THROMBOSIS RESEARCH Printed in the United
Suppl. II, Vol. 8, 1976 Pergamon Press, Inc.
States
SECTION
VII
POLYMOLECULARLAYERS OF FIBRINOGEIJ SYSTEMS AND THE GENESIS OF THROMBOSIS Alfred
L.
Copley and Robert G. King
Laboratoryof Biorheology,Departmentsof Life Science and of Bioengineering,PolytechnicInstituteof New York, Brooklyn,?;.Y. 11201, U.S.A.
ABSTRACT
In 1971 Copley proposed a new theory on the initiationof thrombosis, based on hemorheologicalobservations. A brief summary is given of observationspertainingto the plasmatic zone in relation to the cement fibrin in the proposed endoendotheliallayer as well as of earlier findings on thromboid surface layers. They concern viscous resistance (torquevalues,r, dyne. cm) of layers of systems of fibrinogen and other plasma proteins. New findings are presented of viscous resistanceof surface layers of fibrinogensystems,obtained in steady shear together with findings on the elastic component, secured in oscillatoryshear. A new concept on maintainingthe patency of microvessels,presentedby Copley in 1974,is related to the problem of the initiationof thrombosis. During life, both with the patency of the vascular lumen and with intravascularobstruction, which by necessity are opposite, the stresses on the blood vessel walls are extremelyhigh, particularlyin the capillaryor minute blood vessels. Fibrinogen systemswere therefore exposed to high shear at 1000 set-1 for 3 min prior to measurements. Thereafter, data were secured from lo-3 to 10-l set-1. Highly purified plipoprotein and 8 globulin,which gave no?values also showed none when previouslyexposed to high shear. However, all hitherto tested fibrinogenpreparationsfrom differentmammalian species (human, bovine, sheep, dog, rabbit, and cat) exhibitedhigh rvalues. They usually became higher if subjectedto 3 min of high shearingprior to the low shear testing. The findings are related to a new hypothesis proposed by Blomback and Copley on the transformationof the fibrinogenmolecules, caused by high shearing. Such shearing forces, accordingto this concept,would open up the polymerizationsites of fibrinogenand thus simulate the enzymaticaction of thrombin for the polymerizationof fibrin. The differencebetween the biochemical and hemorheologicalactions is that the fibrinopeptidesare split off by thrombin,while no such cleavagewould occur in fibrinogen, proposed to be altered physicallyby the high shear known to exist 393
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at the vessel wall. 'Ihelayer upon layer deposition ol'such hemorheologically configurated fibrinogen and its polymerization would proceed, resulting in the formation of a thrombus which thus would initiate thrombosis and hemostasis. Copley considers blood cellular aggregation and fibrin coagulation to be secondary processes in the genesis of thrombi in minute or capillary blood vessels.
INTRODUCTION A brief review of earlier observations is given and most recent findings are presented with regard to a new approach to studies of the genesis of throm. bosis.
Fig. 1 Reproduction of Figures in the treatise by Poiseuille, published in 1839(b), pertaining to his studies on the plasmatic zone.
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One of us (A.L.C.)contendedthat it is in the plasmatic zone, which is the cell free part of the circulatingblood in proximity to the blood vessel wall, where the processes occur which initiate thrombosis (1,2). The plasmatic zone, called in the French literature "la zone de Poiseuille",was already described in 1661 by Malpighi in the first in vivo observationson the microcirculation, !lkismarginal or plasmatic zone was also made by him on the frog's lung (3). described in the 18th century by Spallanzaniand Albrecht von Halle (1). Eiowever, it was Poiseuillewho made hemorheologicalmeasurementsand observations on the plasmatic zone (Fig. l), which he reported in 1835 and 1839 (4). Poiseuillecontendedthat it containedtwo major portions,viz., one, an immobile portion next to the endotheliumand the other, the mobile portion. This is diagrammaticallyshown in Figs. 2 and 3.
Fig. 2 Diagrammaticfigure of extrusion for blood accordingto Copley and Staple (6). BASEMEET
MVENTITIAL
%NDOTIIEiIAL CELL
TUXTC
/
Eh?)OENDOTliELIAL FIBRIh' I‘ILM
MXllLE
LAYERS
Fig. 3 Diagram illustratingthe layers of the blood capillarywall and the location of the cement fibrin in the basement membrane and surrounding the endothelialcells (includingthe endoendothelialfibrin layer) in accordancewith Copley's concept. The drawing is meant to convey a general idea without giving scale, dimensionsand without simulatingthe appearanceof elements in the microstructureof the vessel wall (11).
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In 1959, Copley and Staple confirmed the findings of ?oiseuille (5-7). They also made certain observations which, although they do not present definite evidence, suggest that there is a more or less immobile layer next to the endothelium. In 1953, Copley proposed the concept, which has been developed ever since, that fibrin in submicroscopic dimensions lines the inner aspects of the vessel wall in direct contact with the endothelium (8,9). Thus, the circulating blood is separated from the endothelial cells by the so-called endoendothelial fibrin lining. It is the nature of this endoendothelial lining which is of great importance in any considerations regarding the flow of blood. This was discussed recently by Copley (10) in some detail in an article on hemorheological aspects of the endothelium-plasma interface. The proposed identification of this layer with different forms of fibrin, to which he gave the generic term "cement fibrin" (11,12), guided us, as well as other investigators during the past 22 years in numerous experimental studies. The process of thrombus formation begins, according to a new concept by Copley (13), with the adsorption of fibrinogen, followed by a growth process of the adsorption of fibrinogen and other plasma proteins, layer upon layer, until the lumen of the blood vessel involved is either partially or completely obstructed. It is only after this process is initiated that other clotting processes, such as the aggregation of platelets and/or red blood cells, as well as the coagulation of fibrin occur (13,14). Fibrin coagulation means the polymerization of fibrin, followed by its subsequent network formation or gelation and ending in cross-linking. Concepts on the genesis of thrombosis have hitherto been based mainly on two major processes of in vivo clotting, found to occur either separately or mixed, viz., the clumping of blood cellular elements and the coagulation of plasma by the formation of fibrin. THE THREE
MAJOR PROCESSES OF BLOOD CLOTTING
Blood clotting
polymerization
Fibrin
monomer
I Cross-hnklng
Fig. 4 Many years ago, Copley recommended that the word "clotting" be used as a generic term for fibrin coagulation and blood cellular clwnping (15-17). This usage of the non-committal generic term "clotting' was extended recently by him to include the process of time-dependent progressive adsorption of plasma proteins including fibrinogen. This process is considered as a hitherto unrecog-
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nized form of clotting. All three processes of clotting, shown schematically in Fig. 4, are rheological in nature, and any in vivo clotting involving the endothelium and progressing to vascular obstruction may lead to manifestations in various organs comprising the different conditions of thrombosis, which is a purely hemorheological disease or disorder. Any of these processes of clotting can go on separately or mixed. However, it is re-emphasized that the initiation of thrombus formation, both for the development of thrombosis and the arrest of hemorrhage (18) in minute blood vessels, depends mainly upon the proposed primary process of the aggregation of fibrinogen molecules. This concept is based mainly on our rheological findings obtained with surface layers formed on solutions of plasma proteins. TECHNIQUES We used a Weissenberg Rheogoniometer, modified by us to make it more suitable for biological investigations (19,20). The surface layers were measured at shear rates from 1000 to less than 0.1 see-l, using a geometry that is a combined Couette and cone and plate. A guard ring (Fig. 5) is used, which can be easily detached so that the torque (7) derived from surface layers can be separated from that derived from the bulk of the sample. These surface layers contribute added torque, which is particularly high at low shear rates. A similar method was reported earlier by Joly (21). Two measurements were made on each sample, one with the guard ring in place and the second with it removed. 13~subtracting the first value from the second value obtained, the torque contributed by the surface layer can be ascertained.
id level
-A
Fig. 5 Flan view of geometry showing position of removable guard ring A refers to the rotating outer cylinder, while B is the stationary inner cylinder of the accessory to the Weissenberg Rheogoniometer. More recently, we devised a technique of measuring directly the torque generated by the surface layers (20). These values are reported as torque, (7), versus rate of shear. This accessory to the Weissenberg Rheogoniometer is shown diagrammatically in Fig. 6. The device, made of Plexiglass, consists of a ring,9 cm in diameter. The ring just penetrates the surface layers of the test sample, which is held in the lower platen in an annular groove, 0.5 cm in width. The annular groove requires 5 ml of the test sample. This de-
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can also be used for viscoelasticity rneasuements of the surface iayer:: when the oscillatory-mode of the instrment is employed. The thromboid S-~r-t'ace layers of the test sample transmit a torque to the measuring lpper platen whe!the lower platen is rotated.
