MICROVASCULAR
RESEARCH
16, 263-271 (1978)
Microaggregate Formation in Whole Exposed to Shear Stress’
Blood
T. S. DEWITZ,” R. R. MARTIN,~J R. T. SOLIS,? J. D. HELLUMS,* AND L. V. MCINTIRE**~ *Rice Biomedical Laboratory, Rice University, and the department Baylor College of Medicine, Houston, Texas 77001
of Medicine,
Received July 12, 1977 Microaggregate size distributions were measured in fresh blood sheared in a rotational viscometer at 37”. The number, N,, and the total volume, V,, of aggregate particles 13-101 Frn in diameter increased with shear stress at exposures of O-300 dynes/cm* for 5 min. The mean size per aggregate, VA/N,, decreased with shear stress so that V, also decreased at shear stresses greater than 450 dynes/cm* maintained for IO min. Aggregability to 0.2-2.0 M ADP was progressively inhibited after exposure to increasing shear stress. The volume of platelet microaggregates may be increased substantially by the involvement of leukocytes.
INTRODUCTION The formation of thromboemboli has complicated the use of cardiac valves and circulatory prostheses. The appearance of platelet microaggregates has recently been described in association with hemodialysis (Bischel et al., 1973), clinical perfusion (Rittenhouse et al., 1972), and cardiopulmonary bypass (Allardyce et nl., 1966; Turina, 1972; Dutton et al., 1974; McKenna et al., 1975; Solis et ul., 1974a, 1975a,b). These microaggregates have been implicated in neuropathological (Hill et al., 1969) and pulmonary (Reul et al., 1973; Robb, 1973) abnormalities associated with cardiopulmonary bypass. Improved designs for artificial organs and prostheses require more detailed knowledge of thrombogenesis by mechanical trauma and surface exposure. Unfortunately, the complicated and variable shear stress histories of cells in cardiopulmonary bypass and other circulatory support systems are hard to quantitate. Therefore, in vitro studies of shear stress and surface interaction have used simpler flows including stagnation point flow (Morton et al., 1975), flow through tubes (Colantuoni ef al., 1977; Goldsmith et al., 1976), and flows in rotational viscometers (Glover et al., 1974, 1977; Brown et al., 1975; Klose et al., 1975; Hung et al., 1976; Dewitz et al., 1977a). Rotational viscometers have the advantage of applying a known uniform shear stress to the entire sample volume. ’ This investigation was supported by U.S. Public Health Service Grant No. PHS 75-10168,HL 17437, HL 16938, AI-12048, and AI-00446 and U.S. Army Grant No. DADA-17-73-C-3149. i! R. R. M. receives support from a Research Career Development Award (AI-70335) from the National Institutes of Health. 3 Address reprint requests to: L. V. McIntire, Department of Chemical Engineering, Rice University, Houston, Texas 77001. 263
0026.2862/78iOl62-0263$02.0010 Copyright @ 1978 by Adacemic Press, Inc. All rights of reproduction in any form reserved.
264
DEWITZ
ET AL.
Platelets, leukocytes, and erythrocytes may interact by releasing substances that may affect adhesion and aggregation. Shear stress exposure may cause serotonin, norepinephrine, and adenosine diphosphate (ADP) release by platelets (Brown, et al., 1975). These substances stimulate chemotaxis and adhesion of polymorphonuclear leukocytes (PMNs; Banks et al., 1973). Shear stress exposure also stimulates release reactions in leukocytes. Some of the chemicals released into the plasma undoubtedly affect platelet aggregation (Dewitz et al., 1977b). Finally, erythrocytes may release ADP in response to sublethal mechanical trauma. Thus, the response of whole blood to mechanical trauma may be different from that of platelet-rich plasma (PRP) or buffered cell suspensions. This study extends previous investigations of platelet aggregation in plateletrich plasma to examine platelet-leukocyte response in whole blood exposed to shear stress. Instead of using optical density as a measure of platelet aggregation, we measured particle size distributions directly with an electronic particle counter. MATERIALS
AND METHODS
Whole blood samples were obtained from eight normal, human donors who were nonsmokers and were taking no medications. Blood samples were drawn slowly through a heparin lock which was left in place during each experiment. This heparin lock consisted of a 19-gauge needle and butterfly, a three-way polyethylene stopcock and a syringe containing 100 units/ml of sodium heparin. The first few milliliters of blood were discarded with the heparin syringe, and 7.2 ml were drawn into a clear lo-ml polyethylene syringe. Blood obtained by this method was anticoagulated with 1 part of 3.8 wt% sodium citrate to 9 parts of whole blood. The rotational viscometer was designed especially for blood shearing experiments and is described in detail elsewhere (MacCallum et al., 1973). Blood samples were exposed to controlled magnitudes of shear stress in the 0.029-cm gap between a stationary cylindrical bob and a rotating outer cup. Edge effects at the top and bottom of the annular gap were reduced by cone-and-cone (top) and cone-and-plate (bottom) geometries which were designed to reproduce the constant shear stresses of the cylindrical section. Blood-contacting surfaces were siliconized and maintained at 37°C in a Lucite box heated with forced air. Blood samples were introduced into the viscometer immediately after mixing with the citrate. Subsequent rotation of the viscometer cup at 400-3000 rpm produced bulk shear stresses of 75-600 dynes/cm2. Average shear stresses were calculated from the torque transmitted through the blood to the inner platen. Controlled shear stresses were maintained for 300 or 600 sec. Rotation of the viscometer was then halted, and approximately 6 ml of blood was removed via a two-way valve. Each sample was mixed by inversion for 60 sec. Finally, a portion of each sample was counted electronically to establish the particle size distribution. Controls included fresh blood and samples which were exposed to the platen surfaces for the same time as the sheared whole blood, but with no rotation of the outer cup.
