t’ 1-desuifated chat the addition of 0.2 per ce~r Fig. j shows Danishefsky_! did not weaken q's heparin (A) f rom one scource (I, heparin (9) from a second s>mce and Gs, while a 1X1 -desulfated weakened Yiis and Gs by approximarely ir0 per cent, (S.E. Lasker)
F-IG.
Comparisons
j
-of surface
vis-
cosity aI;-d szxface elasticity of O.k$ fibrinogen solution as coatrol, with Tao O.h$ fibrinogen solutions to which 0.2% t>t'-desulfated heparin from two different sources (A gad 3) were added.
of 20 per cent dog plasma considerIn Fig. 5, a preparation ably weakens the surface rigidity (T') of the fibrinogen system. The addition of 0.1 per cent sodium heparin (J. Cnoayj to the plasma system further weakens the surface layer by fibrinogen-dog The addition of 0.1 per cezxz: of rhe same sodabout 60 per cent. ium heparin preparation alone to the fibrinogexz solution caused no change in the Tvalues.
The effect of 0.7 per cent chondr0i:i.n -4 (I. Pa_nis-:?afsk.y) on the surface rigidity of I^ibrinogen surface layers is E~OW-I in Fig, -57 of about 80 per cnn-t was found. in torque values .i reduction Kiq andChien .&is particular fir,ding led to the study by Co?iey, and elastic moduli of surface layers of fibriniJ?f on the viscous ogen system&with chondroitins A, B and C.
7-
Surface rigidity (7') versus rate of shear of 0.4% flbrinagen solution 25 co,ntrol compared with O.i$ fibrj.nogen solution to 7ghic.h O.l$ chondroitin A was added,
2
z j i 1 2 zd
M_
z;
Fig. 8a shows sizy and elasticity and elastic moduli cent, respectively.
the of are
effect surface reduced
of dextran MW 20,000 layers of fibrinogen. by approximately 40
on
l;he -V-iSCOBoth viscous and 50 per
In Fig, 8b viscous and e1asl;i.c moduli of fibrinogen sErface layers as shown to which 0.2 per cent dextran sulfate N% 17,000 was added to the fibrinogen solution. Decreases of approximately j0 per cent in both 7's and Gs were found. The effect of the addition to fibrinogen solution reduced elasticity of these fibrinogen sho-c;llin Fig. 8~.
of 0.2 per cent sodium h?aLuronate the surface viscosity and surface systems by about 50 per ten?;, as
FIGS
1
Sa,Sb?8c
Surface viscosity aiiAdsurface elasticity versus frequency of 0,20/ofibrinogen solution as control, compared with 0.2$ fibrinogen solution to which the following were added: Fig. 8a, 0.2$ dextran, iW 20,000; Fig. 8b, 0.276 dextran sulfate; Fig.8c, 0.2$ sodium hyaluronate.
Fig. 9 shows that depolymerized hyafuronate, when added to a fibrinogen solution, reduces the rigidity of the surface layers of this system by about 60 per cent.
2
;-\
-
:
2
A
A
*
* .
--
z < ,QZj '5 ! = / 2 z a
L.___ ____ -~ _~~~.__ .;' /e.2 lb0 RATE 3F5HEAX SEC-!
FIG.
9
Surface rigidity (7-t versus rate of shear of O.ir-$4 fibrinogen solution as control, compared with 0.47; fibrinogen solution to k;hich S.l$ depolymerized hyaluronate was added.
Copley postuiated that both in the initiation of tb_rom,bosis and of hemostasis the initial Occurrence of a th&rom,bus without thXrombin_ i+ formed by a gro:
The J:lot;i;lg of fibrinogen by minute amounts of ~rotamine sul f a t e z-4 a 3 first described by %
Sew direct evidence for the validity of Copley's theory of in viva fibrinogenin clotting in thrombogenesis, advanced in 1971 (78: studies on fibrin0 was provided by Kitte (32,33) in biomicroscopic 'He demonstrated fib isothiocyanate. gen, la'oeled with fluorescein rinogenin formation which he named "thrombogenesis in statu nascendi" (33). Already in 1954 Zweifach (34) discovered "the accumulation of extensive masses of gelatinous material at the site of injury" as a result of direct inflicted damage to the endothel*Lm. This material could not be digested with hyaluronidase, Fi'brin was not formed due to the heparinization of the animal. The gelatinous material could not have been composed by the clumping of platelets as no embolizing platelet clumps were seen by him at the traumatized site of the vessel vail. He also did not observe platelet aggregation at this site . Zweifach observed the gelatinous material also, lchen he did not heparinize the animal. He considered the gelatinous material as a result of chemical and mechanical tissue trauma. Zweifach stated that he was not certain whether this substance is produced by the endothelial cell or occurs in response to substances released from it which would interact with the piasna.
In Copley's appraisal, Zweifach's evidence that the gelatinous material
findings constitute at the site of the
dlrec;: trauma-
-J-t is emplhasized that the formatln of fibri.nogerLin on the endoendothelial fibrin lining is considered to beoom,e a surface gel subsequent1 y with a large increase in volume (35). Our extra vivum Studies on the elastic modulus of slurface layers of fibrinogen Tke elasziciey of the svstems may mimic the srxuation in viva. regardcrosslinkxig, complex surface layers iS a function of T;ne 'I forces or by covalent bonds. less of ~herlher by van d8r Vaals' ItS measurement provides data concerning the org2nization of the structure &hi& is considered to be a thromboid (thrombus-like) gel. T%e modulus of elasticity is a function of the concentration of cross-iinks within the adsorbed layers of fibrinogen, and, in with other plasma proteins and their Structures. viva f together 'The intertend to accumulate at an interface. Trotein molecules give rise to structures actions of the proteins Kith one another of high viscoelasticity (21),
>zzj!o s t recently Silberberg discussed multilayer Formation of formation as well as macromolecules and related it to fibrinogenin He stated that it iS to the endoendothelial fibrin lining (35). "not surprising and indeed almost to be expected that large molecules like fibrinogen will adsorb both to the endothelium and to Silberberg concluded that the surface of the circulating cells". "they coatings of fibrinogen arise, where thick gel-like surface blood flow to the point where blood can considerably influence flow is essentially closed off" (35)_
2.
blood
forms On the three Their roles in the
of blood prevention
10 shows a diagram Fig. clotting during life,
clotting extra and treatment
of the leading
FIG. A diagram thrombosis
of intravascular blood and of antithrombotic
vivum and in vivo. of thrombosis.
three forms of to thrombosis.
intravascular
IO
clotting during life (incl,antithrombogenic)
leadingto actions,
of polymerizatiori and Ligation in thrombin il?'The processes have beer?_ exrens
On rhe prevention of thrombogenesis fibrinogenin formation
in
relation
50
extra
vivuZ1 -
The consideration of fibrinogenin formation in thrombogenesis is of particular significance in finding agents which inhibit such which xi: are reporting, ap* I. Accordingly, the findings forma+io? pear to be of great importance both in -ithe treatment of thrombosis as well as in its prevention, _k diagram concerning the inhibition to js T'nere appear is given in Fig. 11. Zhe as.mvppation of fibrinogen molecules, QCJ*-D phase of fibrinogenin formation, as there .seguently affect its second phase, viz.,
of flbrinogenil formatioa inhibitors i;fiich affect comprising The first :,Thich subare inhibitors fibrinogenin geiation.
