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Corresponding author at: Research and Development Department, Central Blood Institute, Japanese Red Cross Society, Tatsumi 2-1-67, Koto-ku, Tokyo, 135-8521, Japan.
The thrombus-forming ability is a critical in vitro parameter to assess platelets (PLTs), but flow-based methods using collagen-coated materials generally require multistep, proficiency, and advanced analysis.
Study design and methods
Commercially available collagen-coated bead columns were examined to assess thrombus-forming ability of PLTs. The retention rate as an index of thrombus formation was calculated using the PLT count before and after column passage. Thrombi were imaged by anti-CD41 using a fluorescent microscope. PLT aggregation was measured by light-transmitting aggregometry.
Results
The retention rate was low when apheresis-collected PLT concentrates (PCs) were suspended in plasma either with or without Ca2+. Reconstitution of PCs with red blood cells (RBCs) increased the retention rate with good reproducibility on repeated-measurements, and therefore, PLT samples were reconstructed with RBCs in subsequent experiments. The retention rate of PCs varied widely in a product-dependent manner, and was correlated with the aggregation rate induced by ADP, but not that by collagen. Using platelet-rich-plasma, antagonists of P2Y1 or P2Y12 receptors for ADP reduced both the retention and aggregation of PLTs. Acetylsalicylic acid reduced retention, although it had no effect on ADP-induced aggregation. Prostaglandin E1 significantly inhibited both retention and aggregation. These anti-PLT reagents resulted in reduced or no thrombus formation on the beads.
Conclusion
The collagen-coated bead column was useful to readily examine the thrombus-forming ability of PLTs. Variance of the PLT retention rate was correlated with responsiveness to ADP. Results from anti-PLT reagents revealed that thrombus formation on collagen-coated beads was similar to in vivo thrombus development.
Platelets (PLTs) play critical roles in hemostasis and thrombosis at injured vessel walls by forming thrombi to arrest extravascular bleeding. PLT transfusion has been practiced prophylactically for patients with hemostatic disorders or actively to treat patients with hemorrhagic conditions. Therefore, it is important to evaluate PLT functionality of blood products as an index of hemostatic and thrombotic abilities.
The quality of PLT products for transfusion has been an issue to evaluate preparation methods (buffy coat-derived vs. apheresis) [
Functional characteristics of apheresis-derived platelets treated with ultraviolet light combined with either amotosalen-HCl (S-59) or riboflavin (vitamin B2) for pathogen-reduction.
In vitro effects on platelets irradiated with short-wave ultraviolet light without any additional photoactive reagent using the THERAFLEX UV-Platelets method.
]. The functionality of PLTs was commonly examined by light-transmitting aggregometry under the condition of PLT suspension. On the other hand, some studies addressed PLT functionality to observe adhesion or thrombus formation on collagen-coated materials as a contact phase under flow or static conditions [
Cold storage of platelets in platelet additive solution: an in vitro comparison of two Food and Drug Administration-approved collection and storage systems.
Ultraviolet C light pathogen inactivation treatment of platelet concentrates preserves integrin activation but affects thrombus formation kinetics on collagen in vitro.
]. However, flow-based methods using collagen-coated materials generally require multistep, proficiency, and advanced analyses. Some methods also require fluorescent labeling of PLTs prior to examination, and hence, in addition to the handling, an artifactual effect due to the labeling process cannot be completely ruled out.
In contrast to those methods, the assay using commercially available collagen-coated bead columns is a simple and rapid procedure to assess thrombotic ability of PLTs in clinical settings [
]. The administration of acetylsalicylic acid (ASA) and P2Y12 (ADP recepter) antagonist is a current standard of anti-PLT therapy for patients with cardiovascular disease [
]. The ability of PLT thrombus formation is assessed by measuring PLT counts of samples before and after column passage followed by calculation of the PLT retention rate. The collagen-coated bead column method was originally adapted for whole blood. It was shown that the existence of red blood cells (RBCs), but not neutrophils nor monocytes, was essential for PLT retention [
]. In addition, the bead size was reported to affect the shear stress under flow conditions: small-sized bead columns for high shear stress or conventional columns for low shear stress [
]. Nevertheless, small-sized bead columns are no longer commercially available.
