AN INVESTIGATION INTO THE MECHANISMS OF ROULEAUX FORMATION AND THE DEVELOPMENT OF IMPROVED TECHNIQUES FOR ITS QUANTITATION. Submitted for the degree of Doctor of Philosophy M arch 1996 by: Mark Pearson. B.Sc., A.R.C.S. Department of Physiology & Biophysics Imperial College of Science, Technology & Medicine St. Mary's Hospital Medical School Norfolk Place Paddington London W 2 IPG To Leonard, my mentor and friend ABSTRACT Rouleaux formation (RF) influences a variety of rheological properties of blood (eg shear thinning, thixotropy) and is believed to have effects in-vivo. However, the actual process of RF is little understood, and the primary aim here was to investigate factors that may be involved. There are currently two proposed hypotheses for describing the mechanisms involved in RF, one associated with macromolecular bridging between RBC's, and the second with osmotic forces bringing and holding RBC's together. OifFerentiatwj these two hypotheses, and gm/v/Kj further insight into the mechanisms of RF, was attempted here using two approaches: The first approach involved making measurements, before and after enzyme modification, of RBC aggregation and cellular factors (size, deformability and surface charge), so any changes in the latter could be related to the former. However, many of the enzyme modified RBC's showed extremely high levels of aggregation, which caused problems in obtaining, viscosity and optical measures of aggregation. Hence new experimental procedures and analytical methods had to be developed to deal with this. With these new methods it was possible to show large differences in the aggregating potential of RBC's modified with various enzymes and, as far as dextran-induced-aggregation was concerned, good correlation was found with changes in cellular charge. However, fibrinogen-induced- aggregation followed a different pattern, suggesting that the physico-chemical factors involved differed to dextran. The second approach involved a more direct method for investigating fibrinogen interactions with the RBC surface. Radioactively- and fluorescently-labelled fibrinogen were used in this work in an attempt to directly observe adsorption to the RBC surface, which is an essential feature of the cross-bridging hypothesis. Despite many experimental problems with this approach it was conclusively shown that fibrinogen adsorbed to the RBC surface and was associated with RF. CONTENTS OF THESIS • A B ST R A C T 3 • LIST OF SYMBOLS AND ABBREVIATIONS 5 • LIST OF EQUATIONS 7 • L IST OF T A B L E S 11 • L IST OF FIG U R E S 13 • LIST OF IMAGES (PICTURE SETS) 19 CHAPTER 1. INTRODUCTION 21 CHAPTER 2. METHODS, MATERIALS AND INSTRUMENTATION 61 CHAPTER 3. A STUDY OF THE CONTRAVES LS30 VISCOMETER 85 CHAPTER 4. A STUDY OF THE MYRENNE ERYTHROCYTE AGGREGOMETER (MEA), THE SYLLECTOGRAM AND METHODS OF ITS ANALYSIS 113 CHAPTER 5. HAEMATOCRIT EFFECTS ON VARIOUS BLOOD SUSPENSIONS 151 C H A PT E R 6 . USING ENZYMES TO DEGRADE THE RBC SURFACE TO INVESTIGATE CELLULAR FACTORS OF IMPORTANCE TO ROULEAUX FORMATION / AGGREGATION. 173 CHAPTER 7. A METHOD FOR DIRECTLY INVESTIGATING THE MECHANISMS OF ROULEAUX FORMATION. 210 CHAPTERS. DISCUSSION 231 • • R E FE R E N C E S 237 • ACKNOWLEDGEMENTS 249 • IMAGES (PICTURE SETS) - in sleeve of back cover 250 4 SYMBOLS AND ABBREVIATIONS Y Shear R ate V iscosity H data Viscosity data Hr Relative viscosity 128.5 _ ‘ 1 r Relative viscosity at a high shear rate of 128.5s*1 027, Tir Relative viscosity at a low shear rate of 0.277s*1 A t | Change in viscosity M an(0, 3) a -^S v l Area under aggregating phase of the MEA syllectogram calculated by the MEA at either 0 or 3 s *1 C om pC Y )^^ ^ Area under aggregating phase of the MEA syllectogram calculated by computer analysis program at a shear rate y .