in our surface hemorheoiogical studies, tie cannot differen-tiate between the two phases of fibrinogenin formation. Xevertheless, we propose our method of testing certain agents in the prevention of thrombogenesis and in the treatment of thrombosis. They concern measurements of rigidity (J or torque values) and of viscous moduli (tits ) in steady shear, as well as of elastic modL1l.i (Gs) in osciliatory shear of surface layers of systems of highly purified fibrinogen solutions to which the respective test agents have been added.
ve realize thax surface layers of e. system of highly purified fibrinogen does not fully correspond to the in viva situation, in which we deal -with the circulating wXole blood and izs fluid phase plasma. 1x2 oilr studies we have used systems of surface layers or"
&ig_tll;.:
F;rified
a.~-ions
s&2+ ._uiL
fibrinogen,
as
of
$.zteractigzis
i;here other
wag Fiama
th;i pr:zt&ns
x-,e&
ejminsre
7:~
md
other
the
consti-
in plasma md in whole blood which WO?~ii-j affect fibrinogenin firmation. We already esta'blished that globulins and albumin lowered such formation (38), !t'ealso found that red blood cells lowered the rigidity of surface layers of plasma systems, while plateets increased it markedly (39). Thus I platelezs strengthen fibrinogenin gels and appear to augment thrombogenesis causing mixed thrombi. Thus far, our studies with syszems emgloj-ing highly gurified fibrinogen have provided significant information. However I future studies yill need to deal more and more wirh the viscoelasrrcity of surface layers of different plasmas, as well as of fibrinogen system5, to which different plasma proteins and other constituents of plasma 1411 be added. ‘2er_:s
this experimental survey ‘Throughout the Weissenberg Rheogoniometer, modified by us for surface hemorheolgical studies, was employed. As it is a sophisticated research tool, there will be an urgent need for the development of a more simplified apparatus for clinicai use which measures rigidity, viscous and elastic moduli OF surface layers of fibrinogen systems and of plasma (40,41).
4*
Various
heparins
and
their
antithrombogenic
actions
The heterogeneity of heparin is nov well documented, as deOne of the objectives of this survey was scribed by Lasker (71). that have high USF anticoagulant activity to compare preparations and high anti-Xa activity with fractions that are less effective anticoagulants but retain anti-Xa activity, Also included h;ere preparations that had high affinity for antithrombin III as well as a series of fractions with decreasing USP anticoagulant activity with high anti-Xa activity. The antithrombotic activity of would need to be evaluated regarding and their antithrombogenic action in tion.
preparations, such as heparins, their anticoagulant actions inhibiting fibrinogenin forma-
In our survey of commercial and non-commercial heparins, we which had anticoagulant action did found that most preparations as tested with regard to the not inhibit fibrinogenin formation, The addition of LMW fracrigidity, viscous and elastic moduli. tions of heparin may show lowering of these moduli, while HHVfracmay not. On the other hand, LW+ tions of the same preparations of heparin do not necessarily induce any changes of surfractions Howas shown in Fig. 3a. face viscosity and surface elasticity, studies by Copley, King and Chien$ in 1983 reported (77) ever, marked decreases of surface viscous moduli and surface elastic moduli with heparins os* LMW from 4400 to 5900 daltons. heparin and heparin Kattson and Xilsson (42) studied Holmer, fragments of different molecular weights and with different antiregarding their ability to inhibit factor Xa/APTT activity ratios They found thrombus formation using a rabbit thrombosis model. that neither anti-factor Xa nor the APTT (activated partial thromare good reflectors of antithromboboplastin time) activity alone They also found that a heparin fragment of ?4?44000 tic activity. action. activity but less anticoagulant has the same antithrombotic
ackelfard find
a refatio
L?p+q hsParFr,
7
%-ap . =:-i-G 1
Ed ~1 n-ship 2s
tested
ments
of
in
activit 3.7
orher properzies tic effectiveness' ~~i~;hror;i'o~ti~
(i&j reported between
of
,Taey
thromSosi.s
activity
not
extra
mgdels,
from
contribute
Came to
could
xhat they antithrombotic
viva
concl;lded
L>F~ heparins
I
recently
in
animal
~hgy
efficacy
anti-xia
the
this 21x2);s
af,d extra
vi-
their to
findings that their 'antithrombo-
conclusion, be
could not action 01
predicted
sir;ce
by
ia
viva
measure-
vix7_2m.
reported that low af($j) lii9i-e recently Bzrfoxcliffe ei: al the acTion of high affinity (E-f.) (LX) heparin potentiates finity oiigosaccharides for anzithrombin III (AT-III). They conheparin heparin content of LI'R heparthat the I_,_% cluded from their studies in fractions appears to be an important determinant of their antiThey further stated that "thrombin inhibition thrombotic activisy. Thomas is nor an absolute requirement for antithrombotic activity'!. et ai (46) in their studies assessed the anrithrombotic activity of It apiix heparin oligosaccharides in a rabbit thrombosis model. activity employed by these inpears to us that the antithrombotic action of vestigators may be based in part on zhe antithrombogenic is due to its inhibition of fib+ Their LX34 heparin which we consider formation. rinogenin et al ($8) found that the fractionLindahl ($7) and Rosenberg one with very ation of commercial heparins yielded E:WO fractions, one. Lindahi (12) low Siologicaf activity and the other k*ith 2 high in chemical composition between the could not find any diff erence z;wo fractions. The difference consequently must be very subtle and perhaps depends on the sequence of the sugars in the oligosaccharidc chains. Xe, employed in our surface hemorheological studies therefore, heparins with high and low affinity for supplied by Ulf Lindahl antithrombin. The fraction of heparin, exhibiting high affinity for antithrombin, lowered the viscoelasticity of fibrinogen surface -with low affinity has no such layers (Fig. 3b), rjhile the fraction However, in Fig. 3c there are no changes effect. in surface viscmosity and surface elasticity by the use of another heparin preparation with fractions with low and high affinity for antithrombin,
Of great interest are our findings with preparations of calcium heparin and sodium heparin, supplied by Choay, Paris, on surface layers of highly purified fibrinogen (Fig. ka). The sodium heparin did not decrease the viscous and elastic moduli of fibrinogen surface layers, while calcium heparin increased them markedly, as did Ca2+. There are substances which either promote or inhibit the gelation of fibrin. Such gelation promoting or geloplastic substances and gelation inhibiting or antigeloplastic agents have been found by Copley to be Ca2+ and sodium heparin, respectively ('19we found that Ca2+ Recently, is a geloplastic agent also 51). with regard to fibrinogenin clotting (32). Eowever, only certain sho~-z in this presentation, which have antigeloplasheparins f as
lowering of viscoelasticity of surface layers of fihrinoThe i tbi h--dejulfated hpp~ri~:, gen s>-szems **: J is of intershow2 in Fig. est, beca:use this altered heparin has a 10i;er molecular charge density and minimal anticoamlant action. The X-desuifated heparin preparati0n appears ~0 be another indication that antithrom3ogani.o action is not related to the inhibition Of fibrin formation. The acticsn of heparins,zhich 10~.en the rigidity and viscoelasticity of surface layers of fibrinogen systems, will need to be examined in great detail with regard to the mechanism involved in these actions. Since certain heparin preparations have such actions while others have not, the agent responsible for this activity will need to be isolated from those preparations containing it, We believe that t1he findings which are presented will stimlulate much needed netr' h;ork on heparin and its actions, 3.