In this study, we explored the use of conventional collagen-coated bead columns to assess the thrombus-forming ability of PLTs in apheresis products for transfusion. In addition, we investigated factors correlated with and affecting thrombus formation on collagen-coated beads.
2. Materials and methods
2.1 Reagents and antibodies
ADP and acetylsalicylic acid [ASA: cyclooxygenase (COX) inhibitor] were purchased from Sigma-Aldrich (St. Louis, MO, USA); prostaglandin E1 (PGE1: adenylyl cyclase activator) was from Cayman Chemical (Ann Arbor, IL, USA); MRS2500 (MRS: P2Y1 receptor antagonist) and AR-C66096 (ARC: P2Y12 receptor antagonist) were from Tocris Bioscience (Bristol, UK); Collagen from the equine tendon (Collagenreagent Horm) was from Takeda Austria (Linz, Austria); anti-CD41 Alexa Fluor 594-conjugate (anti-CD41-A594) was from R&D Systems (Minneapolis, MN, USA).
2.2 Blood cell preparation
The study was approved by the institutional research boards of the Japanese Red Cross Society and informed consent was obtained from blood donors. Leuko-reduced apheresis PLT concentrates (PCs) were collected by CCS (MCS+) (Haemonetics Japan, Tokyo, Japan) or Trima (Terumo BCT, Tokyo, Japan) with adenine-citrate-dextrose (ACD) as an anticoagulant followed by X-ray irradiation. To obtain red blood cells (RBCs, type O), whole blood was passed through a leukocyte reduction filter, plasma was removed by centrifugation, and then resulting cells were suspended in mannitol-adenine-phosphate solution [
] followed by X-ray irradiation. Prior to the experiments, RBCs were washed twice with phosphate-buffered saline without calcium and magnesium [PBS (–)] by centrifugation at 1900×g for 10 min at 4 °C. Plasma (type AB) was prepared by centrifugation of PCs at 1900×g for 15 min at 24 °C twice and stored at −30 °C until use. Those blood products were obtained from the Kanto-Koshinetsu Block Blood Center of Japanese Red Cross Society.
To evaluate factors affecting thrombus formation and aggregation, platelet-rich-plasma (PRP: PLTs, 4.9–6.8 × 108/mL; leukocytes, 0.2–2.8 × 105/mL) was prepared from whole blood with ACD (10:1) by centrifugation at 200×g for 15 min at 22 °C as an upper layer.
2.3 Collagen-coated bead column assay
The conventional collagen-coated bead column consists of a polyvinyl tube (12 cm in length) filled with plastic beads (approximately 500 μm in diameter) coated with porcine type I collagen (PURA BEADS COLUMN; ISK, Tokyo, Japan). PLT samples (2.3–2.8 × 108/mL, 2 mL) were prepared by diluting PCs with plasma (type AB) in the presence or absence of Ca2+ (3.3 mM). Alternatively, PLT samples (2 mL) were prepared by diluting PCs with plasma and adding 1 mL of RBCs (type O), which resulted in an approximately 40% hematocrit. PLT samples were prepared at the time of each measurement, not from a single master mix. PLT samples in a 2.5 m L-syringe were incubated at 37 °C for 10 min and then passed through a collagen-coated bead column using a syringe pump (placed in a 37 °C incubator) at a constant speed of 0.4 mL/min. The flow rate was determined using whole blood to obtain an approximately 50% PLT retention rate, which enabled the assessment of an enhanced or reduced thrombus-forming ability. The passed PLT samples were continuously collected in a tube. PLTs before and after column passage were counted using an automated hematology analyzer (XS-800i; Sysmex, Kobe, Japan). The PLT retention rate (%), i.e., adhesion and thrombus formation, was obtained by the following equation: [(PLT count before passage) – (PLT count after passage)]/(PLT count before passage)×100.