STRESS-INDUCED
PLATELET
265
AGGREGATION
Particle counting was done using a Coulter Model T particle size analyzer (Coulter Electronics, Hialeah, Fla.). This equipment counts particles simultaneously in 15 logarithmically scaled channels corresponding to particles of 47.4 to 7.77 x IO5 Frn3 by volume with equivalent spherical diameters of 4.0-102 pm (280-pm aperture). For each count 0.5 ml of blood was diluted with 50 ml of modified Eagle’s solution (Coulter Isoton) and six drops of a stromatolysing agent (Coulter Zap-Isoton). Particle counts were completed within 30 set after this dilution so that leukocyte lysis and aggregate disintegration were minimal. Samples were gently mixed with a small rotary mixer to prevent sedimentation of the microaggregates during the count. Ethylenediaminetetraacetate (EDTA, 1 wt%) was added to portions of the same sheared blood samples. These were allowed to sediment, and electronic and visual (hemocytometer) platelet counts were performed on each sample. Post shear platelet aggregability was tested by stimulating aggregation with two concentrations of exogenous ADP (2 x lop6 and 2 x lop7 M, Sigma Chemical Co., St. Louis, MO.). One part of Verona1 buffer, containing the ADP, was added to nine parts of sheared blood and mixed with a Vortex mixer for 60 sec. The aggregate particles were counted as for the sheared blood samples without ADP addition. Verona1 buffer without ADP was used to prepare control samples for the aggregability studies. Purified suspensions of leukocytes and platelets in phosphate-buffered saline were prepared by centrifugation of blood in a density gradient of Ficoll-Hypaque. Contaminating erythrocytes were lysed with a 0.83% ammonium chloride solution. Preliminary studies with purified platelet, polymorphonuclear leukocyte (PMN), and lymphocyte/monocyte suspensions indicated the approximate sizes of single cells, as shown in Fig. 1. Because leukocytes were generally smaller than 11 pm, particles larger than 13 pm were assumed to be aggregates of platelets and/or leukocytes. Thus, when aggregates are referred to in the results, these represent particles of 13- to 102-pm equivalent spherical diameter. The geometric mean volume of each channel was multiplied by the corresponding particle population to
0 PLATELET/ PMN SUSPENSION 0 PLATELET /LYMkWCCYTE SUSPENSION
IL 0
5 EWIVALENT
FIG. I.
Particle
size distributions
10
15
SPHERICAL
20 DIAMETER
25
30
(PM)
for suspensions of leukocytes and platelets.
266
DEWITZ
ET AL. 0 o D”‘-/ot$, a
I02
IO EQUIVALENT
SPHERICAL
CONTROL
75 w”s/oM2
DIAMETER
[PM)
FIG. 2. Microaggregate formation induced by shear stress: particle size distribution in whole blood’ sheared for 5 min at 37”.
give the total volume of particles. The size distribution of aggregate particles was used to calculate the total volume of aggregates, V,, the total number of aggregates, N,, and the mean volume per aggregate, VA/N,. RESULTS Blood samples from six donors were sheared for 300 set at shear stresses of O-300 dynes/cm2. The mean particle size distribution for the series of experiments is shown in Fig. 2. The modal peak at 8.5 pm corresponds to the leukocyte peak in Fig. 1. Particles of 13- to 102-pm equivalent spherical diameter represent microaggregates. The trend, as seen in all experiments, was that the control blood contained very few aggregates and that the volume of aggregates increased with increased levels of shear stress exposure. The leukocyte modal peak appears to move toward higher volumes with increasing shear stress. The induction of aggregate formation by shear stress is shown more clearly in Fig. 3. The data in this figure represent mean values (+ SEM) for the total volume of aggregates obtained in the six series of experiments. Here, there is a significant (paired t test, P < 0.02) increase in the volume of aggregates after a 5-min exposure to shear stresses as low as 150 dynes/cm2. Another indication of the platelet aggregation alteration by shear stress is the inhibition of further aggregation in response to the addition of exogenous ADP. This inhibition was marked-at an ADP concentration of 0.2 @I, as shown in Fig. 4A. The volume density of aggregates, V,, was significantly reduced by shear stress exposure even in postshear blood samples treated with 2.0 fl ADP. The 0.0 M ADP curve is similar to Fig. 3 and represents data from samples treated exactly as for the ADP-induced samples, except that the Verona1 buffer did not contain ADP. Thus, a measure of the augmentation of aggregation by ADP is the difference between the postshear, baseline curve for no addition of exogenous ADP (0 M) and the aggregate volume curves for 2.0 and 0.2 fl ADP.
STRESS-INDUCED
0
PLATELET
50
loo SHEAR
Is0 STRESS
267
AGGREGATION
200
250
?m
[D”Wct$)
FIG. 3. Microaggregate formation induced by shear stress: total volumes of microaggregates (particles of l3- to IOl-pm diameter) produced by shearing whole blood for 5 min at 37” (means k SEM).
The number of aggregates per microliter larger than 13 Fm in sheared whole blood is shown in Fig. 4B. These data are the means (k SEMs) for the same samples used to compile Fig. 4A. The number density of aggregates induced by shear stress (0 M ADP) increased more rapidly with increasing magnitude of shear stress than did the volume density. Samples with exogenous ADP added contained a number of aggregates which was roughly proportional to the number
SHEAR STRESS
(DYNWCM~)
4. Reduced aggregability following shear stress exposure: additional aggregation caused by 0 M(O), 2 x 10-r&4 (O), or 2 x lO+M (A) exogenous ADP. The ordinates are (A) mean + SEM total volume of aggregates, (B) mean + SEM number of aggregates per microliter, and (C) mean k SEM aggregate size. Blood was sheared 5 min. FIG.
268
DEWITZ
“0 x
ET AL.
,625.
