Alfred L. Copley

Erythrocyte sedimentation of human blood at varying shear rates.

3. Copley, A. L. Rheo/. Acta 1,663, 1961. 4. Copley, A. L. and Scott Blair, G. W. Proc. 8th Int. Congo Blood Transfusion, Tokyo, 1960, p. 6. Basel: S. Karger, Basel 1962. 5. Ruhenstroth-Bauer, C., Brittinger, G., Granzer, E. and Nass, G. Deutsche med. Wchnschr. 85, 808, 1960. 6. Gomulkiewicz, J. Studia Biophysica 35, 21, 1973. 7. Boyd, J. J. M. and Sanderson, J. J. Plasma Dynamics. Barnes and Noble, New York, 1969. 8. Cutler, J. W. Am. J. med. Sci. 183,643, 1932. 9. Reichel, H. Blutk6rperchensenkung., Springer-Verlag, Vienna, 1936. 10. Westergren, A. Acta med. Scand. 54, 247, 1920-1921. II. Whelan, J. A., Huang, C. R. and Copley, A. L. Biorheology 7, 205, 1971. 12. Huang, C. R., Whelan, J. A., Wang, H. H. and Copley, A. L. Biorheology 8, 157, 1971. 13. Copley, A. L., Huang, C. R. and King, R. G. Biorheology 10, 17, 1973. 14. Huang, C. R., King, R. G. and Copley, A. L. Biorheology 10, 23, 1973. 15. Copley, A. L., Huang, C. R. and King, R. G. Symposium Xl: Biorheology, IV. Int. Biophys. Congo Moscow, 1973, p. 438. Pushchino, Academy of Sciences of the U.S.S.R., 1973. 16. Copley, A. L., King, R. G., Chien, S., Usami, S., Skalak, R. and Huang, C. R. Biorheology 12, 257, 1975. 17. Copley, A. L., Luchini, B. W. and Whelan, E. W. In: Hemorheology (Edited by Copley A. L.), p. 375. Pergamon Press, Oxford, 1968. 18. Knisely, M. H. Postgrad. Med. 10, 15, 1951. 19. Fahraeus, R. Rhea/. Acta 1, 656, 1961. 20. Copley, A. L. Rheo/. Acta 1,663, 1961. 21. Leeuwenhoek, A. van (spelled Lewenhoek in the original paper), Phil. Trans. R. Soc., Land. 22, 447, 1700.

Polymolecular layers of fibrinogen systems and the genesis of thrombosis

Abstract In 1971 Copley proposed a new theory on the initiation of thrombosis, based on hemorheological observations. A brief summary is given of observations pertaining to the plasmatic zone in relation to the cement fibrin in the proposed endoendothelial layer as well as of earlier findings on thromboid surface layers. They concern viscous resistance (torque values, τ, dyne. cm) of layers of systems of fibrinogen and other plasma proteins. New findings are presented of viscous resistance of surface layers of fibrinogen systems, obtained in steady shear together with findings on the elastic component, secured in oscillatory shear. A new concept on maintaining the patency of microvessels, presented by Copley in 1974, is related to the problem of the initiation of thrombosis. During life, both with the patency of the vascular lumen and with intravascular obstruction, which by necessity are opposite, the stresses on the blood vessel walls are extremely high, particularly in the capillary or minute blood vessels. Fibrinogen systems were therefore exposed to high shear at 1000 sec −1 for 3 min prior to measurements. Thereafter, data were secured from 10 −3 to 10 −1 sec −1 . Highly purified β lipo-protein and γ globulin, which gave no τ values also showed none when previously exposed to high shear. However, all hitherto tested fibrinogen preparations from different mammalian species (human, bovine, sheep, dog, rabbit, and cat) exhibited high τ values. They usually became higher if subjected to 3 min of high shearing prior to the low shear testing. The findings are related to a new hypothesis proposed by Blomback and Copley on the transformation of the fibrinogen molecules, caused by high shearing. Such shearing forces, according to this concept, would open up the polymerization sites of fibrinogen and thus simulate the enzymatic action of thrombin for the polymerization of fibrin. The difference between the biochemical and hemorheological actions is that the fibrinopeptides are split off by thrombin, while no such cleavage would occur in fibrinogen, proposed to be altered physically by the high shear known to exist at the vessel wall. The layer upon layer deposition of 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.

