Langmuir 2002, 18, 39-45
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Aggregation Mechanism of Blood Platelets Studied by the Time-Resolved Light Scattering Method Katsumi Yabusaki† and Etsuo Kokufuta* Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Received June 12, 2001. In Final Form: September 20, 2001 A laser light-scattering aggregometer was applied to study epinephrine-induced aggregation of human blood platelets. We improved the analytical software for the aggregometer; thus, it has become feasible to use a time-resolved light scattering method (TRLSM) based on Mie’s theory, allowing one to monitor changes in both size and particle concentration as a function of time. By combination of TRLSM with microscopic analysis, it was found that there is a critical condition at which almost all the single platelets aggregate with one another to result in small size aggregates (AGS), at 2 min after addition of epinephrine (1.5 ( 0.5 µM). The resulting AGS was spherical in shape, 15 µm in diameter (as a number-averaged value), and it was composed of about 1900 single platelets closely packed. After the formation of AGS particles went essentially to completion, they aggregated with one another to result in large particles (AGL), with an accompanying decrease in the AGS concentration. The AGL formed is not spherical but consists of irregularly bound AGS particles. The formation of AGL was inhibited upon the addition of aspirin, while there was no critical condition at which the AGL with a characteristic particle size is formed. These physical aspects were discussed in connection with biochemical mechanism for epinephrineinduced platelet aggregation in the presence and the absence of aspirin as the inhibitor.
Introduction Blood platelets play an important role in the formation of thrombi (i.e., thrombosis), through which bleeding is stopped. Thrombosis is regarded as an aggregation process of the platelets induced by biomolecules (i.e., agonist) such as thrombin,1-3 adenosine diphosphate (ADP),4-7 collagen,8,9 and epinephrine.10-12 A failure in thrombosis causes a variety of vascular diseases; thus, the monitoring of an aggregating activity of platelets provides key information in both diagnosis and medication. Measurements of turbidity,13-15 occasionally by a combination of other measurement techniques such microscopic analysis, have been employed to monitor the process of platelet aggregation. It has become apparent that blood * To whom correspondence should be addressed. † Permanent address: Kowa Research Institute, Kowa Co. Ltd., Kannondai, Tsukuba, Ibaraki 305-0856, Japan. (1) Vu, T.-K.; Humg, D. T.; Wheaton, V. I.; Coughlin, S. R. Cell 1991, 64, 1057. (2) Huang, R.-S.; Sorisky, A.; Chrch, W. R.; Simons, E. R.; Rittenhouse, S. E. J. Biol. Chem. 1991, 266, 18435. (3) Lefkowitz, R. J. Nature 1991, 351, 353. (4) Mills, D. C. Thromb. Haemostasis 1996, 76, 835. (5) Daniel, J. L.; Dangelmaier, C.; Jin, J.; Ashby, B.; Smith, J. B.; Kunapuli, S. P. J. Biol. Chem. 1998, 273, 2024. (6) Jantzen, H. M.; Gousset, L.; Bhaskar, V.; Vincent, D.; Tai, A.; Reynolds, E. E.; Conley, P. B. Thromb. Haemostasis 1999, 81, 111. (7) Hollopeter, G.; Jantzen, H. M.; Vincent, D.; Li, G.; England, L.; Ramakrishnan, V.; Yang, R. B.; Nurden, P.; Nurden, A.; Julius, D.; Conley, P. B. Nature 2001, 409, 202. (8) Nieuwenhuis, H. K.; Akkerman, J. W.; Houdijk, W. P.; Sixima, J. J. Nature 1985, 318, 470. (9) Kehrel, B.; Wierwille, S.; Clemetson, K. J.; Anders, O.; Steiner, M.; Knight, C. G.; Farndale, R. W.; Okuma, M.; Barnes, M. J. Blood 1998, 91, 491. (10) Motulsky, H. J.; Insel, P. A. N. Engl. J. Med. 1982, 307, 18. (11) Kobilka, B. K.; Matsui, H.; Kobilka, T. S.; Yang-Feng, T. L.; Francke, U.; Garon, M. G.; Lefkowitz, R. J.; Regan, J. W. Science 1987, 238, 650. (12) Spalding, A.; Vaitkevicius, H.; Dill, S.; MacKenzie, S.; Schmaier, A.; Lockette, W. Hypertension 1998, 31, 603. (13) Born, G. V. R. Nature 1962, 194, 927. (14) Frojmovic, M. M.; Milton, J. G.; Duchastel, A. J. Lab. Clin. Med. 1983, 101, 964. (15) Thompson, N. T.; Scrutton, M. C.; Wallis, R. B. Thomb. Res. 1986, 41, 615.