02 the mation
role of plasma bv heparins
in
the
inhi3ition
.0f fibrinogenin
for-
In 1972 we reported that surface layers of heparinized plasma eshi3ited markedly lower surface rigidity than those of oxalated plasma from the same blood withdrawal from a human subject (40). These findings led us to investigate, over a period of more than ten years, the effect of heparins of different origins on the rigidity and viscoelasticity of surface layers of fibrinogen systems. In the course of this survey, we reported some of our findings(l-3, Instead of plasma we chose highly prrrified human fibrinogen 5,531. of 97-100 per cent purity (9). This choice eliminated the variaplasmas with their varying concentrations of bility of different different constituents. The rigidity of surface layers from a system of 20 per cent dog plasma and 0.2 per cent human fibrinogen, upon the addition of is weakened by approximately 60 per 0.1 per cent sodium heparin, The addition of 0.1 per cent sodium heparin, supplied by J. cent. Choay, to the dog plasma reduced the Tvalues stiil further (Fig. As expected from our earlier investigations with globulins 6). a plasma-fibrinogen system and albumin in 1971 (18) and 1972 (38), showed lower Tvalues than those obtained with a fibrinogen preparation alone. These preliminary findings Open up an entirely new field of agent -which study regarding the efficacy of an antithromSogenic evaluation of suchan agent may further show that the therapeutic action or on its affinity for on its anticoagulant does not depend show that plasma The findings presented in Fig. 5 antithrombin. play 2n import2nt role constituents other than fibrinogen may well clotting +,+.thout thrombin participation. in fibrinogenin
Ve have not yet studied the effect of differeni; purified of fibrinogen and the addition added to solutions plasma proteins of heparin on the rigidity and viscoelastici;y of surface layers In case certain plasma proteins or other plasma of these systems. , tiie lo:"-iering action on surfac.e decrease consZltuents h-i.11 further viscoeiasticity by other glycosaminoglycans, rigidity and surface potential usefulness in the substances, their as ~;ell as by other prevention and therapy of thrombotic conditions will bncacre PX-
trenel_y 6.
importaLzt
*
Inhibition in the formation than certain hnparins
of
fibrinogenin
by
substances
othe
the findings pertaining to We have discussed already above -the discrepancy of antithrombogenic and anti-Xa activities of cerfor which we made their inhibitory action in fitain heparins, 'orinogenin formation responsible. As far as chondroitin A is conet al reported in 1968 (54) marked antithrombotic ce.rned, Morrison in their in vivo findings in rabbits. action of this substance published their extra vivum stu Ejornsson et al (5j), T&ho recently explain its antithrombodies with chondroitin At could not fully tic action on the basis of certain anticoaellant actions which themeasured. The antithrombogenic actions in vivo by the use of the two glycosaminoglycans, chondroitin A and L?fV heparins, reported by Ejornsson et al, Ockelford et al, and Barrowcliffe et al, respectively, can Se explained as the inhibition of fibrinogenin formation b-ythese substances. It appears that the antithrombogenic action in vivo can be equated with the inhibition of fibrinogenin formation in viva. For these reasons, fibrinogenin formation and its inhibition by certain agents can no longer be ignored in co.nsiderations pertaining to thrombogenesis. The recent findings by Copley, King and Chien (17) are of particular importance, as they emphasized the need in employing extra vivum surface hemDrheologica1 testing of aniithrombogenic agents in the inhibition of fibrinogenin formation by heparins L1 5300 and 5900 dalT;ons and by chondroytmoiecular weights of 4400, ins A, B and C. The findings of surface rigidity reduction of about 80 per cent with chondroitin A-fibrinogen systems predates, as already stated, the studies by Copley, King and Chien on the vis coeiasticity of surface layers of fibrinogen surface layers by char. droitins A, 3 and C, resulting in marked reductions of viscous and elastic moduli with all chondroitin-fibrinogen systems, which we tested (77). i;ith substances other than Of special interest are our findings k'e tested sodium hyaluronate 'because of its glycosaminoglycans. it to behave similarly as low mohigh molecular iieight and found
1s
dextra?_
j:;lfase
>&-
17
f :Jf’1io
i
3
.a
J:har.ged
feast tQ .de_xtrarz, >pi 2O.Ot~0, r;e tested their coelasticity of surface lavers of fibrinogen parations have similar surface hemorheological .8b! carxot be responsible for 1, so the charge in surface viscoelasticity.
in
m;ole.~~~.lp,
effec:
or:
solution. aceions the marked
the
cozl-
-%-is-
Both pre(Figs. 8a, decrease
Sor: all of these substances can be applied in the Preiiention Jf thrombogenesis and in thrombosis therapy, alth*eugh they all ca-dse marked decreases in sur face viscoelasticity or in surface rigidity values. Severtheless, some of these substances may prove to be useful in the prevention and therapy of thrombosis.
I;'& Zhan!i the follox*~ing investigators for kindly supplying r
zs
REFEREWCES
i.
The action Gf heparin and other COPLEY, X.L. and KING, R.G. polysaccharides on the rigidity of surface layers of fibrinoASstracts, VI. Internat. Congr. on Thrombosis and iiaegen. of Hemophimostasis and XII. Congr. of the !iorld Federation ~ HaemThrombos P-4, US_4, 26.6.-2.7.1977. lia, Philadelphia, 106, 38 ostas. ( Stuttgart] 1?77. -f
2.
KI?X, R.G. The action of differing heparin and COPLEY, A.L. layers of fibpreparations on the viscoelasticity of surface gy, LaJolla, rinogen. Abstracts, 3. Internat. Congr. Biorheolo Biorheology, 15, 461, 1978. CA, USA, 28.8.-1.9.1978.
3.
szudies of surface Viscoelastic COPLEY, A-L, and KING, R.G. layers of heparin-fibrinogen systems and the antithrombotic Abstracts, VII. action of heparin other than anticoagulation. Internat. Congr. on Thrombosis and Haemostasis, London, EngThrombos. Haemostas. s, 323, 1979. land, 15-20.7.1979.
i:.
The Weissenberg Rheogoniometer COPLEY, _&.L. and KING, R.G. The Karl 1ceissenberg adapted for biorheological studies. In: J. Harris (Ed.), Kampala80th Birthday Celebration Essays. Tairobi-Dar Es Salam, East African Literature Bureau, 1973, PP. 127-143.
3.
Polymolecular layers of fibrinoCOPLEY, A,L, and KISiG, R.G. Hemorheolom gen systems and the genesis of thrombosis. In: (A.L. Copley and S, Okamoto, Eds.) Oxford-Sew and Thrombosis. Pergamon Press, 1976, PP. 393-409; Thrombosis Res. 8, York: Suppl. II, 393-409, 1976.
5.
COPLEY, A-L., KIXG, R.G., KXDRYK, B. elasticity studies of surface layers
and BLOXB~CK, 3. of high2.v Purified
Viscofib-
7 i
I)
8.
9.
10.
The physiological significance of the endoendoCclPLEY, d.L. thelial fibrin lining (EEFL) a3 rhea critical interface in the lvessel-blood organ? and the importance of in s-iv0 rfibrinogenin formation' in health and disease. In: A.LL. Copley, Ed. (7 ), PP* lOj-14j, BLQXB:iCK, B. and ine fibrinogen . LAXER, tionated i966.
BLOW3%X, Y. Purification of Xrkiv Kemi s, t?l5-443, 1956.
S.3:.
and STIVXLX, bovine heparin.
S.S. Arch
human
and
Fhysicochemical studies Biochem. Biophys. 9,
bov-
of frae360-372,
11.
LASKER, S-E. The s, 92-97, 1977.
12.
, G. HOOK, FI., THTXBERG, L. and FR4SSSON, _ _ StrActure binding site on heparin. of the antithrombin L. Proc. Natl, Acad. Sci., E, 3198-3202, 1979.
1" 2.
DINTSHEFSKY, I., EIBER, H.B. and Arch. the cheinistry of heparin. 1960.