PARTICLES> 13/&J
SHEAR
STRESS
( ~N’Wat2
)
FIG. 5. Disruption of microaggregates at higher shear stresses: total volumes of microaggregates produced by shearing whole blood for 10 min at 37”.
induced by mechanical trauma. Because the number of aggregates increased more rapidly than did the total volume of aggregates, the net effect of shear stress exposure was to reduce the mean volume per aggregate. The mean volumes per aggregate are shown in Fig. 4C. The data in this figure are the ratios, VA/N.,,, of the data in Figs. 4A and 4B. The reduction in aggregate size with increasing shear stress is shown clearly. The disintegration of aggregates by higher shear stresses is illustrated by the data shown in Fig. 5. In this figure, the volume of aggregates in two size ranges (13-101 and 32-101 pm) is plotted versus the magnitude of shear stress exposure, up to 600 dynes/cm*. The time of application of the mechanical trauma was 10 min for all samples. The size of aggregates produced by the shear stresses became monotonically smaller as the stress was increased. At shear stresses higher than 25Or
T
SHEAR
STRESS
( “WCM~]
FIG. 6. Platelet attrition during shear stress exposure: fall in platelet count in whole blood sheared 5 min at 37” due to destruction and irreversible aggregation.
STRESS-INDUCED
PLATELET
AGGREGATION
269
450 dynes/cm2 the total volume of particles identified as aggregates actually decreased. An additional factor which may have influenced our results was the attrition of platelets due to shear stress. The resultant fall in postshear platelet counts is shown in Fig. 6. These counts were performed on samples which were treated with EDTA to reduce or eliminate platelet aggregation. This assertion was checked with visual counts which were not significantly different from the electronic counts and showed very few aggregates present. In the preceding data, no correction was made for this slight fall in the number of platelets available for aggregation. DISCUSSION The effects of shear stress on platelet aggregation appear to be twofold. Alteration of the platelets by shear stress exposure stimulates aggregation, while exposure to high shear stress disintegrates large aggregates so that many small aggregates are formed. These results agree qualitatively with those of Brown et al. (1975), who studied particle size distributions in platelet-rich plasma sheared for 5 min at 25”. Much smaller aggregates were observed with PRP, but a maximum total volume of aggregates was identified at shear stresses between 100 and 500 dynes/cm2. The present work indicates a maximum total volume of aggregates for shear stresses of 300 to 450 dynes/cm2. Improved particle dispersion, rapid proliferation of aggregate nuclei, and the disintegration of larger aggregates may all be factors in the reduction of aggregate size by increasing shear stress. The effect of shear stress on the ability of platelets to undergo subsequent aggregation to exogenous ADP has usually been studied using an aggregometer to measure the optical density of the PRP (Glover et al., 1974; Brown et al., 1975; Colantuoni et al., 1977). Because the aggregometer is normally “zeroed” with respect to the postshear plasma, these results correspond to the difference between the baseline and ADP induced curves in Figs. 4A and 4B. An inhibition of the aggregation response to exogenous ADP was observed in sheared PRP which was qualitatively similar to ours in whole blood. Exposure to shear stresses of 100-300 dynes/cm2 reduced the aggregation of PRP in response to 2 x lop6 M ADP by S-77%. The reduction in aggregate volume augmentation by ADP which we observed in whole blood was 20-76% for the same range of shear stresses. Similar aggregometer studies have been done for PRP subjected to oscillatory shear stress in a glass capillary tube (Goldsmith et al., 1976). Inhibition of aggregation in response to 2 x lop6 M ADP was not significant for the shear stresses studied, which were less than 10 dynes/cm2, and thus less than the thresholds observed in our experiments. Visual observations indicated that the aggregates formed during and after shear stress exposure were composed primarily of platelets. However, peripheral involvement of leukocytes might have contributed significantly to the volume of aggregates. The apparent aggregation of leukocytes is indicated by a displacement of the leukocyte modal peak to higher diameters after exposure to increasing shear stresses. This is shown in Fig. 2, but is indicated more clearly in Table 1. Note that
270
DEWITZ
ET AL.
TABLE 1 MOVEMENT OF “LEUKOCYTE” MODEL PEAK” Diameter km) 7.1 9.0 11.3 14.3
Total volume of particles (X IO5 PrnVd) Control
75 dynes/cm2
150 dynes/cm2
300 dynes/cm*
7.97 8.41 1.39 .04
7.60 11.23 5.27 1.12
7.02 9.72 5.93 2.24
5.74 8.58 8.33 7.55
0 Detail of the volume of particles in the neighborhood of the leukocyte modal peak as a function of the magnitude of shear stress exposure (from Fig. 2). Note that as the stress level increases, the modal peak shifts to the “right” (higher equivalent diameters).
the accumulation of platelet aggregates at a given size range could cause the count to increase but not to decrease. Thus the movement of the entire leukocyte peak to the right must be due at least to a passive involvement of leukocytes in microaggregate formation. In a previous study (Dewitz et al., 1977a), increased adhesiveness was observed for PMNs exposed to shear stress. Also, aggregates in films of sheared blood were often observed with PMNs attached to their periphery. Another situation where the involvement of PMNs in aggregate formation has been demonstrated is the formation of microaggregates in stored blood (Solis, 1975b). Our conclusions are: (a) that exposure of whole blood to shear stresses of the order of 75 dynes/cm* for 5 min stimulates aggregation of platelets and leukocytes, (b) that somewhat higher shear stress (about 400 dynes/cm*) tends to disperse aggregates so that they are more numerous and smaller in size than in samples exposed to lower stresses, (c) that platelets develop a refractory state after initial stimulation by shear stress so that additional aggregation to exogenous ADP is inhibited, and (d) that leukocytes are at least passively involved in aggregate formation. These conclusions are of clinical relevance because the study was performed with whole blood at physiological temperatures using shear stresses of the same magnitude as those which are encountered in membrane oxygenators and hemodialysis devices. REFERENCES ALLARDYCE, D. B., YOSHIDA, S. H., AND ASHMORE,P. G. (1966). The importance of microembolism in the pathogenesis and of organ dysfunction caused by prolonged use of the pump oxygenator. J. Thorac. Cardiovasc. Sup. 52, 706-715. BANKS, D. C., AND WITEHALL, J. R. A. (1973). Leukocytes and thrombosis: I, II, and III. Thromb. Diath.