Vice
iig. 6 Plan view of special geometry for direct measurements of rheological properties in steady or oscillatory shear of surface layers. We found during standardization and calibration studies of the new geometry that no torque is contributed by the substrate fluid at shear rates below 10 see-l, and that the torque values obtained are derived exclusively from the surface layers. Above 10 see-l small readings can be obtained from the substrate and we have therefore limited the upper shear rate to 10 set-1 with the geometry employed. SOME FJUUIER FINDINGS Fibrinogen solutions gave the highest torque values when compared with x globulin, while albumin exhibited the least (13). The higher the concentration of each of the tested proteins in solution the higher was the torque value (13,14). This was also the case with 5 to 90 per cent concentrations of platelet-free oxalate plasma and serum obtained from apparently healthy human subjects (14). The addition of 0.4 per cent fibrinogen to 0.25 per cent albumin or to 5 per cent plasma or serum increased viscous resistance. Such addition to 5 per cent albumin or usually to 18 per cent plasma showed no change in torque values (14). A critical concentration of plasma or serum was noted, which ultimately may provide an indicator in the recognition of proneness towards thrombosis and may serve in the management of thrombotic conditions (14). Preliminary data,secured by us,showed that the surface layers of heparinized plasma gave decreased values when compared to the surface layers of oxalate plasma from the same blood withdrawal. There seems to be an antagonistic relationship between the red blood cells and platelets with regard to the formation of thromboid surface layers of fibrinogen and other proteins in plasma (22). We found that the presence of platelets in systems containing fibrinogen, whether or not enriched by other plasma proteins, will increase markedly the viscous resistance of polymolecular protein layers (22). Red blood cells, on the contrary, were found to decrease the viscous resistance of these layers. Thus, red blood cells may well
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have an inhibitingaction in the productionof polymolecularlayers of fibrinogen and other plasma proteins (22). Our findingswith highly purified @lipoprotein and xglobulin, to which 0.4 per cent bovine fibrinogenwas added, did not exhibit viscous resistance from surface layers (23). NEW FINDINGS All recent rheogoniometricmeasurementsof surface layers of fibrinogen and other protein systemswere made with the accessory shown in Fig. 6. There has been some questionwith regard to the air-fibrinogenlayer inter face. We have not found any differencein values if the air was replaced by oil. This is shown in Fig. 7 with a 0.4 per cent human fibrinogen solution. The oil used was a light mineral oil. In a number of other experiments,we always found that there were no changes whether or not we used oil to cover the protein system.
RATE
OF
SHEAR
set-’
Fig. 7 A comparisonof torque values, secured from surface layers of a 0.4 per cent human fibrinogensolution,formed at (-.-) oil-fibrinogenand (-o-) air-fibrinogeninterfaces. In Fig. 8,viscousresistancevalues from surface layers are shown of 0.4 per cent fibrinogen solutionstested at varying shear rates. As can be seen, preshearingat 1000 see-l for 3 min increasedthe torque values for human, bovine, cat, dog, rabbit, and sheep fibrinogens. The largest differenceafter shearingwas shown with the bovine preparation,and the highest values were found with the sheep and human fibrinogens. Since our earlier findings,using highly purified (3lipoprotein,mentioned above, did not show any viscous resistanceupon the addition of fibrinogen,we repeated this experimentin making torque measurementsbefore and after the application of high shear at 1000 see-' for 3 min. Fig. 9 shows that high shearing does not generate torque from surface layers of these preparations,if formed. Surface layers of 0.4 per cent fibrinogensolutionsare measured as controls prior and subsequentto high shearing. In our most recent studies,we made observationson the viscoelasticityof surface layers of fibrinogen systems,using the oscillatorymode of the instrument. Fig. 10 shows typical recorder traces from these experiments. The trace:
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--
r L-_ r-----
I
-----7
I
E 0
e 7?
II
CAT
SHEEP
* 103 I_ -
” y
IO2
L? ?
I 0
-3
IO
-2
RATE
IO OF
-1
SHEAR
IO0
IO2
IO’
(set-’
i-1_ IO
IO
-2
RATE
1
1
1
-3
10-l
OF
I
IO’
IO”
SHEAR
IO2
(SW-‘1
Fig. 8 Viscous resistance values from surface layers of 0.4 per cent solutions of fibrinogen from six species (human, bovine, cat, dog, rabbit,and sheep).
I
10-3
,
I
to-2 RATE
10-l OF
IO0 SHEAR
11 IO'
102
(set-‘1
Fig. 9 Comparative findings of torque values obtained before (-A-) and after (-A-) shearing at 1000 see-1 for 3 min from surface layers of 0.25 per cent highly purified p lipoprotein added to 0.4 per cent highly purified human fibrinogen. As controls, the same fibrinogen samples without the addition of/i lipoprotein were measured before (-o-) and after (-•-) high shearing. exhibit a phase difference of 15 degrees indicating the presence of an elastic component. We have found that the elastic component is increased considerably,
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if a three-minute period of steady high shear at 1000 see-1 is applied prior to measurement. Similar findings were obtained with highly purified ~globulin. The particular gglobulin of 99 per cent purity did not show any torque values from a surface layer, but if this purified &globulin was exposed to high shearing, a weak layer was formed, which faded away after approximately three minutes. This by itself appears to be an interesting phenomenon which invites iurther study.
--
0.0012
radians
3-g. 10 A typical recorder trace, obtained during an oscillatory experiment at a frequency of 0.06 Hertz. The larger trace represents the input motion, while the smaller trace represents the response of the surface layer. The phase difference between the two traces is 15 degrees.
IO -5
lO-4
10-3
FREQUENCY
IO
-2
Id’
(iiartz)
Fig. 11 Comparative elastic modulus data of surface layers of 0.4 per cent solutions of fibrinogen from sheep (-•-), bovine (-o-),and human (-A-) origins. In E'ig.11, data of dynamic tests of surface layers of 0.4 per cent fib, rinogen solutions are shown -pertainingto the elastic modulus. The fibrinogen was from sheep, bovine, and human sources. Both the bovine and human fibrinogens gave nearly identical values of elastic modulus, while the values of sheep fibrinogen were markedly increased. This Figure also shows that the rate of shear has only a small effect on the elastic modulus.
Figure 12 shows data of the surface viscosity in poise calculated from OS, cillatory tests of sheep, bovine, and human fibrinogens at 0.4 per cent coneen,
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tration, plotted against frequency varying from 10"' to 10' Hertz. These sre some differences in viscosity, with sheep fibrinogen showing the highest values. What, however, is most startling, are the extremely high viscosity valvues calculated using a layer thickness of 100 8.
