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Shear-induced activation of platelets
Bmmerhons~
0021-9290’7910201-0113
J Vol 12. pi. IISI25 ci) Pergamon Press Ltd 1979 Printed m Great Bnum
SHEAR-INDUCED
W.00’0
ACTIVATION OF PLATELETS*
J. M. RAMsTAcKt, L. ZUCKERMAKand L. F. MWKROS~ Biomedical Engineering Center, Northwestern University, Evanston, IL 60201, U.S.A.
. and Research Laboratories, Evanston Hospital, Evanston, IL 60201, U.S.A. Abstract - Platelet-rich plasma was subjected to shear in Poiseuille flows through tubes. i.d. = 305,406 and
5Oftm. Bulk-average shear stresses were MO,750 and loo0 dynes/cm’ and the average residence times were 25- 1650 ms. These shears for these residence times did not produce cell lysis but did activate the platelets so that their response to exogenous ADP were reduced and pktekt factor 3 was mkased. Also, thromboelastographic measurements indicated shear-induced hypercoagulabihty. Allowing samples to stand 30-60 mins after being sheared revealed some of the indicated activation was reversibk. Adding the anticoagulant beparin to sheared samples produced an anomalous response. The quantitative results were independent of tube surface-volume ratio, but were dependent on both level of shear stress and on residence
IIVTRODUCDON The production and growth of thrombi depend upon the physical, as well as chemical, milieu. Shear has been observed to inactivate enzymes (Charm et cl., 1970(a)) and cause degradation of fibrinogen (Charm et al., 1970(b)). Roberts et al. (1973) reported that the final stiffness of a fibrin clot is affected by exposing the sample to shear during the clotting process. Spaeth et al. (1973) reported that fluid shear influences the kinetics of the conversion ofprothrombin to thrombin. The present investigation is a study of shear-induced effects on platelets in platelet-rich plasma. A number of studies have dealt with the response of platelets to shear. Collar and Zucker (1970), while studying the et&t of blood handling procedures on platelet retention by glass bead columns, found that platelet adhesion was markedly reduced by inversion in a test tube, slow centrifugation and remixing, or rapid back-and-forth transfer between two plastic or two glass syringes. Retention was restored within one hour after the latter maneuver but could be reduced again by redisturbing the blood. Chang and Robertson (1976) studied platelet aggregation by laminar shear and Brownian motion, both theoretically and experimentally, and reported a critical shear rate, about 75 set- ‘, above which the rate of aggregation becomes independent of shear rates. Glover et al. (1974) reported that clot structure can be affected by exposing platelet-rich plasma to shear for 15 mins prior to clot activation. They followed the development of the clot stiffness. i.e. the storage modulus. as polymerization * Received for publication 22 Augusr 1978. t Present address: Surgikos A. Johnson and Johnson Company, New Brunswick, NJ 08903, U.S.A. $ Reprint requests should be sent to LFM at Northwestern.
proceeded subsequent to recakification. They indicate drastic changes in clot structure if the samples are preconditioned with shear in the range from 30 to 150 dyn/cm’. In addition, shorter clot-initiation times were observed. Their results suggest that shear can activate the coagulation processes. They also indicate that platelets, after being exposed to shear, show reduced aggregability in the presence of colkgen or ADP. Brown et al. (197% using a couette device, subjected platekt-rich plasma to different levels of shear for periods of 2-24 min. Baaed on measures of cell counts, partick size distribution, EM observations, and serotonin release, they concluded that shear as low as SOdyn/cm2 stimulates the platelet rekase reaction and platelet aggregation. Stress as low as 100 dyn/cm’ produced cell lysis. as indicated by acid phosphatase release. The amount of cell iysis appeared to be linearly related to the duration of shear. At low shear, less than 100dyn/cm2, they reported that the reduced aggregobility was reversible and that if allowed to stand the platelets recovered their ability to aggregate in response to exogenous ADP. Hung et al. (1976), using a concentric cylinder device, noted about the same threshold shear, lOO-165dyn/cm’, for cell lysis. The initial rate of lysis increased with increased stress, but as exposure time lengthened the rate of lysis decmased. The present study focuses on changes in platelet physiology induced by short-term exposure to relatively high shear stress, a situation common to ail extracorporeal bypass procedures. This work includes experiments designed to correlate stress exposure to platelet damage and piatekt function. Platelet damage wasinvestigated using platelet counts and measures of plasma acid phosphatase concentration. The platelet. functions investigated were: (i) shape change and
113
114
J. M. RAMSTACK, L.
ZIJCKERMANand
aggregation in the presence of ADP, (ii) release of procoagulant platelet factor 3, PF-3, and (iii) heprin neutralization. In addition, the coagulability of platelet-rich plasma was determined for samples with and without exposure to shearing. MATERIALS AND METHODS
Sample preparation Fresh canine whole blood, collected from the jugular vein using 19G disposable needles and plastic disposable syringes, provided the working material for the study. After collection, only disposable plastic test tubes and pipettes were used for handling. Commonly accepted methodologies were used in measuring plate let shape change, platelet aggregation, plasma acid phosphatase concentration, and platelet factor 3 availability. For most experiments 72 ml of blood were drawn into a citrate anticoagulant, with nine parts of fresh whole blood to one part buffered citrate (three parts 0.1 M sodium citrate and two parts 0.1 M citric acid, pH 4.7-5.0). Four dogs were used in these experiments and the results averaged. In order to avoid the complications associated with combining anticoagulants and to make the results more applicable to in uivo situations, no citrate was used in collecting samples for experiments examining heparin-platelet interactions. Instead, these blood samples were drawn into a low dose heparin solution that permitted handling and sample preparation. This initial dose is called the handling dose. Subsequently, the teat dose of heparin was added. &cause these handling doses were small compared to the test doses and because the anticoagulant effect of heparin is known to decay rapidly, the residual effect of the handling dose on the subsequent tests, performed an hour later, was assumed to be negligible. The heparin doses reported in these tests are based only on the doses added just prior to the tests. This handling heparinization involved the collection of 50 ml of fresh whole blood into 0.2 ml of 4OU/ml of beef-lung heparin (Upjohn Company, Kalamazoo, Michigan) and allowed approximately one hour of careful handling at room temperature before the initiation of clotting or platelet clumping. Two dogs were used for these experiments. Each blood sample was centrifuged, immediately after collection, at 60-140g for 10 min to separate platelet-rich plasma, PRP, from the red cells. After removing the PRP, the remaining sample was spun at 153Og for 10min
to recover
platelet-poor
L. F. MOCKRB
relatively short periods oftime, 25-1600 ms. The entire apparatus consists of a compressed gas supply, a piston-cylinder drive unit, a feed syringe, and a tubular test section. The PRP sample, contained within the syringe, is driven down the tube by the syringe plunger. The plunger, in turn, is driven by the piston and the piston is driven by high pressure from the gas supply. Solenoid valves govern inflow and outflow of gas to and from the piston-cylinder unit. The syringe is encased in an aluminium block and consists of a 10 cc plastic disposable syringe barrel and a specially-made metal plunger. The tubular test sections were made of stainless-steel hypodermic tubing, ranging in length from 15 to 305cm. Three different diameters of tubing were used : 305,406 and 508 pm (i.e. 24.22 and 21 gauge, respectively). The testsection tubes were connected to the outlet of the syringe by means of a special fitting. This tube-syringe fitting was constructed to withstand pressure and to minimize entrance artifacts. The end of the test-section tube was lightly filed and the opening reamed. Epoxy was applied to the exterior of about 5 cm of the tube at one end. Hypodermic needles with luer-lok hubs, either 20 or 18 gauge, were slid over this proximal end of the test-section tube, leaving about 2 mm beyond the entrance to the hub, as shown in Fig. 1. This extension placed the end of the tube at the syringe apex. Air, forced through the opposite opening, blew the epoxy out and left a smooth entrance. After drying, the tube interior was siliconized according to. the instructions of the manufacturer of Siliclad, Clay Adams, Parsippani, NJ.
plasma,
PPP. The PRP and PPP portions were then mixed to synthesize samples with desired platelet concentrations, usually 3@WOO/mm3. Shear apparatus Platelet trauma was induced by the shear stresses generated in iaminar how through circular tubes. The device was chosen primarily for its capacity to generate high average shear stresses, up to 1000dyn/cmz, for
II=
Shearing t&e
id
Fig. 1. Schematic of syringe-tube fitting. This fitting served as the entrance to the uniform-bore tubes used to subject PRP samples to flow stresses.