Haemorrh.
30, 36-71.
BISCHEL,M. D., ORRELL,F. L., SCOLES,B. C., MOHLER,J. G., AND BARBOUR,B. H. (1973). Effects of microemboli blood filtration during hemodialysis. Trans. Amer. Sot. Artif. Intern. Organs 19, 492-497. BROWN, C. H., LEVERETT, L. B., LEWIS, C. W., ALFREY, C. P., AND HELLUMS, J. D. (1975). Morphological, biochemical, and functional changes in human platelets subjected to shear stress. J. Lab. Clin. Med. 86, 462-471.
COLANTUONI, J., HELLUMS, J. D., MOAKE, J. L., AND ALFREY, C. P. (1977). The response of human
STRESS-INDUCED
PLATELET
AGGREGATION
271
platelets to shear stress at short exposure times. Trans. Amer. Sot. Artif. Intern. Orguns 23, 626-630. DEWITZ, T. S., HUNG, T. C., MCINTIRE, L. V., AND MARTIN, R. R. (1977a). Mechanical trauma in leukocytes. J. Lab. C/in. Med. 90, 728-738. DEWITZ, T. S., MCINTIRE, L. V., AND MARTIN, R. R. (1977b). Enzyme release by leukocytes exposed to mechanical trauma. ” Proc. 30th Annual Conference on Engineering in Medicine and Biology,” 19, 151. DUTTON, R. C., EDMUNDS, L. H., HUTCHINSON, J. C., AND ROE, B. B. (1974). Platelet aggregate emboli produced in patients during cardiopulmonary bypass with membrane and bubble oxygenators and blood filters. J. Thorac. Cardiovasc. Surg. 67, 259-264. GLOVER, C. J., MCINTIRE, L. V., LEVERETT, L. B., HELLUMS, J. D., BROWN, C. H., AND NATELSON, E. A. (1974). Effect of shear stress on clot structure formation. Trans. Amer. Sot. Artif. Intern. Organs 20, 462-468. GLOVER, C. F., MCINTIRE, L. V., BROWN, C. H., AND NATELSON, E. A. (1977). Mechanical trauma effect on clot structure formation. Thromb. Res. 10, 11-25. GOLDSMITH, H. L., MARLOW, J. C., AND Yu, S. K. (1976). The effect of oscillatory flow on the release reaction and aggregation of human platelets. Microvasc. Res. 11, 335-359. HILL, J. D., AGUILAR, M. J., BARANCO,A., OSBORN, J. J.,ANDGERBODE, F. (1969). Neuropathological manifestations of cardiac surgery. Ann. Thoruc. Surg. 7, 409-419. HUNG, T. C., HOCHMUTH, R. M., AND JOIST, J. H., et al. (1976). Effects of surface injury and shear stress on platelet aggregation and serotonin release. Trans. Amer. Sot. Artif. Intern. Organs 21, 413-420. KLOSE, H. J., RIEGER, H., AND SCHMID-SCHONBEIN, H. (1975). A rheological method for the quantification of platelet aggregation in vitro and its kinetics under defined flow conditions. Thromb. Res. 7, 261-272. MACCALLUM, R. N., O’BANNON, W., HELLUMS, J. D., ALFREY, C. P., AND LYNCH, E. C. (1973). Viscometric instruments for studies on red blood cell damage. In “Rheology of Biological Systems” (H. L. Gabelnick and M. Litt, eds.), pp. 70-83. Charles C Thomas, Springfield, III. MCKENNA, R., BACHMANN, F., WHITTAKER, B., GILSON, J. R., AND WEINBERG, M. (1975). The hemostatic mechanism after open heart surgery. J. Thorac. Cardiovasc. Surg. 70, 298-308. MORTON, W. A., PERMENTIER, E. M., AND PETSCHEK, H. E. (1975). Study of aggregate formation in region of separated flow. Thromb. Diath. Haemorrh. 34, 840-854. REUL, G. J., JR., GREENBERT, S. D., LEFRAK, E. A., MCCOLLUM, W. B., BEALL, A. C., AND JORDAN, G. (1973). Prevention of post-traumatic pulmonary insufficiency; fine screen filtration of blood. Arch. Surg. 106, 386-394. RITTENHOUSE, E. A., HESSEL, E. A., II, ITO, C. S., AND MERENDINO, K. A. (1972). Effect of dipyridamole on microaggregate formation in the pump oxygenator. Ann. Surg. 175, l-12. ROBB, H. J. The role of micro-embolism in the production of irreversible shock (I%3). Ann. Surg. 158, 685-692. SOLIS, R. T., NOON, G. P., BEALL, A. C., AND DEBAKEY, M. E. (1974a). Particulate microembolism during cardiac operation. Ann. Thorac. Surg. 17, 332-344. SOLIS, R. T., GOLDFINGER, D., GIBBS, M. B., AND ZELLER, J. A. (1974b). Physical characteristics of microaggregates in stored blood. Transfusion 14, 538-550. SOLIS, R. T., BEALL, A. C., NOON, G. P., AND DEBAKEY, M. E. (1975a). Platelet aggregation: Effects of cardiopulmonary bypass. Chest 67, 558-563. SOLIS, R. T., KENNEDY, P. S., BEALL, A. C., NOON, G. P., AND DEB&KEY, M. E. (I975b). Cardiopulmonary bypass: Microembolization and platelet aggregation. Circulation 52, 103-108. TURINA, M. (1972). Die rolle bes blutfilters in der verhutung des lungenschadens nach ganzkoperperfusion. Thoraxchirurgie 20, 122-128.
