Abstract
Shear stress is known to induce platelet activation and aggregation. The red blood cell (RBC) aggregation test requires the application of shear stress for the cells to disaggregate for initialization. We tested the hypothesis that applying shear stress may activate platelets, which can influence RBC aggregation. The present study used a commercial microchip-based aggregometer (RheoSCan-AnD300) with a rotating stirrer for RBC disaggregation. Whole blood samples were exposed to different magnitudes of shear stress with various shearing times. As the rotational speed was increased up to 2800 rpm, the RBC aggregation index (AI) of the whole blood increased by up to 30% (p < 0.05), whereas that of the platelet-excluded blood samples did not show any apparent alteration. The AI also increased in proportion with the stirring time. The data suggest that high shear stress affects RBC aggregation through shear-induced platelet aggregation.
Introduction
The measurement of red blood cell (RBC) aggregation must be accompanied by RBC disaggregation from the application of high shear stress or shear rates regardless of the shearing geometry [2, 14]. For instance, conventional aggregometers have been used to apply high shear rates of as much as 5000 s–1 under the initial settings [4]. If the blood viscosity is assumed to be 3.5 mPa-s at a high shear rate, then the shear stress can be calculated to be about 17.5 Pa. For RBC aggregation measurements, there has been little attention paid to whether high shear stress affects shear-induced platelet activation (SIPA) and alters the aggregation index (AI).
The shear stress is a strong factor for platelet activation compared to other agonists such as ADP, collagen, and fibrinogen. SIPA occurs with a shear stress of greater than 8 Pa, which is high enough to induce platelet activation and aggregation [9, 16]. With high shear stress, platelets are easily activated even for a short exposure time of a few seconds. A shear-induced platelet function analyzer (PFA-100, Siemens, Germany) has been developed and is widely used for clinical diagnosis [5, 7]. Thus, the application of shear stress to disaggregate RBC aggregates should be carefully examined for its potential to affect platelet activation and alter RBC aggregation. In the present study, we hypothesized that a disaggregating shear stress can affect platelet activation and subsequently alter RBC aggregation during stasis. We examined RBC aggregation by varying the applied shear stress.
Materials and methods
Preparation of blood samples
Venous blood was drawn from healthy donors through a 21-gauge butterfly infusion set into two sodium citrate tubes (BD, Franklin Lakes, NJ, USA) as the anticoagulant. To prepare the negative blood sample (i.e., not including platelets), whole blood from one of the two sodium citrate tubes was centrifuged at 800× g for 12 min (UNIEQUIP, Germany), and the buffy coat was removed. The plasma was collected and placed back into a tube containing the negative blood sample in order to make the hematocrit the same as that of the whole blood sample. All measurements were completed within 4 h after the blood samples were collected. All measurements were performed according to the new guidelines for hemorheological measurements provided by the ISCH [1].
Erythrocyte aggregation
The aggregation of RBCs was measured with a light-transmission aggregometer (Rheoscan-AnD 300, RheoMeditech, Seoul, Korea). The microchip (C-01, Rheomeditech, Seoul, Korea) consisted of a flat cylindrical test chamber, sample inlet, air outlet, and stirrer. The dimensions of the test chamber were a 4-mm diameter and 0.3-mm height, whereas those of the stirrer were a 3.4-mm length and 0.15-mm diameter. In order to change the applied shear stress, the rotational speed of the stirrer was varied to 1000, 1800, 2800, and 3800 rpm. The stirring time was set to 10, 30, 60, 120, and 180 s.
As the stirrer rotated, the blood samples rapidly disaggregated and were exposed to the shear force. After the stirrer rotation suddenly stopped, the RBCs in the whole blood tended to aggregate rapidly during the first 10 s. The measurement of the RBC aggregation was completed in 120 s. When expressed as an AI, the RBC aggregation is defined as the ratio of the area below the syllectogram (i.e., plot of transmitted light intensity versus time) to the total area over a 120-s time period; it indicates the normalized degree of accumulated aggregation (see Fig. 1). Further information on RBC aggregation measurements is described elsewhere [14].
Here, the measurement data are reported as the mean ± standard deviation (SD). Statistical testing was performed by using analysis of variance (ANOVA), and the statistical significance was represented with p-values. We considered the results to be statistically significant when p < 0.05. The present study was performed in accordance with the ethical guidelines of the journal Clinical Hemorheology and Microcirculation.
Determination of shear stress
When the stirrer rotated, the shear rate was non-uniform throughout the chamber. The theoretical shear rate at the tip of the stirrer was calculated based on parallel-disk rheometry [6]. The formula is as follows:
Results
In order to elucidate the effect of platelet activation and aggregation on RBC aggregation, we investigated syllectograms (i.e., light intensity vs. time curves) for different rotational speeds and times. To amplify the effect of stirring, we extended the stirring time up to 180 s at different rotational speeds (1000–3800 rpm).
Effect of rotational speed on RBC aggregation
Figure 2A shows the syllectogram of the negative blood samples. In the stirring phase, the transmitted light intensity increased with the rotational speed of the stirrer. The variation in the light intensity may be associated with the RBC deformability. Depending on the applied shear stress, the RBCs showed different degrees of deformation, which resulted in different light intensities. A higher deformation of the RBCs resulted in a higher transmitted light intensity. After the stirrer was suddenly stopped, the light intensity curves of the four different rotational speeds became coincident over 120 s. The negative samples did not show any difference as the stirring speed was increased up to 2800 rpm. However, the syllectograms of the whole blood, which included platelets, showed somewhat different results from those of the negative samples, as shown in Fig. 2B. Note that minimum for each curve was not exactly matched each other. These results shown in Fig. 2A and B were further analyzed with conventional aggregation indices: the AI, M-index, half-time (t1/2), M-index, and amplitude (Amp). These are given in Tables 1 and 2.