115
Shear-induced activation of platelets One of the disadvantages of using a tubular shearing device is that all elements of the fluid are not exposed to the same shear stress nor do they all have the same transit time within the tube. The shear stress and transit time vary with radial position. The platelet trauma is correlated, therefore, with flow-weighted averages and for this study the radial distribution is assumed to be Poiseuillian. The “flow-averaged”, “cup-mixed”, or “bulk” shear stress of the exiting sample, r‘), is defined as R TUT
(2x
dr)/Q
J*0
and for Poiseuille flow is 2(pi - p,)D/lSL, in which r and D are the fluid shear stress and velocity, respectively, at a radius r, R = tube radius, D = tube diameter, Q = flow rate, L = tube length, pi = pressure at inlet, and pO = pressure at outlet. Note that rb is slightly larger than one half, or 8/15, of Tat the wall. The residence time, tr,, or bulk average time of shear exposure, is defined as R (L/u)ur
(2n
i
dr)/Q
0
and for Poiseuille flow is 32&‘/(pi p = fluid viscosity. As seen below, interest is the product of shear rate The bulk average of this variable,
- po)D2, in which another variable of and exposure time. (~t)~, is defined as
R (2x
ytur dr)/Q
s0
and for Poiseuille flow is 16L/3D, in which y = shearstrain-rate = T/F. This parameter is useful for comparing the results of this work with that previously reported by others. Note that in studies such as this, one cannot distinguish whether a measured variable is a function of r or y. Separating the dependence on Tor y requires running tests with different viscosity; changing the viscosity of the suspending medium would probably result in a changed chemical environment for the platelets and a new set of variables, however. In each experiment, using a particular tube, the driving pressure was adjusted so as to achieve a desired value for 5) and thereby also determining the value of t,,. Other values of tb for a given TVwere achieved using tubes of different lengths. The tube inlet pressure, pi, was assumed to be that in the syringe and the outlet pressure, pot was assumed to be atmospheric. Since the exit was open, the latter is true. The former assumption, however, neglects all entrance effects. This, of course, is not strictly correct, and the Ap’s used are larger than should be associated with Poiseuille viscous stresses. The error is due to (i) the conversion of pressure energy within the syringe to kinetic energy within the tube and (ii) the extra viscous stresses in the entrance due to thin boundary layers. Because of the bell-shaped entrance, the latter is probably small, however. A calculation of the former indicates an error in Ap of less than 10% in most cases, RMf2_‘---”
with a few exceptions as high as 13% in the short tubes. This means the calculated rb is higher than the true value and the error would have its largest effect on the data associated with the shorter tubes, i.e. those with short residence times. The general validity of the method, on the other hand, was tested by using the device to measure the viscosity of water. Using tubes of all three diameters, several lengths and several driving pressures, ten tests gave an average value of the viscosity of 0.89 f 0.01 cp, i.e. within 3”/,,of the handbook value at 24°C. The general procedure for shearing the platelets was as follows: About 8 ml of the PRP was poured into a fresh plastic disposable syringe and the plunger inserted. Air in the syringe was expelled and the syringe connected to the tube. Some test Ruid was manually pushed through the tube lumen to force out all air. The syringe was inserted into the aluminium block and the piston head moved to contact with the plunger head. The pressure control was set to the proper value. The bow was initiated by triggering the solenoid valve between the compressed air source and the cylinder. Steady state, as indicated by the pressure gauge, was established within a second. The PRP was gathered in a test tube at the distal end of the tube, discarding the first ml. After each use, the tubing was flushed with a milk-soap solution followed by distilled water and then air. The tubing was subsequently dried and stored in a heated incubator until used again. Platelet-lysis
tests
(i) Platelet concentrations were determined by the usual method of counting under direct phase microscopy. Besides those counts made for purposes of sample preparation, platelet concentrations weue determined before and after each shear exposure. (ii) Plasma acid phosphatase, AP, was measured utilizing the calorimetric assay developed by Boehringler Mannheim. The results are presented in terms of percent AP released, in which % AP released is defined such that AP in unsheared PPP is used to indicate 00; release and AP in the plasma of thrice frozen and thawed PRP is used to indicate lOOo/,release. A5 with the platelet counts, plasma acid phosphatase was determined both before and after exposure to shear. Platelet function
tests
(i) Measurements of platelet shape change in response to exogenous ADP, as well as measurements of platekt aggregation, were made using standard procedures and a commercial aggregometer (Bio/Data PAP-ZA, Willow Grove, Penn.). These measurements are based upon recordings of changes in light transmission through a PRP sample relative to transmission through a PPP sample after the addition of the aggregating agent (ADP). Because of a substantial voltage offset in the aggregometer output, the display of the shape change is rather small on the aggregometer chart recorder. A more manageable recording was made by connecting the aggregometer output and
116
J. M. RAMSTACK. L. ZUCKERMAN and L. F. MO~KROS
a suitable voltage source to the differential inputs of an instrumentation amplifier and the amplifier to an auxiliary recorder. The voltage source was adjusted so that the quiescent output of the amplifier was zero and the gain of the amplifier was adjusted to produce an appropriate deflection on the recorder. The methods used for determining shape-change function were similar to those of Born (1970). Ten lambda ofa 0.5 M EDTA solution (pH adjusted to 7.4 with NaOH) was added to 1 ml of PRP (concentration about 300,000/mm3). After transferring the plasma specimens to aggregomcter test tubes and inserting the tubes into the unit, O.OSmi of 2(1O)‘s M ADP was added to 0.45 ml of PRP. Two measurements were made from the shape change pattern : the slope or rate of-shape-change and the maximal amount of shape change. These measurements also were recorded for aiiquots both before and after shear exposure. (ii) Platelet aggregation in response to exogenous ADP was measured using an unmodified Bio/Data Aggregometer. Following recommended procedures, 0.05 ml of 2(10)-’ M ADP was added to 0.45 ml of PRP (platelet concentration, 300,000/mm3). Two measure ments were recorded: the final percentage of aggregation and the time, ts,,, to M”/, of this aggregation, with the latter indicating the rate of gggregation. Again, data was collected on samples with and without shear exposure. (iii) Platelet factor 3 availability, another indication of platekt stimulation, was measured by observing the decrease, below baseline, of the clotting time. Using the method of Rabiner and Hrodek (1968)O.l ml of PRP, with 50,000 platelets per mm3, was incubated with 0.1 ml of a 1.5% celite solution in veronal buffer for 2 min. After incubation, 0.1 ml of 0.03 M CaCI, was added and the clotting time recorded using the Coagulation Profiler, Bio/Data, Model CP-8. The clotting time of an unsheared PRP aliquot is used to represent the baseline, or zero percent, PF-3 availability. The clotting time of a thrice frozen-and-thawed specimen is used to represent loOY, PF-3 availability. The clotting times ofsheard samples were converted to percent PF-3 available, i.e. % PF-3 =(c.t. of unsheared minus c.t. of available sheared)/(c.t. of unsheared minus c.t. of frozen and thawed). Duplicate samples were measured and averaged. (iv) Platelets are known to have a strong ability to neutralize the effect of the anticoagulant heparin (e.g. Walsh et al., 1974). The influence of shear on this ability of platelets to neutralize heparin was measured by observing the time-changes of the thromboelastographic r-value of samples that were heparinized after being sheared and comparing the results to the time changes for unsheared samples. The lightly heparinized PRP sample was prepared, a portion sheared, the test dose of heparin added, and sequential aliquots tested for thromboelastographic r-value (a measure of clotting time) as the sample was incubated at 37°C. Another portion was left unsheared and similarly tested. To determine if the effects shown were due to plasma trauma or platelet trauma, two identical
portions of a PRP sample (797,0CtO/mm3),which had been lightly heparinized, were diluted with PPP (one part PRP to 5.75 parts PPP). The first portion was diluted with PPP plasma that had been subjected to shear of tb = 750 dyn/cm2 for t, = 337 ms ; the second portion was diluted with unsheared PPP plasma. Heparin was then added to both portions and the rvalues measured on sequential aliquots. (v) Finally, some preliminary tests were conducted to test the reversibility of shear-induced changes in platelet function. All of the above tests of changes in platelet function were conducted, except for the tests of heparin neutralization, within five minutes after exposure to shear. As an indication of reversibility, samples were allowed to stand at room temperature, 25”C, for 30 or 6Omin, post shearing exposure, prior to tests for platekt function. Coagulability rests The objective of these tests is to determine if coagulability of PRP is affected by shear. Samples of PRP were divided into two portions. One portion was subjected to shear in the tubular device and the other portion left undisturbed. Immediately after collecting the flow out of the tube, the two portions were tested for coagulability using a thromboelastograph (see, for instance, Caprini ef al., 1974). RESULl?J
All experiments, except those for reversibility and heparin neutralization, were conducted using PRP from four dogs and the data presented represents the average of the results. Figure 2(a-b) show normalized data for platelet count, acid phosphatase release, and degree of platelet aggregation following stimulation with exogenous ADP. All data is normalized with the corresponding values for unstressed samples of the same PRP and plotted as a function of bulk residence time, t, Three levels of bulk stress were examined. The results indicate no significant changes with level of stress or with time ofexposure. The scatter is within the normal experimental variations for these tests. The data on platelet counts and release ofacid phosphatase indicate the platelets were not stressed severely enough to cause cell lysis. Furthermore, the data of Fig. 2(c) indicate that the stressed platelets could aggregate, in response to ADP, to the same degree as unstressed platelets. These stress levels and stress durations, thus, did not cause severe trauma to the cells. Figures 3-6, however, indicate some platelet functions are severely affected by these exposures to stress. Figures 3 and 4 show the effect of shear stress on the ability of platelets to change shape after being stimulated by exogenous ADP. Figure 3 shows the maximum amount of shape change and Fig. 4 shows the rate of shape change. In both cases, the measures were normalized with values from unsheared samples. The results show, for each value of TV,that the amount and rate of shape change induced by ADP is markedly
Shear-induced activation of platelets
to -A
0
0.2
0.4
Bulk-Average
0.6
1.0
1.2
Time,
lb,
0.6
Residence
1.4
1.6
SeC
Fig. 2(a). Platelet counts after exposure to shear. Data is normalized with prc-exposure platelet counts. Average value I: 1.00 f 0.02 SEM. (b) Per cent ofacid phosphatase released after exposure to shear. Average value 1 2.277; f 1.20%SEM. (c) Degree of platelet aggregation induced by ADP after exposure to shear. Data is normalized by degree of aggregation for samples unexposed to shear. Average value = 1.04 & 0.02 SEM. A: T*= 1000 dynlcm’, tube id = 305pm; 0: rb = 7Rdyn/cm2; tube id = 305F; 750 dyn/cm’ ; tube id = 406 m ; V: rb = 300 dynfcm’, tube id = 305 m ; V : TV= 300dyn/cm’, .:r,= tubeid=WfJm.
lessened if the sample is exposed to shear and the degree- of reduction is dependent on the duration of exposure. The decrease for any one exposure time, on the other hand, is greater for the larger level of stress. A 50”,;,reduction in the maximum shape change occurs with exposure times of only 300 and 175 ms for stresses of 300 and loo0 dynjcn?, respectively. The compar-
Bulk - Average
able reduction in rate of shape change occufi at 5SXland 170ms. Just a few milliseconds of shear exposure causes large reductions in these platelet respnses. With longer exposure times, the additional reduction is lessened. The dependence on level of stress is more pronounced on the rate of shape change than on the amount of shape change.
Residence
Time,
t
b ’
.sec
Fig. 3. Maximum shape change of stressed platelets following the addition of 20 @vl ADP. The measured magnitude of shape change is normaiii by the measured maximum shape change occurring with unstr~& Platelets. All flows were in tubes having an id = 305 w. A: rb = 1000 dyn/cm2 ; 0: tb = 300 dyn/cm’. Solid lines are nonlinear least squares fit of data to an exponential decay. (See Discussion.)
118
J. M. RAMSTACK, L. ZUCKERMAN
00
0,s
and L. F. MOWROS
I.0
Bulk - Average
Residence
lime,
I.S
t
SSC
b’
Fig. 4. Initial rate of shape change for stressed platelets following the addition of 20 PM ADP. The measured rate of shape change is normalized by,the measured rate of shape change occurring with unstressed platelets. All flows werein tubes having an id = 305~. A: r, = fOOOdyn/crn2; V: r, = 3OOdynlcm’. Solid lines are nonlinear least squares fit of data to an exponential decay. (See Discussion.)
Figure 5 shows that the rate at which platelets aggregate, when stimulated by exogenous ADP, is markedly affected by prior exposure to shear, even though the total degree of aggregation was unaffected (Fig. 2c, above). The ratio of t5,, of the sheared samples to that of the unsheared samples is indicated as a function of shearing duration for three values of bulk stress and for three tube sixes. The data, quite scattered, indicate t,,, is approximately linearly related to t,, with the slope increasing with q. For two levels of rb, 750 and 300dyn/cm2, the surface-to-volume ratio of the test section was altered. An increase, for instance, in measured effect with an increase in surface-to-volume
ratio in’@& if rb snd tb are COnStant, that the stimulation is, at least, partly due to an interaction of platelets with the tube walls. If, on the other hand, a change of surface-to-volume ratio produces no change in the results, as is indicated in Fig. 5, the effects on the platelets can be attributed to the shear alone. Thus, the rate of platelet aggregation, after the addition of exogenous ADP, is reduced if the platelets are previously exposed to a shear stress, even though the final amount of aggregation is unaflected. Figure 6 shows the PF-3 availability in the PRP as a function of average time of shear for the three levels of bulk shear stress. The results indicate an increase in
s ._ ‘d
,” L
z 0
* 0.0
-
*.
1
-
-a.
0,s
Bulk - Average
Residence
1 * 1.0
Time,
-
s
*
10 1.5
t
b ’
set
Fig. 5. Time required for 50%of the aggregation to occur following the addition of 20 PM ADP to samples of stressed platelets. Measured time is normalized with corresponding time for samples of unstressed platelets. A:~,~lo()()d~/~~,tubCid=305~;~:~~=7750dyn/cm~,tubeid=305~;~:r,~750dyn/cm~, tubeid=406~;V:r,=U)Odyn/cm2,tubeid=U)S~;~:r,=300dyn/cm2,tubeid~K)8~.Solid lines arc least squares fit of data to be straight line. (See Discussion.)
