Maximum Aggregation Rate Research Articles

The effect of agitating buffy coats on platelet quality before soft spin

BackgroundPlatelet plays a vital role in both physiological and pathological processes. However, the limited storage time of platelet in vitro poses an immense challenge for its applications because of the increased risk of bacterial contamination and platelet storage lesions. Agitation can inhibit lesions by facilitating continuous oxygenation of platelets and permitting excess carbon dioxide to be removed during storage. However, it is still not known whether agitating BCs gives a positive effect on platelet quality. Objectives: To evaluate the quality difference between platelet concentrates (PCs) from buffy coats (BCs) held rest and agitation. MethodsSamples were withdrawn for cell count, blood gas analysis, free hemoglobin level, hypotonic shock response, maximum aggregation rate, activation marker expression (CD62P and CD42b) and coagulation function. Results: We found the PCs prepared from the agitating BCs had fewer residual WBCs, exhibited a better gas exchange ability, slower metabolism (higher pH, higher content glucose, and lower lactic acid levels), better hypotonic shock response, and lower levels of CD62P. The TEG-PC assays showed no difference in coagulation function. Conclusion: Our findings showed that BC can be agitated overnight before a soft spin.

Kinetic evidence for multiple aggregation pathways in antibody light chain variable domains.

Aggregation of antibody light chain proteins is associated with the progressive disease light chain amyloidosis. Patient-derived amyloid fibrils are formed from light chain variable domain residues in non-native conformations, highlighting a requirement that light chains unfold from their native structures in order to aggregate. However, mechanistic studies of amyloid formation have primarily focused on the self-assembly of natively unstructured peptides, and the role of native state unfolding is less well understood. Using a well-studied light chain variable domain protein known as WIL, which readily aggregates in vitro under conditions where the native state predominates, we asked how the protein concentration and addition of pre-formed fibril "seeds" alter the kinetics of aggregation. Monitoring aggregation with thioflavin T fluorescence revealed a distinctly non-linear dependence on concentration, with a maximum aggregation rate observed at 8 μM protein. This behavior is consistent with formation of alternate aggregate structures in the early phases of amyloid formation. Addition of N- or C-terminal peptide tags, which did not greatly affect the folding or stability of the protein, altered the concentration dependence of aggregation. Aggregation rates increased in the presence of pre-formed seeds, but this effect did not eliminate the delay before aggregation and became saturated when the proportion of seeds added was greater than 1 in 1600. The complexity of aggregation observed in vitro highlights how multiple species may contribute to amyloid pathology in patients.

KRAS-mutant colorectal cancer cell lines cause a prothrombotic state through the upregulation of thrombin: experimental study

Background: The KRAS genotype status is strongly associated with a prothrombotic state in colorectal cancer, and hypercoagulability and cancer-related thrombosis are among the significant events leading to poor prognosis. However, this correlation has not been confirmed at the cellular level. This study aimed to assess the maximum platelet aggregation rate and thrombin expression induced by colorectal cancer cells under different KRAS genotypes. Materials and methods: Platelet aggregation rate assay and western blotting analysis were used to detect platelet aggregation and thrombin expression induced by four colorectal cancer cells with different KRAS genotypes, including RKO, HCT116, SW480, and SW620. FVIIa/tissue factor and thrombin inhibitors were added to explore changes in platelet aggregation rates induced by colorectal cancer cells and the association between KRAS genotype status and hypercoagulable state. Results: KRAS-mutant cells were more likely to increase maximal platelet aggregation, with RKO, HCT116, SW480, and SW620 inducing 34.7%, 55.4%, 44.4%, and 63.8% of platelet aggregation, respectively. The maximum platelet aggregation rate was higher in the metastatic rectal cancer tumour strain SW620 than in the primary rectal cancer strain SW480. RKO cells had lower thrombin expression than the other three cells. Furthermore, the addition of thrombin inhibitors caused a more significant decrease in the platelet aggregation rate in KRAS-mutant cell lines compared to KRAS wild-type cell lines. Conclusion: Compared to KRAS wild-type colorectal cancer cells, KRAS-mutant colorectal cancer cell lines were more likely to be hypercoagulable through the upregulation of thrombin expression, which was mainly achieved through the TF-thrombin pathway.