14.
DANISHEFSKY, I., STEIEER, Investigations on the A. 17&l-174j, 1969. 224,
1j.
K4RRIS, "I., HERP, A. and PIGX4N, 14. Metal catalysis in the depolymerization of hyaluronic acid autoxidants. J. Am.Chem, sot. 94, 7ji'O-7572, 1972.
16.
Some LXSKER, S,E. and their component ular Functions and New York, Academic
77.
COPLEY, X.L., KING, R.G. and CHIEX7 S. On action of low molecular weight heparin and B and C. Biorheoloffvc,20, 697-704, 1983. -
78.
Non-Newtonian behavior of surface COPLEY, A.L. and a nerj concept of the plasma protein systems thrombosis. Biorheolog-y, 8, 79-84, 1971.
19.
COPLEY, account.
30.
COPLEY, fibrin
=?.L. In: X.L. lining
heterogeneity
of
heparins.
Federation
Proc,
Investigations CARR, J.J. Biochem, Biophys, B,llbi20,
H., BELLA, chemistry
JR. A. and of heparin.
011
FRIEDLANDER, J. Biol. Cherry,
structure-function relationships of heparins fractions. In: Heparin: Structure, CellClinical Applications. M.X. McDuffie (Ed.7 Press, 1979, pp. 143-lj7.
The endoendothelial A.L. Copley, Ed.
the antithrombogenic of chondroitins A,
fibrin lining: (I), pp.!-26.
Hemorheological aspects of the and of fibrinogen gel clotting. .
layers ofhuman initiation of
X historicai
endoendothelial Their import-
7 .,
__I
23,
24,
25,.
26,
STEi
H.5,
Fibrins. Heidelberg,
and XIEWIAROKSKI, S. by protanine sulfate. 7963.
polymerization Biophys. Acta,
None~~~ymatic
Biochim,
Unrersuchungen zur Strluktur dea Fibrinogens und thesis, Ruprecht Karl Universit~t Heidelberg, 1963, pp* 58-67. Cited by Gollwitzer et al (27).
Doctor
27.
R. I BODE, Y, SOLLVITZER, of fibrinogen molecules.
28.
GOHEX, C., Polymorphism 1966. 388,
29.
TOONEY, N.X. fibrinogen.
and :K_4RGES, H,E. In: A.L. Copley,
XUCERL, 2. and mLL, J. Xol. Biol. 22,
SiAYTER, H., GOLDSTEIR, L. in fibrinogen aggregates.
aild J.
On the aggregation Ed, (7), pg. 41-53.
GOHEX', C, Crystalline Mol. Sioi. 2, 363-385,
states 1977. C. of
of
C. 385-
a modified
30.
KEISEL, J.V., PHILLIPS, G.?:. and COHEM, fibrinogen and fibrin: II. Architecture Ann. N.Y, Acad. Sci. 408, 367-379 7 7983.
31.
GOLLWITZER, R., BODE, Xi., SCHRWM, diffraction of BERGER, R, Laser 21$-235, ?z.Y. Acad ~ sci * 9, Am.
32.
Eiveissaustausch VITTE, S. phology, 2, 79-85, 1979.
33.
i;ber das VerhalWITTE, S. Vitalmikroskopische Unter sur,hungen ten van Fibrinogen: in der Mikrozirkulation. In : Fibrinogen, Side Effects of Therapy with Clotting Fibrin and Fibrin Glue. Factor Concentrates. K. Schimpf Stuttgart-New York: F. (Ed.), ~erlag, 1980, pp. 35-89. K. Schactauer
34.
The exchange of materials betKeen blood vessels ZF/'EIF$CH, B,:i. on Connective Tissues. and lymph compartments. Trans. 5+ Conf. Josiah &lacy, Jr. Foundation, 1954, pp. 38-77. Sew York,
H.J., oriented 1983.
zwischen
Blut
The the
strucwre of fibrin clot.
TYPKE, D. and GUCKENfibrinogen molecules.
und
Lymphe.
J.
L5;m-
;ihrombosis A, L, In : illaries. Easel. ference, Benno Sch-dabe, i9jj, pp.
COFLZY,
azd
in
throoboembolization
biood
cap-
&j2-
<3n the fibrin forming S, and DEVI, A. COPLEY, A +L * , BATERJEE, of electrophorosed ho-vine thrombin activities and geloplastic ;974. preparations. Thrombosis Res. 2, 37 _j:, resisza.nce of thromboid. ViSCOUS C;7PLE>-, X.L. znd KIXG, R.G. sr,rface layers in systems of plasima proteins (thrambus-like) Thrombosis Res. L, j-17, 1972. fi'brino.gen. including
CCPLEY, A.L. and KING, R.G. Tlie action cells and plateiets on viscous resistance terns. Biorheolo,?, 10, 533-539, 1973. COPLEY, gravity
Certain A.L. environment.
of human red blood .of plasma protein
aspects of hemorheol "-7 Biorheology-, 5, 37-49,
in a near 1979.
SyS-
zero
KING, R.G.. CHIEF, S., USXXI, S. and COPLEY, A.L. Biorheologicaf methods employing the Ueissenberg Rneogoniumeter. In: New Methods in Biorheology. J.F. Stbltz (Ed.). Biorheoiogv, Sup= 2, 1982, in press. HOLMER , E., NATTSOV, C. and YILSSON, S. Anticoagulant and antithrombotic effects of heparin and lo;- molecular weight heparin fragments in rabbits. Thrombosis Res. 3, 475-485, 1982. CARTER, C.J., KELTON, J.G., HIRSH, J. and GEXT, >I. Relationship between the antithrombotic and anticoagulant effects of iow molecular weight heparin. Thrombosis Res. 21, 169-174, 1981
OCKELFORD, P.A., CARTER, C.J., MITCHELL., L. and HIRSil, J. Discordance between the anti-Xa activity and the antithrombotic activity of an uitra-lo:d molecular weight heparin fraction, ThrombosisRes. 28, 401-kO9, 1982. BARRCWCLIFFE, T.\<., SIERTOX, R.E., * L .) LIXDAHL, E'. and THONAS, D.P. iates the action of high-affinity Thrombosis Res. 2, 12j-133, 1984
HAVERCROFT, S.J., THXXBERG, Low-affinity heparin potentheparin oligosaccharides.
THOMAS, D.P., NERTOX, R.E., BARROWCLIFFE, T.V., THUNBERG, L. and LISDAHL, U. Effects of heparin 01 igosaccharides uith high affinity for antithrombin III in experimental venous thrombctsis. Haemostas. 9, 244-228, 1982. Thrombos. Structural basis for the biological effects of LIXDAHL, U. Structure, Cellular Functions, and heparin. In: Heparin, iEd.) Xew York, Academic X.X. NcDuffie Clinical Application. Press, 1979, pp. 167-180. -
ROSE~;BERG, R.D.,
ARANX~X,
G,
and
LAM,
5.
Structure
function
COTLE'i, A,L..
antagonistic
The
arin on The gefaricn ?roc , l&27, Yyj3, Ci>PLE‘i, X,L. prepared from 3olopie et de
phase
effects of
blood
!&I zh&x Ehrombop1asti.c human
placenta
and
nf
calcium
coagulation.
ions and hepFedrratior-,
component: of geloplastin -de Pathnnevue Beige plasma. '4, ?26-135, 19jJ.
3+ on the effect of Ca on A riote COPLEY, X.L. and RIXG, R.C. she viscous and el.zstic mudull< 05 szr face layers of fibrinogen. Cotloid arid Pofvmer Sci. 63, 7?1-133, PI-O@-. 1983. COPLEY,
X.L,
On
biorheolo,gy: Rheologica
1973;
Joint @enary lecture. Acta x1 845, 1974.