119
Shear-induced activation of platelets
Bulk - Average
Resadence
Time
, t
b
t 9e.C
Fig. 6. Fraction of PF3 released in samples of platelet-rich plasma subjected to flow stresses. A: ?b = 1000dyn/cm2, tube id -p 305 w; 0: 750dyn/cm2, tube id = 305~; 0: Q = 750dyn/cm2, tube id = 406 pm; V: ~~ = 300 dyn/cm2, tube id = 305 m, (I : ~~= 300dyn/cm’, tube id = 508 m. Solid lines are nonlinear least squares fit of data to an exponential function. (See Discussion.)
procoagulant activity with increasing t,, and for a particular tb the percentage of PF-3 that is availabk increases with increased 7’b.The incremental changes in
PF-3 availability for incremental changes in tb monotonically decrease as tb increases. Varying the tube diameter, or surface-to-volume ratio, for two bulk shearing stresses, 750 and 300 dyn/cm2, has littk e&t on the results, again indicating the trauma is shear related and not surface related. The above results indicate that four measures of platelet functions were influenced by both shear stress and time of exposure to the shear: the rate and maximum amount of shape change subsequent to addition of exogenous ADP, rate of platelet aggregation subsequent to addition of ADP, and PF-3 availability. These effects were all measured on the PRP within five minutes after shear exposure. The results shown in Table 1 are from similar tests but on samples allowed to stand 30 or 60 min after shearing. The data indicate that the effect on the amount of
shape change is essentially reversed in less than 30 min after shear exposure. The effects on the rate-of-shape change show partial reversibility for TV = 1000 and 750 dyn/cmz and total reversibility for 300 dyn/cm’. In all three cases, though, essentially ail of the reversibility occurs in less than 30min with little additional etfect during the 30-60 min period. Measurement of tso aggregation time indicate only modest reversibility for all three stress levels. The availability of PF-3 is essentially not reversible, as might be expected, even after 60min. The data of Table 1 indicate, for all four measures of platelet function, that whatever degree of reversibility takes place it is essentially complete in less than 30 min. Table 2 indicate-s the effect of shear on the coagulability of PRP. Those samples that were more severely sheared, i.e. larger values of 7* or tb or both, consistently show a reduced thromboelastographic rvalue, akin to a reduced clotting time. The rate clot stiffening, a-value, and the final clot stiffness, a,,,-value,
Table 1. Effects of allowing samples to quiescently stand for 30-W min following shear stimulation : Data is from one dog only Maximum shape change, normalized
Rate of shape change, nonnaliied
Aggregation half-time, t 1o normal&d
Fraction of PF3 released
TV= 1000 d/cm2, tb = 0.49 s 0+ min + 30 min +6Qmin
0.23 1.00 0.85
0.28 0.79 0.85
2.25 2.01 -
0.27 0.37
T*= 750d/cm2, tb -- 0.64s 0+ min +3Omin +6Omin
0.50 1.03 1.00
0.22 0.65 0.64
1.40 1.20 1.28
0.41 0.23 0.40
~~= 3OOd/cm*, t* = 1.03 s 0+ mm +3Omin +6Omin
0.00 0.96 0.80
0.00 1.09 1.04
1:75 1.56 -
0.68 0.51 0.49
I20
J. M. RA~STACK. L. ZUCKERMANand L. F. MOCKROS Table 2. Coagulability (TEG) measurements of sheared and unsheared platelet rich plasma Platelet concentration (platelets/mm”)
110800
r-value* (mm)
a-valuet (rad)
a,-value1 (mm)
Unsheared Sheared : 750 dyn/cm2, 644 ms Unsheared Sheared: 75Odyn/cm’, 337 ms
5.0 3.0
1.43 1.45
72.5 73.5
4.0 2.5
1.40 1.43
72.0 72.5
100,000
Unsheared Sheared: 440dyn/cm*. 350 ms
4.5 3.5
1.40 1.40
72.0 74.0
100,000
Unsheared Sheared: 440 dyn/cm’, 140 ms
3.5 3.5
1.40 1.43
68.0 76.0
100,000
Unsheared Sheared: 440 dyn/cm’. 70 ms
4.0 4.0
1.26 1.38
72.0 72.0
100,000
Unsheared Sheared : 440dyn/cm’, 35 ms
5.5 5.5
1.22 1.22
72.0 72.0
200,000
l A measure of clotting time. t A m&sure of the rate of clot stifiening. $ A measure of final clot stitTness.
normalized values of the measured r-time minus the rtime, r,,, for samples that had received only the small handling dose of heparin, i.e. essentially the native samples by this time. The figures, for test doses of 2.11 u/ml and 0.35 u/ml, respectively, show the commonly-observed exponential decay of anticoagulant effect for unsheared samples. The sheared samples, however, indicate an altogether different and unexpected result. Five minutes after shearing and after heparin addition the clotting times are much shorter than those of the unsheared samples and are
show some consistent increases associated with the shear, although the changes are not really significant for this test. Far from dramatic, these tests nevertheless indicate shear-induced hypercoagulability and are consistant with the PF-3 tests. Figures 7 and 8 show the results of shearing on the effectiveness of heparin in producing prolonged clotting times, as represented by thromboelastographic rvalues. A sample was sheared, the test dose of heparin added and then aliquots from the sample tested for clotting time at sequential times. The plot shows also
7.0
concanlration 8 2OO,OOO/mms concrntrotion * 2.11u/ml 7, 8 750 dynrr/oms t, = 337 mr
6R
6D
40
3.0
2D
LC
0
0
20
40
60
60
100
120
140
Tima after addition of hrparin,minufrs
Fig. 7. Normalized thromboelastographic r-values following the addition of heparin to unsheared 0 and sheared l samples of platelet-rich plasma. r, = thromboelastographic value for an unsheared and unheparinized, i.e. native, aliquot. Platelet concentration = 200,000/mm3, heparin concentration = 2.11 u/ml, r6 = 750 dyn/cm’, tb = 337 ms, tube diameter = 305pm.
Shear-induced
activation
Plotalot Hemrin 2.5
121
of platelets
concentration * 110.000~~’ concentration = 0.35 u/ml T, = 750 dynes/cm’ t, * 644 mr
2.0
1.5
1.0
0.5
0 0
20 Time
40
60
00
of tar oddition
of
100
120
140
heporin, minutrs
Fig. 8. Normalized thromboelastographic r-value foliowing the addition of heparin to unsheared 0 and sheared l samples of platelet-rich plasma. rn = thromboelastographic value for an unsheared and unheparinized, i.e. native aliquot. Platelet concentration = 110,000/mm3, hcparin concentration = 0.35 u/ml, rb = 750 dyn/cm’, t), = 644 ms, tube diameter = 305 pm.
to the rn values, indicating almost no anticoagulant effect due to the heparin. Subsequently, however, the clotting times increase to a peak at 37 min in Fig. 7 and 25 min in Fig. 8 and then decrease along the same exponential decay curves of the unsheared samples. Additional tests were conducted to determine whether these shear-related effects on heparin potency are due to plasma trauma (e.g. protein or enzyme denaturation, especially antithrombin III, could produce such a result) or due to platelet trauma (e.g. hypercoaguiabiiity and increased availability of PF-3 as reported above). The experiments, testing samples of PRP that had been diluted with sheared PPP and comparing the results with samples diluted with unsheared PPP, indicate the sheared plasma did not produce results shown in Fig. 7 and 8. After adding 1.0 u/ml of heparin, the r-values of both samples show essentially the same exponential decay. Thus, the tests imply that shearing the platelets, probably associated with producing the hypercoaguiabiiity indicated by the PF-3 experiments, is responsible for the retarded rise and subsequent decay of anticoagulability. Finally, an auxiliary experiment was conducted to test the maximum effect of platelet components on heparin neutralization. Platelet-rich plasma with 100,000 platelets/mm3 was frozen and thawed three times prior to the addition of heparin. Aiiquots from the sample were tested for coagulability with the TEG at 4,8, 13, 24, 30 and 39 min after the addition of 0.53 units of heparin per ml and compared to tests on an unheparinized aiiquot. The r, &and a, values ail indicated little change from the unheparized values, thereby close
implying that the total release of platelet contents is sufficient to completely neutralize this concentration of heparin and that the effect is permanent. DISCUSSION
No significant platelet lysis, as indicated by the ceil counts and measurements for released acid #hasphatase, occurred in these experiments with bulk shear stresses 300 < rb < 1000 dyn/cm’ and bulk-averaged exposure times 25 < tb -C 1650 ms. Although the present experiments did not produce ceil iysis, these stresses and exposure times did induce significant sublethal trauma to the platelets. In response to exogenous ADP, the sheared platelets did not change shape as quickly or to the extent of unsheared plameiets. Additionally, the rate of platelet aggregation was depressed by the imposition of shear. A number of possible mechanisms might explain the effect of shear on the platelet response to ADP. Shearing may iniitiate release of endogenous ADP from a few of the cells. At low levels of plasma ADP, platelets are known to become refractory (see, for example, Zucker, et al. 1972) and their response to subsequent higher concentrations becomes inhibited. Shear also may alter the platelet membrane in such a way that the reactive sites for ADP (see, for example, Booyse and Rafeison. 1972) are damaged, leading to an inhibit& response to ADP. Shearing the platelets also increased the availability of PF-3, as shown in Fig. 6. Similarly, thromboeiastographic measurements indicated hypercoagulability
122
J. M. RAKSTACK, L. ZUCKERMAN and L. F. MO~KRCS
after shearing that is consistent with Glover’s (1974) observation of shortened clotting times after exposure to shear. Platelet factor 3, the coagulation accelerator, most likely is a membrane component. The increased availability of this membrane component contributes to the argument for membrane trauma induced by shear. Shearing not only caused platelets to become hypercoagulable and refractory, but also alters, in an interesting way, the ability of platelets to neutralize heparin. Instead of the usual initially very prolonged clotting time followed by an exponential decrease, the sheared samples showed an initial clotting time like that of unheparinized samples, subsequent clotting times increasing to a peak degree of anticoagulation 20-40min after the heparin addition, and finally clotting times decreasing along the exponential decay curve ofthe unsheared samples. (Note that the time for disappearance of a shear effect on heparin action is about the same length of time needed for reversibility of the shear effect on platelet response to ADP). The additional tests on unsheared PRP diluted with sheared and unsheared plasma indicate the effect is due to platelet stimulation and not plasma-factor alteration. An explanation of this phenomenon is not obvious. These samples contain procoagulant activity produced by shearing the platelets and anticoagulant activity produced by the heparin. The bell-shaped curves represent the time changes of the net effect of these two activities. The procoagulant effect of shear on unheparinized samples, as indicated by the thromboelastography, however, was relatively mild compared to the anticoagulant effect of heparin on unsheared samples, The nearly complete neutralization at short times may indicate, therefore. the strong effect of shear in releasing platelet factor 4, the antiheparin. Whatever the mechanism, the result has important implications on the use of heparin during extracorporeal bypass. The surface-to-volume ratio of the tubing was varied in a number of experiments and the results were unchanged for comparable stresses and exposure times. This suggests that shear and not interactions between the PRP and the tube inner surface, either physical or chemical, are responsible for the platelet stimulation. Figures 3,4 and 6, which represent, respectively, the extent and rate of platelet shape-change and PF-3 availabiIity,indicate that for each shear stress theslope of the curves in these graphs decrease with increased exposure times. Hung et al. (1976) noted the same decrease in rate of thrombocyte lysis with exposure time. One possible explanation of this nonlinear phenomenon is that a fraction of the platelet sample is immediately influenced by shear with the remainder more resistive and less affected, even after long exposure times. Alternatively, of course, platelet stimulation could be a nonlinear phenomenon itself, a little stress for a short time causing significant platelet stimulation and the same stress for an additional