Predictive value of the maximum aggregation rate of platelet for septic shock and septic shock with disseminated intravascular coagulation

To investigate the predictive value of the maximum aggregation rate (MAR) of platelet for septic shock and septic shock with disseminated intravascular coagulation (DIC). A retrospective case-control study enrolled patients with sepsis admitted to department of critical care medicine of the First Affiliated Hospital of Zhengzhou University from January 2021 to November 2022. The basic data, dynamic platelet aggregation rate, blood routine, inflammation indicators, sequential organ failure assessment (SOFA) and other clinical indicators within 24 hours after admission were collected. Septic patients were divided into the shock group and the non-shock group according to the presence of septic shock; then refer to the International Society on Thrombosis and Hemostasis (ISTH) standard, patients with septic shock were divided into the shock DIC group and the shock non-DIC group according to the presence of dominant DIC. Compared the differences in platelet aggregation function between these groups, and the receiver operator characteristic curve (ROC curve) was drawn to evaluate the predictive value of the MAR for septic shock and septic shock with DIC. Spearman correlation analysis was used to analyze the correlation of MAR with inflammation indicators and the severity of illness in patients with sepsis. A total of 153 sepsis patients were included and 61 with septic shock (including 17 with dominant DIC and 44 without dominant DIC). Compared with the non-shock group, the level of procalcitonin (PCT), C-reactive protein (CRP), and SOFA score were significantly higher in the shock group [PCT (mg/L): 6.90 (2.50, 23.50) vs. 0.87 (0.26, 5.75), CRP (mg/L): 156.48 (67.11, 230.84) vs. 90.39 (46.43, 182.76), SOFA score: 11.00 (8.00, 14.00) vs. 5.00 (3.00, 8.00), all P < 0.05]. The platelet count (PLT) and the MAR induced by adenosine diphosphate (ADP), adrenaline (A), collagen (COL), and arachidonic acid (AA; ADP-MAR, A-MAR, COL-MAR, AA-MAR) in the shock group were significantly decreased [PLT (×109/L): 101.00 (49.00, 163.50) vs. 175.50 (108.25, 254.50), ADP-MAR: 28.50% (22.00%, 38.05%) vs. 45.90% (33.98%, 60.28%), A-MAR: 38.90% (30.00%, 55.40%) vs. 65.15% (54.38%, 72.53%), COL-MAR: 27.90% (20.85%, 36.55%) vs. 42.95% (33.73%, 54.08%), AA-MAR: 24.70% (16.40%, 34.20%) vs. 46.55% (28.33%, 59.20%), all P < 0.05]. Subgroup analysis revealed that, compared with the shock non-DIC group, the SOFA scores were significantly higher in patients in the shock DIC group (13.29±5.23 vs. 10.39±3.58, P < 0.05), the PLT and COL-MAR in the shock DIC group were significantly reduced [PLT (×109/L): 36.00 (22.00, 67.50) vs. 115.50 (84.25, 203.75), COL-MAR: 21.50% (17.85%, 32.60%) vs. 30.95% (22.98%, 38.53%), all P < 0.05]. ROC curve analysis showed that A-MAR had a higher predictive value for septic shock, and the area under the ROC curve (AUC) was 0.814 [95% confidence interval (95%CI) was 0.742-0.886, P = 0.000]. When the optimal cut-off value was 51.35%, the sensitivity was 68.9%, the specificity was 82.6%, the positive predictive value was 0.724 and the negative predictive value was 0.800. COL-MAR had some predictive value for septic shock with DIC, and the AUC was 0.668 (95%CI was 0.513-0.823, P = 0.044). When the optimal cut-off value was 21.90%, the sensitivity was 52.9%, the specificity was 79.5%, the positive predictive value was 0.500, and the negative predictive value was 0.813. Spearman correlation analysis showed that the MAR induced by each inducer was negatively correlated with inflammatory indicators and SOFA scores in sepsis patients, with A-MAR showing the strongest correlation with SOFA score (r = -0.327, P = 0.000). MAR, an indicator of platelet aggregation function, shows predictive value for septic shock and septic shock with DIC, and it could be used to for evaluating the severity of patients with sepsis. In addition, tt also can be used as a monitoring index to predict the changes of sepsis patients and to guide the treatment.

Improvement of platelet preservation by inhibition of TRPC6.

The preservation of platelets (PLTs) by room temperature (RT) oscillation limits their shelf life to between 4 and 7 days because of the decrease in PLT function. TRPC6 is a non-selective mechanically sensitive cation channel that has been shown to mediate Ca2+ signalling, implying a role in PLT activation during preservation by RT oscillation. This study was designed to investigate whether inhibition of TRPC6 can improve the RT preservation of PLTs and the possible underlying mechanism. Human PLTs from whole blood were stored at 22 ± 2°C with oscillation in plasma or M-sol (mixture of solutions). BI-749327, a specific TRPC6 inhibitor, was administered throughout the preservation period. PLT distribution width (PDW), mean platelet volume (MPV), maximum platelet aggregation rate (MAR) and average aggregation rate (AAR) were measured. The MTT method was used to assess the relative viability of PLTs. Flow cytometry was used to measure the changes of Ca2+ concentration in PLTs and phosphatidylserine (PS) exposure on the PLT surface, and western blotting was used to assess the expression changes of platelet TRPC6 and CD62P proteins. Compared with the control group, inhibition of TRPC6 with BI-749327 significantly reduced the PDW, MPV and Ca2+ concentration, the MAR and AAR were significantly increased, the expression of TRPC6 and CD62P protein was significantly down-regulated in PLTs, and the PS exposure was significantly reduced on the PLT surface. However, these effects were all reversed by activation of TRPC6. Inhibition of TRPC6 improves the quality of PLT preservation by inhibiting the Ca2+ signal mediated by TRPC6.