The endoendothelial fibrin lining as the crucial barrier and the role of fibrin(ogenin) gels in controlling transcapillary transport.

The interface between the two portions of the vessel-blood organ, viz., the vessel wall and the circulating blood, is considered by the author to be the endoendothelial fibrin lining (EEFL). The view that the endothelium, consisting of the endothelial cells and the interendothelial cement substance, is the primary filtration barrier in capillary permeability (CP) is no longer tenable. There is considerable evidence that the primary barrier is an endocapillary protein layer, originally postulated by Danielli in 1940. Copley considered this layer to be identical with the EEFL formed in the more or less immobile portion of the plasmatic zone in close proximity to the vessel wall. Processes of fibrin formation and fibrinolysis can occur there homeostatically, undisturbed by the flow of blood. The fibrinopeptides and plasminopeptides, freed at this site by the conversion of fibrinogen to fibrin and of plasminogen to plasmin, respectively, were reported by Copley et al in 1966 to augment CP. These peptides thus take part in the steadily occurring normal physiological CP. This is facilitated by the porosity of the EEFL due to the network or gel structure of fibrin strands. The authors concept that the EEFL acts as the primary barrier, controlling transendothelial transport and transport across the basement membrane (BM), is discussed on the basis of older and recent findings by several investigators. In particular, the BM is dealt with in some detail as a barrier. Emphasis is placed on the existence of fibrin as a main constituent of the BM, hitherto not generally known. This was demonstrated by direct evidence in the production of (non-thrombocytopenic) vascular purpura with fibrin antiserum. Numerous tiny foci of fibrin(ogenin) gels are expected to stud the BM. Augmented capillary fragility (CF) due to increased fibrinolysis of many of these focal fibrin gels result in petechial hemorrhages. CF and CP are physical properties of the blood capillary wall which behave antagonistically and are controlled by fibrin formation and fibrinolysis, steadily occurring in the vascular layers including the BM. This barrier secures the integrity of the capillary wall by preventing extravasation of blood or hemorrhages. New experimental approaches to verify the detection of fibrin in the microstructure of the capillary wall are proposed. Moreover, hemorheological experimentation, models and treatments are needed to establish whether or not the EEFL is the crucial, critical barrier in CP, as proposed.

The physiological significance of the endoendothelial fibrin lining (EEFL) as the critical interface in the ‘vessel-blood organ’ and the importance of in vivo ‘fibrinogenin formation’ in health and disease