platelets exhibit different aggregation profiles depending on the species of agonist; for example, in epinephrineinduced aggregation there are two stages in which single platelets (monomer) yield small aggregates (AGS) and large aggregates (AGL).16 Several studies10-12,17-20 have focused on the biochemical aspects for the aggregation of platelets by epinephrine. From turbidity measurements, however, it is not possible to learn about changes in the number and the size of platelet aggregates. In addition, the turbidimetric method is not readily applied to study AGS, especially at low particle concentrations. These would be the main reasons the study of platelet aggregation has not yet progressed in the interdisciplinary area of biochemistry and colloid science. Recently, a laser light-scattering aggregometer (Kowa PA-100) was developed with the intention of diagnostic uses,16,21-25 where particular attention was paid to the sensitivity for detecting a very low concentration of AGS at the initial stage of the platelet aggregation process. The number concentration of aggregated particles was given solely as counts of scattering signals/time, while an attempt was made to estimate the particle size from scattering intensity on the basis of Mie’s theory.26 In these (16) Ozaki, Y.; Sato, K.; Yatomi, Y.; Yamamoto, T.; Shirasawa. Y.; Kume, S. Anal. Biochem. 1994, 218, 284. (17) Bennett, J. S.; Vilaire, G.; Burch, J. W. J. Clin. Invest. 1981, 68, 981. (18) Harris, D. N.; Greenberg, R.; Phillips, M. B.; Michel, I. M.; Goldenberg, H. J.; Haslanger, M. F.; Steinbacher, T. E. Eur. J. Pharmacol. 1984, 103, 9. (19) Oliver, J. A.; Albrecht, R. M. Scanning Microsc. 1987, 1, 745. (20) Lalau Keraly, C.; Kinlough-Rathbone, R. L.; Packham, M. A.; Suzuki, H.; Mustard, J. F. Thromb. Haemostasis 1988, 60, 209. (21) Satoh, K.; Ozaki, Y.; Qi, R.-M.; Yang, L.-B.; Asazuma, N.; Yatomi, Y.; Kume, S. Thomb. Res. 1996, 81, 515. (22) Tohgi, H.; Takahashi, H.; Watanabe, K.; Kuki, H.; Shirasawa, Y. Thromb. Haemostasis 1996, 75, 838. (23) Qi, R.-M.; Ozaki, Y.; Satoh, K.; Yang, L.-B.; Asazuma, N.; Yatomi, Y.; Kume, S. J. Cardiovasc. Pharmacol. 1996, 28, 215. (24) Eto, K.; Takeshita, S.; Ochiai, M.; Ozaki, Y.; Sato, T.; Isshiki, T. Cardiovasc. Res. 1998, 40, 223. (25) Nomura, S.; Tandon, N. N.; Nakamura, T.; Kambayashi, J. Haemostasis 2000, 30, 174. (26) Mie, G. Ann. Phys. 1908, 25, 377.
10.1021/la010879b CCC: $22.00 © 2002 American Chemical Society Published on Web 12/08/2001
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Figure 1. Outline of laser-light scattering aggregometer (a), scheme for light scattering in a measuring cuvette (b), and example of output signals from a pair of adjacent photodiodes (c). The output signal consists of an ac component (due to large particles such as aggregated platelets) and a dc component (due to small particles such as single platelets). A broken line in (c) indicates a threshold voltage to eliminate the dc signal. Actually, this elimination was performed with a preamplifier in which the output signals from both photodiodes were subtracted from each other.