RESEARCH
16, 263-271 (1978)
Microaggregate Formation in Whole Exposed to Shear Stress’
Blood
T. S. DEWITZ,” R. R. MARTIN,~J R. T. SOLIS,? J. D. HELLUMS,* AND L. V. MCINTIRE**~ *Rice Biomedical Laboratory, Rice University, and the department Baylor College of Medicine, Houston, Texas 77001
of Medicine,
Received July 12, 1977 Microaggregate size distributions were measured in fresh blood sheared in a rotational viscometer at 37”. The number, N,, and the total volume, V,, of aggregate particles 13-101 Frn in diameter increased with shear stress at exposures of O-300 dynes/cm* for 5 min. The mean size per aggregate, VA/N,, decreased with shear stress so that V, also decreased at shear stresses greater than 450 dynes/cm* maintained for IO min. Aggregability to 0.2-2.0 M ADP was progressively inhibited after exposure to increasing shear stress. The volume of platelet microaggregates may be increased substantially by the involvement of leukocytes.
INTRODUCTION The formation of thromboemboli has complicated the use of cardiac valves and circulatory prostheses. The appearance of platelet microaggregates has recently been described in association with hemodialysis (Bischel et al., 1973), clinical perfusion (Rittenhouse et al., 1972), and cardiopulmonary bypass (Allardyce et nl., 1966; Turina, 1972; Dutton et al., 1974; McKenna et al., 1975; Solis et ul., 1974a, 1975a,b). These microaggregates have been implicated in neuropathological (Hill et al., 1969) and pulmonary (Reul et al., 1973; Robb, 1973) abnormalities associated with cardiopulmonary bypass. Improved designs for artificial organs and prostheses require more detailed knowledge of thrombogenesis by mechanical trauma and surface exposure. Unfortunately, the complicated and variable shear stress histories of cells in cardiopulmonary bypass and other circulatory support systems are hard to quantitate. Therefore, in vitro studies of shear stress and surface interaction have used simpler flows including stagnation point flow (Morton et al., 1975), flow through tubes (Colantuoni ef al., 1977; Goldsmith et al., 1976), and flows in rotational viscometers (Glover et al., 1974, 1977; Brown et al., 1975; Klose et al., 1975; Hung et al., 1976; Dewitz et al., 1977a). Rotational viscometers have the advantage of applying a known uniform shear stress to the entire sample volume. ’ This investigation was supported by U.S. Public Health Service Grant No. PHS 75-10168,HL 17437, HL 16938, AI-12048, and AI-00446 and U.S. Army Grant No. DADA-17-73-C-3149. i! R. R. M. receives support from a Research Career Development Award (AI-70335) from the National Institutes of Health. 3 Address reprint requests to: L. V. McIntire, Department of Chemical Engineering, Rice University, Houston, Texas 77001. 263
0026.2862/78iOl62-0263$02.0010 Copyright @ 1978 by Adacemic Press, Inc. All rights of reproduction in any form reserved.
264
DEWITZ
ET AL.
Platelets, leukocytes, and erythrocytes may interact by releasing substances that may affect adhesion and aggregation. Shear stress exposure may cause serotonin, norepinephrine, and adenosine diphosphate (ADP) release by platelets (Brown, et al., 1975). These substances stimulate chemotaxis and adhesion of polymorphonuclear leukocytes (PMNs; Banks et al., 1973). Shear stress exposure also stimulates release reactions in leukocytes. Some of the chemicals released into the plasma undoubtedly affect platelet aggregation (Dewitz et al., 1977b). Finally, erythrocytes may release ADP in response to sublethal mechanical trauma. Thus, the response of whole blood to mechanical trauma may be different from that of platelet-rich plasma (PRP) or buffered cell suspensions. This study extends previous investigations of platelet aggregation in plateletrich plasma to examine platelet-leukocyte response in whole blood exposed to shear stress. Instead of using optical density as a measure of platelet aggregation, we measured particle size distributions directly with an electronic particle counter. MATERIALS
AND METHODS
Whole blood samples were obtained from eight normal, human donors who were nonsmokers and were taking no medications. Blood samples were drawn slowly through a heparin lock which was left in place during each experiment. This heparin lock consisted of a 19-gauge needle and butterfly, a three-way polyethylene stopcock and a syringe containing 100 units/ml of sodium heparin. The first few milliliters of blood were discarded with the heparin syringe, and 7.2 ml were drawn into a clear lo-ml polyethylene syringe. Blood obtained by this method was anticoagulated with 1 part of 3.8 wt% sodium citrate to 9 parts of whole blood. The rotational viscometer was designed especially for blood shearing experiments and is described in detail elsewhere (MacCallum et al., 1973). Blood samples were exposed to controlled magnitudes of shear stress in the 0.029-cm gap between a stationary cylindrical bob and a rotating outer cup. Edge effects at the top and bottom of the annular gap were reduced by cone-and-cone (top) and cone-and-plate (bottom) geometries which were designed to reproduce the constant shear stresses of the cylindrical section. Blood-contacting surfaces were siliconized and maintained at 37°C in a Lucite box heated with forced air. Blood samples were introduced into the viscometer immediately after mixing with the citrate. Subsequent rotation of the viscometer cup at 400-3000 rpm produced bulk shear stresses of 75-600 dynes/cm2. Average shear stresses were calculated from the torque transmitted through the blood to the inner platen. Controlled shear stresses were maintained for 300 or 600 sec. Rotation of the viscometer was then halted, and approximately 6 ml of blood was removed via a two-way valve. Each sample was mixed by inversion for 60 sec. Finally, a portion of each sample was counted electronically to establish the particle size distribution. Controls included fresh blood and samples which were exposed to the platen surfaces for the same time as the sheared whole blood, but with no rotation of the outer cup.