Association between P2Y1 and P2Y12 polymorphisms and acute myocardial infarction and ADP-induced platelet aggregation

BackgroundThe objective of this study was to investigate the relationship between P2Y1 and P2Y12 genotypes and the risk of acute myocardial infarction (AMI) in the Quanzhou population and to determine associations between P2Y1 and P2Y12 genotypes and ADP-induced platelet aggregation in this population.MethodsAll subjects were screened for P2Y1 (c.1622A > G) and P2Y12 (H1/H2, c.34C > T) polymorphisms by direct DNA sequencing. The maximal platelet aggregation rate (MAR) in AMI patients (n = 61) and healthy control subjects (n = 50) was measured by a PL-12 platelet function analyzer, and adenosine diphosphate (ADP) (5 μmol/L) was used as an agonist.ResultsThe haploid H2 allele in the P2Y12 gene was more frequent in patients with AMI than in control subjects (OR 1.887, P = 0.005). The P2Y12 H2 haplotype was significantly associated with AMI in the codominant (P = 0.008), dominant (OR 2.103, P = 0.003), and overdominant models (OR 2.133, P = 0.003). After adjusting for potential confounders, H2 haplotype carriers had a 2.132-fold increased risk for AMI (OR 2.132, P = 0.012) compared with noncarriers. Moreover, we observed that the ADP-induced MAR in the carriers of the H2 haplotype from the control group was somewhat higher than that in noncarriers of this group (P = 0.020). However, we failed to demonstrate that the P2Y1 H1/H2 polymorphism affected ADP-induced MAR in AMI patients. Additionally, P2Y1 c.1622A > and P2Y12 c.34C > T polymorphisms were not associated with the risk of AMI or ADP-induced MAR in either group.ConclusionsTherefore, our results suggest that the P2Y12 H2 haplotype was associated with a higher risk of AMI, while its effect on increased ADP-induced platelet aggregation remains to be investigated. Thus, the P2Y12 H2 haplotype may be a potential marker for AMI.

Independent association of PCSK9 with platelet reactivity in subjects without statin or antiplatelet agents.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) levels could predict cardiovascular event in patients with well-controlled LDL-C levels, suggesting an LDL-independent mechanism of PCSK9 on the cardiovascular system. Accumulating evidence suggests PCSK9 might be associated with increased platelet reactivity. This study aimed to assess the relationship between PCSK9 levels and platelet reactivity in subjects not taking statins or antiplatelet agents. A cross-sectional study was conducted to investigate the independent contribution of PCSK9 to platelet activity by controlling for the potential confounding factors. The study population included 89 subjects from a health examination centre who underwent routine annual health check-ups or had an examination before a selective operation. Subjects taking statins or antiplatelet agents were excluded. Adenosine diphosphate (ADP)-induced platelet aggregation was determined by PL-11 platelet analyzer using impedance aggregometry and plasma PCSK9 levels were determined using an ELISA. Serum Lipid profile was assessed by measuring the concentration of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG), with low-density lipoprotein cholesterol (LDL-C) being directly measured using enzymatic techniques. The association between PCSK9 and platelet reactivity was investigated. The study subjects were composed of 53 males and 36 females with an average age of 55 (±11) years old. The univariate correlation analysis showed significant correlation between ADP-induced maximal aggregation rate (MAR) and PCSK9 (r = 0.55, p < 0.001) as well as TC (r = 0.23, p = 0.028), LDL-C (r = 0.27, p < 0.001), and PLT (r = 0.31, p = 0.005). Being male (41.2% vs. 46.6, p = 0.04) and smoking (37.4 vs. 46.2%, p = 0.016) were associated with lower ADP-induced MAR than being female and non-smoking. However, there is no correlation between PCSK9 and AA-induced platelet maximal aggregation rate (r = 0.17, p = 0.12). Multiple regression analysis suggested that PCSK9 contributed independently to ADP-induced maximal aggregation rate (β = 0.08, p = 0.004) after controlling for the effect of TC, LDL-C, PLT, being male, and smoking. PCSK9 is positively associated with platelet reactivity, which may partly account for the beneficial effect of PCSK9 inhibition in reducing the risk of major adverse cardiovascular events after acute coronary syndrome (ACS).

Low-dose and standard-dose ticagrelor compared with clopidogrel in patients with acute coronary syndromes: A cohort study from china.