The authors theory of the endoendothelial fibrin lining (EEFL), first advanced in 1953 and developed by him ever since, localizes the homeostasis between steady fibrin formation and deposition, or ‘fibrination’, and continuous fibrinolysis in the more or less immobile portion of the plasmatic zone next to the vessel wall. In 1971, the author advanced, in relation to the EEFL, the theory of fibrinogen gel clotting without thrombin action or ‘fibrinogenin’ formation in vivo. Considerable direct and indirect experimental evidence, secured by the author and by several other investigators, advanced markedly the knowledge of the normal physiology and the pathophysiology of various disease processes involving the vessel wall and blood circulation. n nThe information presented is an extension to that given in the authors recent overview (Clin. Hemorheology 1, 9–72, 1981). It deals both with new data by several investigators including those by the author, as well as with older data from the literature. The author maintained already in 1960 that the blood together with the blood vessels, in which it circulates, constitute ‘an entity’. In 1981 he postulated this entity to be a very special organ, named conveniently ‘vessel-blood organ’, which is ubiquitous and penetrates all other organs and adjacent tissues. The EEFL of the vessel-blood organ is considered by the author as the crucial critical interface between the blood and the vessel wall. It is the primary barrier, followed by the endothelium (comprising the endothelial cells and the interendothelial cement substance which contains or is identical with ‘cement fibrin’) and the basement membrane for the exchanges between the blood, the vessel wall and its surrounding tissues and spaces. The EEFL acts as anticoagulant, is antithrombogenic, maintains vascular patency and aids cardiac action by decreasing significantly the apparent viscosity of blood, referred to in the literature as the ‘Copley-Scott Blair phenomenon’. n nA new concept of leukocyte emigration traversing the capillary wall is presented, affecting focal fibrinolysis of the EEFL and of fibrin contained in the interendothelial cement substance and in the basement membrane. n nThe physical property of capillary (or vascular) permeability is related to the existence of the EEFL, since, as found by Copley et al, both fibrinopeptides, liberated in the transition of fibrinogen to fibrin, and plasminopeptides, freed in the conversion of plasminogen to plasmin, enhance capillary permeability. Capillary fragility, which is antagonistic to capillary permeability, is in great part due to fibrinolytic action on fibrin as a constituent of the basement membrane. Pseudohemorrhages occur as minute microaneurysms in augmented capillary permeability, when the basement membrane remains intact and blood escapes through the interendothelial junctions into the exoendothelial space between the endothelium and the basement membrane. The occurrence of petechiae can be either due to focal lesions in the basement membrane leading to extravasation of blood or due to these pseudohemorrhages. Thus, petechiae are not necessarily identical with capillary hemorrhages. n nNew surface hemorheological findings, obtained by Chien, King and Copley with a modified Weissenberg rheogoniometer, are presented. We found Ca2+ to increase markedly the viscous and elastic moduli of fibrinogen surface layers. Other studies concern marked decreases of these surface rheological moduli by the addition to fibrinogen solution of the following substances: heparins of low molecular weights of 4400, 5300, and 5900; chondroitin A, B and C; dextran MW 20,000; dextran sulfate MW 17,000; and sodium hyaluronate. The author considers these findings of inhibition of fibrinogenin formation to mirror antithrombogenic actions of these different agents, which are expected to prevent thrombogenesis. n nThe pathogenesis of different disease processes, such as thrombosis, hyperthermia therapy of cancer, (non-thrombocytopenic) vascular purpura, and atherogenesis is briefly discussed. The author relates these disease processes to the theories of the EEFL and of in vivo fibrinogenin formation. In this conclusions he suggests a number of studies to be made towards the advancement of knowledge pertaining to these theories involving health and disease.

On biorheology. Joint Plenary Lecture12

SummaryDescriptions of biorheological phenomena go back into antiquity, but studies of flow properties of living matter began probably with the discovery of the circulation in blood capillary vessels byMalpighi in 1686 and the streaming in plant cells byCorti in 1774. Biorheology comprizes the study of the deformation and flow of living organisms and inanimate biological systems or of materials directly derived from living organisms. The term biorheology was introduced in 1948 (A. L. Copley, Proc. Internat. Congress on Rheology, Scheveningen, Holland, 1948, North-Holland Publ. Co., Amsterdam and Interscience Publishers, New York, 1949, Vol. 1, p. 47). Ever since, biorheology offered a framework to connect the sciences of biology with rheology. This frame, which proved to be secure, permits the application of a number of rheological treatments to biological systems. The different fields of biorheology, including hemorheology, have grown rapidly and to such an extent that any abridged survey will remain inadequate.Jean-Léonard-Marie Poiseuille first reported in 1835 hisin vivo studies “Recherches sur les causes du mouvement du sang dans les vaisseaux capillaires”, which led to the application of rheological treatments to the flow of blood. The discovery of the laws of flow was based on experiments whichPoiseuille was stimulated to make from his observations in living blood capillaries, resulting in his studies “Recherches expérimentales sur le mouvement des liquides dans les tubes de très petits diamètres”. These reports, published from 1840 to 1842, were accepted throughout Europe. On the basis ofPoiseuilles findings,Maxwell, Jacobson Mathieu and others deduced from the fundamental equation ofNewton the well known formula for viscosity, which was later named afterPoiseuille. It will be demonstrated that there are other reciprocal stimuli from biology and rheology, which promise to continue to be fruitful. Numerous biological phenomena and processes await a rheological approach for the characterization of the flow properties involved and for quantitative studies. It will be shown that biorheology, in spite of its brief history as an organized science, is of growing importance in the biological and medical sciences. New knowledge gained in biorheology, as applied to the practice of medicine and surgery, will serve the well-being of the human species.