increment of time causing relatively less stimulation. All the experiments indicating shear-induced alterations in platelet function show that the changes are dependent on both shear stress and exposure times. Thus, the effects are a function of both TVand t,. The nature of the functional dependence could be many. The data of Fig. 3,4 and 6 may be approximated by exponential or hyperbolic functions of bulk-averaged residence time. Furthermore, if the amount and rate of shape-change are subtracted from unity, all three functions could be fitted by s,, = 1 - a exp( -kt,) or, alternatively, s0 = t&k + ts), in which s,, is the observed stimulation (e.g. fractional reduction in shape change or fractional PF3 release) and k is a decay coefficient. The coefficient k, in turn, depends on the level of shear stress r, and on which test is being considered. The maximum number of stress levels used in any of these tests is three and, consequently, the functional dependence of k on K*cannot be determined with any confidence. A simple first approximation, perhaps not unreasonable for this range of stress, is that k depends linearly on T) or y& The shear-induced stimulation of a particular platelet, on the other hand, should depend on the local value of shear and local time of exposure. Assume, as the above arguments suggest, that the amount of stimulation, s, experienced by a platelet is s = 1 - exp[-Kyt],
(1)
in which K is the decay. constant for a particular platelet function, y is the local shearing strain-rate and t is the time of exposure to the shear for that platelet. (The question of whether stimulation is due to physical stress, r, or to strain rate, y, cannot be determined from these experiments, as mentioned above.) Both y and t depend on radial position for tubular flow. The measured data on a bulk sample, however, should be Sb = (2n
R [l - exp( - Kyt)] x (ur dr)lQ. s0
Letting y = r/R, substituting Poiseuille expressions for 9, t and V, and integrating by parts, this expression reduces to s, = (4KLID)
C~+Y’]~Y, (2)
in which L and D are the tube length and diameter, respectively. A closed-form integration of equation (2) is not apparent, but the results of numerical integration are shown in Fig. 9, in which sb is plotted as a function of KL/D. This curve, for all practical purposes, could be approximated, as shown on the figure, by a hyperbola or an exponential function. The two curves shown are the best-fit curves to the curve representing the integral. Thus, if the local stimulation is given by equation (l), the observed stimulation of a bulk sample may be approximated by s,, = 1 - 0.904exp[-2.41KL/D],
(3)
123
Shear-induced activation of platelets
three tests correlate quite well, whereas the f,,, data is somewhat scattered. A test for whether or not the entrance effects were minimal in these tests would be whether or not the observed data extrapolates to zero for zero residence time. The least squares curves fit to the PF3 data do indeed extrapolate to about 0.0 at 1, = 0. The corresponding curves for the shape-change and rate of shape change, however, extrapolate to about 0.1 or 0.2. This does not necessarily imply a significant entrance effect, though. As shown in Fig. 9, fitting theoretically perfect data would give an intercept of about 0.1. Charm et al. (1970b). in a study of the denaturation of fibrinogen induced by shear flow through tubes, suggested that the data be compared on the basis of product of shear rate and exposure time, yt. CombinKL/D ing the data for all levels of rb, an attempt was made to Fig. 9. The theoretically observable bulk-averaged stimuof a cupmixed sample subjected to Poiscuillc fit the shape change and PF-3 data of the present lation, -, flow and assuming the local stimulation is given by s = 1 study, using a nonlinear least squares technique, to - exp[ - Kyt]. The non-linear least-squares fit of this curve functions of the form a exp[ - br,tJ, a exp[ -bri ‘lb], to two parameter exponential function, 1 - IIexp[ - bq,tb], and a exp[ - bt,]. Table 4 gives the r2 values of these -, and to a one parameter hyperbolic function td fits. Thus, the maximum shape change and PF-3 data (k + r*). -----. are best fit by functions of ri”tb and the rate of shape change by a function of r&,. The difference between the fits to r,t, and T&, however, are small, considering in which the 0.904 and 2.41 are the result of a nonlinear the amount of data available. For simplicity further least squares fit of the exponential to equation (2) for 0 comparison is made on the basis of dependence on ?bfb 5 KL/D I 1. For Poiseuille flow r,,tb = 4(rt),,/S and or, equivalently, (rth or (yth &r)s = 16rL/3D. Thus, L/D = 15r,t,/64p and Figure 10 shows the percent of PF-3 available, 2.41KL/D = 0.565Ks,t,/F = 0.565K,r,t,, i.e. reduction in maximum shape-change,and reductkn in so = I - 0.904exp[ -0.565K1r&J, (4) rate of shape change as a function of (yth, For clarity, the t,, aggregation data was excluded from the graph in which K 1 = KJp. The data of Figs. 3,4 and 6 were because of its large scatter, but the data fell between the fitted, using a nonlinear least squares technique, to equations of the form so = 1 - Qexp[ -0.565K,t,], in shape-change and PF-3 data. Although the present .which K1 = K lob. The curves drawn on Figs. 3,4 and 6 tests did not indicate any platelet lysis at these levels of are the result of this fitting. Table 3 gives the values of (I shear and exposure times, Brown er al. (1975) and and K2 along with the correlation measures, ?, Hung et al. (1976) reported lysis with shear stresses as obtained from the analysis. Also given are the paralow as 15Odyn/cm’. Their exposure times, however, were in the order of minutes. Also plotted on Fig. 10 meters of linear fit of the t50 data. The data of the first
Table 3. Two-parameter curve tits to data on platelet stimulation. First three tests are nonlinearly fitted to oexp[-O.%SK,r,]. linearly fit to II + Kltb dy$m’
Rate of shape change Platelet factor 3 r&%%
Rate of aggregation, rsO
103 or K,
K,X
a
Maximum shape change
The ls,, data is
r2
ICQO
0.78
300
0.81
4.42 2.95
0.96 0.95
loo0 300
0.18 0.92 ’
4.53 2.23
0.93 0.95
1000 750 300
1.12 1.02 0.97
3.09 1.69 0.98
0.96 0.94 0.95
1000 750 300
0.90 1.11 1.19
3.56 0.55 0.22
.0.94 0.30 0.26
124
J. M. RAMSTACK, L.
ZUCKERMAN
and L. F.
MXKROS
Table 4. Comparison of data fits to exponential functions of r,tc r:“r, and tb for sbapechange and PF-3 data. Table gives rJ measures of fit 0
exP(- br,f*)
(Icxp(- br:‘?,)
aexp(-bt&
Medium shape-change
0.87
0.95
0.92
Rate of shape-change
0.92
0.89
0.76
PF-3 release
0.86
0.91
0.82
are the data from Hung et al. (1976) for the release of lactic dehydrogenasc induced by shear in concentric cylinder device, the data from Brown et al. (1975) for the release of acid phosphatase also induced by shear in concentric cylinder device, and the data of Charm et ui. (1970) for the percent of unclottable fibrinogen induced by shear in tube flow. Thus, fairly low values of yt will alter the ability of platelets to respond to exogenous ADP and slightly larger values increase the availability qf PF-3. These stimulations occur at (ytJb of lo’-105. Fibrinogen, on the other hand, becomes denatured at (ytk of I@-10’ and, finally, the platelets are lysed at yr values of 106-t09. The results of this investigation has application in many areas of medical practice, from drawing blood samples into syringes for clotting tests to extracorporeal circulation and artificial organs. These results, however, are based on experiments with canine platelets and some caution is in order when applying the conclusions to situations involving humans.
Differences between platelets of different species are likely and further investigative work is necessary. Acknowledgements - This work was supported by funds awarded to Northwestern University by the National Institutes of Health under Grant numbers HL 16582 and GM 00874. The authors thank Dr. Joseph Caprinifor his generous offering of laboratory facilities, Mr. Paul Vagher for his expert assistance with laboratory methods, Mr. John Mitterling for his help with the animals, and Messrs. Mark !&ntag and Steve Swan who helped with the lab tests. REFERENCES Boojse, F. A. and Rafclson, M. E. (1972) Regulation and
mechanism of phtckt aggregation. Amr. N.Y: Acad. Sci. 201, 37. Born, G. V. R. (1970) Observations on the change in shape of blood platelets brought about by adenosine diphosphate. J. J’hysiol. Z#, 487. Brown, C. H, Leverett, L. B.. Lewis, C. W., Alfrey, C. P., Hellums, J. D. (1975) Response of human platelets to shear stress. Trans. Am.Sot. crrtif.internal Organs 21,35.
loo
60
Fig. 10. The per cent stimulation as a function of the product of shearing strain-rate, y. and time subjected to the shear, t. For samples sheared in tubes, the bulk-average values of yt are used. For samples sheared between concentric cylinders, the actual yt values are used. 0 = data from measurements of maximum shape change following addition of ADP,samples sheared in tubes; q = data from measurements of the rate of shape change following addition of ADP, samples sheared in tubes; m = data from measurements of the per cent PF-3 released, samples sheared in tubes; l = data from measurements of per cent unclottable fibrinogen (Charm et al., 1970), samples sheared in tubes: A = data from measurements of LDH release (Hung et al., 1976). samples sheared between concentric cylinders ; and A = data from measurements of acid phosphatase release (Brown et al., 1975), samples sheared between concentric cylinders.
Shear-induced activation of platelets Caprini, J. A., Echenhoff, J. B., Ramstack, J. M., Zuckerman, L., Mockros, L. F. (1974) Contact activation ofheparinized plasma. Thrumb. Res. 5, 379. Chang, H. N. and Robertson, C. R. (1976) Platelet aggregation by laminar shear and brownian motion. Ann. Biomed. Enging 4, 151. Charm, S. E. and Wong. B. L. (1970(a)) Enzyme inactivation with shearing, Biotech. Bioengng 12, 1103. Charm, S. E. and Wong, B. L. (1970(b)) Shear degradation of fibrinogen in the circulation. Science 170, 466. Caller, B. S. and Zucker, M. B. (1971) Reversible decrease in platelet retention by glass bead columns (adhesiveness) induced by disturbing the blood. Proc. Sot. exp. Biol. Med. 136, 769. Glover. C. J., M&tire. L. V.. Leverett. L. B.. Hellurns. J. D.. Brown, C. H. and Natelson, E. A. (1974) ‘Effects of shear stress on clot formation. Trans. Am. Sac. ortif. internal Organs 20,463. Hung, T. C., Hochmuth, R. M.. Joist, J. H., and Sutera, S. P.