PurposePrevious trials have demonstrated that ticagrelor was superior to clopidogrel in acute coronary syndrome (ACS) patients. However, several recent studies showed that ticagrelor was associated with a significantly higher risk of bleeding compared with clopidogrel, especially in East Asian patients. Low-dose ticagrelor might improve the safety of ACS patients in the Chinese population. Therefore, this study mainly explored the low-dose ticagrelor in Chinese ACS patients.MethodsA total of 199 ACS patients were enrolled in this study. The maximum platelet aggregation rate induced by adenosine-5-diphosphate (ADP) was detected by light transmittance aggregometry (LTA). Platelet aggregation rate induced by ADP of more than or equal to 42.9% was defined as high on-treatment platelet reactivity (HPR) to P2Y12 inhibitors. All patients were followed up for at least 12 months. Clinical outcomes, changes of antiplatelet regimen, medication compliance and adverse reactions were collected.ResultsPatients were divided into three groups according to the P2Y12 inhibitors, including 87 cases in clopidogrel (75 mg once a day) group, 41 cases in ticagrelor 60 mg (twice a day) group, and 71 cases in ticagrelor 90 mg (twice a day) group. ADP-induced platelet aggregation rates in ticagrelor 60 mg group and 90 mg group were 28.4 (19.6, 42.9) and 22.33 (15.1, 34.7) respectively, which were significantly lower than those in clopidogrel group 49.3 (36.5, 61.0) with adjusted P < 0.001. At the same time, there was no significant difference in ADP-induced platelet aggregation rate between ticagrelor 60 mg and 90 mg group (adjusted P = 0.105). Compared with clopidogrel, the proportion of normal on-treatment platelet reactivity (NPR) of ticagrelor 60 mg and ticagrelor 90 mg were significantly higher than that of clopidogrel, and the proportion of NPR of ticagrelor 90 mg group was significantly higher than that of ticagrelor 60 mg group.ConclusionsPatients of ticagrelor 60 mg and ticagrelor 90 mg had comparable platelet aggregation rates induced by ADP, and both of them had significantly more potent antiplatelet aggregation activity detected by LTA than clopidogrel.

Study on the antithrombotic effect and physiological mechanism of okanin

The objectives of this study were to investigate the antithrombotic effect and physiological mechanism of okanin, a flavonoid monomer in Coreopsis tinctoria Nutt. The antithrombotic effects of okanin were determined by the anticoagulant activity test in vitro and in vivo, the venous thrombosis and arterial thrombosis test in rats. To study the antithrombotic physiological mechanisms of okanin, UV spectrophotometer and enzyme-linked immunosorbent assay (ELISA) were used to determine the effects of three concentrations of okanin on the contents of 6-keto-prostaglandin F1α (6-Keto-PGF1α), thromboxane B2 (TXB2), endothelin-1 (ET-1), antithrombin III (AT-Ⅲ), protein C (PC) and von willebrand factor (vWF) in the plasma of rats with arterial thrombosis; ELISA was used to detect the effects of okanin on the contents of plasminogen (PLG), tissue plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in the plasma of mice and Chinese white rabbits. The results showed that okanin could prolong the coagulation time in vitro and in vivo of animals (P < 0.01 in the high dose group) and the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) of human venous blood (ATPP of medium dose group P < 0.01; PT, TT P < 0.05. P < 0.01 in the high dose group); inhibit the maximum platelet aggregation rate of rabbits (P < 0.05 in the low dose group; P < 0.01 in the medium and high dose groups), decrease the dry and wet weight of venous thrombosis and the wet weight of common carotid artery thrombosis in rats (low dose group, P < 0.05; medium and high dose groups, P < 0.01); increase the levels of 6-Keto-PGF1α, AT-Ⅲ, PLG and t-PA in animal plasma; decrease the levels of TXB2, ET-1, vWF and PAI-1 in animal plasma. It is concluded that okanin can significantly inhibit thrombosis, and its physiological mechanisms were related to affecting the activation of related coagulation factors in endogenous and exogenous coagulation pathways, affecting the physiological characteristics of platelets, repairing damaged vascular endothelial cells and enhancing the activity of the fibrinolytic system.