,05upu 10-5
10-4
10-3
10-z
FREQUENCY
IO-
IO0
(Hertz)
Fig. 12 Plot of viscosity (dynes.sec/cm2) versus frequency of 0.4 per cent solutions of fibrinogen from sheep (-•-), bovine (-o-_)and human (-&) origins. de chose arbitrarily the value of 100 1 as the layer thickness and used it for all our calculations of findings presented in Figs. 11 and 12, although the surface layers may have varying and higher values of thickness. In case the thickness of these layers is increased, there will be a decrease in the viscosity values; however, they would still be extremely high. Recently, Smith, Morrissey,et a1.(25) found the thickness of fibrinogen surface layers to differ, under certain conditions, from 200 to 600 X. Nevertheless, the values for the elastic moduli measured are extremely high, in the neighborhood of 60,000 to about 25O,OOO dynes/cm* over the different rates of shear with the three preparations tested. These high values suggest the presence of very strong intermolecular bonds of these polyrnolecularfibrinogen layers.
ON THE FORMATION OF POLYMOLECULAR LAYERS OF FIBRIN~G~
(CLOTTING OF FIBRINOGEN WITHOUT THROMBIN)
In 1971, Copley referred to a non-homogeneous distribution of the adsorbed proteins and a decrease and/or imbalance of protective colloids (13). He defined "non-homogeneous distribution" as time-dependent, progressive adsorption of the plasma proteins from the solution at the interface with the vessel wall and the free surface of the adsorbed proteins (13). This non-homogeneous distribution of the adsorbed proteins may be related to considerations of Copley and Staple (7) as to whether a suspension of macromolecules would show a radial distribution when flowing through a capillary tube in which the velocity gradient from the axis to the wall of the tube was steep. A paper on electrical field-flow fractionation of proteins by Caldwell et al (24) appears to support the earlier considerations by Copley and Staple. Field-flow fractionation is a separation method in which various applied fields, working in conjunction with cross sectional flow non-uniformities in a narrow tube, cause the differential migration of molecules and ions. It is possible that there is a higher transport rate for larger molecules than for smaller ones. It appears that their findings would substantiate the contention of a non-uniform distribution of particles across a flow channel. As an alternative to the production of
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these polymolecular layers, Copley (10) proposed the occurrence of changes in the solubility of fibrinogen and of other plasma proteins in the affected part of the circulation. This occurrence can be brought about either by alteration of the plasma proteins including fibrinogen, or by certain additives, which may be present or released in the affected areas changing the solubility of these proteins. Nevertheless, any of these alternative explanations are proposed to concur with alterations in the flow properties of blood in these affected regions and the induction of the formation of polymolecular films or layers of the plasma proteins, particularly of fibrinogen.
WALL SBl!tAR FATES AND THE BLOMEifiCK-COPLEY HYFOTHESIS OF CONFORMATI0NA.L CHANGE OF FIBRINOGEPJ The shear rates are known to be very high at the wall of the different blood vessels. They are particularly high in the small or minute vessels. There is some discrepancy in the literature of wall shear rates estimated by different authors (26-28). The values,which Whitmore (26), Chien (27),and Charm and Kurland (28) reported, are compiled by us in a composite Figure (Fig. 13).
I AORTA
L
ARTERIES
I
ARTERIOLES
1
CAPILLARIES
1
VENULES
I
VEINS
.
VEh
CAVA
Fig. 13 Compilation of values of wall shear rates in different blood vessels from data by Whitmore (26), Chien (27),and Charm and Kurland (28). In the capillaries these values are particularly high, as reported by all authors. However, there appear to be some marked differences in values reported for arterioles by Chien (27), which show values of about 1000 see-l, while those reported by Charm and Kurland are 8000 set-l. Such a value of nearly 8000 set-1 is given for capillaries by Chien, while Charm and Kurland give a value of 1000 see-l. The values by Chien also differ from those by Charm and Kurland with regard to the venules. The latter authors ive a value of 800, while Chien's values differ between about 70 to 300 set-B . As pointed out recently by Copley (lo), there is a need for re-investigating the wall shear rates to find out the reason for these discrepancies. Nevertheless, it is clear that wall shear rates are particularly high. The values of shear rates at the wall of the aorta are similar in the data presented by Whitmore,and by Charm and Kurland, as are the values at the wall of arteries, as far as human subjects are concerned. Whitmore reported values in the dog which for the aorta are appreciably higher than in man. However, in the arteries and capillaries, there is not much of a difference. The wall shear rates are much
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lower in the veins than in the capillaries, as reported by all three groups of investigators. In discussions on these high wall shear forces, Blomb&k and Copley developed a working hypothesis that the high shear forces present at the blood vessel wall may bring about conformational changes of fibrinogen molecules at certain sites in the circulation. Recently it has been demonstrated by Kudryk, gen contains polymerization sites in its fragment The sites residing in N-DSK become activated upon thrombin-like enzymes. This appears to be due to tion sites or their exposure after release of the
BlombZck et al that fibrinoD and N-DSK portions ;29,30). treatment with thrombin or unfolding of the polymerizafibrinopeptides.
It is likely that all polymerization processes, whether enzymatic or nonenzymatic, always involve conformational change in N-DSK, whereby polymerization sites are exposed. Stewart and Niewiarowski (31,32) described the polymerization of Pibrinogen by protsmine sulfate without the action of thrombin. Their findings can be explained by neutralization of the acidic charges on the fibrinopeptides, bringing about a similar conformational change as brought about by the release of these peptides by enzymes. It is according to the Blomback-Copley working hypothesis that these conformational changes csn also be induced by shearing forces with subsequent exposure to polymerization. ON VASCULAR PATENCY The problem of vascular patency is of utmost importance with regard to certain rheological properties of the vessel wall which prevent the blood vessel from collapsing. In recent .years, Fung et al (33,34) explored this side of the problem of vascular patency, particularly as the rigidity of the capillary wall is concerned. Fung claimed on the basis of experimental observations that the rigidity of blood capillaries is due to the gel-like materials surrounding the wall of the blood vessel. In measurements of the stress-strain relation of the mesentery, Fung et al could show that the material is nonlinear elastic with a shear modulus that varies linearly with the shear stress. According to Fung (35),the patency of blood capillaries is on the basis that mechanically they are like "tunnels in gels ':.A muscle capillary or a mesentery capillary blood vessel will not collapse when the entire tissue is subjected to hydrostatic pressure. The elastic behavior of a blood capillary depends also on the mechanical properties of the surrounding medium. Thus, the elastic rigidity of the different surrounding media, as proposed by Fung, appears to be an important factor in capillary patency. A new concept advanced last year by Copley (10,35) offers a hitherto unknown factor for vascular patency and provides a different explanation for the elastic rigidity of blood vessels and their patency. This factor concerns the elastic rigidity of the proposed endoendothelial fibrin lining and is based on our recent viscous resistance and viscoelasticity studies of surface layers of fibrinogen. The contention is that, since the proposed endoendothelial fibrin layer is continuously exposed to the very high shearing at the vessel wall, particularly of blood capillaries, the endoendothelial layers on these walls would make them highly rigid. Thus, it is this factor of endoendothelial fibrin rigidity which is considered important in maintaining the patency of all capillary blood vessels. Since in our viscoelasticity studies of polymolecular