Biorhe-
Prolongation FIORRISOX, L.Y., RI'CKER, P.G. and ERSHOFF, B,H. of thrombus-formation time in rabbits given chondroitin sulface A. J. Atheroscler, Res. & 319-327, 1968. BJfJfiSS30?;,
effect
of
T.D., NASH, P.V. and chondroitin-k-sulfate.
SCK4TES, R. Thrombosis
The anticoagulant Res. 27, ?j-21,1982
of
Copley
Biorheoloa-, Brooklyn,
(Received
and
R,G.
King
Polytechnic Institute 7 1207 , i_;‘s& 8-S
3-2.1984; Accepted in revised by Editor S.E. Laskerf
fom
of
Ye:; York?
2.4.1984
\
JiORDS:
Fibrinogen, fibrinogenin, surface bemorheology, antithrombogenic action, heparins, chondroitins, hyalilro-, nate, de_utrans 237
period The main ob.jec Give o f this survey, m-2-6e Q~ST .an exre=ded of more +ihan tez years (l-3), is to compare the surface effects of different DfeFafations of heparin of varying a=ticoagulzxt and antithrom3otic actions. These heparins were added to sollutions of PT! human fibrinogen and hemorheological properties highly ourif?.,_ of the surface layers formed on these solutions were measured for comparison. The properties investigated xere rigidity, viscosity and elasticity. The other objective :r;as to study these surface properties of fibrinogen systems containing substances of knows and The paraunknown antithrombotic activity other than heparins. meters for testing antithrombotic activity estra vivum, r;hich are This is dealt ?
f-03?
There have been several pathways suggested by different groups of investigators for the action of heparin. They concern the treatment of experimental thrombosis in animals and the treatment of thrombosis in patients, in cases where there was no significant A satisfactory anticoagulant action but an antithrombotic effect, explanation has thus far not been given by different investigators for the incongruity between antithrombotic and anticoagulant actions of certain agents in thrombosis therapy. significance as Our extra vivum findings are of particular to be applicable to the prevention and treatment of they appear This is based on Copley's concept of thromthrombotic conditions. of fibrinogen molebogenesis due to the aggregation and gelation without the oarticipation forming so-called l'fibrinogenin", cules, i The inhibition of fibrinogenin formation by Cerof thrombin (7,8): tain heparin preparations and other substancesis considered to be an indicator of their antithrombogenic activity, whichis disc*ussed.
Highly clottability)
purified human prepared by
and bovine the method
fibrinogen of BlombZck
(97-100 per cent and Blomb'ick (9)
HzFL)hepafin prepafations h-ere sac12red I”rom different 5oLfrces. r;ere manuThe com>mercial sodium heparins employed in this survey _&bbott, Chicago? 1ll.(USX); companies: factured by the following X'.J, jUS_?i); Laboratojres Choay, Paris (France); Organon, Orange, _a Xich. (USA.). and Upjohn, Kalamazoo, Kort'hridge , Cal.(USX) Riker, were supplied by L.B. Jaques, Univeroreparations Some of these L Saskatooa (Canada). sity of Saskatchewan,
:iere obtained Tram Xon-commercial preparations of heparin Fractionated heparin from U, investigators as follo:
We were informed that heparin fractions Tjere prepared by alcofractionation (lo), depolymerization with heparinase followed by affinity to antithrombin by fractionation (11) and fractionation Derivatives were also prepared by acid X-desulfation III (72). Dextran sulfate was obtained from Schwarz-Mann Labora(13,!4). tories, Orangeberg, NY (USA). Sodium hyaluronate was secured from Miles Laboratories, Elkhart, Ind. (USA) and depolymerized hyaluronate x-as made according to Harris et al (I?).
hOl
The blood samples, obtained from the jugular veins of dogs, drawn into evacuated collecting tubes containing sodium hepaof the plasma, rin. For the preparation the blood xas centrifuged at 3000 r-pm for 15 min.
were
A Keissenberg F&eogoniometer modified for surface hemorheologistudies (h-6) was used for measurements of surface rigidity viscosity ( ls) and surface elasticity (G,). The ifi, surface volume of sample require 1 for each measurement was 4 ml. All measurements were made at 37 2 0.5OC. cal
It should be noted that detergents should not be used to clean parts with which the test material comes in contact. The meaSUfiIlg geometry device should preferably be rinsed at least three times in physiologic saline to remove residual fibrinogen and other components of the test sample.
any
RESULTS I\r'o surface layers alone were tested as
could be control.
detected
when
heparin
solutions
1 shows the viscous and elastic moduli of surface layers Fig. of 0.4 per cent fibrinogen solutions, to xhich two pfeparatiOnS of sodium heparin from two commercial sources, A, B, in 0.2 per cent concentrations, were added. Both preparations did not change the values of surface viscosity (q's) and surface elasticity (GS) from those of the control.
Ir; Fig. are ShO%v?l of CO which 0.2 heparin were viscosixy and
2 the surface viscosit:; and surface elasticixv values surface layers of 0.4 per cent fibrinogen soi;tions per cent high and low molecular weight fractions of added. Tkese particular hepariDs did not affect the elasticity of the fibrinogen surface layers.
;9
-
q.__a.
+
:cO__~___~--p~,~-a u yz _ .% z T f. z ; 5 : 15. -! -*.;-'9 -z r t:0. -2. r2 _-
a-
FIG.
i
z= z,
;; 1 -'I& iz ! z2 j sn I-. ,C’
2
Surface
r
_.
.- __~ ._~ ,(y :’ .I __~__.. i‘qfC:t’d:Y ,+r91z
viscosity and surversus freface elasticity silency of ssrface layers of t-J solution as * &$ fibrizogen control f comzared with O.k$ fibrinogen to krhich 0.2$ low XV heparin fraction and 0.2$ high M< fraction have beer, added.
the other hand, shoris thar 0.2 -,er cent of a difFig. 3a,on reduces?ts lox molecular weight (LX'!!) heparin fraction. ferent CA) The LAX% heparin was prepared by Lasand Gs by about 60 per cent. ker (11,16). Fig.3b demonstrates that a 0.1 per cent; heparin fraction, which has a high affinity for antithrombin, ioxered 'q's and Gs -by while another heparin fraction which had a loi< about 90 per cent, affinity for antithrombin showed no change in either 7's or Gs. The heparins, shohn in Fig. li,were received from Vlf Lindahl. Fig. and high heparins
3~ demonstrates zhat different heparin fractions of 1074 The affinity for antithrombin did noz weakes:Q', and Gs. shown in this Figure were also received from Elf Lizdahl,
rii~h high affinity for Fig. 3d shows that a heparin preparation layers of 0.4 the elastic modulus of surface antithrombin reduces However, fibrinogen solution by abour. JO per cent. per cent bovine viscosity moduli did not change significantly.
FIGS .