Stability, aggregation, and sedimentation behaviors of typical nano metal oxide particles in aqueous environment

The wide use of nano metal oxide particles (NMOPs) brings about their inevitable release into the water environment, affecting the environment and human health. Therefore, the stability, aggregation, and sedimentation process of four typical NMOPs (ZnO NPs, CeO2 NPs, TiO2 NPs, and CuO NPs) were investigated in artificial water and real municipal sewage to reveal their complicated behavior. Results showed that NMOPs aggregated at the pH of zero-charge point, and their hydrodynamic diameters and aggregation rates could reach the maximum values. The hydrodynamic diameters and aggregation rates of ZnO NPs, CeO2 NPs, TiO2 NPs, and CuO NPs at the zero-charge point were 617, 1760, 870, 1502 nm, and 31.7, 1158.1, 48.3, 115.7 nm/min, respectively. In addition, the dissolution of NMOPs led to the sedimentation rates under acidic conditions being much lower than those under neutral and alkaline conditions. The aggregation and sedimentation performance of NMOPs were affected by not only pH but also ionic strength (IS) and species. The aggregation rates of NMOPs increased with the increase of IS (0–10 mM), and the maximum aggregation rate of CeO2 NPs was 470.1 nm/min (pH = 7 and CaCl2 = 10 mM). According to Coulomb's law, divalent cations (Mg2+, Ca2+) were more competitively adsorbed on the surface of NMOPs than monovalent cations (K+, Na+), which increased the zeta potential and aggregation rate of NMOPs. Furthermore, the NMOPs were easier to aggregate in municipal sewage because of the homogeneous aggregation between nanoparticles and heterogeneous aggregation with natural colloids. The total interaction energy between NMOPs was calculated by the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theoretical formula, which was consistent with the experimental results.

Early predictive value of platelet function for clinical outcome in sepsis

Sepsis is the leading course of morbidity and mortality in critically ill patients. This study aimed to evaluate the predictive value of the platelet aggregation for mortality in patients with sepsis. In addition, the relationship between impaired mitochondria and the platelet aggregation was explored. This was a prospective, observational cohort study. The platelet aggregation rate in response to adenosine diphosphate (ADP) was assessed. The primary outcome was 28-day mortality. Platelet mitochondrial parameters, including adenosine triphosphate(ATP), mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) opening, were measured. Platelet mitochondrial ultrastructure was observed using transmission electron microscopy. 86 patients with 65 survivors and 21 non-survivors were enrolled. Platelets of non-survivors with sepsis were hyporesponsive to ADP, in terms of maximal aggregation rate (P<0.001). Receiver operating characteristic curves analysis demonstrated that the AUC estimated 28-day mortality for platelet aggregation rate was 0.814. At the optimal cut-off value of 35.8% for platelet aggregation rate, the sensitivity was 86.2% and the specificity was 66.7%. Kaplan-Meier analysis showed that a platelet aggregation rate of less than 35.8% was associated closely with poor survival. After adjusting for lactate by Cox regression analysis, platelet aggregation rate was identified as an independent predictor for the probability of 28-day mortality. Compared with survivors, non-survivors showed a significant reduction in platelet ATP and MMP-index (both P<0.001), and a remarkable increase in mPTP opening (P<0.001). ATP and MMP-index were positively correlated with platelet aggregation rate (R square=0.75, R square=0.44, respectively). Platelet aggregation rate could be an early predictive biomarker for mortality in sepsis. Impaired platelet mitochondrial activity affects platelet aggregation and correlates with the severity of sepsis.

The effects of fructose diphosphate on routine coagulation tests in vitro

To evaluate the effects of fructose diphosphate (FDP) on routine coagulation tests in vitro, we added FDP into the mixed normal plasma to obtain the final concentration of 0, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 30 and 35 mg/mL of drug. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (FBG) and thrombin time (TT) of samples were analyzed with blood coagulation analyzers from four different manufacturers(Sysmex, Stago, SEKISUI and Werfen) and their corresponding reagents, respectively. Before the experiment, we also observed whether there were significant differences in coagulation test results of different lots of reagents produced by each manufacturer. At the same time as the four routine clotting tests, the Sysmex blood coagulation analyzer and its proprietary analysis software were used to detect the change of maximum platelet aggregation rate in platelet-rich plasma after adding FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL). The results of PT, aPTT and TT showed a FDP (0–35 mg/mL) concentration-dependent increase and a FBG concentration-dependent decrease. The degree of change (increase or decrease) varied depending on the assay system, with PT and aPTT being more affected by the Sysmex blood coagulation testing instrument reagent system and less affected by CEKISUI, TT less affected by CEKISUI and more affected by Stago, and FBG less affected by Stago and more affected by Sysmex. The results of PT, aPTT and TT were statistically positively correlated with their FDP concentrations, while FBG was negatively correlated. The correlation coefficients between FDP and the coagulation testing systems of Sysmex, Stago, Werfen and SEKISUI were 0.975, 0.988, 0.967, 0.986 for PT, and 0.993, 0.989, 0.990 and 0.962 for aPTT, 0.994, 0.960, 0.977 and 0.982 for TT, − 0.990, − 0.983, − 0.989 and − 0.954 for FBG, respectively. Different concentrations of FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL) had different effects on the maximum aggregation rate of platelet induced by the agonists of adenosine diphosphate (ADP, 5 µmol/L), arachidonic acid (Ara, 1 mmol/L), collagen (Col, 2.5 µg/mL) and epinephrine (Epi,10 µmol/L), but the overall downward trend was consistent, that is, with the increase of FDP concentration, the platelet aggregation rate decreased significantly. Our experimental study demonstrated a possible effect of FDP on the assays of coagulation and Platelet aggregation, which may arise because the drug interferes with the coagulation and platelet aggregation detection system, or it may affect our in vivo coagulation system and Platelet aggregation function, the real mechanism of which remains to be further verified and studied.