125
(1976) Shear induced aggregation and lysis of platelets. Trans. Am. Sot. art% internal Organs 22, 285. Rabiner, S. F. and Hrodek, 0. (1968) Platelet factor 3 in normal subjects and patients with renal failure. J. clin. Invest. 41, 901. Roberts, W. W., Lorand, L. and Mockros. L. F. (1973) Viscoelastic properties of fibrin clots. Biorheology 10, 29. Spaeth, E. E., Roberts, G. W., Yadwadker, S. R., Ng, P. K. and Jackson, C. M. (1973) The influence of fluid shear on the kinetics of blood coagulation reactions. Trans. Am. Sot. artif. internal Organs 19, 179. Walsh, P. N., B&s, R. and Gagnatelli, G. (1974) Platelet anitherapin activity : assay based on factor X,, inactivation by heparin and anti-fact& X,. Br. J. Haekt. 26, 405. Zucker. M. B.. R&in. P. L.. Friedbern. N. M. and Caller. B.S. (1972) Mechanisms of platelet fun:tion as revealed by the retention of platelets in glass bead columns. Ann. N.2’. Acad. Sci. 201, 138.
0021-9290’7910201-0113
J Vol 12. pi. IISI25 ci) Pergamon Press Ltd 1979 Printed m Great Bnum
SHEAR-INDUCED
W.00’0
ACTIVATION OF PLATELETS*
J. M. RAMsTAcKt, L. ZUCKERMAKand L. F. MWKROS~ Biomedical Engineering Center, Northwestern University, Evanston, IL 60201, U.S.A.
. and Research Laboratories, Evanston Hospital, Evanston, IL 60201, U.S.A. Abstract - Platelet-rich plasma was subjected to shear in Poiseuille flows through tubes. i.d. = 305,406 and
5Oftm. Bulk-average shear stresses were MO,750 and loo0 dynes/cm’ and the average residence times were 25- 1650 ms. These shears for these residence times did not produce cell lysis but did activate the platelets so that their response to exogenous ADP were reduced and pktekt factor 3 was mkased. Also, thromboelastographic measurements indicated shear-induced hypercoagulabihty. Allowing samples to stand 30-60 mins after being sheared revealed some of the indicated activation was reversibk. Adding the anticoagulant beparin to sheared samples produced an anomalous response. The quantitative results were independent of tube surface-volume ratio, but were dependent on both level of shear stress and on residence
IIVTRODUCDON The production and growth of thrombi depend upon the physical, as well as chemical, milieu. Shear has been observed to inactivate enzymes (Charm et cl., 1970(a)) and cause degradation of fibrinogen (Charm et al., 1970(b)). Roberts et al. (1973) reported that the final stiffness of a fibrin clot is affected by exposing the sample to shear during the clotting process. Spaeth et al. (1973) reported that fluid shear influences the kinetics of the conversion ofprothrombin to thrombin. The present investigation is a study of shear-induced effects on platelets in platelet-rich plasma. A number of studies have dealt with the response of platelets to shear. Collar and Zucker (1970), while studying the et&t of blood handling procedures on platelet retention by glass bead columns, found that platelet adhesion was markedly reduced by inversion in a test tube, slow centrifugation and remixing, or rapid back-and-forth transfer between two plastic or two glass syringes. Retention was restored within one hour after the latter maneuver but could be reduced again by redisturbing the blood. Chang and Robertson (1976) studied platelet aggregation by laminar shear and Brownian motion, both theoretically and experimentally, and reported a critical shear rate, about 75 set- ‘, above which the rate of aggregation becomes independent of shear rates. Glover et al. (1974) reported that clot structure can be affected by exposing platelet-rich plasma to shear for 15 mins prior to clot activation. They followed the development of the clot stiffness. i.e. the storage modulus. as polymerization * Received for publication 22 Augusr 1978. t Present address: Surgikos A. Johnson and Johnson Company, New Brunswick, NJ 08903, U.S.A. $ Reprint requests should be sent to LFM at Northwestern.
proceeded subsequent to recakification. They indicate drastic changes in clot structure if the samples are preconditioned with shear in the range from 30 to 150 dyn/cm’. In addition, shorter clot-initiation times were observed. Their results suggest that shear can activate the coagulation processes. They also indicate that platelets, after being exposed to shear, show reduced aggregability in the presence of colkgen or ADP. Brown et al. (197% using a couette device, subjected platekt-rich plasma to different levels of shear for periods of 2-24 min. Baaed on measures of cell counts, partick size distribution, EM observations, and serotonin release, they concluded that shear as low as SOdyn/cm2 stimulates the platelet rekase reaction and platelet aggregation. Stress as low as 100 dyn/cm’ produced cell lysis. as indicated by acid phosphatase release. The amount of cell iysis appeared to be linearly related to the duration of shear. At low shear, less than 100dyn/cm2, they reported that the reduced aggregobility was reversible and that if allowed to stand the platelets recovered their ability to aggregate in response to exogenous ADP. Hung et al. (1976), using a concentric cylinder device, noted about the same threshold shear, lOO-165dyn/cm’, for cell lysis. The initial rate of lysis increased with increased stress, but as exposure time lengthened the rate of lysis decmased. The present study focuses on changes in platelet physiology induced by short-term exposure to relatively high shear stress, a situation common to ail extracorporeal bypass procedures. This work includes experiments designed to correlate stress exposure to platelet damage and piatekt function. Platelet damage wasinvestigated using platelet counts and measures of plasma acid phosphatase concentration. The platelet. functions investigated were: (i) shape change and
113
114
J. M. RAMSTACK, L.
ZIJCKERMANand
aggregation in the presence of ADP, (ii) release of procoagulant platelet factor 3, PF-3, and (iii) heprin neutralization. In addition, the coagulability of platelet-rich plasma was determined for samples with and without exposure to shearing. MATERIALS AND METHODS
Sample preparation Fresh canine whole blood, collected from the jugular vein using 19G disposable needles and plastic disposable syringes, provided the working material for the study. After collection, only disposable plastic test tubes and pipettes were used for handling. Commonly accepted methodologies were used in measuring plate let shape change, platelet aggregation, plasma acid phosphatase concentration, and platelet factor 3 availability. For most experiments 72 ml of blood were drawn into a citrate anticoagulant, with nine parts of fresh whole blood to one part buffered citrate (three parts 0.1 M sodium citrate and two parts 0.1 M citric acid, pH 4.7-5.0). Four dogs were used in these experiments and the results averaged. In order to avoid the complications associated with combining anticoagulants and to make the results more applicable to in uivo situations, no citrate was used in collecting samples for experiments examining heparin-platelet interactions. Instead, these blood samples were drawn into a low dose heparin solution that permitted handling and sample preparation. This initial dose is called the handling dose. Subsequently, the teat dose of heparin was added. &cause these handling doses were small compared to the test doses and because the anticoagulant effect of heparin is known to decay rapidly, the residual effect of the handling dose on the subsequent tests, performed an hour later, was assumed to be negligible. The heparin doses reported in these tests are based only on the doses added just prior to the tests. This handling heparinization involved the collection of 50 ml of fresh whole blood into 0.2 ml of 4OU/ml of beef-lung heparin (Upjohn Company, Kalamazoo, Michigan) and allowed approximately one hour of careful handling at room temperature before the initiation of clotting or platelet clumping. Two dogs were used for these experiments. Each blood sample was centrifuged, immediately after collection, at 60-140g for 10 min to separate platelet-rich plasma, PRP, from the red cells. After removing the PRP, the remaining sample was spun at 153Og for 10min
to recover
platelet-poor
L. F. MOCKRB
relatively short periods oftime, 25-1600 ms. The entire apparatus consists of a compressed gas supply, a piston-cylinder drive unit, a feed syringe, and a tubular test section. The PRP sample, contained within the syringe, is driven down the tube by the syringe plunger. The plunger, in turn, is driven by the piston and the piston is driven by high pressure from the gas supply. Solenoid valves govern inflow and outflow of gas to and from the piston-cylinder unit. The syringe is encased in an aluminium block and consists of a 10 cc plastic disposable syringe barrel and a specially-made metal plunger. The tubular test sections were made of stainless-steel hypodermic tubing, ranging in length from 15 to 305cm. Three different diameters of tubing were used : 305,406 and 508 pm (i.e. 24.22 and 21 gauge, respectively). The testsection tubes were connected to the outlet of the syringe by means of a special fitting. This tube-syringe fitting was constructed to withstand pressure and to minimize entrance artifacts. The end of the test-section tube was lightly filed and the opening reamed. Epoxy was applied to the exterior of about 5 cm of the tube at one end. Hypodermic needles with luer-lok hubs, either 20 or 18 gauge, were slid over this proximal end of the test-section tube, leaving about 2 mm beyond the entrance to the hub, as shown in Fig. 1. This extension placed the end of the tube at the syringe apex. Air, forced through the opposite opening, blew the epoxy out and left a smooth entrance. After drying, the tube interior was siliconized according to. the instructions of the manufacturer of Siliclad, Clay Adams, Parsippani, NJ.
plasma,
PPP. The PRP and PPP portions were then mixed to synthesize samples with desired platelet concentrations, usually 3@WOO/mm3. Shear apparatus Platelet trauma was induced by the shear stresses generated in iaminar how through circular tubes. The device was chosen primarily for its capacity to generate high average shear stresses, up to 1000dyn/cmz, for
II=
Shearing t&e
id
Fig. 1. Schematic of syringe-tube fitting. This fitting served as the entrance to the uniform-bore tubes used to subject PRP samples to flow stresses.