3a,3b,3c,3d
elasticity viscosity and surface Comparisons between the surface , of 0.476 fibrinogen solution as control, and 0.4$ fibrinogen to which Fig. 321~ 0,2$ of a iow Mk heparin fraction (A) and a 0.2$ (3) have been added; Fig. 3b, fraction of another low MW beparin n.l$ of a heparin h;ith low affinity for antithrombin, and O.l$ Of a heparin with high affinity for antithrombin have been added; Fig. 3c, 0.2$ of heparin with low affinity for antithrzmbin, and affinity for antithrombin have been 0.27/o of a heuarin with high Pig. jd, 0.1% of a heparin preparation of high affinity added; for antithrombin has been added. Figs. ha and kb are data curves of the surface viscosir;y ar_d elaszicity, respectively of 0.4 per cent fibrinogen as control, to which (a) 0.1 per cent sodium heparin, (b) 0.1 per cent calcium heparin and (c), as control, SmM CaC12 were added. Both the calcium he?arin and the calcium ions increased both T's and Gs by almost 50 per cent.
viscosity (Fig. ha) and surface elasxicity (Fig. 45) Surface versus frequency of O.tig fibrinogen solution as 32.ontrol I comsolutions of 0.576 fibrinogen fo which O.l$ pared -with three O,l$ calcium heparin and jmX C-a?+, respectivesodium heparin, 17~. have been added.
i _t’ 1-desuifated chat the addition of 0.2 per ce~r Fig. j shows Danishefsky_! did not weaken q's heparin (A) f rom one scource (I, heparin (9) from a second s>mce and Gs, while a 1X1 -desulfated weakened Yiis and Gs by approximarely ir0 per cent, (S.E. Lasker)
F-IG.
Comparisons
j
-of surface
vis-
cosity aI;-d szxface elasticity of O.k$ fibrinogen solution as coatrol, with Tao O.h$ fibrinogen solutions to which 0.2% t>t'-desulfated heparin from two different sources (A gad 3) were added.
of 20 per cent dog plasma considerIn Fig. 5, a preparation ably weakens the surface rigidity (T') of the fibrinogen system. The addition of 0.1 per cent sodium heparin (J. Cnoayj to the plasma system further weakens the surface layer by fibrinogen-dog The addition of 0.1 per cezxz: of rhe same sodabout 60 per cent. ium heparin preparation alone to the fibrinogexz solution caused no change in the Tvalues.
The effect of 0.7 per cent chondr0i:i.n -4 (I. Pa_nis-:?afsk.y) on the surface rigidity of I^ibrinogen surface layers is E~OW-I in Fig, -57 of about 80 per cnn-t was found. in torque values .i reduction Kiq andChien .&is particular fir,ding led to the study by Co?iey, and elastic moduli of surface layers of fibriniJ?f on the viscous ogen system&with chondroitins A, B and C.
7-
Surface rigidity (7') versus rate of shear of 0.4% flbrinagen solution 25 co,ntrol compared with O.i$ fibrj.nogen solution to 7ghic.h O.l$ chondroitin A was added,
2
z j i 1 2 zd
M_
z;
Fig. 8a shows sizy and elasticity and elastic moduli cent, respectively.
the of are
effect surface reduced
of dextran MW 20,000 layers of fibrinogen. by approximately 40
on
l;he -V-iSCOBoth viscous and 50 per
In Fig, 8b viscous and e1asl;i.c moduli of fibrinogen sErface layers as shown to which 0.2 per cent dextran sulfate N% 17,000 was added to the fibrinogen solution. Decreases of approximately j0 per cent in both 7's and Gs were found. The effect of the addition to fibrinogen solution reduced elasticity of these fibrinogen sho-c;llin Fig. 8~.
of 0.2 per cent sodium h?aLuronate the surface viscosity and surface systems by about 50 per ten?;, as
FIGS
1
Sa,Sb?8c
Surface viscosity aiiAdsurface elasticity versus frequency of 0,20/ofibrinogen solution as control, compared with 0.2$ fibrinogen solution to which the following were added: Fig. 8a, 0.2$ dextran, iW 20,000; Fig. 8b, 0.276 dextran sulfate; Fig.8c, 0.2$ sodium hyaluronate.
Fig. 9 shows that depolymerized hyafuronate, when added to a fibrinogen solution, reduces the rigidity of the surface layers of this system by about 60 per cent.
2
;-\
-
:
2
A
A
*
* .
--
z < ,QZj '5 ! = / 2 z a
L.___ ____ -~ _~~~.__ .;' /e.2 lb0 RATE 3F5HEAX SEC-!
FIG.
9
Surface rigidity (7-t versus rate of shear of O.ir-$4 fibrinogen solution as control, compared with 0.47; fibrinogen solution to k;hich S.l$ depolymerized hyaluronate was added.
Copley postuiated that both in the initiation of tb_rom,bosis and of hemostasis the initial Occurrence of a th&rom,bus without thXrombin_ i+ formed by a gro:
The J:lot;i;lg of fibrinogen by minute amounts of ~rotamine sul f a t e z-4 a 3 first described by %
Sew direct evidence for the validity of Copley's theory of in viva fibrinogenin clotting in thrombogenesis, advanced in 1971 (78: studies on fibrin0 was provided by Kitte (32,33) in biomicroscopic 'He demonstrated fib isothiocyanate. gen, la'oeled with fluorescein rinogenin formation which he named "thrombogenesis in statu nascendi" (33). Already in 1954 Zweifach (34) discovered "the accumulation of extensive masses of gelatinous material at the site of injury" as a result of direct inflicted damage to the endothel*Lm. This material could not be digested with hyaluronidase, Fi'brin was not formed due to the heparinization of the animal. The gelatinous material could not have been composed by the clumping of platelets as no embolizing platelet clumps were seen by him at the traumatized site of the vessel vail. He also did not observe platelet aggregation at this site . Zweifach observed the gelatinous material also, lchen he did not heparinize the animal. He considered the gelatinous material as a result of chemical and mechanical tissue trauma. Zweifach stated that he was not certain whether this substance is produced by the endothelial cell or occurs in response to substances released from it which would interact with the piasna.
In Copley's appraisal, Zweifach's evidence that the gelatinous material
findings constitute at the site of the
dlrec;: trauma-
-J-t is emplhasized that the formatln of fibri.nogerLin on the endoendothelial fibrin lining is considered to beoom,e a surface gel subsequent1 y with a large increase in volume (35). Our extra vivum Studies on the elastic modulus of slurface layers of fibrinogen Tke elasziciey of the svstems may mimic the srxuation in viva. regardcrosslinkxig, complex surface layers iS a function of T;ne 'I forces or by covalent bonds. less of ~herlher by van d8r Vaals' ItS measurement provides data concerning the org2nization of the structure &hi& is considered to be a thromboid (thrombus-like) gel. T%e modulus of elasticity is a function of the concentration of cross-iinks within the adsorbed layers of fibrinogen, and, in with other plasma proteins and their Structures. viva f together 'The intertend to accumulate at an interface. Trotein molecules give rise to structures actions of the proteins Kith one another of high viscoelasticity (21),
>zzj!o s t recently Silberberg discussed multilayer Formation of formation as well as macromolecules and related it to fibrinogenin He stated that it iS to the endoendothelial fibrin lining (35). "not surprising and indeed almost to be expected that large molecules like fibrinogen will adsorb both to the endothelium and to Silberberg concluded that the surface of the circulating cells". "they coatings of fibrinogen arise, where thick gel-like surface blood flow to the point where blood can considerably influence flow is essentially closed off" (35)_
2.
blood
forms On the three Their roles in the
of blood prevention
10 shows a diagram Fig. clotting during life,
clotting extra and treatment
of the leading
FIG. A diagram thrombosis
of intravascular blood and of antithrombotic
vivum and in vivo. of thrombosis.
three forms of to thrombosis.