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fibrinogen layers we found them to exhibit an elastic component, the latter is considered to contribute to the elasticity of the blood vessel wall (10). ON NON-EXZYMATIC INDUCED POLYMERIZATION OF FIBRINOGEN AND THE INITIATION OF THROMBUS FORMATION IN THROMBOSIS AND BEMOSTASIS Several observations were reported in the literature which support Copley's new concept of the initiation of thrombosis (36-40). They have a bearing on our findings of thromboid layers of fibrinogen and other proteins. The observations by Hartert using his method of rheo-simulation, reported three years ago (38) and this year (jg), as well as his new findings employing the rheosimulation, reported at this Seminar (ho), appear to provide direct evidence in support of Copley's concept. The possible role of fibrinogen layers on the vessel wall in connection with the production of wound thrombi in the mechanisms of hemostasis was reIt is possible that with the onset of bleedcently discussed by Copley (18). ing the altered hemorheological situation will alter the even distribution of plasma proteins at the site of extravasation. Consequently, a dynamic process occurs, viz., the initial adsorption of fibrinogen and other plasma proteins: followed by a growth process of the adsorption of layer upon layer of these proteins, constituting thrombus formation. This is then followed by platelet deposition and clumping, as well as by fibrin coagulation. Caro, Fitz-Gerald and Schroter (41) made observations with regard to the genesis of atherosclerosis which appear to relate to our findings on viscous resistance of fibrinogen surface layers following exposure to high shearing forces. Caro et al observed that the distribution of fatty streaking and early plaques may be strongly influenced by high shear rates at the arterial wall and also at the sites of bifurcations of arteries when there is turbulence and the shear rate is high. The primary process of fibrinogen aggregation (preceeding the secondary processes of blood cellular clumping and/or of fibrin coagulation) is considered by one of us (A.L.C.) to occur most rapidly (18) and possibly within several seconds. The sequence of these processes needs to be investigated both extra vivum and in vivo. Our results appear to demonstrate that high shear forces, which are known to be present at a vessel wall, have induced an alteration in fibrinogen, and it is believed that this represents an unfolding of the polymerization sites of the fibrinogen molecule in accordance with the Blomback-Copley working hypothesis. In this way these high shearing forces would bring about the unfolding which otherwise, as is well established, is the result of partial proteolysis by thrombin. We do not yet know whether the increase in torque values is due to thickened polymer layers being formed or to extra bonds forming within the layers. IN CONCLUSION As students of thrombosis and of related phenomena affecting the circulation of blood, we are preoccupied with the obstruction of the lutninaof blood vessels due to clotting processes and with processes which keep the vascular lumina patent. These latter processes either inhibit the various forms of clotting or, after clotting already has set in, lead to the lysis or disintegration of clots of different origins. These occurrences will, of course, continue to be a main concern of studies related to thrombosis subsequent to its initiation. Patency depends also on the elastic rigidity ofvesselwalls.
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The phenomena of the polymerizationof fibrinogenmolecules and of intravascular aggregationof fibrinogen,layer upon layer, will need to be studied at many levels in a variety of scientificfields and disciplines. In the case of blood cellular clumping and of blood coagulation,such studies have been ongoing for more than one hundred years,an enterprisein which many investigators continue to be active. New develolpnents in fibrinogenresearch, in ultrastrucmicroscopictechniques,inimmunochemistryand in surtural including elecf;ron face chemistry,as well as in hemorheology,provide us with new tools for experimental scrutinityof Copley's concept or theory of the genesis of thrombosis. ACKNOWLEDGEMENTS The studies were supportedin part by the Office of Naval Research conNOOO-14-67-A-0449and NOOO-14-75-C-0221,and most recenttracts NR 2754 (03), ly also by the National Institutesof Health, grant HL 17556-01. We are indebted to Dr. M. Burstein, Paris, France, for supplyingus with his highly purified preparationof /3lipoprotein. REFERENCES 1.
COPLEY, A. L. Adherence and viscosity of blood contactingforeign surfaces, and the plasmatic zone in blood circulation. Nature I&, 551, 1958.
2.
COPLEY, A. L. The endoendothelialfibrin film and fibrinolysis. Proc.VIII. Internat.Congr. Hematol. Tokyo, 1960. Tokyo, Pan-FacificPress, 1962, vol. 3, P. 1648.
3. MALPIGHI, M. De Pulmonibus,Bononia, 1661, Translatedby James Young. PYOC. Royal Sot. Med. 3, 7, 1929. i+. POISEUILLE,J. I,.M. Recherchessur les causes du mouvement du sang dans les vaisseaux capillaires.C.R. Acad. Sci. 1, 554, 1835; Tome VII des Savants etrangers, iinpr.Royale, Paris , (1839). 5. COPLEY, A. L., and STAPLE, P. H. The plasmatic zone and velocity of blood flow in the microcirculationof the hamster's cheek pouch. Fed. Proc.(Fed. Amer. Sot. Exp. Biol.)18, 30, 1959. 6.
COPLEY, A. L., SCOTT BLAIR, G. W., BALEA, T., and STAPLE, P. H. The marginal plasmatic zone and the flow of blood in living capillaryvessels.In:Flow Propertiesof Blood and Other BiologicalSystems, A. L. Copley and I;.Stainsby,Eds. , Pergamon Press, London, 1960,p. 418.
7.
COPLEY, A. L. and STAPLE, P. H.: Haemorheologicalstudies on the plasmatic zone in the microcirculationof the cheek pouch of Chinese and Syrian hsmsters. Biorheology5, 3, 1962.
8.
COPLEY, A. L. On a new physiologicrole of fibrin and the capillorrhagic effect of fibrinolysinin normal and X-irradiatedrabbits. Abstracts Internat. Physiol. Congress,Montreal 1953, p. 280. Communic.,a
9.
COPLEY, A. L. Effet capillorrhagiquede la fibrinolysineet de l'antifibrin. olysine sur la membrane nictitsnte du lapin normal et expose aux rayons X. Arch. int. pharmacodyn.Ther. B, 426, 1954.
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COPLEY, A. L. Hemorheologicalaspects of the endothelium-plasmainterface MicrovascularRes. g, 192, 1974.
11. COPLEY, A. L. On the anticoagulantaction of fibrin in the prevention of thrombosis.Proc. IX. Congr. Internat.Sot. Haemat.,MexicoCity, 1962. Mexico City: UniversidadNational Autonoma de Mexico, 1964, vol. 2, p.
367. 12. COPLEY, A. L. On cement fibrin, a proposed constituentof the capillary basement membrane, and on lesions in the capillarywall in diabetesmellitus. In: Diabetes. Woe. 6. Congr. Internat.Diabetes Fed., Stockholm 1967. J. Ostman and R. D. G. Milner (Eds.)Amsterdam:Excerpta Medica Gdation, 1969,p. 606.
13. COPLEY, A. L. Non-Newtonianbehavior of surface layers of human plasma protein systems and a new concept of the initiationof thrombosis. Biorheology8, 79, 1971.
14. COPLEY, A. I,.and KING, R. G. Viscous resistanceof thromboid (thrombuslike) surface layers in systems of plasma proteins includingfibrinogen, ThrombosisResearch 1, 5, 1972.
15. COPLEY, A. L. Neue Auffassungenuber Hamorrhagie,H&mostase und Thrombotie ‘tiztlicheForschung.ll, I/114, 1957. 16. COPLEY, A. L. Le r&e de la fibrine et de la fibrinolysedans l'integrite de la paroie vasculaire.He?mostase(Paris).3, 13, 1963.