115
Shear-induced activation of platelets One of the disadvantages of using a tubular shearing device is that all elements of the fluid are not exposed to the same shear stress nor do they all have the same transit time within the tube. The shear stress and transit time vary with radial position. The platelet trauma is correlated, therefore, with flow-weighted averages and for this study the radial distribution is assumed to be Poiseuillian. The “flow-averaged”, “cup-mixed”, or “bulk” shear stress of the exiting sample, r‘), is defined as R TUT
(2x
dr)/Q
J*0
and for Poiseuille flow is 2(pi - p,)D/lSL, in which r and D are the fluid shear stress and velocity, respectively, at a radius r, R = tube radius, D = tube diameter, Q = flow rate, L = tube length, pi = pressure at inlet, and pO = pressure at outlet. Note that rb is slightly larger than one half, or 8/15, of Tat the wall. The residence time, tr,, or bulk average time of shear exposure, is defined as R (L/u)ur
(2n
i
dr)/Q
0
and for Poiseuille flow is 32&‘/(pi p = fluid viscosity. As seen below, interest is the product of shear rate The bulk average of this variable,
- po)D2, in which another variable of and exposure time. (~t)~, is defined as
R (2x
ytur dr)/Q
s0
and for Poiseuille flow is 16L/3D, in which y = shearstrain-rate = T/F. This parameter is useful for comparing the results of this work with that previously reported by others. Note that in studies such as this, one cannot distinguish whether a measured variable is a function of r or y. Separating the dependence on Tor y requires running tests with different viscosity; changing the viscosity of the suspending medium would probably result in a changed chemical environment for the platelets and a new set of variables, however. In each experiment, using a particular tube, the driving pressure was adjusted so as to achieve a desired value for 5) and thereby also determining the value of t,,. Other values of tb for a given TVwere achieved using tubes of different lengths. The tube inlet pressure, pi, was assumed to be that in the syringe and the outlet pressure, pot was assumed to be atmospheric. Since the exit was open, the latter is true. The former assumption, however, neglects all entrance effects. This, of course, is not strictly correct, and the Ap’s used are larger than should be associated with Poiseuille viscous stresses. The error is due to (i) the conversion of pressure energy within the syringe to kinetic energy within the tube and (ii) the extra viscous stresses in the entrance due to thin boundary layers. Because of the bell-shaped entrance, the latter is probably small, however. A calculation of the former indicates an error in Ap of less than 10% in most cases, RMf2_‘---”
with a few exceptions as high as 13% in the short tubes. This means the calculated rb is higher than the true value and the error would have its largest effect on the data associated with the shorter tubes, i.e. those with short residence times. The general validity of the method, on the other hand, was tested by using the device to measure the viscosity of water. Using tubes of all three diameters, several lengths and several driving pressures, ten tests gave an average value of the viscosity of 0.89 f 0.01 cp, i.e. within 3”/,,of the handbook value at 24°C. The general procedure for shearing the platelets was as follows: About 8 ml of the PRP was poured into a fresh plastic disposable syringe and the plunger inserted. Air in the syringe was expelled and the syringe connected to the tube. Some test Ruid was manually pushed through the tube lumen to force out all air. The syringe was inserted into the aluminium block and the piston head moved to contact with the plunger head. The pressure control was set to the proper value. The bow was initiated by triggering the solenoid valve between the compressed air source and the cylinder. Steady state, as indicated by the pressure gauge, was established within a second. The PRP was gathered in a test tube at the distal end of the tube, discarding the first ml. After each use, the tubing was flushed with a milk-soap solution followed by distilled water and then air. The tubing was subsequently dried and stored in a heated incubator until used again. Platelet-lysis
tests
(i) Platelet concentrations were determined by the usual method of counting under direct phase microscopy. Besides those counts made for purposes of sample preparation, platelet concentrations weue determined before and after each shear exposure. (ii) Plasma acid phosphatase, AP, was measured utilizing the calorimetric assay developed by Boehringler Mannheim. The results are presented in terms of percent AP released, in which % AP released is defined such that AP in unsheared PPP is used to indicate 00; release and AP in the plasma of thrice frozen and thawed PRP is used to indicate lOOo/,release. A5 with the platelet counts, plasma acid phosphatase was determined both before and after exposure to shear. Platelet function
tests
(i) Measurements of platelet shape change in response to exogenous ADP, as well as measurements of platekt aggregation, were made using standard procedures and a commercial aggregometer (Bio/Data PAP-ZA, Willow Grove, Penn.). These measurements are based upon recordings of changes in light transmission through a PRP sample relative to transmission through a PPP sample after the addition of the aggregating agent (ADP). Because of a substantial voltage offset in the aggregometer output, the display of the shape change is rather small on the aggregometer chart recorder. A more manageable recording was made by connecting the aggregometer output and
116
J. M. RAMSTACK. L. ZUCKERMAN and L. F. MO~KROS
a suitable voltage source to the differential inputs of an instrumentation amplifier and the amplifier to an auxiliary recorder. The voltage source was adjusted so that the quiescent output of the amplifier was zero and the gain of the amplifier was adjusted to produce an appropriate deflection on the recorder. The methods used for determining shape-change function were similar to those of Born (1970). Ten lambda ofa 0.5 M EDTA solution (pH adjusted to 7.4 with NaOH) was added to 1 ml of PRP (concentration about 300,000/mm3). After transferring the plasma specimens to aggregomcter test tubes and inserting the tubes into the unit, O.OSmi of 2(1O)‘s M ADP was added to 0.45 ml of PRP. Two measurements were made from the shape change pattern : the slope or rate of-shape-change and the maximal amount of shape change. These measurements also were recorded for aiiquots both before and after shear exposure. (ii) Platelet aggregation in response to exogenous ADP was measured using an unmodified Bio/Data Aggregometer. Following recommended procedures, 0.05 ml of 2(10)-’ M ADP was added to 0.45 ml of PRP (platelet concentration, 300,000/mm3). Two measure ments were recorded: the final percentage of aggregation and the time, ts,,, to M”/, of this aggregation, with the latter indicating the rate of gggregation. Again, data was collected on samples with and without shear exposure. (iii) Platelet factor 3 availability, another indication of platekt stimulation, was measured by observing the decrease, below baseline, of the clotting time. Using the method of Rabiner and Hrodek (1968)O.l ml of PRP, with 50,000 platelets per mm3, was incubated with 0.1 ml of a 1.5% celite solution in veronal buffer for 2 min. After incubation, 0.1 ml of 0.03 M CaCI, was added and the clotting time recorded using the Coagulation Profiler, Bio/Data, Model CP-8. The clotting time of an unsheared PRP aliquot is used to represent the baseline, or zero percent, PF-3 availability. The clotting time of a thrice frozen-and-thawed specimen is used to represent loOY, PF-3 availability. The clotting times ofsheard samples were converted to percent PF-3 available, i.e. % PF-3 =(c.t. of unsheared minus c.t. of available sheared)/(c.t. of unsheared minus c.t. of frozen and thawed). Duplicate samples were measured and averaged. (iv) Platelets are known to have a strong ability to neutralize the effect of the anticoagulant heparin (e.g. Walsh et al., 1974). The influence of shear on this ability of platelets to neutralize heparin was measured by observing the time-changes of the thromboelastographic r-value of samples that were heparinized after being sheared and comparing the results to the time changes for unsheared samples. The lightly heparinized PRP sample was prepared, a portion sheared, the test dose of heparin added, and sequential aliquots tested for thromboelastographic r-value (a measure of clotting time) as the sample was incubated at 37°C. Another portion was left unsheared and similarly tested. To determine if the effects shown were due to plasma trauma or platelet trauma, two identical
portions of a PRP sample (797,0CtO/mm3),which had been lightly heparinized, were diluted with PPP (one part PRP to 5.75 parts PPP). The first portion was diluted with PPP plasma that had been subjected to shear of tb = 750 dyn/cm2 for t, = 337 ms ; the second portion was diluted with unsheared PPP plasma. Heparin was then added to both portions and the rvalues measured on sequential aliquots. (v) Finally, some preliminary tests were conducted to test the reversibility of shear-induced changes in platelet function. All of the above tests of changes in platelet function were conducted, except for the tests of heparin neutralization, within five minutes after exposure to shear. As an indication of reversibility, samples were allowed to stand at room temperature, 25”C, for 30 or 6Omin, post shearing exposure, prior to tests for platekt function. Coagulability rests The objective of these tests is to determine if coagulability of PRP is affected by shear. Samples of PRP were divided into two portions. One portion was subjected to shear in the tubular device and the other portion left undisturbed. Immediately after collecting the flow out of the tube, the two portions were tested for coagulability using a thromboelastograph (see, for instance, Caprini ef al., 1974). RESULl?J
All experiments, except those for reversibility and heparin neutralization, were conducted using PRP from four dogs and the data presented represents the average of the results. Figure 2(a-b) show normalized data for platelet count, acid phosphatase release, and degree of platelet aggregation following stimulation with exogenous ADP. All data is normalized with the corresponding values for unstressed samples of the same PRP and plotted as a function of bulk residence time, t, Three levels of bulk stress were examined. The results indicate no significant changes with level of stress or with time ofexposure. The scatter is within the normal experimental variations for these tests. The data on platelet counts and release ofacid phosphatase indicate the platelets were not stressed severely enough to cause cell lysis. Furthermore, the data of Fig. 2(c) indicate that the stressed platelets could aggregate, in response to ADP, to the same degree as unstressed platelets. These stress levels and stress durations, thus, did not cause severe trauma to the cells. Figures 3-6, however, indicate some platelet functions are severely affected by these exposures to stress. Figures 3 and 4 show the effect of shear stress on the ability of platelets to change shape after being stimulated by exogenous ADP. Figure 3 shows the maximum amount of shape change and Fig. 4 shows the rate of shape change. In both cases, the measures were normalized with values from unsheared samples. The results show, for each value of TV,that the amount and rate of shape change induced by ADP is markedly
Shear-induced activation of platelets
to -A
0
0.2
0.4
Bulk-Average
0.6
1.0
1.2
Time,
lb,
0.6
Residence
1.4
1.6
SeC
Fig. 2(a). Platelet counts after exposure to shear. Data is normalized with prc-exposure platelet counts. Average value I: 1.00 f 0.02 SEM. (b) Per cent ofacid phosphatase released after exposure to shear. Average value 1 2.277; f 1.20%SEM. (c) Degree of platelet aggregation induced by ADP after exposure to shear. Data is normalized by degree of aggregation for samples unexposed to shear. Average value = 1.04 & 0.02 SEM. A: T*= 1000 dynlcm’, tube id = 305pm; 0: rb = 7Rdyn/cm2; tube id = 305F; 750 dyn/cm’ ; tube id = 406 m ; V: rb = 300 dynfcm’, tube id = 305 m ; V : TV= 300dyn/cm’, .:r,= tubeid=WfJm.
lessened if the sample is exposed to shear and the degree- of reduction is dependent on the duration of exposure. The decrease for any one exposure time, on the other hand, is greater for the larger level of stress. A 50”,;,reduction in the maximum shape change occurs with exposure times of only 300 and 175 ms for stresses of 300 and loo0 dynjcn?, respectively. The compar-
Bulk - Average
able reduction in rate of shape change occufi at 5SXland 170ms. Just a few milliseconds of shear exposure causes large reductions in these platelet respnses. With longer exposure times, the additional reduction is lessened. The dependence on level of stress is more pronounced on the rate of shape change than on the amount of shape change.
Residence
Time,
t
b ’
.sec
Fig. 3. Maximum shape change of stressed platelets following the addition of 20 @vl ADP. The measured magnitude of shape change is normaiii by the measured maximum shape change occurring with unstr~& Platelets. All flows were in tubes having an id = 305 w. A: rb = 1000 dyn/cm2 ; 0: tb = 300 dyn/cm’. Solid lines are nonlinear least squares fit of data to an exponential decay. (See Discussion.)
118
J. M. RAMSTACK, L. ZUCKERMAN
00
0,s
and L. F. MOWROS
I.0
Bulk - Average
Residence
lime,
I.S
t
SSC
b’
Fig. 4. Initial rate of shape change for stressed platelets following the addition of 20 PM ADP. The measured rate of shape change is normalized by,the measured rate of shape change occurring with unstressed platelets. All flows werein tubes having an id = 305~. A: r, = fOOOdyn/crn2; V: r, = 3OOdynlcm’. Solid lines are nonlinear least squares fit of data to an exponential decay. (See Discussion.)
Figure 5 shows that the rate at which platelets aggregate, when stimulated by exogenous ADP, is markedly affected by prior exposure to shear, even though the total degree of aggregation was unaffected (Fig. 2c, above). The ratio of t5,, of the sheared samples to that of the unsheared samples is indicated as a function of shearing duration for three values of bulk stress and for three tube sixes. The data, quite scattered, indicate t,,, is approximately linearly related to t,, with the slope increasing with q. For two levels of rb, 750 and 300dyn/cm2, the surface-to-volume ratio of the test section was altered. An increase, for instance, in measured effect with an increase in surface-to-volume
ratio in’@& if rb snd tb are COnStant, that the stimulation is, at least, partly due to an interaction of platelets with the tube walls. If, on the other hand, a change of surface-to-volume ratio produces no change in the results, as is indicated in Fig. 5, the effects on the platelets can be attributed to the shear alone. Thus, the rate of platelet aggregation, after the addition of exogenous ADP, is reduced if the platelets are previously exposed to a shear stress, even though the final amount of aggregation is unaflected. Figure 6 shows the PF-3 availability in the PRP as a function of average time of shear for the three levels of bulk shear stress. The results indicate an increase in
s ._ ‘d
,” L
z 0
* 0.0
-
*.