To evaluate factors affecting thrombus formation, PLT samples (2.2–2.5 × 108/mL) consisting of 1 mL of PRP and 1 mL of RBCs were incubated with either vehicle [PBS (–)], MRS (10 μM), ARC (10 μM), ASA (0.5 mM), or PGE1 (10 μM) for 10 min at 37 °C prior to column passage.
2.4 Platelet aggregation
PLTs in plasma (3 × 108/mL) supplemented with CaCl2 (3.3 mM) were incubated at 37 °C with stirring at 1000 rpm in a light-transmitting aggregometer (HemaTracer 313 M, TAIYO Instruments, Osaka, Japan). After 1.5-min incubation, collagen (2.5 μg/mL) or ADP (10 μM) was added, and then transmittance was recorded for 7 min. Light transmittance at the maximum level was defined as the platelet aggregation rate.
To evaluate factors affecting aggregation, PRP (3 × 108/mL) diluted with plasma was incubated with either vehicle [PBS (–)], MRS (10 μM), ARC (10 μM), ASA (0.5 mM), or PGE1 (10 μM) for 3 min at 37 °C, and then stimulated by ADP (10 μM).
2.5 Fluorescence imaging of PLTs on collagen-coated beads
After the passage of PLT samples, the column tube was cut and the beads were extruded by lysing and fixing (1% formaldehyde) solution (BD Biosciences, San Jose, CA, USA). After 10-min hemolysis and fixation, the beads were washed with and suspended in Tris-buffered saline containing 0.05% Tween 20 (TBS-T). PLTs were probed for 15 min with anti-CD41-A594. After washing with TBS-T, autofluorescence from collagen and elastin was quenched using the TrueView quenching kit (Vector Laboratories, CA, USA). Fluorescence images were obtained using a fluorescence-image microscope with the Z-stack and sectioning mode (BZ-X700, KEYENCE, Tokyo, Japan).
2.6 Statistical analysis
The correlation between retention and aggregation rates was analyzed using Prism 7 (GraphPad, La Jolla, caCA. USA). Differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. A p-value of less than 0.05 was considered significant.
3. Results
3.1 Reproducibility of repeated measurements of PLT retention rate
Three PCs [PC1 (day 2), PC2 (day 3), and PC3 (day 2)] were passed through the collagen-coated bead columns for ten repeated measurements at the indicated compositions (Fig.1 and Table 1). In the absence of RBCs, small amounts of PLTs of PC1 were retained in the collagen-coated bead columns regardless of the presence of Ca2+. When PLT samples from PC1 were prepared with RBCs, the retention rate increased to 65%, indicating that RBCs were critical for PLT retention in the collagen-coated bead columns. PLT retention was also observed using PC2 and PC3 with better reproducibility in the presence of RBCs. Therefore, PLT samples for the collagen-coated bead columns were prepared by mixing PCs or PRP with RBCs at a hematocrit of 40% in the subsequent experiments.
Fig. 1PLT retention rate of ten-times repeated measurements by collagen-coated bead columns. One PLT sample (3 × 108/mL, PC1) was prepared with plasma in the presence or absence of Ca2+, or with plasma and RBCs. Two other PLT samples (PC2 and PC3) were prepared with plasma and RBCs. PLT sample passage through the collagen-coated bead column was repeated ten times.
3.2 Correlation of inter-product variance between retention rate and aggregability
As seen in PC1, 2, and 3 (Fig.1 and Table 1), the retention rate varied by products and probably by storage days. To explore factors affecting inter-product variance, PCs (day 1, n = 40) underwent both collagen-coated bead column passage and light-transmitting aggregometry. The retention rate varied from 4.4 to 80.4% in Fig. 2A and from 8.3 to 80.4% in Fig. 2B (20 out of 40 PCs overlapped between A and B). The retention rate was significantly correlated with ADP aggregation (p = 0.003; Fig. 2B), but not with collagen (p = 0.117; Fig. 2A).