A novel scale-up strategy for cultivation of BHK-21 cells based on similar hydrodynamic environments in the bioreactors

The scale-up of animal cell cultivation is important but remains complex and challenging. In the present study, we propose a novel scale-up strategy for baby hamster Syrian kidney-21 (BHK-21) cell cultivation based on similar hydrodynamic environments. The hydrodynamic characteristics of the different scale bioreactors were determined by computational fluid dynamics (CFD) and further correlated with the agitation speed. The optimal hydrodynamic environment for cell cultivation and vaccine production was determined from the cultivation experiments of BHK-21 cells in 5-L laboratory-scale bioreactors equipped with different impellers at various agitation speeds. BHK-21 cell cultivation was scaled up from 5-L to 42-, 350-, and 1000-L bioreactors by adjusting the agitation speed to make the hydrodynamic features similar to those in the 5-L bioreactor, especially for the shear rate in the impeller zone (γimp) and energy dissipation rate in the tank bulk zone (εtan). The maximum cell density and cell aggregation rate in these scaled-up bioreactors were in the range of 4.6 × 106 ~ 4.8 × 106 cells/mL and 16 ~ 20%, which are comparable to or even better than those observed in the 5-L bioreactor (maximum cell density 4.8 × 106 cells/mL, cell aggregation rate 21%). The maximum virus titer of 108.0 LD50/mL achieved in the 1000-L bioreactor was close to 108.3 LD50/mL that obtained in the 5-L bioreactor. Hence, the scale-up strategy proposed in this study is feasible and can efficiently facilitate the scale-up processes of animal cell cultivation.

Characteristics of cerebral ischemic stroke based on moyamoya disease and atherosclerosis-associated intracranial arterial stenosis

To analyze the characteristics of acute ischemic stroke (AIS) resulting from moyamoya disease (MMD) and intracranial large artery atherosclerotic stenosis (LAS). This real-world case control study enrolled imaging-confirmed AIS patients owing to MMD or LAS hospitalized from January 2015 through September 2020 consecutively. The features of risk factors, peripheral blood, and imaging presentations were compared between the two cohorts. A total of 191 eligible patients entered into final analysis, including 70 cases with MMD stroke and 121 with LAS stroke. LAS stroke vs. MMD stroke, the ratios of hyperlipidemia, hypertension, diabetes, and hyperhomocysteinemia were higher in the former (65.3 vs.12.9%, 65.3% vs. 4.3%, 39.7% vs. 2.9%, and 43.8% vs.12.9%; all p < 0.01) as well as baseline plasma arachidonic acid (AA) and adenosine diphosphate (ADP)-stimulated maximum platelet aggregation rates (75.3% vs. 60.8% and 73.1% vs.64.9%, respectively, all p < 0.01), which were positively correlated with triglycerides and cholesterol levels, blood glucose, age, and platelet counts (all p < 0.01). Classical watershed infarction (WSI) accounted for 87.14% in MMD stroke and 40.49% in LAS stroke, respectively (p < 0.01). Almost all of the patients with LAS showed plaques in arterial walls on CTA maps and non-homogeneous thickening with irregular luminal narrowing on HRMRI, while plaques were seldom found in MMD besides homogeneous thickening with regular luminal narrowing. Differing from LAS stroke, MMD stroke mainly presents with WSI and does not feature with platelet hyper-aggregation and fragmentation of ulcer plaque. Whereby, focusing on perfusion improvement rather than antiplatelets and statins may be the predominant step in MMD-stroke correction.

Performance Metrics of the AGGRESTAR PL-12® Platelet Function Analyser in Patients with TIA or Ischaemic Stroke on Commonly-Prescribed Antiplatelet Regimens