For the negative samples given in Table 1, none of the aggregation indices showed any apparent alteration as the rotational speed was increased up to 2800 rpm. When the stirrer speed reached 3800 rpm, the aggregation indices then showed apparent increases (p < 0.05). Note that the AI increased with the stirring speed, whereas the M-index decreased. In general, the AI and M-index show the same trends. The opposing trends of the AI and M-index in Table 1 were due to the alteration of Amp. Recall that the AI is similar to the ratio of M to Amp. As given in Table 1, the decrease in Amp was larger than that in the M-index. Subsequently, the AI increased with the stirring speed. At 3800 rpm, all aggregation indices showed apparent alterations from their values at 1000 rpm, even though the platelets were removed from the blood.
Table 2 gives the AI results for the whole blood samples, which included platelets. Note that all four aggregation indices showed significant alterations at 2800 rpm rather than at 3800 rpm. In fact, the M-index showed the first apparent alteration even at 1800 rpm (p = 0.01). The AI increased with the rotational speed and reached its maximum at 2800 pm. Further increasing the stirring speed resulted in a slight decrease of the AI at 3800 rpm. Similar to the negative samples, the M-index decreased with the rotational speed, whereas Amp decreased further. Even though the half-time decreased as the stirring speed was increased, even up to 3800 rpm, it did not show any significant changes.
Effect of stirring time on RBC aggregation
As discussed in the previous section, we examined the factors that affect RBC aggregation due to shearing at rotational speeds of 1000–3800 rpm over a fixed stirring time of 180 s. In our recent report [15], we found that 180 s maximizes SIPA at 2800 rpm. These results were consistent with those of a previous report [7]. Now, the effect of the shearing time at a fixed shear stress needed to be determined. Figure 3 compares the AI values with different shearing times at two given rotational speeds (1000 and 1800 rpm). The shearing durations were set to be 10, 30, 60, and 120 s.
For a relatively low shear (1000 rpm), the AI values gradually increased with the shearing time but did not show significant changes up to 60 s. However, even for low shear, shearing for 120 s resulted in an apparent difference in the AI (38.5) compared to shearing for 10 s. Furthermore, the maximum value of the AI (39.7) was reached at 180 s. For the rotational speed of 1800 rpm, the AI value rapidly increased with the shearing time and reached its maximum at 60 s. Further increasing the shearing time caused a slight decrease in the AI at 120 and 180 s, but the value was still significantly higher than the AI at 10 s.
Discussion
A high shear stress is known to be able to activate platelets and cause them to aggregate [10]. A previous study reported that the threshold value of the shear stress to avoid platelet activation is 8 Pa [11]. In conventional aggregometers, however, the shear stress is applied with an approximate range of 15–20 Pa, which is much greater than the threshold shear stress to activate platelets. In the present study, we found that the RBC aggregation indices are significantly dependent on the applied shear stress and shearing time. These alterations to the RBC aggregation indices were minimized with negative samples that excluded platelets. Therefore, shear-dependent RBC aggregation is caused by SIPA rather than RBCs.
Of course, a shear stress of 5–20 Pa may trigger the active regulation of RBC deformability associated with calcium ions [8]. In addition, slight alterations to the RBC deformability have been used to alter RBC aggregation [18]. The negative samples that excluded platelets showed a slight increase in AI with the shear stress, as listed in Table 1. Negative sample experiments were designed to exclude the SIPA effect on RBC aggregation. Thus, the increasing trend of the AI with shear for negative samples can be directly associated with shear-dependent RBC rheology. Ulker et al. [17] reported that a mechanical shear stress can activate functional nitric oxide (NO) synthesizing mechanisms in RBCs and that the export of NO from RBCs is enhanced by mechanical stress. Meram et al. [8] clearly demonstrated that the shear stress increases RBC deformability. However, the shear-dependent RBC aggregation in negative samples was not observed at moderate shear stresses (4–12 Pa) but at high shear stresses (15 Pa).
The whole blood sample including platelets showed shear-dependent results for the RBC aggregation indices, as shown in Fig. 3. As discussed above, the shear dependence of RBC aggregation is mainly caused by shear-activated platelets, not by the RBCs themselves. Because most conventional aggregometers are used with whole blood for disaggregating shear stress, the magnitude of the shear stress and the shearing time should be carefully selected. As shown in Fig. 3, even at low shear stresses (1000 rpm or 4 Pa), a long duration (t≥120 s) of shearing would cause significant changes to the RBC aggregation. Of course, applying a high shear stress (1800 rpm or 7 Pa) caused significant changes to the RBC aggregation within a short period of time (t≥60 s). Commercial devices for the platelet function (PFA-200, Siemens) have adopted a very short shearing time to activate platelets with a pressure-driven capillary flow system. The wall shear stress in a capillary have been estimated to be about 10–15 Pa. Nesbitt at al. [10] comprehensively demonstrated platelet activation and aggregation at high shear rates (≥20,000 s–1) in stenosed arteries. Thus, no disaggregating shear stress should be greater than 8 Pa, and the shearing time should be efficiently short to avoid any platelet activation.
Footnotes
Acknowledgments
This study was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (No. HI14C0670).