Fig. 2Relationship between PLT retention rate and PLT aggregation stimulated by collagen or ADP. (A) PCs underwent collagen-coated bead column passage and light-transmitting aggregometry using collagen (2.5 μg/mL) or ADP (10 μM) as the stimulus (n = 30). (B) PLTs on the collagen-coated beads showing three kinds of retention rate (RtR) were probed with anti-CD41-AF594 and observed using a fluorescence-image microscope at ×10 magnification. Scale bar: 500 μm.
Typical fluorescence images of PLTs on the collagen-coated beads are shown in Fig. 2C. According to the increase of the retention rate, the PLT thrombi became larger and stacking of PLTs also became evident on the beads.
3.3 Effects of anti-PLT reagents on PLT thrombus formation and ADP-induced aggregation
The retention rate of PRP (day 0) in the presence of vehicle was 57.6 ± 7.1% (mean ± SD, n = 5, range = 49.1-67.0%; Fig. 3A), and large thrombi with PLT stacking were evident on the collagen-coated beads (Fig. 3B). ADP receptor antagonists, MRS and ARC, significantly reduced the retention rate to 20.7 and 25.8%, respectively, with smaller PLT thrombi (Fig. 3A and B). The retention rate was further reduced to 15.1% by the combination of MRS and ARC. ASA and PGE1 also reduced the retention rate. Although PGE1 showed a 12.8% retention rate, no thrombi were detected on the beads (Fig. 3B).
Fig. 3Factors affecting thrombus formation on collagen-coated beads. (A) PLT samples (PRP) containing RBCs and either vehicle, MRS (10 μM), ARC (10 μM), MRS + ARC (10 μM each), ASA (0.5 mM), or PGE1 (10 μM) were passed through the collagen-coated bead columns. Mean ± SD, n = 5. Asterisks above error bars indicate significance compared with the vehicle. *p < 0.05, **p < 0.01. (B) Beads after passage of PLT samples were probed with anti-CD41-AF594. Typical images of beads obtained by a fluorescence-image microscope at ×20 magnification are presented. Scale bar: 50 μm.
The ADP-induced aggregation of PRP (day 0) in the presence of vehicle was 75.4 ± 4.6% (mean ± SD, n = 5; Fig. 4A). The ADP receptor antagonists MRS and ARC significantly reduced the ADP-induced aggregation to 34.8 and 24.2% (Fig. 4A), respectively, with quite different traces of aggregation curves (Fig. 4B). MRS, an antagonist of the P2Y1 receptor, attenuated the aggregation with sustained aggregate formation, while ARC, an antagonist of the P2Y12 receptor, attenuated the aggregation, but the aggregates were subsequently disaggregated. In contrast to PLT thrombus formation, the combination of MRS and ARC markedly inhibited ADP-induced aggregation (Fig. 4A and B). Furthermore, ASA had no effect on ADP-induced aggregation, while PGE1 significantly inhibited the aggregation response.
Fig. 4Factors affecting PLT aggregation induced by ADP. PLT (PRP) aggregation induced by ADP (10 μM) was measured by light-transmitting aggregometry. (A) PLT aggregation in the presence of the indicated reagents: vehicle, MRS (10 μM), ARC (10 μM), MRS + ARC (10 μM each), ASA (0.5 mM), or PGE1 (10 μM). Mean ± SD, n = 5. Asterisks above error bars indicate significance compared with the vehicle. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Typical traces of the aggregation curves in the presence of the indicated reagents.