studies,21-25 thus, a quantitative description for timedependent platelet aggregation with respect to the size and the number concentration of aggregates had not yet been successful. As a result, the previous laser lightscattering aggregometer is excellent in diagnostic and biomedical uses, but for use in a quantitative study of the platelet aggregation there is still great room for improvement in the way of analyzing scattering data. In this work, we have improved the analytical software for the aggregometer, to estimate both size and number concentration of aggregated platelets as a function of time. This improvement made it possible for us to employ a time-resolved light scattering method (TRLSM) in monitoring the time course of platelet aggregation. Then we focused on the epinephrine-induced aggregation system, for which we have some comparative information as follows: (i) A few microscopic studies19,20 have demonstrated a morphological change of platelets, i.e., discoid into spherical form. (ii) It has been predicted that such spherical particles would aggregate with one another to result in AGS; after that AGL would be formed via either of further aggregation of the monomers or aggregation of AGS.16 (iii) In the presence of aspirin the AGL formation did not take place.21 Since AGS and AGL may be observed with a microscope, TRLSM seems to bring its ability into full play in a quantitative study of the platelet aggregation with the aid of microscopic analysis. Our goal in this study is to provide a detailed mechanism of epinephrine-induced platelet aggregation from both physical and biochemical standpoints. Experimental Section Materials. Blood from healthy donor was mixed with an aqueous trisodium citrate solution (3.8%; anticoagulant; Wako Pure Chemical Co., Osaka, Japan) at a ratio of blood:anticoagulant ) 9:1 by volume. The mixture was then subject to centrifugation (800g; 10 min) by which we obtained two aqueous layers,
i.e., a yellowish upper layer containing platelets as the desired constituent and a red bottom layer containing red blood cells. The upper layer was collected and used as the platelet-rich plasma (PRP). The concentration of platelets was 1.5 × 108 particles/ mL, as estimated by microscopic analysis. Epinephrine and aspirin (acetylsalicylic acid), respectively used as an inducer and inhibitor of platelet aggregation, were obtained from Sigma Chem. Co. Specimens (i.e., PRP including platelets and their aggregates) for microscopic observations were obtained by fixation with formaldehyde (4%, Wako Pure Chemical Co., Osaka, Japan), followed by mitochondrial staining with a fluorophore (1 µM DiOC(6)3, Molecular Probes); both procedures were continued for 10 min at room temperature. Apparatus. A laser light-scattering aggregometer (Kowa PA-100, Kowa Co., Nagoya, Japan) was employed with newly improved analytical computer software. Figure 1 shows the outline of our instrument. A fine laser beam with wavelength (λ) of 675 nm from a 20 mW laser diode (Toshiba, Tokyo, Japan) was passed through a lens system (LS1) to focus it on a point, the position of which was adjusted to locate ca. 1 mm from the inner surface of a cylindrical glass cuvette to avoid multiple scattering. The scattered light from particles in a small volume27 (ca 10-4 µL) around the focal point was passed through another lens system (LS2) and detected by a 2-channel photodiode array. The optic axis of the LS2 was fixed at 90° to the axis of laser light path, but the detection of scattering light through LS2 was made over a range of 90 ( 23° due to the following reason: According to Mie’s theory for large particles characterized by the size parameter (πd/λ, where d is diameter) more than 2-5, the angular distribution of the scattered light intensity exhibits an irregular oscillation, but the mean of a series of the intensities at different scattering angles converges at a certain value which is proportional to the particle size. The output signals from a photodiode consist of an alternating current (ac) component due to aggregated platelets and a direct current (dc) component due to nonaggregated single platelets; thus, a pair of the photodiodes was used to subtract the dc component. After this subtraction, the output (27) This is commonly referred to as an observation volume16 and an instrument constant determinable via a calibration with a sample whose size and concentration are known.
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Figure 2. Typical calibration curve for determining particle concentration (CN) from peak counts (N). The results were obtained using PSL with a diameter of 10 µm. All the data points were well fitted by log N ) log CN - 2.52 (r2 ) 0.993). Thus, we obtained that k ∼ 3.0 × 10-3 in eq 1. Note that our instruments need counting peaks for 10 s to determine CN with a full precision. signals in voltage are digitized to 2048 steps by an analog/digital (A/D) converter. As a result, we may record the scattered light intensity as a function of time (t) (see Figure 1). Measurements. PRP (300 µL) was admitted into a glass cuvette, together with a magnetic stirring bar. The cuvette was then placed in a holder with a temperature controlling device and incubated at 37 °C with stirring at 1000 rpm. Data for background corrections were recorded for 30 s before addition of epinephrine to initiate the aggregation throughout all the measurements. When the inhibitory effect of aspirin was studied, PRP was incubated for 10 min in the presence of aspirin. After that, the measurements were carried out as mentioned above.