intravascular
IO
clotting during life (incl,antithrombogenic)
leadingto actions,
of polymerizatiori and Ligation in thrombin il?'The processes have beer?_ exrens
On rhe prevention of thrombogenesis fibrinogenin formation
in
relation
50
extra
vivuZ1 -
The consideration of fibrinogenin formation in thrombogenesis is of particular significance in finding agents which inhibit such which xi: are reporting, ap* I. Accordingly, the findings forma+io? pear to be of great importance both in -ithe treatment of thrombosis as well as in its prevention, _k diagram concerning the inhibition to js T'nere appear is given in Fig. 11. Zhe as.mvppation of fibrinogen molecules, QCJ*-D phase of fibrinogenin formation, as there .seguently affect its second phase, viz.,
of flbrinogenil formatioa inhibitors i;fiich affect comprising The first :,Thich subare inhibitors fibrinogenin geiation.
in our surface hemorheoiogical studies, tie cannot differen-tiate between the two phases of fibrinogenin formation. Xevertheless, we propose our method of testing certain agents in the prevention of thrombogenesis and in the treatment of thrombosis. They concern measurements of rigidity (J or torque values) and of viscous moduli (tits ) in steady shear, as well as of elastic modL1l.i (Gs) in osciliatory shear of surface layers of systems of highly purified fibrinogen solutions to which the respective test agents have been added.
ve realize thax surface layers of e. system of highly purified fibrinogen does not fully correspond to the in viva situation, in which we deal -with the circulating wXole blood and izs fluid phase plasma. 1x2 oilr studies we have used systems of surface layers or"
&ig_tll;.:
F;rified
a.~-ions
s&2+ ._uiL
fibrinogen,
as
of
$.zteractigzis
i;here other
wag Fiama
th;i pr:zt&ns
x-,e&
ejminsre
7:~
md
other
the
consti-
in plasma md in whole blood which WO?~ii-j affect fibrinogenin firmation. We already esta'blished that globulins and albumin lowered such formation (38), !t'ealso found that red blood cells lowered the rigidity of surface layers of plasma systems, while plateets increased it markedly (39). Thus I platelezs strengthen fibrinogenin gels and appear to augment thrombogenesis causing mixed thrombi. Thus far, our studies with syszems emgloj-ing highly gurified fibrinogen have provided significant information. However I future studies yill need to deal more and more wirh the viscoelasrrcity of surface layers of different plasmas, as well as of fibrinogen system5, to which different plasma proteins and other constituents of plasma 1411 be added. ‘2er_:s
this experimental survey ‘Throughout the Weissenberg Rheogoniometer, modified by us for surface hemorheolgical studies, was employed. As it is a sophisticated research tool, there will be an urgent need for the development of a more simplified apparatus for clinicai use which measures rigidity, viscous and elastic moduli OF surface layers of fibrinogen systems and of plasma (40,41).
4*
Various
heparins
and
their
antithrombogenic
actions
The heterogeneity of heparin is nov well documented, as deOne of the objectives of this survey was scribed by Lasker (71). that have high USF anticoagulant activity to compare preparations and high anti-Xa activity with fractions that are less effective anticoagulants but retain anti-Xa activity, Also included h;ere preparations that had high affinity for antithrombin III as well as a series of fractions with decreasing USP anticoagulant activity with high anti-Xa activity. The antithrombotic activity of would need to be evaluated regarding and their antithrombogenic action in tion.
preparations, such as heparins, their anticoagulant actions inhibiting fibrinogenin forma-
In our survey of commercial and non-commercial heparins, we which had anticoagulant action did found that most preparations as tested with regard to the not inhibit fibrinogenin formation, The addition of LMW fracrigidity, viscous and elastic moduli. tions of heparin may show lowering of these moduli, while HHVfracmay not. On the other hand, LW+ tions of the same preparations of heparin do not necessarily induce any changes of surfractions Howas shown in Fig. 3a. face viscosity and surface elasticity, studies by Copley, King and Chien$ in 1983 reported (77) ever, marked decreases of surface viscous moduli and surface elastic moduli with heparins os* LMW from 4400 to 5900 daltons. heparin and heparin Kattson and Xilsson (42) studied Holmer, fragments of different molecular weights and with different antiregarding their ability to inhibit factor Xa/APTT activity ratios They found thrombus formation using a rabbit thrombosis model. that neither anti-factor Xa nor the APTT (activated partial thromare good reflectors of antithromboboplastin time) activity alone They also found that a heparin fragment of ?4?44000 tic activity. action. activity but less anticoagulant has the same antithrombotic
ackelfard find
a refatio
L?p+q hsParFr,
7
%-ap . =:-i-G 1
Ed ~1 n-ship 2s
tested
ments
of
in
activit 3.7
orher properzies tic effectiveness' ~~i~;hror;i'o~ti~
(i&j reported between
of
,Taey
thromSosi.s
activity
not
extra
mgdels,
from
contribute
Came to
could
xhat they antithrombotic
viva
concl;lded
L>F~ heparins
I
recently
in
animal
~hgy
efficacy
anti-xia
the
this 21x2);s
af,d extra
vi-
their to
findings that their 'antithrombo-
conclusion, be
could not action 01
predicted
sir;ce
by
ia
viva
measure-
vix7_2m.
reported that low af($j) lii9i-e recently Bzrfoxcliffe ei: al the acTion of high affinity (E-f.) (LX) heparin potentiates finity oiigosaccharides for anzithrombin III (AT-III). They conheparin heparin content of LI'R heparthat the I_,_% cluded from their studies in fractions appears to be an important determinant of their antiThey further stated that "thrombin inhibition thrombotic activisy. Thomas is nor an absolute requirement for antithrombotic activity'!. et ai (46) in their studies assessed the anrithrombotic activity of It apiix heparin oligosaccharides in a rabbit thrombosis model. activity employed by these inpears to us that the antithrombotic action of vestigators may be based in part on zhe antithrombogenic is due to its inhibition of fib+ Their LX34 heparin which we consider formation. rinogenin et al ($8) found that the fractionLindahl ($7) and Rosenberg one with very ation of commercial heparins yielded E:WO fractions, one. Lindahi (12) low Siologicaf activity and the other k*ith 2 high in chemical composition between the could not find any diff erence z;wo fractions. The difference consequently must be very subtle and perhaps depends on the sequence of the sugars in the oligosaccharidc chains. Xe, employed in our surface hemorheological studies therefore, heparins with high and low affinity for supplied by Ulf Lindahl antithrombin. The fraction of heparin, exhibiting high affinity for antithrombin, lowered the viscoelasticity of fibrinogen surface -with low affinity has no such layers (Fig. 3b), rjhile the fraction However, in Fig. 3c there are no changes effect. in surface viscmosity and surface elasticity by the use of another heparin preparation with fractions with low and high affinity for antithrombin,
Of great interest are our findings with preparations of calcium heparin and sodium heparin, supplied by Choay, Paris, on surface layers of highly purified fibrinogen (Fig. ka). The sodium heparin did not decrease the viscous and elastic moduli of fibrinogen surface layers, while calcium heparin increased them markedly, as did Ca2+. There are substances which either promote or inhibit the gelation of fibrin. Such gelation promoting or geloplastic substances and gelation inhibiting or antigeloplastic agents have been found by Copley to be Ca2+ and sodium heparin, respectively ('19we found that Ca2+ Recently, is a geloplastic agent also 51). with regard to fibrinogenin clotting (32). Eowever, only certain sho~-z in this presentation, which have antigeloplasheparins f as
lowering of viscoelasticity of surface layers of fihrinoThe i tbi h--dejulfated hpp~ri~:, gen s>-szems **: J is of intershow2 in Fig. est, beca:use this altered heparin has a 10i;er molecular charge density and minimal anticoamlant action. The X-desuifated heparin preparati0n appears ~0 be another indication that antithrom3ogani.o action is not related to the inhibition Of fibrin formation. The acticsn of heparins,zhich 10~.en the rigidity and viscoelasticity of surface layers of fibrinogen systems, will need to be examined in great detail with regard to the mechanism involved in these actions. Since certain heparin preparations have such actions while others have not, the agent responsible for this activity will need to be isolated from those preparations containing it, We believe that t1he findings which are presented will stimlulate much needed netr' h;ork on heparin and its actions, 3.