17. COPLEY; A. L. The genesis of thrombosis.Pat. Fiziol. eksper. Ter. (J. Pathol. Physiol. Exper. Therapy,Moscow). 8, 3, 1964. 18. COPLEY, A. L. Bleeding time, other in vivo tests and the arrest of hemorrhage. ThrombosisResearch 2, 1, 1974. 19. KING: R. G. and COPLEY, A. L. Modificationsto the Weissenbergrheogoniometer for hemorheologicaland other biorheologicalstudies.Biorheology 1: 1, 1970. 20. COPLEY, A. L. and KING, R. G. The WeissenbergRheogoniometeradapted for biorheologicalstudies. 1n:The Karl Weissenberg80th Birthday Celebration Essays J. Harris,(Ed.),p. 127. East African Literature Bureau, KsmpalaNairobi-Dar Es Salam,
1973.
21.
JOLY, M. Dispositif pour la viscosimetrie precise de syst'emescontenant des proteines. Biorheology 1, 15, 1962.
22.
COPLEY, A. L. and KING, R. G. The action of human red blood cells and platelets on viscous resistance of plasma protein systems. Biorheology lo, 533, 1973.
23. COPLEY, A. L
and KING, R. G. The reducing action of highly purified y globulin and filipoprotein on the viscous resistance of surface layers of fibrinogen: Thrombosis Research 5, 193, 1974.
24.
CALDWELL, K. D., KESNER, L. F., MYERS, M. N. and GIDDINGS, J. C. Electrical field-flow fractionation of proteins. Science l.& 296, 1972.
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25. SMITH, L.E. and MORRISSEY, B-W. Personal communications, 1975. 26. WHITMORE, R.L. Rheology of the Circulation, Oxford-New York, Pergamon Press, 1968. 27. CHIEN, S. Present state of blood rheology. In: Hemodilution. Theoretical Basis and Clinical Application. K. Messmer and H. S&mid-Schonbein, Basel, S. Karger, 1972, p. 1. 28.
Eds.
CHARM, S.E. and KURLAND, G.S. Blood Flow and Microcirculation. New York, John Wiley & Sons, 1974.
29. KUDRYK, B.,REUTERBY, J. and BLOMBACK, B. Adsorption of plasmic fragment D to thrombin modified fibrinogen-Sepharose. Thrombosis Research 1, 297,ly73. 30. KUDRYK, B., COLLEN, D., WOODS, K.R. and BLOMBACK, B. Evidence for localization of polymerization sites in fibrinogen. J.Biol.Chem. 249,3325, 1974. 31. STEWART, G. J. and NIEWIAROWSKI, S. Nonenzymatic polymerization of fibrinogen by protsmine sulfate: An electron microscopic study. Biochim. Biophys. Acta 194, 462, 1969.
32. NIEWIAROWSKI, S., STEWART, G. J. and MARDER, U. J. Formation of highly ordered polymers from fibrinogen and fibrin degradation products. Biochim. Biophys. Acta 221, 326, 1971.
33. FUNG, Y. C., ZWEIFACH, B. W. and INTAGLIETTA, M. Elastic environment of the capillary bed. Circul. Res. 19, 441, 1966. 34. FUNG, Y. C. Stochastic flow in capillary blood vessels. Microvasc. Res. 2, 34, 1973. 35. COPLEY, A. L. A new concept of capillary patency based on viscous resistance studies of surface layers of fibrinogen systems exposed to high shear. Abstracts, 8. European Conference of Microcirculation, Le Touquet. France, June 17-22, 1974, Paper No. 1.
36. SCARBOROUGH, D. E., MASON, R. G., DALLDORF, F. G. and BRINKHOUS, K. M. Morphologic manifestations of blood-solid interfacial reactions. A scanning and transmission electron microscopic study. Laboratory Investigation 20,164, 1969.
37. BLOOM, .4. L., GIDDINGS, J. C. and WILKS, C. J. Factor VIII on the vascular intima: Possible importance in haemostasis and thrombosis. Nature, ?JewBioloB 3, 217, 1973.
38. HARTERT, H. Rheo-simulation. A new method for the assay of clotting process and factor XIII. A preliminary report. Biorheology
l-l,355, 1974.
39. HARTERT, H. Clotting in layers in the rheosimulator.Biorheology g,249,1975 40. HARTERT, H. H. New aspects of blood clotting and thrombosis. Investigations with the method of rheosimulation.
This volume, p. 381.
41. CARO, C. G., FITZ-GERALD, J. M. and SCHROTER, R. C. Arterial wall shear and distribution of early atheroma in man. Nature 223, 1159, 1969.
Suppl. II, Vol. 8, 1976 Pergamon Press, Inc.
States
SECTION
VII
POLYMOLECULARLAYERS OF FIBRINOGEIJ SYSTEMS AND THE GENESIS OF THROMBOSIS Alfred
L.
Copley and Robert G. King
Laboratoryof Biorheology,Departmentsof Life Science and of Bioengineering,PolytechnicInstituteof New York, Brooklyn,?;.Y. 11201, U.S.A.
ABSTRACT
In 1971 Copley proposed a new theory on the initiationof thrombosis, based on hemorheologicalobservations. A brief summary is given of observationspertainingto the plasmatic zone in relation to the cement fibrin in the proposed endoendotheliallayer as well as of earlier findings on thromboid surface layers. They concern viscous resistance (torquevalues,r, dyne. cm) of layers of systems of fibrinogen and other plasma proteins. New findings are presented of viscous resistanceof surface layers of fibrinogensystems,obtained in steady shear together with findings on the elastic component, secured in oscillatoryshear. A new concept on maintainingthe patency of microvessels,presentedby Copley in 1974,is related to the problem of the initiationof thrombosis. During life, both with the patency of the vascular lumen and with intravascularobstruction, which by necessity are opposite, the stresses on the blood vessel walls are extremelyhigh, particularlyin the capillaryor minute blood vessels. Fibrinogen systemswere therefore exposed to high shear at 1000 set-1 for 3 min prior to measurements. Thereafter, data were secured from lo-3 to 10-l set-1. Highly purified plipoprotein and 8 globulin,which gave no?values also showed none when previouslyexposed to high shear. However, all hitherto tested fibrinogenpreparationsfrom differentmammalian species (human, bovine, sheep, dog, rabbit, and cat) exhibitedhigh rvalues. They usually became higher if subjectedto 3 min of high shearingprior to the low shear testing. The findings are related to a new hypothesis proposed by Blomback and Copley on the transformationof the fibrinogenmolecules, caused by high shearing. Such shearing forces, accordingto this concept,would open up the polymerizationsites of fibrinogenand thus simulate the enzymaticaction of thrombin for the polymerizationof fibrin. The differencebetween the biochemical and hemorheologicalactions is that the fibrinopeptidesare split off by thrombin,while no such cleavagewould occur in fibrinogen, proposed to be altered physicallyby the high shear known to exist 393
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at the vessel wall. 'Ihelayer upon layer deposition ol'such hemorheologically configurated fibrinogen and its polymerization would proceed, resulting in the formation of a thrombus which thus would initiate thrombosis and hemostasis. Copley considers blood cellular aggregation and fibrin coagulation to be secondary processes in the genesis of thrombi in minute or capillary blood vessels.
INTRODUCTION A brief review of earlier observations is given and most recent findings are presented with regard to a new approach to studies of the genesis of throm. bosis.
Fig. 1 Reproduction of Figures in the treatise by Poiseuille, published in 1839(b), pertaining to his studies on the plasmatic zone.
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One of us (A.L.C.)contendedthat it is in the plasmatic zone, which is the cell free part of the circulatingblood in proximity to the blood vessel wall, where the processes occur which initiate thrombosis (1,2). The plasmatic zone, called in the French literature "la zone de Poiseuille",was already described in 1661 by Malpighi in the first in vivo observationson the microcirculation, !lkismarginal or plasmatic zone was also made by him on the frog's lung (3). described in the 18th century by Spallanzaniand Albrecht von Halle (1). Eiowever, it was Poiseuillewho made hemorheologicalmeasurementsand observations on the plasmatic zone (Fig. l), which he reported in 1835 and 1839 (4). Poiseuillecontendedthat it containedtwo major portions,viz., one, an immobile portion next to the endotheliumand the other, the mobile portion. This is diagrammaticallyshown in Figs. 2 and 3.