Results Calibrations. The number concentration of particles is directly proportional to the peak counts per fixed time (see Figure 1). As mentioned in the Experimental Section, however, the observation volume is an instrument constant. Therefore, we made a careful calibration using authentic samples with various concentrations of poly(styrene) latexes (PSL) with different sizes. As can be seen from Figure 2, there is a good linear relation between the peak counts (N in counts/10 s) and particle concentration (CN in particles/mL). Thus we obtained the following empirical formula:
CN ) kN
(1)
Here k (∼3.0 × 10-3) is an instrument constant relating to the observation volume. We may estimate the diameter (d in µm) of a particle from the scattering intensity (I) of a peak. Since the size parameter for a platelet is about 10, we must consider Mie’s theory for estimating d from I. At present, several faster Mie’s algorithms have been reported;28-30 thus, we may calculate a relation between I and d. As can be seen from Figure 3a, however, I at a fixed θ is not linear against d. This causes the errors inherent in the use of Mie’s theory for estimating the size for a large particle (πd/λ > 5). To overcome this disadvantage, we attempted to employ the mean (Ih) of a series of I values I1, I2, ..., In which are determined at different θs; that is, n
I ) (1/n)
Ii ∑ i)1
(2)
where n is the replication number of intensity measure(28) Lentz, W. J. Appl. Opt. 1976, 15, 668. (29) Wiscombe, W. J. Appl. Opt. 1980, 19, 1505.
Figure 3. Calculated results of scattering light intensities (SLI): (a) SLI denotes I at θ ) 90°, the value of which was calculated as a function of d according to ref 30, using refractive indexes of medium (µ1; 1.33) and particle (µ2; 1.59) for PSL. (b) SLI denotes hI by eq 2 for PSL with d ) 10 µm; in the calculation, n times of the random sampling of θ were performed in the range of 67° to 113°. (c) SLI denotes hI∞, which was approximated by hI at n ) 10 000. In (c), L and U respectively denote the lower limit (10 µm) and upper limit (70 µm), within which a good linear relation between hI∞ and d was obtained. Table 1. Comparison of Scattering Light Intensities Observed and Calculated by Eq 3 for Different Authentic Samples intensity × 103 (mV) sample
d (µm)
obsd
calcda,b
PSL(1)c
5.1 10.4 20.3 25.0 30.2 41.7 7.0
0.65 2.7 7.2 13.0 16.6 29.0 0.12
0.69 2.6 7.3 12.6 16.1 29.5 0.11
PSL(2) PSL(3) PSL(4) PSL(5) PSL(6) red cellc
a Results are given by h I by n ) 10 000 (instead of hI∞; see Figure 3) through a conversion of arbitrary unit into mV using a relation 2 of hI∞ ) k′d , where k′ (0.669 mV/µm) is an instrument constant. bRefractive indexes of medium (µ ) and particle (µ ) for the 1 2 calculation were µ1 ) 1.33 and µ2 ) 1.59 for PSL(1) to PSL(6); µ1 c ) 1.35 and µ2 ) 1.40 for red cell. Used as a reference sample although its size is lower than the lower limit (10 µm).
ments at different θs for the particle with a fixed size. Then, we randomly choose θ in a range of θ ) 90 ( 23° and calculated hI by eq 2 for a particle with d ) 10 µm as an example. From Figure 3b, it is clear that hI converges at a value (Ih∞) when increasing n. As a result, the calculations of hI∞ values as a function of d gave a good linear relationship at a limited range of d (i.e., 10 µm < d < 70 µm) (see Figure 3c), the relation of which can be given by
I∞ ) d2
(3)
In our measurements the scattering intensities at θ ) 90 ( 23° were detected through LS2 (see Figure 1). Therefore, the measured intensity would be equivalent to hI∞ in eq 3 and proportional to d2. To confirm this prediction, the experimental data for several PSL samples with known
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Figure 4. Changes in diameter (d) and number concentration (CN) with time (t) for platelet aggregation induced by epinephrine. Epinephrine concentrations were given in parentheses of each three-dimensional graph.