Viscoelasticity studies of surface layers of highly purified fibrinogen (Fg) systems and the role of pH on the rigidity of such layers.

Abstract Studies are reported on the viscosity (η) and elastic modulus (γ) in dynamic shear and on the rigidity (τ) in steady shear of surface layers of highly purified human and bovine fibrinogen. The measurements were made with a modified Weissenberg rheogoniometer. Preparations of highly purified human and bovine fibrinogen of 98 to 100 per cent coagulability were employed. High values of viscosity and viscoelasticity were found with varying concentrations of human and bovine fibrinogen from 0.4 to 0.00004. The significance of these findings is discussed in relation to blood-bubble generation or the embolization of fibrinogen or plasma coated air bubbles in decompression sickness. The rigidity of the fibrinogen surface layers was found to depend on the pH. Two different buffers were employed at pH from 5.4 to 8.7. The τ values between pH 6.5 and 7.6 did not show changes in rigidity. At pH below 6.5, τ values increased markedly and at pH above 7.6, decreases in τ were recorded. The findings, obtained at pH below 6.5, may be of special significance in the understanding of what may trigger the initiation of thrombus formation in the circulation, in particular the microcirculation, by fibrinogen clotting without thrombin participation, i.e., the aggregation and gelation of fibrinogen on the endothelium, both in thrombogenesis and incipient hemostasis.

The endoendothelial fibrin lining, fibrinogen gel clotting, and the endothelium-blood interface.

The concept of the endoendothelial fibrin lining (EEFL) was first advanced by the author in 1953 and developed ever The concept of in vivo fibrinogen gel clotting, an offspring of the EEFL, first presented by Copley (4) in 1971, concerns in vivo clotting of fibrinogen without the participation of thrombin. As the author cannot deal here with all aspects of this large and growing research field, the reader should see the overview, published in 1981 in the new journal Clinical Hemorheo1ogy.l Additional information can be obtained from the papers presented at the symposium entitled The Endoendothelial Fibrin Lining, held in September 1981 in Jerusalem as part of the XII. European Conference on Microcirculation, which are to be published as a supplement to Thrombosis Re~ e a r c h . ~ Since several speakers deal with the EEFL andfor fibrinogen gel clotting in their presentations at this conference, the authors remarks regarding their previous studies and findings are limited. The term blood capillary is used as a generic designation, similar to Landis;6 for any minute vessel from 5 to 20 1.1 or even more in diameter, since no morphologic criteria exist for establishing precise boundaries between the capillaries and the smallest arterioles and venules. After a particular blood capillary can be clearly identified, it should be given the proper term in accordance with the nomenclature introduced by Chambers and Zweifach.

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

In earlier studies using a modified Weissenberg Rheogoniometer, we found decreased rigidity or torque values (tau) in surface layers of heparin plasma, when compared to tau of oxalate plasma from the same blood withdrawal (Thrombosis Res. 1, 1-17, 1972). In subsequent studies of the viscoelasticity of surface layers of highly purified fibrinogen (97-100% clottability) of human and bovine origin, we found, with some heparins, marked lowering of surface viscous moduli (etas) and of surface elastic moduli (Gs). With some heparins no changes in tau, etas and Gs occurred. Certain low molecular weight (LMW) preparations of heparins showed decreases, but some did not. This is also the case with heparins of low and high affinity for antithrombin. Calcium heparin and Ca2+ alone always increased etas and Gs, when added to the fibrinogen system. N-desulfated heparin both decreased or did not change etas and Gs. Preparations of fibrinogen in dog plasma, to which sodium heparin was added, resulted in a decrease of tau values. These results appear to emphasize that plasma proteins other than fibrinogen, and other plasma constituents, may affect surface hemorheological values. These findings suggest needed interface studies of fibrinogen systems to which plasma or plasma constituents are added. We found also that other substances, i.e., dextran MW 20,000; dextran sulfate MW 17,000; sodium hyaluronate and depolymerized hyaluronate decreased tau, etas and Gs markedly. Recent findings in the literature are discussed in relation to thrombogenesis in which fibrinogenin gelation is considered as the initial phase of blood clotting. Fibrinogenin is the new term for initial fibrinogen aggregation and subsequent fibrinogen gelation without thrombin participation. The inhibition of fibrinogenin formation extra vivum is considered to be a valid indicator of antithrombogenic activity of substances which play a significant role in investigations on the therapy and prevention of thrombotic conditions.