Fig. 2 Diagrammaticfigure of extrusion for blood accordingto Copley and Staple (6). BASEMEET
MVENTITIAL
%NDOTIIEiIAL CELL
TUXTC
/
Eh?)OENDOTliELIAL FIBRIh' I‘ILM
MXllLE
LAYERS
Fig. 3 Diagram illustratingthe layers of the blood capillarywall and the location of the cement fibrin in the basement membrane and surrounding the endothelialcells (includingthe endoendothelialfibrin layer) in accordancewith Copley's concept. The drawing is meant to convey a general idea without giving scale, dimensionsand without simulatingthe appearanceof elements in the microstructureof the vessel wall (11).
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In 1959, Copley and Staple confirmed the findings of ?oiseuille (5-7). They also made certain observations which, although they do not present definite evidence, suggest that there is a more or less immobile layer next to the endothelium. In 1953, Copley proposed the concept, which has been developed ever since, that fibrin in submicroscopic dimensions lines the inner aspects of the vessel wall in direct contact with the endothelium (8,9). Thus, the circulating blood is separated from the endothelial cells by the so-called endoendothelial fibrin lining. It is the nature of this endoendothelial lining which is of great importance in any considerations regarding the flow of blood. This was discussed recently by Copley (10) in some detail in an article on hemorheological aspects of the endothelium-plasma interface. The proposed identification of this layer with different forms of fibrin, to which he gave the generic term "cement fibrin" (11,12), guided us, as well as other investigators during the past 22 years in numerous experimental studies. The process of thrombus formation begins, according to a new concept by Copley (13), with the adsorption of fibrinogen, followed by a growth process of the adsorption of fibrinogen and other plasma proteins, layer upon layer, until the lumen of the blood vessel involved is either partially or completely obstructed. It is only after this process is initiated that other clotting processes, such as the aggregation of platelets and/or red blood cells, as well as the coagulation of fibrin occur (13,14). Fibrin coagulation means the polymerization of fibrin, followed by its subsequent network formation or gelation and ending in cross-linking. Concepts on the genesis of thrombosis have hitherto been based mainly on two major processes of in vivo clotting, found to occur either separately or mixed, viz., the clumping of blood cellular elements and the coagulation of plasma by the formation of fibrin. THE THREE
MAJOR PROCESSES OF BLOOD CLOTTING
Blood clotting
polymerization
Fibrin
monomer
I Cross-hnklng
Fig. 4 Many years ago, Copley recommended that the word "clotting" be used as a generic term for fibrin coagulation and blood cellular clwnping (15-17). This usage of the non-committal generic term "clotting' was extended recently by him to include the process of time-dependent progressive adsorption of plasma proteins including fibrinogen. This process is considered as a hitherto unrecog-
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nized form of clotting. All three processes of clotting, shown schematically in Fig. 4, are rheological in nature, and any in vivo clotting involving the endothelium and progressing to vascular obstruction may lead to manifestations in various organs comprising the different conditions of thrombosis, which is a purely hemorheological disease or disorder. Any of these processes of clotting can go on separately or mixed. However, it is re-emphasized that the initiation of thrombus formation, both for the development of thrombosis and the arrest of hemorrhage (18) in minute blood vessels, depends mainly upon the proposed primary process of the aggregation of fibrinogen molecules. This concept is based mainly on our rheological findings obtained with surface layers formed on solutions of plasma proteins. TECHNIQUES We used a Weissenberg Rheogoniometer, modified by us to make it more suitable for biological investigations (19,20). The surface layers were measured at shear rates from 1000 to less than 0.1 see-l, using a geometry that is a combined Couette and cone and plate. A guard ring (Fig. 5) is used, which can be easily detached so that the torque (7) derived from surface layers can be separated from that derived from the bulk of the sample. These surface layers contribute added torque, which is particularly high at low shear rates. A similar method was reported earlier by Joly (21). Two measurements were made on each sample, one with the guard ring in place and the second with it removed. 13~subtracting the first value from the second value obtained, the torque contributed by the surface layer can be ascertained.
id level
-A
Fig. 5 Flan view of geometry showing position of removable guard ring A refers to the rotating outer cylinder, while B is the stationary inner cylinder of the accessory to the Weissenberg Rheogoniometer. More recently, we devised a technique of measuring directly the torque generated by the surface layers (20). These values are reported as torque, (7), versus rate of shear. This accessory to the Weissenberg Rheogoniometer is shown diagrammatically in Fig. 6. The device, made of Plexiglass, consists of a ring,9 cm in diameter. The ring just penetrates the surface layers of the test sample, which is held in the lower platen in an annular groove, 0.5 cm in width. The annular groove requires 5 ml of the test sample. This de-
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can also be used for viscoelasticity rneasuements of the surface iayer:: when the oscillatory-mode of the instrment is employed. The thromboid S-~r-t'ace layers of the test sample transmit a torque to the measuring lpper platen whe!the lower platen is rotated.
Vice
iig. 6 Plan view of special geometry for direct measurements of rheological properties in steady or oscillatory shear of surface layers. We found during standardization and calibration studies of the new geometry that no torque is contributed by the substrate fluid at shear rates below 10 see-l, and that the torque values obtained are derived exclusively from the surface layers. Above 10 see-l small readings can be obtained from the substrate and we have therefore limited the upper shear rate to 10 set-1 with the geometry employed. SOME FJUUIER FINDINGS Fibrinogen solutions gave the highest torque values when compared with x globulin, while albumin exhibited the least (13). The higher the concentration of each of the tested proteins in solution the higher was the torque value (13,14). This was also the case with 5 to 90 per cent concentrations of platelet-free oxalate plasma and serum obtained from apparently healthy human subjects (14). The addition of 0.4 per cent fibrinogen to 0.25 per cent albumin or to 5 per cent plasma or serum increased viscous resistance. Such addition to 5 per cent albumin or usually to 18 per cent plasma showed no change in torque values (14). A critical concentration of plasma or serum was noted, which ultimately may provide an indicator in the recognition of proneness towards thrombosis and may serve in the management of thrombotic conditions (14). Preliminary data,secured by us,showed that the surface layers of heparinized plasma gave decreased values when compared to the surface layers of oxalate plasma from the same blood withdrawal. There seems to be an antagonistic relationship between the red blood cells and platelets with regard to the formation of thromboid surface layers of fibrinogen and other proteins in plasma (22). We found that the presence of platelets in systems containing fibrinogen, whether or not enriched by other plasma proteins, will increase markedly the viscous resistance of polymolecular protein layers (22). Red blood cells, on the contrary, were found to decrease the viscous resistance of these layers. Thus, red blood cells may well
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have an inhibitingaction in the productionof polymolecularlayers of fibrinogen and other plasma proteins (22). Our findingswith highly purified @lipoprotein and xglobulin, to which 0.4 per cent bovine fibrinogenwas added, did not exhibit viscous resistance from surface layers (23). NEW FINDINGS All recent rheogoniometricmeasurementsof surface layers of fibrinogen and other protein systemswere made with the accessory shown in Fig. 6. There has been some questionwith regard to the air-fibrinogenlayer inter face. We have not found any differencein values if the air was replaced by oil. This is shown in Fig. 7 with a 0.4 per cent human fibrinogen solution. The oil used was a light mineral oil. In a number of other experiments,we always found that there were no changes whether or not we used oil to cover the protein system.