In this study, we tested a convenient method to monitor thrombus formation by PLT products using a commercially available device, the collagen-coated bead column. In accordance with a previous study [
], it was reconfirmed that thrombus formation by PLTs required the coexistence of RBCs (Fig. 1). RBCs would rheologically promote PLT homing to the surface of beads. In addition, it was suggested that RBCs contribute to PLT activation by generating thrombin through their membrane phosphatidylserine (PS) [
]. Therefore, it is likely that PLTs attached to collagen-coated beads through glycoprotein VI (GPVI) were activated by an outside-in signal mediated by GPVI-collagen interaction and/or by thrombin generated through PS-exposing RBCs to form a p-selectin-positive thrombus core [
]. Recently, direct interaction between PLTs and RBCs through FasL/FasR was shown to be important in thrombus formation by facilitating PS exposure on RBCs [
Because the retention rate of PCs on the collagen-coated beads varied widely depending on products, the variance was initially postulated to correlate with the responsiveness of PLTs to collagen. However, no correlation of the retention rate was observed with the aggregation response to collagen (Fig. 2A), but it was significantly correlated with ADP aggregation (Fig. 2B). As shown in Fig. 2C, PLT thrombi became bigger and not wider than the adhesion area in accordance with an increasing retention rate. These findings were explained by the model generated by the previous study that PLTs were stacked and thrombi were grown on an initially generated PLT core in response to TXA2 and ADP released from stacked PLTs [
]. Namely, the growth of thrombi was controlled by the responsiveness of PLTs to ADP. As shown in Fig. 3, thrombus formation was attenuated by the block of ADP signaling (MRS, ARC, and MRS + ARC) and TXA2 generation (ASA). This observation was in good agreement with the previous in vivo study showing that the administration of cangrelor (P2Y12 antagonist) and/or ASA resulted in smaller thrombus formation compared with a vehicle group involving mice with laser-injured vessel walls [
]. These findings suggest that the collagen-coated bead column method reflects a similar mechanism to that observed in vivo. However, the difference between a small retention rate (Fig. 3A) and no thrombus formation (Fig. 3B) in PGE1 should be clarified in further experiments.
The retention rate widely varied in PCs (n = 40) from 4.4 to 80.4% (Fig. 2A and B), while PRP (n = 5) showed a small variance from 49.1 to 67.0% (Fig. 3). This difference might be due to the preparation method: PCs were collected by apheresis machines, while PRP were prepared by gentle centrifugation once, and hence, PLTs in PCs would be physiologically modulated with regard to responsiveness to ADP in addition to individual differences.
In contrast to the collagen-coated bead column assay, anti-PLT reagents quite differently affected ADP-induced aggregation (Fig. 4). The combination of P2Y1 (MRS) and P2Y12 (ARC) antagonists synergistically inhibited the aggregation completely, although COX inhibitor (ASA) showed no effect on the aggregability, indicating that ADP-induced aggregation did not require TXA2 production and its outside-in signaling. This inconsistency might be due to the difference of the assay system: the collagen-coated bead column method relied on contact with PLTs, affected by other outside-in signals in addition to the existence of RBCs, while the light-transmitting aggregometry was carried out under PLT suspension conditions without RBCs and the aggregation reaction was evoked solely by ADP.
In conclusion, we revealed that the difference in the thrombus-forming ability of PCs on collagen-coated beads was correlated with PLT responsiveness to ADP. Thrombus formation on the beads depended on not only signaling downstream of ADP receptors but also TXA2 production. This method will be useful to assess the functionality of PLT products as a similar model of in vivo thrombosis from the additional point of view of evidence supporting anti-PLT therapy.
Conflict of interest
The authors declare to have no conflict of interest.
Author contributions
Hideki Abe conducted the study, performed experiments and wrote the manuscript; Kimika Endo performed experiments; Masayuki Shiba and Masahiro Satake reviewed and revised the manuscript.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or non-for-profit sectors.
References
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Platelet concentrates derived from buffy coat and apheresis: biochemical and functional differences.
Functional characteristics of apheresis-derived platelets treated with ultraviolet light combined with either amotosalen-HCl (S-59) or riboflavin (vitamin B2) for pathogen-reduction.
In vitro effects on platelets irradiated with short-wave ultraviolet light without any additional photoactive reagent using the THERAFLEX UV-Platelets method.
Cold storage of platelets in platelet additive solution: an in vitro comparison of two Food and Drug Administration-approved collection and storage systems.
Ultraviolet C light pathogen inactivation treatment of platelet concentrates preserves integrin activation but affects thrombus formation kinetics on collagen in vitro.
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