STRESS-INDUCED
PLATELET
265
AGGREGATION
Particle counting was done using a Coulter Model T particle size analyzer (Coulter Electronics, Hialeah, Fla.). This equipment counts particles simultaneously in 15 logarithmically scaled channels corresponding to particles of 47.4 to 7.77 x IO5 Frn3 by volume with equivalent spherical diameters of 4.0-102 pm (280-pm aperture). For each count 0.5 ml of blood was diluted with 50 ml of modified Eagle’s solution (Coulter Isoton) and six drops of a stromatolysing agent (Coulter Zap-Isoton). Particle counts were completed within 30 set after this dilution so that leukocyte lysis and aggregate disintegration were minimal. Samples were gently mixed with a small rotary mixer to prevent sedimentation of the microaggregates during the count. Ethylenediaminetetraacetate (EDTA, 1 wt%) was added to portions of the same sheared blood samples. These were allowed to sediment, and electronic and visual (hemocytometer) platelet counts were performed on each sample. Post shear platelet aggregability was tested by stimulating aggregation with two concentrations of exogenous ADP (2 x lop6 and 2 x lop7 M, Sigma Chemical Co., St. Louis, MO.). One part of Verona1 buffer, containing the ADP, was added to nine parts of sheared blood and mixed with a Vortex mixer for 60 sec. The aggregate particles were counted as for the sheared blood samples without ADP addition. Verona1 buffer without ADP was used to prepare control samples for the aggregability studies. Purified suspensions of leukocytes and platelets in phosphate-buffered saline were prepared by centrifugation of blood in a density gradient of Ficoll-Hypaque. Contaminating erythrocytes were lysed with a 0.83% ammonium chloride solution. Preliminary studies with purified platelet, polymorphonuclear leukocyte (PMN), and lymphocyte/monocyte suspensions indicated the approximate sizes of single cells, as shown in Fig. 1. Because leukocytes were generally smaller than 11 pm, particles larger than 13 pm were assumed to be aggregates of platelets and/or leukocytes. Thus, when aggregates are referred to in the results, these represent particles of 13- to 102-pm equivalent spherical diameter. The geometric mean volume of each channel was multiplied by the corresponding particle population to
0 PLATELET/ PMN SUSPENSION 0 PLATELET /LYMkWCCYTE SUSPENSION
IL 0
5 EWIVALENT
FIG. I.
Particle
size distributions
10
15
SPHERICAL
20 DIAMETER
25
30
(PM)
for suspensions of leukocytes and platelets.
266
DEWITZ
ET AL. 0 o D”‘-/ot$, a
I02
IO EQUIVALENT
SPHERICAL
CONTROL
75 w”s/oM2
DIAMETER
[PM)
FIG. 2. Microaggregate formation induced by shear stress: particle size distribution in whole blood’ sheared for 5 min at 37”.
give the total volume of particles. The size distribution of aggregate particles was used to calculate the total volume of aggregates, V,, the total number of aggregates, N,, and the mean volume per aggregate, VA/N,. RESULTS Blood samples from six donors were sheared for 300 set at shear stresses of O-300 dynes/cm2. The mean particle size distribution for the series of experiments is shown in Fig. 2. The modal peak at 8.5 pm corresponds to the leukocyte peak in Fig. 1. Particles of 13- to 102-pm equivalent spherical diameter represent microaggregates. The trend, as seen in all experiments, was that the control blood contained very few aggregates and that the volume of aggregates increased with increased levels of shear stress exposure. The leukocyte modal peak appears to move toward higher volumes with increasing shear stress. The induction of aggregate formation by shear stress is shown more clearly in Fig. 3. The data in this figure represent mean values (+ SEM) for the total volume of aggregates obtained in the six series of experiments. Here, there is a significant (paired t test, P < 0.02) increase in the volume of aggregates after a 5-min exposure to shear stresses as low as 150 dynes/cm2. Another indication of the platelet aggregation alteration by shear stress is the inhibition of further aggregation in response to the addition of exogenous ADP. This inhibition was marked-at an ADP concentration of 0.2 @I, as shown in Fig. 4A. The volume density of aggregates, V,, was significantly reduced by shear stress exposure even in postshear blood samples treated with 2.0 fl ADP. The 0.0 M ADP curve is similar to Fig. 3 and represents data from samples treated exactly as for the ADP-induced samples, except that the Verona1 buffer did not contain ADP. Thus, a measure of the augmentation of aggregation by ADP is the difference between the postshear, baseline curve for no addition of exogenous ADP (0 M) and the aggregate volume curves for 2.0 and 0.2 fl ADP.
STRESS-INDUCED
0
PLATELET
50
loo SHEAR
Is0 STRESS
267
AGGREGATION
200
250
?m
[D”Wct$)
FIG. 3. Microaggregate formation induced by shear stress: total volumes of microaggregates (particles of l3- to IOl-pm diameter) produced by shearing whole blood for 5 min at 37” (means k SEM).
The number of aggregates per microliter larger than 13 Fm in sheared whole blood is shown in Fig. 4B. These data are the means (k SEMs) for the same samples used to compile Fig. 4A. The number density of aggregates induced by shear stress (0 M ADP) increased more rapidly with increasing magnitude of shear stress than did the volume density. Samples with exogenous ADP added contained a number of aggregates which was roughly proportional to the number
SHEAR STRESS
(DYNWCM~)
4. Reduced aggregability following shear stress exposure: additional aggregation caused by 0 M(O), 2 x 10-r&4 (O), or 2 x lO+M (A) exogenous ADP. The ordinates are (A) mean + SEM total volume of aggregates, (B) mean + SEM number of aggregates per microliter, and (C) mean k SEM aggregate size. Blood was sheared 5 min. FIG.
268
DEWITZ
“0 x
ET AL.
,625.