1
-
-a.
0,s
Bulk - Average
Residence
1 * 1.0
Time,
-
s
*
10 1.5
t
b ’
set
Fig. 5. Time required for 50%of the aggregation to occur following the addition of 20 PM ADP to samples of stressed platelets. Measured time is normalized with corresponding time for samples of unstressed platelets. A:~,~lo()()d~/~~,tubCid=305~;~:~~=7750dyn/cm~,tubeid=305~;~:r,~750dyn/cm~, tubeid=406~;V:r,=U)Odyn/cm2,tubeid=U)S~;~:r,=300dyn/cm2,tubeid~K)8~.Solid lines arc least squares fit of data to be straight line. (See Discussion.)
119
Shear-induced activation of platelets
Bulk - Average
Resadence
Time
, t
b
t 9e.C
Fig. 6. Fraction of PF3 released in samples of platelet-rich plasma subjected to flow stresses. A: ?b = 1000dyn/cm2, tube id -p 305 w; 0: 750dyn/cm2, tube id = 305~; 0: Q = 750dyn/cm2, tube id = 406 pm; V: ~~ = 300 dyn/cm2, tube id = 305 m, (I : ~~= 300dyn/cm’, tube id = 508 m. Solid lines are nonlinear least squares fit of data to an exponential function. (See Discussion.)
procoagulant activity with increasing t,, and for a particular tb the percentage of PF-3 that is availabk increases with increased 7’b.The incremental changes in
PF-3 availability for incremental changes in tb monotonically decrease as tb increases. Varying the tube diameter, or surface-to-volume ratio, for two bulk shearing stresses, 750 and 300 dyn/cm2, has littk e&t on the results, again indicating the trauma is shear related and not surface related. The above results indicate that four measures of platelet functions were influenced by both shear stress and time of exposure to the shear: the rate and maximum amount of shape change subsequent to addition of exogenous ADP, rate of platelet aggregation subsequent to addition of ADP, and PF-3 availability. These effects were all measured on the PRP within five minutes after shear exposure. The results shown in Table 1 are from similar tests but on samples allowed to stand 30 or 60 min after shearing. The data indicate that the effect on the amount of
shape change is essentially reversed in less than 30 min after shear exposure. The effects on the rate-of-shape change show partial reversibility for TV = 1000 and 750 dyn/cmz and total reversibility for 300 dyn/cm’. In all three cases, though, essentially ail of the reversibility occurs in less than 30min with little additional etfect during the 30-60 min period. Measurement of tso aggregation time indicate only modest reversibility for all three stress levels. The availability of PF-3 is essentially not reversible, as might be expected, even after 60min. The data of Table 1 indicate, for all four measures of platelet function, that whatever degree of reversibility takes place it is essentially complete in less than 30 min. Table 2 indicate-s the effect of shear on the coagulability of PRP. Those samples that were more severely sheared, i.e. larger values of 7* or tb or both, consistently show a reduced thromboelastographic rvalue, akin to a reduced clotting time. The rate clot stiffening, a-value, and the final clot stiffness, a,,,-value,
Table 1. Effects of allowing samples to quiescently stand for 30-W min following shear stimulation : Data is from one dog only Maximum shape change, normalized
Rate of shape change, nonnaliied
Aggregation half-time, t 1o normal&d
Fraction of PF3 released
TV= 1000 d/cm2, tb = 0.49 s 0+ min + 30 min +6Qmin
0.23 1.00 0.85
0.28 0.79 0.85
2.25 2.01 -
0.27 0.37
T*= 750d/cm2, tb -- 0.64s 0+ min +3Omin +6Omin
0.50 1.03 1.00
0.22 0.65 0.64
1.40 1.20 1.28
0.41 0.23 0.40
~~= 3OOd/cm*, t* = 1.03 s 0+ mm +3Omin +6Omin
0.00 0.96 0.80
0.00 1.09 1.04
1:75 1.56 -
0.68 0.51 0.49
I20
J. M. RA~STACK. L. ZUCKERMANand L. F. MOCKROS Table 2. Coagulability (TEG) measurements of sheared and unsheared platelet rich plasma Platelet concentration (platelets/mm”)
110800
r-value* (mm)
a-valuet (rad)
a,-value1 (mm)
Unsheared Sheared : 750 dyn/cm2, 644 ms Unsheared Sheared: 75Odyn/cm’, 337 ms
5.0 3.0
1.43 1.45
72.5 73.5
4.0 2.5
1.40 1.43
72.0 72.5
100,000
Unsheared Sheared: 440dyn/cm*. 350 ms
4.5 3.5
1.40 1.40
72.0 74.0
100,000
Unsheared Sheared: 440 dyn/cm’, 140 ms
3.5 3.5
1.40 1.43
68.0 76.0
100,000
Unsheared Sheared: 440 dyn/cm’. 70 ms
4.0 4.0
1.26 1.38
72.0 72.0
100,000
Unsheared Sheared : 440dyn/cm’, 35 ms
5.5 5.5
1.22 1.22
72.0 72.0
200,000
l A measure of clotting time. t A m&sure of the rate of clot stifiening. $ A measure of final clot stitTness.
normalized values of the measured r-time minus the rtime, r,,, for samples that had received only the small handling dose of heparin, i.e. essentially the native samples by this time. The figures, for test doses of 2.11 u/ml and 0.35 u/ml, respectively, show the commonly-observed exponential decay of anticoagulant effect for unsheared samples. The sheared samples, however, indicate an altogether different and unexpected result. Five minutes after shearing and after heparin addition the clotting times are much shorter than those of the unsheared samples and are
show some consistent increases associated with the shear, although the changes are not really significant for this test. Far from dramatic, these tests nevertheless indicate shear-induced hypercoagulability and are consistant with the PF-3 tests. Figures 7 and 8 show the results of shearing on the effectiveness of heparin in producing prolonged clotting times, as represented by thromboelastographic rvalues. A sample was sheared, the test dose of heparin added and then aliquots from the sample tested for clotting time at sequential times. The plot shows also
7.0
concanlration 8 2OO,OOO/mms concrntrotion * 2.11u/ml 7, 8 750 dynrr/oms t, = 337 mr
6R
6D
40
3.0
2D
LC
0
0
20
40
60
60
100
120
140
Tima after addition of hrparin,minufrs
Fig. 7. Normalized thromboelastographic r-values following the addition of heparin to unsheared 0 and sheared l samples of platelet-rich plasma. r, = thromboelastographic value for an unsheared and unheparinized, i.e. native, aliquot. Platelet concentration = 200,000/mm3, heparin concentration = 2.11 u/ml, r6 = 750 dyn/cm’, tb = 337 ms, tube diameter = 305pm.
Shear-induced
activation
Plotalot Hemrin 2.5
121
of platelets
concentration * 110.000~~’ concentration = 0.35 u/ml T, = 750 dynes/cm’ t, * 644 mr
2.0
1.5
1.0
0.5
0 0
20 Time
40
60
00
of tar oddition
of
100
120
140
heporin, minutrs
Fig. 8. Normalized thromboelastographic r-value foliowing the addition of heparin to unsheared 0 and sheared l samples of platelet-rich plasma. rn = thromboelastographic value for an unsheared and unheparinized, i.e. native aliquot. Platelet concentration = 110,000/mm3, hcparin concentration = 0.35 u/ml, rb = 750 dyn/cm’, t), = 644 ms, tube diameter = 305 pm.
to the rn values, indicating almost no anticoagulant effect due to the heparin. Subsequently, however, the clotting times increase to a peak at 37 min in Fig. 7 and 25 min in Fig. 8 and then decrease along the same exponential decay curves of the unsheared samples. Additional tests were conducted to determine whether these shear-related effects on heparin potency are due to plasma trauma (e.g. protein or enzyme denaturation, especially antithrombin III, could produce such a result) or due to platelet trauma (e.g. hypercoaguiabiiity and increased availability of PF-3 as reported above). The experiments, testing samples of PRP that had been diluted with sheared PPP and comparing the results with samples diluted with unsheared PPP, indicate the sheared plasma did not produce results shown in Fig. 7 and 8. After adding 1.0 u/ml of heparin, the r-values of both samples show essentially the same exponential decay. Thus, the tests imply that shearing the platelets, probably associated with producing the hypercoaguiabiiity indicated by the PF-3 experiments, is responsible for the retarded rise and subsequent decay of anticoagulability. Finally, an auxiliary experiment was conducted to test the maximum effect of platelet components on heparin neutralization. Platelet-rich plasma with 100,000 platelets/mm3 was frozen and thawed three times prior to the addition of heparin. Aiiquots from the sample were tested for coagulability with the TEG at 4,8, 13, 24, 30 and 39 min after the addition of 0.53 units of heparin per ml and compared to tests on an unheparinized aiiquot. The r, &and a, values ail indicated little change from the unheparized values, thereby close
implying that the total release of platelet contents is sufficient to completely neutralize this concentration of heparin and that the effect is permanent. DISCUSSION
No significant platelet lysis, as indicated by the ceil counts and measurements for released acid #hasphatase, occurred in these experiments with bulk shear stresses 300 < rb < 1000 dyn/cm’ and bulk-averaged exposure times 25 < tb -C 1650 ms. Although the present experiments did not produce ceil iysis, these stresses and exposure times did induce significant sublethal trauma to the platelets. In response to exogenous ADP, the sheared platelets did not change shape as quickly or to the extent of unsheared plameiets. Additionally, the rate of platelet aggregation was depressed by the imposition of shear. A number of possible mechanisms might explain the effect of shear on the platelet response to ADP. Shearing may iniitiate release of endogenous ADP from a few of the cells. At low levels of plasma ADP, platelets are known to become refractory (see, for example, Zucker, et al. 1972) and their response to subsequent higher concentrations becomes inhibited. Shear also may alter the platelet membrane in such a way that the reactive sites for ADP (see, for example, Booyse and Rafeison. 1972) are damaged, leading to an inhibit& response to ADP. Shearing the platelets also increased the availability of PF-3, as shown in Fig. 6. Similarly, thromboeiastographic measurements indicated hypercoagulability
122
J. M. RAKSTACK, L. ZUCKERMAN and L. F. MO~KRCS
after shearing that is consistent with Glover’s (1974) observation of shortened clotting times after exposure to shear. Platelet factor 3, the coagulation accelerator, most likely is a membrane component. The increased availability of this membrane component contributes to the argument for membrane trauma induced by shear. Shearing not only caused platelets to become hypercoagulable and refractory, but also alters, in an interesting way, the ability of platelets to neutralize heparin. Instead of the usual initially very prolonged clotting time followed by an exponential decrease, the sheared samples showed an initial clotting time like that of unheparinized samples, subsequent clotting times increasing to a peak degree of anticoagulation 20-40min after the heparin addition, and finally clotting times decreasing along the exponential decay curve ofthe unsheared samples. (Note that the time for disappearance of a shear effect on heparin action is about the same length of time needed for reversibility of the shear effect on platelet response to ADP). The additional tests on unsheared PRP diluted with sheared and unsheared plasma indicate the effect is due to platelet stimulation and not plasma-factor alteration. An explanation of this phenomenon is not obvious. These samples contain procoagulant activity produced by shearing the platelets and anticoagulant activity produced by the heparin. The bell-shaped curves represent the time changes of the net effect of these two activities. The procoagulant effect of shear on unheparinized samples, as indicated by the thromboelastography, however, was relatively mild compared to the anticoagulant effect of heparin on unsheared samples, The nearly complete neutralization at short times may indicate, therefore. the strong effect of shear in releasing platelet factor 4, the antiheparin. Whatever the mechanism, the result has important implications on the use of heparin during extracorporeal bypass. The surface-to-volume ratio of the tubing was varied in a number of experiments and the results were unchanged for comparable stresses and exposure times. This suggests that shear and not interactions between the PRP and the tube inner surface, either physical or chemical, are responsible for the platelet stimulation. Figures 3,4 and 6, which represent, respectively, the extent and rate of platelet shape-change and PF-3 availabiIity,indicate that for each shear stress theslope of the curves in these graphs decrease with increased exposure times. Hung et al. (1976) noted the same decrease in rate of thrombocyte lysis with exposure time. One possible explanation of this nonlinear phenomenon is that a fraction of the platelet sample is immediately influenced by shear with the remainder more resistive and less affected, even after long exposure times. Alternatively, of course, platelet stimulation could be a nonlinear phenomenon itself, a little stress for a short time causing significant platelet stimulation and the same stress for an additional