Experimentally-induced petechial hemorrhage and white embolization in the rabbit's nictitating membrane

Abstract 1. 1. Various hemorrhagic agents were injected intradermally into ear, abdomen, conjunctiva, and nictitating membrane of about three hundred nonanesthetized male rabbits, and biomicroscopic observations were made at the site of injection. 2. 2. Different areas of the skin of the same animal differed in the degree of petechial production with the same test agent. Because of this finding these regions were not considered conducive to any approach of a quantitative nature. The best region found for observational studies of petechial hemorrhage was the nictitating membrane. 3. 3. A new technique which utilizes both nictitating membranes of the rabbits eyes was developed and is described to test the fragility of the minute blood vessels within a three-hour test period by the time of occurrence and number of petechiae. 4. 4. At a given dosage administered, a correlation was found between the number and the time of occurrence of petechiae. When different dosages were used, the number produced correlated directly with the dosage. Physiologic saline, distilled water, and mineral oil served as controls and did not produce petechiae. 5. 5. It was possible to divide the animals into two groups, those susceptible and those resistant to induced hemorrhage by a given test agent. 6. 6. Individual variations were found in the reparability of the damaged vessels. Some animals repaired these vessels in a few hours, while others required days. 7. 7. The following agents were used locally to produce petechial hemorrhage in the nictitating membrane: Merthiolate, Shiga protein toxin, moccasin snake venom, croton oil in mineral oil, sodium heparin, heparin plasma, saline plasma, citrate plasma, serum, clostridium welchii toxin, clostridium septicum toxin, streptolysin streptococcus pyogenes Group A, cysteine hydrochloride, and tricresol. 8. 8. Capillary ruptures induced by croton oil are probably due to the formation of large particles which embolize. The embolization of these vessels leads to their rupture and petechial manifestation. 9. 9. The local or systemic injection of heparin results in the constant occurence of white emboli in the nictitating membrane. It appeared that these emboli were of a different nature than those produced by croton oil. 10. 10. The local injection of heparin always resulted in petechiae in the nictitating membrane, although the systemic injection of heparin alone did not necessarily result in petechiae. Systemic heparinization combined with Merthiolate always produced petechiae in the nictitating membrane. 11. 11. When local application of Merthiolate or croton oil was preceded by the systemic injection of sodium heparin, more petechial hemorrhages resulted. 12. 12. The first occurrence of white emboli preceded or was observed simultaneously with the first occurrence of petechiae. Such emboli were not, or only rarely, observed following injections of moccasin snake venom and Merthiolate into the nictitating membrane, although innumerable petechiae occurred with these agents. 13. 13. Serum, heparin plasma, and citrate plasma, from rabbit origin, produced white emboli and petechiae following local injection in the nictitating membrane. With the exception of citrate plasma, petechiae were always produced. The latter appeared to diminish petechial formation and also the appearance of white emboli, both of which were lacking in the nictitating membrane of one animal but present in the four membranes of two other animals.

Biorheological methods employing the Weissenberg rheogoniometer.

The theories and spatial concepts of Karl Weissenberg have been applied to the science of biorheology by employing the Weissenberg Rheogoniometer. Two main types of experimental methods have been used for the characterization of the bulk shear properties of biorheological fluids: (A) In continuous laminar shearing motion, the tangential and normal components of stress are measured at a series of rates of shear. From these parameters, the apparent viscosity, an elastic modulus and a recoverable strain, are calculated as functions of the rate of shear. In this continuous shear experiment, the physical structure present in the material at any given rate of shear may be quite different from the material in its rest state or at other rates of shear. (B) In harmonic oscillatory motion, the material is subjected to a harmonic laminar shear about its rest state at a number of frequencies and small strain amplitudes. From these experiments the dynamic moduli of viscosity and elasticity are calculated. Preparations of biological materials, such as whole blood and systems of blood components, both in health and disease states, have been investigated using methods A and B, together with studies of surface layers of plasma proteins at interfaces.