sizes were compared with the results of calculations by eq 3 (see Table 1). There is a good agreement between the experimental and theoretical d values. This clearly indicates that the sizes of particles with d values from 10 µm (lower limit) to 70 µm (upper limit) can be estimated by eq 3. Time Sequential Changes in Size Distribution of Platelet Aggregates. By uses of eqs 1 and 3, we may estimate both concentration and size of platelet aggregates as a function of time. Figures 4 and 5 show the results by TRLSM for the epinephrine-induced platelet aggregation in the absence and the presence of aspirin, respectively. It should be noted that in our measurements the lower limit of d is 10 µm and the upper limit is 70 µm. Also note that we must count peaks for 10 s in determining particle concentration. Thus, each of the data points on the x axis as well as on y axis in the three-dimensional diagram was given at 10 s intervals and connected with a straight line. Several interesting aspects were observed in platelet aggregation with epinephrine in the absence and the presence of aspirin: (i) Small aggregates (AGS) whose sizes are in a range of d < 20 µm are formed at a low dose (0.3 µM) of epinephrine. (ii) An increase in the epinephrine dose causes a increase in the size as well as the concentration of the aggregates, meaning the formation of large aggregates (AGL) with d > 30 µm. (iii) At a dose level (3 µM) of epinephrine, this trend becomes more clear with the elapse of time. (iv) At a very high epinephrine dose (10 µm), the AGL formation accompanies a decrease in the concentration of AGS. (v) The aggregation of platelets is depressed by aspirin (see Figure 5). (vi) In particular, aspirin inhibits the formation of AGL (d > 30 µm) but not of AGS (d < 20 µm); thus, the reverse change is observable in the diagrams when increasing the aspirin concentration (30) van de Hulst, H. C. Light Scattering by Small Particles; Dover Publications: New York, 1981; pp 114-130.
and the epinephrine concentration (compare Figures 4 with Figure 5). Microscopic Observations. Figure 6 shows typical photographs obtained via microscopic observations at different stages of epinephrine-induced platelet aggregation. The small aggregates (indicated by arrowhead) were observed in whole stages for the platelet aggregation with epinephrine, while large particles (by asterisk) appeared at high epinephrine doses (>3 µM). The nonaggregated free platelets as the monomer cannot be seen under this magnification. Detailed microscopic analyses showed that the presence of epinephrine at different dose levels turns discoid platelets (with a diameter of 2 µm) into spherical particles (with a diameter of 1.2 µm). The remaining concentration of nonaggregated free platelets after the addition of epinephrine was ca. 3.0 × 107 particles/mL, the value of which was independent of epinephrine concentration and was held constant at all the stages of the microscopic observation. Because our PRP sample contained 1.5 × 108 particles/mL of monomer platelets, this result means that about 80% of the monomers were consumed in forming AGS and/or AGL. Microscopic analysis permits us to determine the size and the concentration of AGS as well as of AGL; however, it is actually impossible to make the analysis as a function of time at different dose levels of epinephrine and aspirin. Thus, the microscopic analysis was made at fixed conditions, i.e., at 2 min after the addition of 1 µM epinephrine for AGS and at 4 min after the addition of 10 µM epinephrine for AGL. Then we obtained the following data: diameter ∼ 15 ( 6 µm and total concentration ∼ 5.2 × 105 particles/mL for AGS; total concentration ∼ 7.9 × 104 particles/mL for AGL. (Note that in counting the number of the aggregates with a microscope, the size distribution was not considered.) The size of AGL was not
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be understand as an interparticle cross-linking among single platelets via the binding of fibrinogen molecules to GPIIb/IIIa on the plasma membrane. However, another biochemical factor has been pointed out on the basis of the following facts: (i) Thromboxane A2 (TXA2), a metabolite from arachidonic acid generated by endogenous cyclooxygenase in the platelet, plays an important role in the platelet aggregation.17,18,34,35 (ii) Aspirin has a selective inhibitory action on cyclooxygenase activity.36 (iii) In the presence of aspirin the epinephrineinduced AGL formation was inhibited. Taking this into account, we may say that AGL is not formed via the aggregation of single platelets but via the binding of several AGS particles, the processes of which may be expressed as
Figure 5. Inhibitory effect of aspirin on platelet aggregation induced by 10 µM epinephrine. The concentration of aspirin was given in parentheses of each three-dimensional graph.