increment of time causing relatively less stimulation. All the experiments indicating shear-induced alterations in platelet function show that the changes are dependent on both shear stress and exposure times. Thus, the effects are a function of both TVand t,. The nature of the functional dependence could be many. The data of Fig. 3,4 and 6 may be approximated by exponential or hyperbolic functions of bulk-averaged residence time. Furthermore, if the amount and rate of shape-change are subtracted from unity, all three functions could be fitted by s,, = 1 - a exp( -kt,) or, alternatively, s0 = t&k + ts), in which s,, is the observed stimulation (e.g. fractional reduction in shape change or fractional PF3 release) and k is a decay coefficient. The coefficient k, in turn, depends on the level of shear stress r, and on which test is being considered. The maximum number of stress levels used in any of these tests is three and, consequently, the functional dependence of k on K*cannot be determined with any confidence. A simple first approximation, perhaps not unreasonable for this range of stress, is that k depends linearly on T) or y& The shear-induced stimulation of a particular platelet, on the other hand, should depend on the local value of shear and local time of exposure. Assume, as the above arguments suggest, that the amount of stimulation, s, experienced by a platelet is s = 1 - exp[-Kyt],
(1)
in which K is the decay. constant for a particular platelet function, y is the local shearing strain-rate and t is the time of exposure to the shear for that platelet. (The question of whether stimulation is due to physical stress, r, or to strain rate, y, cannot be determined from these experiments, as mentioned above.) Both y and t depend on radial position for tubular flow. The measured data on a bulk sample, however, should be Sb = (2n
R [l - exp( - Kyt)] x (ur dr)lQ. s0
Letting y = r/R, substituting Poiseuille expressions for 9, t and V, and integrating by parts, this expression reduces to s, = (4KLID)
C~+Y’]~Y, (2)
in which L and D are the tube length and diameter, respectively. A closed-form integration of equation (2) is not apparent, but the results of numerical integration are shown in Fig. 9, in which sb is plotted as a function of KL/D. This curve, for all practical purposes, could be approximated, as shown on the figure, by a hyperbola or an exponential function. The two curves shown are the best-fit curves to the curve representing the integral. Thus, if the local stimulation is given by equation (l), the observed stimulation of a bulk sample may be approximated by s,, = 1 - 0.904exp[-2.41KL/D],
(3)
123
Shear-induced activation of platelets
three tests correlate quite well, whereas the f,,, data is somewhat scattered. A test for whether or not the entrance effects were minimal in these tests would be whether or not the observed data extrapolates to zero for zero residence time. The least squares curves fit to the PF3 data do indeed extrapolate to about 0.0 at 1, = 0. The corresponding curves for the shape-change and rate of shape change, however, extrapolate to about 0.1 or 0.2. This does not necessarily imply a significant entrance effect, though. As shown in Fig. 9, fitting theoretically perfect data would give an intercept of about 0.1. Charm et al. (1970b). in a study of the denaturation of fibrinogen induced by shear flow through tubes, suggested that the data be compared on the basis of product of shear rate and exposure time, yt. CombinKL/D ing the data for all levels of rb, an attempt was made to Fig. 9. The theoretically observable bulk-averaged stimuof a cupmixed sample subjected to Poiscuillc fit the shape change and PF-3 data of the present lation, -, flow and assuming the local stimulation is given by s = 1 study, using a nonlinear least squares technique, to - exp[ - Kyt]. The non-linear least-squares fit of this curve functions of the form a exp[ - br,tJ, a exp[ -bri ‘lb], to two parameter exponential function, 1 - IIexp[ - bq,tb], and a exp[ - bt,]. Table 4 gives the r2 values of these -, and to a one parameter hyperbolic function td fits. Thus, the maximum shape change and PF-3 data (k + r*). -----. are best fit by functions of ri”tb and the rate of shape change by a function of r&,. The difference between the fits to r,t, and T&, however, are small, considering in which the 0.904 and 2.41 are the result of a nonlinear the amount of data available. For simplicity further least squares fit of the exponential to equation (2) for 0 comparison is made on the basis of dependence on ?bfb 5 KL/D I 1. For Poiseuille flow r,,tb = 4(rt),,/S and or, equivalently, (rth or (yth &r)s = 16rL/3D. Thus, L/D = 15r,t,/64p and Figure 10 shows the percent of PF-3 available, 2.41KL/D = 0.565Ks,t,/F = 0.565K,r,t,, i.e. reduction in maximum shape-change,and reductkn in so = I - 0.904exp[ -0.565K1r&J, (4) rate of shape change as a function of (yth, For clarity, the t,, aggregation data was excluded from the graph in which K 1 = KJp. The data of Figs. 3,4 and 6 were because of its large scatter, but the data fell between the fitted, using a nonlinear least squares technique, to equations of the form so = 1 - Qexp[ -0.565K,t,], in shape-change and PF-3 data. Although the present .which K1 = K lob. The curves drawn on Figs. 3,4 and 6 tests did not indicate any platelet lysis at these levels of are the result of this fitting. Table 3 gives the values of (I shear and exposure times, Brown er al. (1975) and and K2 along with the correlation measures, ?, Hung et al. (1976) reported lysis with shear stresses as obtained from the analysis. Also given are the paralow as 15Odyn/cm’. Their exposure times, however, were in the order of minutes. Also plotted on Fig. 10 meters of linear fit of the t50 data. The data of the first
Table 3. Two-parameter curve tits to data on platelet stimulation. First three tests are nonlinearly fitted to oexp[-O.%SK,r,]. linearly fit to II + Kltb dy$m’
Rate of shape change Platelet factor 3 r&%%
Rate of aggregation, rsO
103 or K,
K,X
a
Maximum shape change
The ls,, data is
r2
ICQO
0.78
300
0.81
4.42 2.95
0.96 0.95
loo0 300
0.18 0.92 ’
4.53 2.23
0.93 0.95
1000 750 300
1.12 1.02 0.97
3.09 1.69 0.98
0.96 0.94 0.95
1000 750 300
0.90 1.11 1.19
3.56 0.55 0.22
.0.94 0.30 0.26
124
J. M. RAMSTACK, L.
ZUCKERMAN
and L. F.
MXKROS
Table 4. Comparison of data fits to exponential functions of r,tc r:“r, and tb for sbapechange and PF-3 data. Table gives rJ measures of fit 0
exP(- br,f*)
(Icxp(- br:‘?,)
aexp(-bt&
Medium shape-change
0.87
0.95
0.92
Rate of shape-change
0.92
0.89
0.76
PF-3 release
0.86
0.91
0.82
are the data from Hung et al. (1976) for the release of lactic dehydrogenasc induced by shear in concentric cylinder device, the data from Brown et al. (1975) for the release of acid phosphatase also induced by shear in concentric cylinder device, and the data of Charm et ui. (1970) for the percent of unclottable fibrinogen induced by shear in tube flow. Thus, fairly low values of yt will alter the ability of platelets to respond to exogenous ADP and slightly larger values increase the availability qf PF-3. These stimulations occur at (ytJb of lo’-105. Fibrinogen, on the other hand, becomes denatured at (ytk of I@-10’ and, finally, the platelets are lysed at yr values of 106-t09. The results of this investigation has application in many areas of medical practice, from drawing blood samples into syringes for clotting tests to extracorporeal circulation and artificial organs. These results, however, are based on experiments with canine platelets and some caution is in order when applying the conclusions to situations involving humans.
Differences between platelets of different species are likely and further investigative work is necessary. Acknowledgements - This work was supported by funds awarded to Northwestern University by the National Institutes of Health under Grant numbers HL 16582 and GM 00874. The authors thank Dr. Joseph Caprinifor his generous offering of laboratory facilities, Mr. Paul Vagher for his expert assistance with laboratory methods, Mr. John Mitterling for his help with the animals, and Messrs. Mark !&ntag and Steve Swan who helped with the lab tests. REFERENCES Boojse, F. A. and Rafclson, M. E. (1972) Regulation and
mechanism of phtckt aggregation. Amr. N.Y: Acad. Sci. 201, 37. Born, G. V. R. (1970) Observations on the change in shape of blood platelets brought about by adenosine diphosphate. J. J’hysiol. Z#, 487. Brown, C. H, Leverett, L. B.. Lewis, C. W., Alfrey, C. P., Hellums, J. D. (1975) Response of human platelets to shear stress. Trans. Am.Sot. crrtif.internal Organs 21,35.
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Fig. 10. The per cent stimulation as a function of the product of shearing strain-rate, y. and time subjected to the shear, t. For samples sheared in tubes, the bulk-average values of yt are used. For samples sheared between concentric cylinders, the actual yt values are used. 0 = data from measurements of maximum shape change following addition of ADP,samples sheared in tubes; q = data from measurements of the rate of shape change following addition of ADP, samples sheared in tubes; m = data from measurements of the per cent PF-3 released, samples sheared in tubes; l = data from measurements of per cent unclottable fibrinogen (Charm et al., 1970), samples sheared in tubes: A = data from measurements of LDH release (Hung et al., 1976). samples sheared between concentric cylinders ; and A = data from measurements of acid phosphatase release (Brown et al., 1975), samples sheared between concentric cylinders.
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(1976) Shear induced aggregation and lysis of platelets. Trans. Am. Sot. art% internal Organs 22, 285. Rabiner, S. F. and Hrodek, 0. (1968) Platelet factor 3 in normal subjects and patients with renal failure. J. clin. Invest. 41, 901. Roberts, W. W., Lorand, L. and Mockros. L. F. (1973) Viscoelastic properties of fibrin clots. Biorheology 10, 29. Spaeth, E. E., Roberts, G. W., Yadwadker, S. R., Ng, P. K. and Jackson, C. M. (1973) The influence of fluid shear on the kinetics of blood coagulation reactions. Trans. Am. Sot. artif. internal Organs 19, 179. Walsh, P. N., B&s, R. and Gagnatelli, G. (1974) Platelet anitherapin activity : assay based on factor X,, inactivation by heparin and anti-fact& X,. Br. J. Haekt. 26, 405. Zucker. M. B.. R&in. P. L.. Friedbern. N. M. and Caller. B.S. (1972) Mechanisms of platelet fun:tion as revealed by the retention of platelets in glass bead columns. Ann. N.2’. Acad. Sci. 201, 138.