< <1 vi Change in area of the syllectogram ^ 1 1 2 5 Iodine (radioactively) labelled fibrinogen ^ R h -la b Rhodamine (fluorescently) labelled fibrinogen ^ U n la b Unlabelled fibrinogen B Brom elain CT a-Chymotrypsin CV Coefficient of variation ds deci seconds ( 10*1 s) ESR Erythrocyte Sedimentation Rate F Formaldehyde G Glutaraldehyde H ct Haematocrit (or Packed Cell Volume) HSR High Shear Rate LSR Low Shear Rate LT Light Transmission MCV Mean cell volume MEA Myrenne Erythrocyte Aggregometer MSM The microscope slide method of fixation MW Mean weight molecular weight 5 M n Mean number molecular weight NA Neuraminidase P Paraformaldehyde MS PerioJ/c fe.i&£cktf RBC Red blood cell (erythrocyte) R®CSusp Red blood cell suspension RF Rouleaux formation SD Standard deviation T Trypsin T h Throm bin TTM The test tube method of fixation WBC White blood cell (leukocyte) 6 LIST OF EQUATIONS: Eqn 1.1: Smoluchowski's equation. UE . C (-) 30 n Eqn 1.2: Relation of electrophoretic mobility ( UE) to Debye-Hiickel length (k 1) and surface charge UE - (—r\K ) 30 density ( a). Eqn 1.3: Describes velocity profile of Newtonian liquid — ■ 1 - ( — )2 31 flow through a tube. v max r max dv Eqn 1.4: Definition of shear rate ( y). Y ■ —dr . 31 Ft Eqn 1.5: Definition of shear stress (r). T - ----A 31 X E q n 1 . 6 : Definition of viscosity ( 77). v - — 32 Y Eqn 1.7: Darcy's Law. 0 « AP - R— 32 Eqn 1.8: Poiseuille-Hagen equation. Q - LF 11 r< 32 8 / Eqn 1.9: Combination of equations 1.7 and 1.8. K . 8 7 ’l- 32 3i r 4 Eqn 1.10: Einsteins equation for rigid particles. * 1 ♦ a.C 35 Eqn 1.11: Definition of apparent relative T\g of the blood sample ^ or ' of the suspending phase 35 viscosity (q j Eqn 1.12: Change in r|a with hct (H) between log T\a - k1 * k"*H 39 30-60% at a fixed y. Eqn 1.13: rj changes with temperature (T) for C r 40 Newtonian fluids and blood )-Ae T 7 Eqn 1.14: Forces involved in the aggregation F.'Fh-Ft -F,- Fn 51 o f RBC's. Eqn 2.1: Calculation of the concentration (C) of a Wi 63 dialysed substance. FIN E q n 2 .2 : Calculation of fibrinogen concentration [<M • 15.3 • y - • 4 280 64 [<p], by Rampling-Gaffney method. Eqn 2.3: Loge-linear relationship between r) of Ln (?\susp) ■ 65 RBCSusp and polymer concentration [P]. Eqn 2.4: Calculation of the volume of enzyme 5 H stock solution needed to give 5mg/ml v ■ V' 68 EN2' ^c STOCK'm 'rsAS{P of RBC's at a hct H. Eqn 2.5 : Calculation of the volume of stock aggregating agent solution needed to / AGG H . y 69 produce a specified concentration per AGG UC STOCK C 1UUIOC) ^ ml of supernatant. Eq" 2.6 : Calculation of the supernatant volume change aAV y SUSP . AC -yv SOL 70 needed to correct a blood sample to 45% hct. 45 H l Eqn 2.7: Calculation for RBC size (MCV). MCI' - too N, 72 Eqn 2.8: Relation of torque (T) and shear stress (x) in the 2 2 T rl*r2 gap between the bob (radius r l5 height h) and T - — .[— 81 A *h rl2 -r22J cup (radius r2) of the Viscometer. Eq" 3.1 : Conversion equation of torque t) - EXP [-4.5627 .(R. 1.6095 ) 91 readings to r\ values. -Ln (y)] . Torque Reading 8 Eq" 3.2 : Power law relationship between q and y valid for blood in an aggregation phase (Phase-A) ti - c.yG 91 and in a deformation phase (Phase-D). Eqn 3.3: Equation 3.2 manipulated to give the equation of a straight line representing shear thinning ^ Ln(x ir) - G A'L n (y ) ♦ C A g \ characteristics of LSR q ^ - ie aggregation phase (A-Phase). Eqn 3.4: As for equation 3.3, but for HSR q ^ - ie L n 0lr) - G D«Ln(y) ♦ CD 91 deformation phase (D-Phase). Eqn 3.5: Definition of 0-277q r; an index of • RBC Susp Viscosity at 0.277 s A 92 aggregation Suspending Phase Viscosity RBC Viscosity at j Eqn 3.6: Definition of 1285qr; an index of 128.5 _ Susp__________ ' ___________________ 128 .5 '1 g j 'If* ** deformation. Suspending Phase Viscosity Eqn 4.1: Monoexponential curve fitting equation for fitting to the aggregation phase of LT - K m - b m,ExP [-<v(r-/min)] 117 the syllectogram obtained from the MEA. Eq" 4.2 : As for equation 4.1, but the curve fitting l t - K b- a b.Exp [-&fc»(r-'min)] 117 equation is biexponential. ’ c^Exp Eq" 5.1 : Modified form of equation 1.12 that assumes Ln(T\J - mx • H 157 k' equals logc(supernatant q) at all y's. Eq" 5.2: Relationship between nq and y for whole (mj) - -0.171 .Ln(y) - 2.79 158 blood between y's 0.277-27.7s*1. Eq" 5.3 : As for equation 5.2 but between y's L n (m j) - -0.0895 *Ln{ y) - 3.13 158 69.5 and 128.5s'1. 9 Eq" 5.4: Authors' equation relating r|p H and y. In(T,r) - H.iA'X x 158 E q n 5.5: Whittington et al's equation linking q p H i"(n P - H.Ln(.— ) and y based on a Weavers et al's linear-log Ay) 170 * relationship between ml and y . Ay) E q n 5.6: Matrais' equation derived from removing irij 45 , \H~ 170 from equation 5.1. ^ r 45 ’ f ^ r not* 10 LIST OF TABLES: T ab 1.1: Some blood vessels with their average diameter and wall thickness.