expressed numerically due to its irregularity. Nevertheless, we see a good agreement between the results from microscopic observations (Figure 6) and TRLSM (see Figures 4 and 5). Discussion Biochemical Understanding of Epinephrine-Induced Platelet Aggregation in the Absence and the Presence of Aspirin. Epinephrine (i.e., adrenaline) is a neurotransmitter appearing in the sympathetic nervous system. The circulating concentration of epinephrine in blood rises when the sympathetic nervous system has been activated.31 Then, epinephrine molecules bind to R2adrenergic receptors on the plasma membrane of a platelet. This biochemical change activates the fibrinogen binding site composed of a complex of glycoproteins GPIIb and GPIIIa, the complex of which is usually abbreviated as GPIIb/IIIa.32,33 Thus, the overall aggregation process may (31) Yamaguchi, N.; de Champlain, J.; Nadeau, R. Circ. Res. 1975, 36, 662. (32) Plow, E. F.; Ginsberg, M. H. Prog. Hemostasis Thromb. 1989, 9, 117. (33) Newman, P. J.; Seligsohn, U.; Lyman, S.; Coller, B. S. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 3160.
RM f MR
(4a)
βMR f (MR)β
(4b)
where MR represents AGS formed via aggregation of R single platelets (as monomer M) and (MR)β is AGL formed from β particles of AGS. Although a few studies37-41 have focused on the reason the AGS formation induces an increase in TXA2 concentration to further activate the fibrinogen-binding GPIIb/IIIa complex, the biochemical understanding for the AGL formation has not yet been completed. Nevertheless, our TRLSM experiments strongly support the twostage aggregation mechanism given by eqs 4a and 4b, since (i) aggregation of AGS accompanies a decrease in its concentration and (ii) this process is inhibited by addition of aspirin. This is supported by the results of the microscopic analysis; that is, (i) the remaining concentration of free platelets is almost unchanged in both AGS and AGL formation processes and (ii) the observed AGS is spherical in shape, while AGL consists of irregularly bound spherical particles. Examination of Aggregates by Use of NumberAveraged Particle Diameter. Our data in Figures 4 and 5 are useful for analyzing the time-dependent platelet aggregation process with respect to the size and the concentration. However, it is preferable to use an index from which the size distribution at a fixed time can be calculated. Each distribution pattern in Figures 4 and 5 seems to resemble the χ2 distribution rather than the Gaussian distribution; the former is often used in characterizing the molecular-weight distributions for many macromolecular systems. Thus, we attempted to estimate a number-averaged particle diameter (d h ) using
d ) Σdini/Σni
(5)
Here, di and ni denote the diameter and the number of ith particle, respectively. The information about the sizes and (34) Houslay, M. D.; Bojanic, D.; Wilson, A. Biochem. J. 1986, 234, 737. (35) Hirata, M.; Hayashi, Y.; Ushikubi, F.; Yokota, Y.; Kageyama, R.; Nakanishi, S.; Narumiya, S. Nature 1991, 349, 617. (36) Burch, J. W.; Stanford, N.; Majerus, P. W. J. Clin. Invest. 1978, 61, 314. (37) Lipfert, L.; Haimovich, B.; Schaller, M. D.; Cobb, B. S.; Parsons, J. T.; Brugge, J. S. J. Cell Biol. 1992, 119, 905. (38) Yamaguchi, K.; Nomura, S.; Kido, H.; Kawakatsu, T.; Fukuroi, T.; Suzuki, M.; Hamamoto, K.; Yanabu, M.; Kokawa, T.; Yasunaga, K. Am. J. Hematol. 1993, 44, 106. (39) Clark, E. A.; Shattil, S. J.; Ginsberg, M. H.; Bolen, J.; Brugge, J. S. J. Biol. Chem. 1994, 269, 28859. (40) Shattil, S. J.; Kashiwagi, H.; Pampori, N. Blood 1998, 91, 2645. (41) Paul, B. Z.; Jin, J.; Kunapuli, S. P. J. Biol. Chem. 1999, 274, 29108.