02 the mation
role of plasma bv heparins
in
the
inhi3ition
.0f fibrinogenin
for-
In 1972 we reported that surface layers of heparinized plasma eshi3ited markedly lower surface rigidity than those of oxalated plasma from the same blood withdrawal from a human subject (40). These findings led us to investigate, over a period of more than ten years, the effect of heparins of different origins on the rigidity and viscoelasticity of surface layers of fibrinogen systems. In the course of this survey, we reported some of our findings(l-3, Instead of plasma we chose highly prrrified human fibrinogen 5,531. of 97-100 per cent purity (9). This choice eliminated the variaplasmas with their varying concentrations of bility of different different constituents. The rigidity of surface layers from a system of 20 per cent dog plasma and 0.2 per cent human fibrinogen, upon the addition of is weakened by approximately 60 per 0.1 per cent sodium heparin, The addition of 0.1 per cent sodium heparin, supplied by J. cent. Choay, to the dog plasma reduced the Tvalues stiil further (Fig. As expected from our earlier investigations with globulins 6). a plasma-fibrinogen system and albumin in 1971 (18) and 1972 (38), showed lower Tvalues than those obtained with a fibrinogen preparation alone. These preliminary findings Open up an entirely new field of agent -which study regarding the efficacy of an antithromSogenic evaluation of suchan agent may further show that the therapeutic action or on its affinity for on its anticoagulant does not depend show that plasma The findings presented in Fig. 5 antithrombin. play 2n import2nt role constituents other than fibrinogen may well clotting +,+.thout thrombin participation. in fibrinogenin
Ve have not yet studied the effect of differeni; purified of fibrinogen and the addition added to solutions plasma proteins of heparin on the rigidity and viscoelastici;y of surface layers In case certain plasma proteins or other plasma of these systems. , tiie lo:"-iering action on surfac.e decrease consZltuents h-i.11 further viscoeiasticity by other glycosaminoglycans, rigidity and surface potential usefulness in the substances, their as ~;ell as by other prevention and therapy of thrombotic conditions will bncacre PX-
trenel_y 6.
importaLzt
*
Inhibition in the formation than certain hnparins
of
fibrinogenin
by
substances
othe
the findings pertaining to We have discussed already above -the discrepancy of antithrombogenic and anti-Xa activities of cerfor which we made their inhibitory action in fitain heparins, 'orinogenin formation responsible. As far as chondroitin A is conet al reported in 1968 (54) marked antithrombotic ce.rned, Morrison in their in vivo findings in rabbits. action of this substance published their extra vivum stu Ejornsson et al (5j), T&ho recently explain its antithrombodies with chondroitin At could not fully tic action on the basis of certain anticoaellant actions which themeasured. The antithrombogenic actions in vivo by the use of the two glycosaminoglycans, chondroitin A and L?fV heparins, reported by Ejornsson et al, Ockelford et al, and Barrowcliffe et al, respectively, can Se explained as the inhibition of fibrinogenin formation b-ythese substances. It appears that the antithrombogenic action in vivo can be equated with the inhibition of fibrinogenin formation in viva. For these reasons, fibrinogenin formation and its inhibition by certain agents can no longer be ignored in co.nsiderations pertaining to thrombogenesis. The recent findings by Copley, King and Chien (17) are of particular importance, as they emphasized the need in employing extra vivum surface hemDrheologica1 testing of aniithrombogenic agents in the inhibition of fibrinogenin formation by heparins L1 5300 and 5900 dalT;ons and by chondroytmoiecular weights of 4400, ins A, B and C. The findings of surface rigidity reduction of about 80 per cent with chondroitin A-fibrinogen systems predates, as already stated, the studies by Copley, King and Chien on the vis coeiasticity of surface layers of fibrinogen surface layers by char. droitins A, 3 and C, resulting in marked reductions of viscous and elastic moduli with all chondroitin-fibrinogen systems, which we tested (77). i;ith substances other than Of special interest are our findings k'e tested sodium hyaluronate 'because of its glycosaminoglycans. it to behave similarly as low mohigh molecular iieight and found
1s
dextra?_
j:;lfase
>&-
17
f :Jf’1io
i
3
.a
J:har.ged
feast tQ .de_xtrarz, >pi 2O.Ot~0, r;e tested their coelasticity of surface lavers of fibrinogen parations have similar surface hemorheological .8b! carxot be responsible for 1, so the charge in surface viscoelasticity.
in
m;ole.~~~.lp,
effec:
or:
solution. aceions the marked
the
cozl-
-%-is-
Both pre(Figs. 8a, decrease
Sor: all of these substances can be applied in the Preiiention Jf thrombogenesis and in thrombosis therapy, alth*eugh they all ca-dse marked decreases in sur face viscoelasticity or in surface rigidity values. Severtheless, some of these substances may prove to be useful in the prevention and therapy of thrombosis.
I;'& Zhan!i the follox*~ing investigators for kindly supplying r
zs
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2.
KI?X, R.G. The action of differing heparin and COPLEY, A.L. layers of fibpreparations on the viscoelasticity of surface gy, LaJolla, rinogen. Abstracts, 3. Internat. Congr. Biorheolo Biorheology, 15, 461, 1978. CA, USA, 28.8.-1.9.1978.
3.
szudies of surface Viscoelastic COPLEY, A-L, and KING, R.G. layers of heparin-fibrinogen systems and the antithrombotic Abstracts, VII. action of heparin other than anticoagulation. Internat. Congr. on Thrombosis and Haemostasis, London, EngThrombos. Haemostas. s, 323, 1979. land, 15-20.7.1979.
i:.
The Weissenberg Rheogoniometer COPLEY, _&.L. and KING, R.G. The Karl 1ceissenberg adapted for biorheological studies. In: J. Harris (Ed.), Kampala80th Birthday Celebration Essays. Tairobi-Dar Es Salam, East African Literature Bureau, 1973, PP. 127-143.
3.
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ARANX~X,
G,
and
LAM,
5.
Structure
function
COTLE'i, A,L..
antagonistic
The
arin on The gefaricn ?roc , l&27, Yyj3, Ci>PLE‘i, X,L. prepared from 3olopie et de
phase
effects of
blood
!&I zh&x Ehrombop1asti.c human
placenta
and
nf
calcium
coagulation.
ions and hepFedrratior-,
component: of geloplastin -de Pathnnevue Beige plasma. '4, ?26-135, 19jJ.
3+ on the effect of Ca on A riote COPLEY, X.L. and RIXG, R.C. she viscous and el.zstic mudull< 05 szr face layers of fibrinogen. Cotloid arid Pofvmer Sci. 63, 7?1-133, PI-O@-. 1983. COPLEY,
X.L,
On
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Joint @enary lecture. Acta x1 845, 1974.
Biorhe-
Prolongation FIORRISOX, L.Y., RI'CKER, P.G. and ERSHOFF, B,H. of thrombus-formation time in rabbits given chondroitin sulface A. J. Atheroscler, Res. & 319-327, 1968. BJfJfiSS30?;,
effect
of
T.D., NASH, P.V. and chondroitin-k-sulfate.
SCK4TES, R. Thrombosis
The anticoagulant Res. 27, ?j-21,1982