RATE
OF
SHEAR
set-’
Fig. 7 A comparisonof torque values, secured from surface layers of a 0.4 per cent human fibrinogensolution,formed at (-.-) oil-fibrinogenand (-o-) air-fibrinogeninterfaces. In Fig. 8,viscousresistancevalues from surface layers are shown of 0.4 per cent fibrinogen solutionstested at varying shear rates. As can be seen, preshearingat 1000 see-l for 3 min increasedthe torque values for human, bovine, cat, dog, rabbit, and sheep fibrinogens. The largest differenceafter shearingwas shown with the bovine preparation,and the highest values were found with the sheep and human fibrinogens. Since our earlier findings,using highly purified (3lipoprotein,mentioned above, did not show any viscous resistanceupon the addition of fibrinogen,we repeated this experimentin making torque measurementsbefore and after the application of high shear at 1000 see-' for 3 min. Fig. 9 shows that high shearing does not generate torque from surface layers of these preparations,if formed. Surface layers of 0.4 per cent fibrinogensolutionsare measured as controls prior and subsequentto high shearing. In our most recent studies,we made observationson the viscoelasticityof surface layers of fibrinogen systems,using the oscillatorymode of the instrument. Fig. 10 shows typical recorder traces from these experiments. The trace:
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--
r L-_ r-----
I
-----7
I
E 0
e 7?
II
CAT
SHEEP
* 103 I_ -
” y
IO2
L? ?
I 0
-3
IO
-2
RATE
IO OF
-1
SHEAR
IO0
IO2
IO’
(set-’
i-1_ IO
IO
-2
RATE
1
1
1
-3
10-l
OF
I
IO’
IO”
SHEAR
IO2
(SW-‘1
Fig. 8 Viscous resistance values from surface layers of 0.4 per cent solutions of fibrinogen from six species (human, bovine, cat, dog, rabbit,and sheep).
I
10-3
,
I
to-2 RATE
10-l OF
IO0 SHEAR
11 IO'
102
(set-‘1
Fig. 9 Comparative findings of torque values obtained before (-A-) and after (-A-) shearing at 1000 see-1 for 3 min from surface layers of 0.25 per cent highly purified p lipoprotein added to 0.4 per cent highly purified human fibrinogen. As controls, the same fibrinogen samples without the addition of/i lipoprotein were measured before (-o-) and after (-•-) high shearing. exhibit a phase difference of 15 degrees indicating the presence of an elastic component. We have found that the elastic component is increased considerably,
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if a three-minute period of steady high shear at 1000 see-1 is applied prior to measurement. Similar findings were obtained with highly purified ~globulin. The particular gglobulin of 99 per cent purity did not show any torque values from a surface layer, but if this purified &globulin was exposed to high shearing, a weak layer was formed, which faded away after approximately three minutes. This by itself appears to be an interesting phenomenon which invites iurther study.
--
0.0012
radians
3-g. 10 A typical recorder trace, obtained during an oscillatory experiment at a frequency of 0.06 Hertz. The larger trace represents the input motion, while the smaller trace represents the response of the surface layer. The phase difference between the two traces is 15 degrees.
IO -5
lO-4
10-3
FREQUENCY
IO
-2
Id’
(iiartz)
Fig. 11 Comparative elastic modulus data of surface layers of 0.4 per cent solutions of fibrinogen from sheep (-•-), bovine (-o-),and human (-A-) origins. In E'ig.11, data of dynamic tests of surface layers of 0.4 per cent fib, rinogen solutions are shown -pertainingto the elastic modulus. The fibrinogen was from sheep, bovine, and human sources. Both the bovine and human fibrinogens gave nearly identical values of elastic modulus, while the values of sheep fibrinogen were markedly increased. This Figure also shows that the rate of shear has only a small effect on the elastic modulus.
Figure 12 shows data of the surface viscosity in poise calculated from OS, cillatory tests of sheep, bovine, and human fibrinogens at 0.4 per cent coneen,
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tration, plotted against frequency varying from 10"' to 10' Hertz. These sre some differences in viscosity, with sheep fibrinogen showing the highest values. What, however, is most startling, are the extremely high viscosity valvues calculated using a layer thickness of 100 8.
,05upu 10-5
10-4
10-3
10-z
FREQUENCY
IO-
IO0
(Hertz)
Fig. 12 Plot of viscosity (dynes.sec/cm2) versus frequency of 0.4 per cent solutions of fibrinogen from sheep (-•-), bovine (-o-_)and human (-&) origins. de chose arbitrarily the value of 100 1 as the layer thickness and used it for all our calculations of findings presented in Figs. 11 and 12, although the surface layers may have varying and higher values of thickness. In case the thickness of these layers is increased, there will be a decrease in the viscosity values; however, they would still be extremely high. Recently, Smith, Morrissey,et a1.(25) found the thickness of fibrinogen surface layers to differ, under certain conditions, from 200 to 600 X. Nevertheless, the values for the elastic moduli measured are extremely high, in the neighborhood of 60,000 to about 25O,OOO dynes/cm* over the different rates of shear with the three preparations tested. These high values suggest the presence of very strong intermolecular bonds of these polyrnolecularfibrinogen layers.
ON THE FORMATION OF POLYMOLECULAR LAYERS OF FIBRIN~G~
(CLOTTING OF FIBRINOGEN WITHOUT THROMBIN)
In 1971, Copley referred to a non-homogeneous distribution of the adsorbed proteins and a decrease and/or imbalance of protective colloids (13). He defined "non-homogeneous distribution" as time-dependent, progressive adsorption of the plasma proteins from the solution at the interface with the vessel wall and the free surface of the adsorbed proteins (13). This non-homogeneous distribution of the adsorbed proteins may be related to considerations of Copley and Staple (7) as to whether a suspension of macromolecules would show a radial distribution when flowing through a capillary tube in which the velocity gradient from the axis to the wall of the tube was steep. A paper on electrical field-flow fractionation of proteins by Caldwell et al (24) appears to support the earlier considerations by Copley and Staple. Field-flow fractionation is a separation method in which various applied fields, working in conjunction with cross sectional flow non-uniformities in a narrow tube, cause the differential migration of molecules and ions. It is possible that there is a higher transport rate for larger molecules than for smaller ones. It appears that their findings would substantiate the contention of a non-uniform distribution of particles across a flow channel. As an alternative to the production of
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these polymolecular layers, Copley (10) proposed the occurrence of changes in the solubility of fibrinogen and of other plasma proteins in the affected part of the circulation. This occurrence can be brought about either by alteration of the plasma proteins including fibrinogen, or by certain additives, which may be present or released in the affected areas changing the solubility of these proteins. Nevertheless, any of these alternative explanations are proposed to concur with alterations in the flow properties of blood in these affected regions and the induction of the formation of polymolecular films or layers of the plasma proteins, particularly of fibrinogen.
WALL SBl!tAR FATES AND THE BLOMEifiCK-COPLEY HYFOTHESIS OF CONFORMATI0NA.L CHANGE OF FIBRINOGEPJ The shear rates are known to be very high at the wall of the different blood vessels. They are particularly high in the small or minute vessels. There is some discrepancy in the literature of wall shear rates estimated by different authors (26-28). The values,which Whitmore (26), Chien (27),and Charm and Kurland (28) reported, are compiled by us in a composite Figure (Fig. 13).
I AORTA
L
ARTERIES
I
ARTERIOLES
1
CAPILLARIES
1
VENULES
I
VEINS
.