Introduction: The optimal time interval after venepuncture to perform platelet function/reactivity testing at low shear stress on the novel AGGRESTAR PL-12® platelet function analyser in non-Chinese Cerebrovascular Disease (CVD) patients is unknown. Methods: Twelve TIA/ischaemic stroke patients were recruited to this cross-sectional, methodological study: 3 on aspirin monotherapy, aspirindipyridamole combination therapy, clopidogrel monotherapy and aspirinclopidogrel combination therapy, respectively. The PL-12 (‘mode 2’) was used to calculate the % maximum aggregation rate to fixed doses of arachidonic acid (%MARAA) and adenosine diphosphate (%MARADP). Samples were analysed every 15 minutes from 30-135 minutes, and every 30 minutes between 165-225 minutes after venepuncture to calculate the time interval providing optimal interassay Coefficients of Variation (CVs). Results: Mean CVs were ≤ 7.37% for the %MARAA assay in patients on aspirin monotherapy or combination therapy, and ≤ 10.24% for the %MARADP assay in patients on clopidogrel monotherapy or combination therapy if assays were performed between 90-120 minutes post-venepuncture. CVs ≤ 10% were also obtained from assays performed between 90-165 minutes postvenepuncture on aspirin monotherapy or combination therapy. Discussion: Reliable and reproducible platelet function/reactivity data can be obtained with the AGGRESTAR PL-12 analyser in non-Chinese CVD patients on commonly-prescribed antiplatelet monotherapy or combination therapy regimens between 90-120 minutes post-venepuncture.

The variation of serum uric acid levels among patients with myocardial infarction treated with ticagrelor and the association between serum uric acid and platelet reactivity

Objectives: To compare the impact of ticagrelor or clopidogrel on serum uric acid levels among patients with ST-segment elevation myocardial infarction (STEMI) who underwent primary percutaneous coronary intervention (PCI) and further evaluate the effects of variation of serum uric acid levels on platelet reactivity. Methods: STEMI patients who admitted to Fuwai Hospital from April 2017 to January 2020, and underwent primary PCI and discharged alive with aspirin and ticagrelor or clopidogrel were included in this study. Patients were divided into ticagrelor group and clopidogrel group. The baseline clinical data were collected. Serum uric acid and creatinine levels at baseline and 30 days post-PCI were measured. Light transmittance aggregometry was used to assess maximum aggregation rate induced by adenosine diphosphate and arachidonic acid. The changes of serum uric acid and creatinine were compared between the two groups. Multivariate logistic regression was performed to evaluate independent related factors for rise in the uric acid levels, and the effect of variation of serum uric acid level on platelet reactivity was analyzed. Results: A total of 967 patients were included, the age was (59.4±12.1) years, and 163 case were female. There were 550 cases in ticagrelor group (56.9%) and 417 cases in clopidogrel group (43.1%). Baseline serum uric acid and creatinine levels were similar between the 2 groups. At 30 days, the serum uric acid level [(347.2±96.5) mmol/L vs. (341.2±105.3) mmol/L, P=0.009] and absolute [46.4 (-2.4, 88.1) mmol/L vs. 25.0 (-21.9, 73.0) mmol/L, P=0.001] and percentage [13.2 (-0.01, 29.0) % vs. 7.9 (-5.7, 25.0) %, P=0.007] increase in the serum uric acid levels were significantly higher in ticagrelor group than in clopidogrel group. The level of serum creatinine at 30 days was significantly lower in ticagrelor group than in clopidogrel group [(89.7±21.3) μmol/L vs. (94.4±43.9) μmol/L, P<0.05], whereas there were no differences in absolute [8.0 (-1.4, 16.6) μmol/L vs. 7.8 (-2.0, 16.6) μmol/L] and percentage [10.5 (-1.7%, 22.6%) vs. 9.8 (-2.4%, 22.1%)] change in the serum creatinine between the 2 groups (all P>0.05). Logistic regression analysis showed that, after adjusting for confounding factors, ticagrelor therapy was an independent related factor of serum uric acid elevation (OR=1.582, 95% CI:1.023-2.447, P=0.039). The variation of the serum uric acid levels did not affect platelet aggregation and the percentage of high platelet reactivity in both groups. Conclusions: Ticagrelor use is related to a significant increase in the serum uric acid levels at 30 days post-PCI in this patient cohort. The variations in the uric acid levels do not increase the percentage of high platelet reactivity in STEMI patients treated with ticagrelor or clopidogrel.

Association between insulin resistance and aspirin or clopidogrel resistance in Chinese patients with recent ischemic stroke/TIA

ABSTRACT Objective: This study aims to discover whether insulin resistance is an independent predictor for antiplatelet drug resistance in patients with ischemic cerebrovascular disease. Methods: This study used a prospective cohort method. Patients diagnosed as minor ischemic stroke or transient ischemic attack (TIA) were enrolled successively. All patients have been administrated aspirin and/or clopidogrel and were tested for fasting glucose and insulin and platelet aggregation inhibition tests which was detected by light transmission aggregometry (LTA). The maximum platelet aggregation rate (AA) of ≥20% or the maximum platelet aggregation rate (ADP) of ≥50% was defined as antiplatelet drug resistance. Multivariable logistic regression was performed to estimate the association between HOMA-IR and antiplatelet drug resistance. Results: This study recruited successively 237 patients with mild-to-moderate ischemic stroke or TIA in Beijing Tiantan Hospital from 2018 to 2019. Of them 60 cases were recognized as having insulin resistance. There are 46 patients in insulin resistance group (76.7%) developed antiplatelet drug resistance, which was significantly more frequent than patients without insulin resistance (35%, P < 0.0001). Insulin resistance was an independent risk factor for antiplatelet drug resistance in patients with recent ischemic stroke/TIA after adjusted for confounding factors (Odds Ratio 5.281; 95%CI, 2.15 to 13.01, P < 0.0001). Conclusions: Insulin resistance was an independent predictor for development of antiplatelet drug resistance in patients with recent minor ischemic stroke or TIA. More attention should be paid to recognize these patients and antithrombotic effect should be monitored when antiplatelet drugs were applied to these patients.