PARTICLES> 13/&J
SHEAR
STRESS
( ~N’Wat2
)
FIG. 5. Disruption of microaggregates at higher shear stresses: total volumes of microaggregates produced by shearing whole blood for 10 min at 37”.
induced by mechanical trauma. Because the number of aggregates increased more rapidly than did the total volume of aggregates, the net effect of shear stress exposure was to reduce the mean volume per aggregate. The mean volumes per aggregate are shown in Fig. 4C. The data in this figure are the ratios, VA/N.,,, of the data in Figs. 4A and 4B. The reduction in aggregate size with increasing shear stress is shown clearly. The disintegration of aggregates by higher shear stresses is illustrated by the data shown in Fig. 5. In this figure, the volume of aggregates in two size ranges (13-101 and 32-101 pm) is plotted versus the magnitude of shear stress exposure, up to 600 dynes/cm*. The time of application of the mechanical trauma was 10 min for all samples. The size of aggregates produced by the shear stresses became monotonically smaller as the stress was increased. At shear stresses higher than 25Or
T
SHEAR
STRESS
( “WCM~]
FIG. 6. Platelet attrition during shear stress exposure: fall in platelet count in whole blood sheared 5 min at 37” due to destruction and irreversible aggregation.
STRESS-INDUCED
PLATELET
AGGREGATION
269
450 dynes/cm2 the total volume of particles identified as aggregates actually decreased. An additional factor which may have influenced our results was the attrition of platelets due to shear stress. The resultant fall in postshear platelet counts is shown in Fig. 6. These counts were performed on samples which were treated with EDTA to reduce or eliminate platelet aggregation. This assertion was checked with visual counts which were not significantly different from the electronic counts and showed very few aggregates present. In the preceding data, no correction was made for this slight fall in the number of platelets available for aggregation. DISCUSSION The effects of shear stress on platelet aggregation appear to be twofold. Alteration of the platelets by shear stress exposure stimulates aggregation, while exposure to high shear stress disintegrates large aggregates so that many small aggregates are formed. These results agree qualitatively with those of Brown et al. (1975), who studied particle size distributions in platelet-rich plasma sheared for 5 min at 25”. Much smaller aggregates were observed with PRP, but a maximum total volume of aggregates was identified at shear stresses between 100 and 500 dynes/cm2. The present work indicates a maximum total volume of aggregates for shear stresses of 300 to 450 dynes/cm2. Improved particle dispersion, rapid proliferation of aggregate nuclei, and the disintegration of larger aggregates may all be factors in the reduction of aggregate size by increasing shear stress. The effect of shear stress on the ability of platelets to undergo subsequent aggregation to exogenous ADP has usually been studied using an aggregometer to measure the optical density of the PRP (Glover et al., 1974; Brown et al., 1975; Colantuoni et al., 1977). Because the aggregometer is normally “zeroed” with respect to the postshear plasma, these results correspond to the difference between the baseline and ADP induced curves in Figs. 4A and 4B. An inhibition of the aggregation response to exogenous ADP was observed in sheared PRP which was qualitatively similar to ours in whole blood. Exposure to shear stresses of 100-300 dynes/cm2 reduced the aggregation of PRP in response to 2 x lop6 M ADP by S-77%. The reduction in aggregate volume augmentation by ADP which we observed in whole blood was 20-76% for the same range of shear stresses. Similar aggregometer studies have been done for PRP subjected to oscillatory shear stress in a glass capillary tube (Goldsmith et al., 1976). Inhibition of aggregation in response to 2 x lop6 M ADP was not significant for the shear stresses studied, which were less than 10 dynes/cm2, and thus less than the thresholds observed in our experiments. Visual observations indicated that the aggregates formed during and after shear stress exposure were composed primarily of platelets. However, peripheral involvement of leukocytes might have contributed significantly to the volume of aggregates. The apparent aggregation of leukocytes is indicated by a displacement of the leukocyte modal peak to higher diameters after exposure to increasing shear stresses. This is shown in Fig. 2, but is indicated more clearly in Table 1. Note that
270
DEWITZ
ET AL.
TABLE 1 MOVEMENT OF “LEUKOCYTE” MODEL PEAK” Diameter km) 7.1 9.0 11.3 14.3
Total volume of particles (X IO5 PrnVd) Control
75 dynes/cm2
150 dynes/cm2
300 dynes/cm*
7.97 8.41 1.39 .04
7.60 11.23 5.27 1.12
7.02 9.72 5.93 2.24
5.74 8.58 8.33 7.55
0 Detail of the volume of particles in the neighborhood of the leukocyte modal peak as a function of the magnitude of shear stress exposure (from Fig. 2). Note that as the stress level increases, the modal peak shifts to the “right” (higher equivalent diameters).
the accumulation of platelet aggregates at a given size range could cause the count to increase but not to decrease. Thus the movement of the entire leukocyte peak to the right must be due at least to a passive involvement of leukocytes in microaggregate formation. In a previous study (Dewitz et al., 1977a), increased adhesiveness was observed for PMNs exposed to shear stress. Also, aggregates in films of sheared blood were often observed with PMNs attached to their periphery. Another situation where the involvement of PMNs in aggregate formation has been demonstrated is the formation of microaggregates in stored blood (Solis, 1975b). Our conclusions are: (a) that exposure of whole blood to shear stresses of the order of 75 dynes/cm* for 5 min stimulates aggregation of platelets and leukocytes, (b) that somewhat higher shear stress (about 400 dynes/cm*) tends to disperse aggregates so that they are more numerous and smaller in size than in samples exposed to lower stresses, (c) that platelets develop a refractory state after initial stimulation by shear stress so that additional aggregation to exogenous ADP is inhibited, and (d) that leukocytes are at least passively involved in aggregate formation. These conclusions are of clinical relevance because the study was performed with whole blood at physiological temperatures using shear stresses of the same magnitude as those which are encountered in membrane oxygenators and hemodialysis devices. REFERENCES ALLARDYCE, D. B., YOSHIDA, S. H., AND ASHMORE,P. G. (1966). The importance of microembolism in the pathogenesis and of organ dysfunction caused by prolonged use of the pump oxygenator. J. Thorac. Cardiovasc. Sup. 52, 706-715. BANKS, D. C., AND WITEHALL, J. R. A. (1973). Leukocytes and thrombosis: I, II, and III. Thromb. Diath.