VEh
CAVA
Fig. 13 Compilation of values of wall shear rates in different blood vessels from data by Whitmore (26), Chien (27),and Charm and Kurland (28). In the capillaries these values are particularly high, as reported by all authors. However, there appear to be some marked differences in values reported for arterioles by Chien (27), which show values of about 1000 see-l, while those reported by Charm and Kurland are 8000 set-l. Such a value of nearly 8000 set-1 is given for capillaries by Chien, while Charm and Kurland give a value of 1000 see-l. The values by Chien also differ from those by Charm and Kurland with regard to the venules. The latter authors ive a value of 800, while Chien's values differ between about 70 to 300 set-B . As pointed out recently by Copley (lo), there is a need for re-investigating the wall shear rates to find out the reason for these discrepancies. Nevertheless, it is clear that wall shear rates are particularly high. The values of shear rates at the wall of the aorta are similar in the data presented by Whitmore,and by Charm and Kurland, as are the values at the wall of arteries, as far as human subjects are concerned. Whitmore reported values in the dog which for the aorta are appreciably higher than in man. However, in the arteries and capillaries, there is not much of a difference. The wall shear rates are much
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lower in the veins than in the capillaries, as reported by all three groups of investigators. In discussions on these high wall shear forces, Blomb&k and Copley developed a working hypothesis that the high shear forces present at the blood vessel wall may bring about conformational changes of fibrinogen molecules at certain sites in the circulation. Recently it has been demonstrated by Kudryk, gen contains polymerization sites in its fragment The sites residing in N-DSK become activated upon thrombin-like enzymes. This appears to be due to tion sites or their exposure after release of the
BlombZck et al that fibrinoD and N-DSK portions ;29,30). treatment with thrombin or unfolding of the polymerizafibrinopeptides.
It is likely that all polymerization processes, whether enzymatic or nonenzymatic, always involve conformational change in N-DSK, whereby polymerization sites are exposed. Stewart and Niewiarowski (31,32) described the polymerization of Pibrinogen by protsmine sulfate without the action of thrombin. Their findings can be explained by neutralization of the acidic charges on the fibrinopeptides, bringing about a similar conformational change as brought about by the release of these peptides by enzymes. It is according to the Blomback-Copley working hypothesis that these conformational changes csn also be induced by shearing forces with subsequent exposure to polymerization. ON VASCULAR PATENCY The problem of vascular patency is of utmost importance with regard to certain rheological properties of the vessel wall which prevent the blood vessel from collapsing. In recent .years, Fung et al (33,34) explored this side of the problem of vascular patency, particularly as the rigidity of the capillary wall is concerned. Fung claimed on the basis of experimental observations that the rigidity of blood capillaries is due to the gel-like materials surrounding the wall of the blood vessel. In measurements of the stress-strain relation of the mesentery, Fung et al could show that the material is nonlinear elastic with a shear modulus that varies linearly with the shear stress. According to Fung (35),the patency of blood capillaries is on the basis that mechanically they are like "tunnels in gels ':.A muscle capillary or a mesentery capillary blood vessel will not collapse when the entire tissue is subjected to hydrostatic pressure. The elastic behavior of a blood capillary depends also on the mechanical properties of the surrounding medium. Thus, the elastic rigidity of the different surrounding media, as proposed by Fung, appears to be an important factor in capillary patency. A new concept advanced last year by Copley (10,35) offers a hitherto unknown factor for vascular patency and provides a different explanation for the elastic rigidity of blood vessels and their patency. This factor concerns the elastic rigidity of the proposed endoendothelial fibrin lining and is based on our recent viscous resistance and viscoelasticity studies of surface layers of fibrinogen. The contention is that, since the proposed endoendothelial fibrin layer is continuously exposed to the very high shearing at the vessel wall, particularly of blood capillaries, the endoendothelial layers on these walls would make them highly rigid. Thus, it is this factor of endoendothelial fibrin rigidity which is considered important in maintaining the patency of all capillary blood vessels. Since in our viscoelasticity studies of polymolecular
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fibrinogen layers we found them to exhibit an elastic component, the latter is considered to contribute to the elasticity of the blood vessel wall (10). ON NON-EXZYMATIC INDUCED POLYMERIZATION OF FIBRINOGEN AND THE INITIATION OF THROMBUS FORMATION IN THROMBOSIS AND BEMOSTASIS Several observations were reported in the literature which support Copley's new concept of the initiation of thrombosis (36-40). They have a bearing on our findings of thromboid layers of fibrinogen and other proteins. The observations by Hartert using his method of rheo-simulation, reported three years ago (38) and this year (jg), as well as his new findings employing the rheosimulation, reported at this Seminar (ho), appear to provide direct evidence in support of Copley's concept. The possible role of fibrinogen layers on the vessel wall in connection with the production of wound thrombi in the mechanisms of hemostasis was reIt is possible that with the onset of bleedcently discussed by Copley (18). ing the altered hemorheological situation will alter the even distribution of plasma proteins at the site of extravasation. Consequently, a dynamic process occurs, viz., the initial adsorption of fibrinogen and other plasma proteins: followed by a growth process of the adsorption of layer upon layer of these proteins, constituting thrombus formation. This is then followed by platelet deposition and clumping, as well as by fibrin coagulation. Caro, Fitz-Gerald and Schroter (41) made observations with regard to the genesis of atherosclerosis which appear to relate to our findings on viscous resistance of fibrinogen surface layers following exposure to high shearing forces. Caro et al observed that the distribution of fatty streaking and early plaques may be strongly influenced by high shear rates at the arterial wall and also at the sites of bifurcations of arteries when there is turbulence and the shear rate is high. The primary process of fibrinogen aggregation (preceeding the secondary processes of blood cellular clumping and/or of fibrin coagulation) is considered by one of us (A.L.C.) to occur most rapidly (18) and possibly within several seconds. The sequence of these processes needs to be investigated both extra vivum and in vivo. Our results appear to demonstrate that high shear forces, which are known to be present at a vessel wall, have induced an alteration in fibrinogen, and it is believed that this represents an unfolding of the polymerization sites of the fibrinogen molecule in accordance with the Blomback-Copley working hypothesis. In this way these high shearing forces would bring about the unfolding which otherwise, as is well established, is the result of partial proteolysis by thrombin. We do not yet know whether the increase in torque values is due to thickened polymer layers being formed or to extra bonds forming within the layers. IN CONCLUSION As students of thrombosis and of related phenomena affecting the circulation of blood, we are preoccupied with the obstruction of the lutninaof blood vessels due to clotting processes and with processes which keep the vascular lumina patent. These latter processes either inhibit the various forms of clotting or, after clotting already has set in, lead to the lysis or disintegration of clots of different origins. These occurrences will, of course, continue to be a main concern of studies related to thrombosis subsequent to its initiation. Patency depends also on the elastic rigidity ofvesselwalls.
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The phenomena of the polymerizationof fibrinogenmolecules and of intravascular aggregationof fibrinogen,layer upon layer, will need to be studied at many levels in a variety of scientificfields and disciplines. In the case of blood cellular clumping and of blood coagulation,such studies have been ongoing for more than one hundred years,an enterprisein which many investigators continue to be active. New develolpnents in fibrinogenresearch, in ultrastrucmicroscopictechniques,inimmunochemistryand in surtural including elecf;ron face chemistry,as well as in hemorheology,provide us with new tools for experimental scrutinityof Copley's concept or theory of the genesis of thrombosis. ACKNOWLEDGEMENTS The studies were supportedin part by the Office of Naval Research conNOOO-14-67-A-0449and NOOO-14-75-C-0221,and most recenttracts NR 2754 (03), ly also by the National Institutesof Health, grant HL 17556-01. We are indebted to Dr. M. Burstein, Paris, France, for supplyingus with his highly purified preparationof /3lipoprotein. REFERENCES 1.
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