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Figure 6. Microscopic photographs of typical aggregation processes of platelets induced by epinephrine: (a) at 2 min after addition of 1 µM epinephrine; (b) at 4 min after addition of 10 µM epinephrine. Further details were given in text.
min after addition of epinephrine. Also found from Figure 7 is that d h ∼ 15 µm for this size. These strongly suggest the two-stage aggregation mechanism in which AGL results from AGS. Structures of AGS and AGL. It would be interesting to discuss the morphological structure of AGS as well as of AGL on the basis of the data from TRLSM and to compare it with microscopically observed results. For this purpose, we focused on a fractal dimension, in particular on the dimension (D) of an object which can be divided into arbitrary small NS pieces, each of which is a small replica of the entire set and is scaled down by a factor of r from the whole. Then D is defined as NsrD, where D ) 1 for length, D ) 2 for area, and D ) 3 for volume. When the object is a sphere with diameter d1 and consists of small spheres with diameter d2, Ns is given by
Ns ) (d1/d2)3
(6)
For a spherical object containing x small spheres, eq 6 becomes
Ns ) x/(1 - f)
Figure 7. Changes in number-averaged particle diameter (d h) with time (t) for epinephrine-induced platelet aggregation in the absence (a) and the presence (b) of aspirin. Numbers in parentheses for graphs a and b show the concentrations of epinephrine and aspirin, respectively. To study the inhibitory effect of aspirin, the aggregation was induced by 10 µM epinephrine.
the concentrations of all the particles in a system should be required for estimating d h by eq 5, while in our TRLSM there were the lower and the upper limit for measurable particle sizes. Nevertheless, we calculated d h by eq 5 from data in Figures 4 and 5, because the microscopic analysis showed that the contents of particles in the ranges d > 70 µm and d < 10 µm were less than 5%. It seems that neglecting this would not cause a serious error for our present purpose, which is to make clear the two-stage aggregation mechanism. Figure 7 shows the time dependence of d h as a function of epinephrine concentration in the absence and the presence of aspirin. We may see a critical concentration of epinephrine to yield the AGS with a maximum size. From the curves in the absence and the presence of different dose levels of aspirin, it is found that such a characteristic AGS particle results from a limited epinephrine concentration ranging from 1 to 3 µM and at 2
(7)
Here, f denotes the “defective” fraction of the small spheres in the object, and at the closest packing f should be zero. h ) 15 µm, we may assume that x ∼ R in For AGS with d eq 4a; thus, its value (1850) is obtained by dividing the concentration (1.2 × 108 particles/mL) of single platelets consumed in forming AGS by the concentration (6.5 × 104 particles/mL) of the resulting AGS. When d1 is taken as 15 µm (i.e., d h ) and d2 as 1.2 µm, the combination of eqs 6 and 7 gives f ) 0.05, indicating that the AGS is physically regarded as a spherical particle into which many of single platelet spheres are closely packed. This is consistent with the results obtained by microscopic analysis. Similarly we studied the structure of AGL, but for this we focused on an aggregate formed at 4 min after addition of 10 µM epinephrine. The reason is that there is no convergent value in the d h vs time curves in Figure 7, even at a very high epinephrine dose (10 µM). Under such a fixed condition, we obtained d h ∼ 28 µm and concentration ∼ 1.6 × 104 particles/mL. From these data, x (equivalent to β of eq 4b) becomes 4, so that f ) 0.39, meaning that an AGL is formed via aggregation of a few AGS particles spherical in shape. For AGL, therefore, an agreement between results by TRLSM and by microscopy was also obtained. As a result, we were able to look at physical aspects for AGS formation from single platelets as well as for AGL formation from AGS, both processes of which have been
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Figure 8. Epinephrine-induced aggregation process of platelets given by schematic drawings and microscopic photographs. The photographs of AGS and AGL were taken from Figure 6, while that of activated platelet was obtained from the same sample as shown in Figure 6 but at a more high magnification. The photograph of intact platelet was obtained before the addition of epinephrine.
expressed by eqs 4a and 4b. Taking this into account, the overall process of epinephrine-induced platelet aggregation can be understood via the schematic drawing in Figure 8, in which several of the biochemical aspects were also considered.
existence. With the aid of a microscope, TRLSM provides significant data, from which we may look at several of physical aspects in colloid interactions. When biocolloids such as blood platelets were studied by TRLSM, we could discuss quantitatively their interaction mechanism from both physical and biochemical standpoints.
Conclusions This study has demonstrated that TRLSM based on Mie’s theory is useful for examining a system in which microscopically observable large colloid particles are in
Acknowledgment. Financial support from Kowa Co. Ltd. to this project is gratefully acknowledged. LA010879B