Haemorrh.
30, 36-71.
BISCHEL,M. D., ORRELL,F. L., SCOLES,B. C., MOHLER,J. G., AND BARBOUR,B. H. (1973). Effects of microemboli blood filtration during hemodialysis. Trans. Amer. Sot. Artif. Intern. Organs 19, 492-497. BROWN, C. H., LEVERETT, L. B., LEWIS, C. W., ALFREY, C. P., AND HELLUMS, J. D. (1975). Morphological, biochemical, and functional changes in human platelets subjected to shear stress. J. Lab. Clin. Med. 86, 462-471.
COLANTUONI, J., HELLUMS, J. D., MOAKE, J. L., AND ALFREY, C. P. (1977). The response of human
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platelets to shear stress at short exposure times. Trans. Amer. Sot. Artif. Intern. Orguns 23, 626-630. DEWITZ, T. S., HUNG, T. C., MCINTIRE, L. V., AND MARTIN, R. R. (1977a). Mechanical trauma in leukocytes. J. Lab. C/in. Med. 90, 728-738. DEWITZ, T. S., MCINTIRE, L. V., AND MARTIN, R. R. (1977b). Enzyme release by leukocytes exposed to mechanical trauma. ” Proc. 30th Annual Conference on Engineering in Medicine and Biology,” 19, 151. DUTTON, R. C., EDMUNDS, L. H., HUTCHINSON, J. C., AND ROE, B. B. (1974). Platelet aggregate emboli produced in patients during cardiopulmonary bypass with membrane and bubble oxygenators and blood filters. J. Thorac. Cardiovasc. Surg. 67, 259-264. GLOVER, C. J., MCINTIRE, L. V., LEVERETT, L. B., HELLUMS, J. D., BROWN, C. H., AND NATELSON, E. A. (1974). Effect of shear stress on clot structure formation. Trans. Amer. Sot. Artif. Intern. Organs 20, 462-468. GLOVER, C. F., MCINTIRE, L. V., BROWN, C. H., AND NATELSON, E. A. (1977). Mechanical trauma effect on clot structure formation. Thromb. Res. 10, 11-25. GOLDSMITH, H. L., MARLOW, J. C., AND Yu, S. K. (1976). The effect of oscillatory flow on the release reaction and aggregation of human platelets. Microvasc. Res. 11, 335-359. HILL, J. D., AGUILAR, M. J., BARANCO,A., OSBORN, J. J.,ANDGERBODE, F. (1969). Neuropathological manifestations of cardiac surgery. Ann. Thoruc. Surg. 7, 409-419. HUNG, T. C., HOCHMUTH, R. M., AND JOIST, J. H., et al. (1976). Effects of surface injury and shear stress on platelet aggregation and serotonin release. Trans. Amer. Sot. Artif. Intern. Organs 21, 413-420. KLOSE, H. J., RIEGER, H., AND SCHMID-SCHONBEIN, H. (1975). A rheological method for the quantification of platelet aggregation in vitro and its kinetics under defined flow conditions. Thromb. Res. 7, 261-272. MACCALLUM, R. N., O’BANNON, W., HELLUMS, J. D., ALFREY, C. P., AND LYNCH, E. C. (1973). Viscometric instruments for studies on red blood cell damage. In “Rheology of Biological Systems” (H. L. Gabelnick and M. Litt, eds.), pp. 70-83. Charles C Thomas, Springfield, III. MCKENNA, R., BACHMANN, F., WHITTAKER, B., GILSON, J. R., AND WEINBERG, M. (1975). The hemostatic mechanism after open heart surgery. J. Thorac. Cardiovasc. Surg. 70, 298-308. MORTON, W. A., PERMENTIER, E. M., AND PETSCHEK, H. E. (1975). Study of aggregate formation in region of separated flow. Thromb. Diath. Haemorrh. 34, 840-854. REUL, G. J., JR., GREENBERT, S. D., LEFRAK, E. A., MCCOLLUM, W. B., BEALL, A. C., AND JORDAN, G. (1973). Prevention of post-traumatic pulmonary insufficiency; fine screen filtration of blood. Arch. Surg. 106, 386-394. RITTENHOUSE, E. A., HESSEL, E. A., II, ITO, C. S., AND MERENDINO, K. A. (1972). Effect of dipyridamole on microaggregate formation in the pump oxygenator. Ann. Surg. 175, l-12. ROBB, H. J. The role of micro-embolism in the production of irreversible shock (I%3). Ann. Surg. 158, 685-692. SOLIS, R. T., NOON, G. P., BEALL, A. C., AND DEBAKEY, M. E. (1974a). Particulate microembolism during cardiac operation. Ann. Thorac. Surg. 17, 332-344. SOLIS, R. T., GOLDFINGER, D., GIBBS, M. B., AND ZELLER, J. A. (1974b). Physical characteristics of microaggregates in stored blood. Transfusion 14, 538-550. SOLIS, R. T., BEALL, A. C., NOON, G. P., AND DEBAKEY, M. E. (1975a). Platelet aggregation: Effects of cardiopulmonary bypass. Chest 67, 558-563. SOLIS, R. T., KENNEDY, P. S., BEALL, A. C., NOON, G. P., AND DEB&KEY, M. E. (I975b). Cardiopulmonary bypass: Microembolization and platelet aggregation. Circulation 52, 103-108. TURINA, M. (1972). Die rolle bes blutfilters in der verhutung des lungenschadens nach ganzkoperperfusion. Thoraxchirurgie 20, 122-128.