Antiplatelet Loading Effects Prior to Endovascular Treatment: Carotid Artery Stenosis versus Unruptured Cerebral Aneurysms.

Carotid artery stenosis and cerebral aneurism may have different platelet functions and antiplatelet responses because these diseases have different etiologies. In this study, we compared the antiplatelet loading effects prior to endovascular treatment between carotid artery stenosis and unruptured cerebral aneurysm (UCA) patients. Nine patients with asymptomatic carotid artery stenosis (ACS), 14 with symptomatic carotid artery stenosis (SCS), and 20 with unruptured cerebral aneurysms were enrolled in this study. Antiplatelet (aspirin + clopidogrel) loading effects prior to endovascular treatment were evaluated using light transmission aggregometry and platelet aggregate detection methods. Although there are differences in the prevalence of atherosclerosis risk factors, maximum aggregation rates in light transmission aggregometry and platelet aggregation-prone properties were not different in the three disease groups. Preoperative dual antiplatelet therapy with aspirin and clopidogrel may be appropriate for both carotid artery stenosis and cerebral aneurism patients even though their conditions and background factors differ.

Conversion and outcome of dual antiplatelet therapy after bleeding in patients with acute coronary syndrome

Abstract Objective To explore the conversion and outcome of antiplatelet therapy in patients with acute coronary syndrome (ACS) who have suffered bleeding after receiving aspirin combined with ticagrelor. Methods Patients who diagnosed with ACS and given aspirin 100 mg once daily combined with ticagrelor 90 mg twice daily for dual antiplatelet therapy (DAPT) were selected. The patients were divided into bleeding group and non-bleeding group according to whether bleeding events occurred within one year after DAPT. The bleeding group was divided into the next four groups according to the converted antiplatelet treatment strategy decided by the doctor and the patient. Patients in group A to D were treated with ticagrelor 90mg twice a day plus aspirin 0–50mgonce a day, ticagrelor 60mg twice a day plus aspirin 100mgonce a day, clopidogrel 75mgonce a day plus aspirin 100mg once a day and ticagrelor 90mg twice a day plus aspirin 100mg once a day, respectively. All patients were followed up for 1 year after DAPT. The primary outcome is change of bleeding degree by BARC. The secondary outcomes includes BARC bleeding, all-cause mortality and MACE. Results A total of 752 cases were enrolled, including 250 in the bleeding group and 502 in the non-bleeding group. The bleeding group was divided into four treatment groups, which were 63, 43, 38 and 95 cases among A, B, C and D group. There was a significant difference in the improvement of bleeding among the four groups. To be noted, patients in group A has the highest improvement rate of hemorrhage (65.1% vs 62.8% vs 47.4% vs 24.2%, P&amp;lt;0.01). There were no significant differences in the incidence of MACE and all-cause mortality (P&amp;gt;0.05). The incidence of major MACE in patients in the bleeding group was 14.4%, which was higher than that in the non-bleeding group (9.2%, P=0.03).The result of multivariate logistic regression analysis showed that female (OR=5.18, 95% CI: 1.61–16.65, P=0.006), low body weight (OR=7.73, 95% CI: 2.46–24.49, P&amp;lt;0.001) and low AA-induced maximum platelet aggregation rate (OR=2.72, 95% CI:1.09–6.84, P=0.033) were independent risk factors for hemorrhage. Conclusion Patients with acute coronary syndrome who have suffered bleeding after receiving aspirin combined with ticagrelor were at a higher risk of MACE than those who did not. Low-dose aspirin (0–50mg/d) combined with ticagrelor or even ticagrelor alone has the best effect on reducing bleeding without increasing the risk of ischemia. Female, low body mass and low AA-induced maximum platelet aggregation rate are independent risk factors for bleeding. Funding Acknowledgement Type of funding source: None

Aggregation behavior of zinc oxide nanoparticles and their biotoxicity to Daphnia magna: Influence of humic acid and sodium alginate

The widespread applications of zinc oxide nanoparticles (ZnO NPs) have raised increasing concerns due to their adverse environmental effects. The ubiquitous natural organic matter in natural aqueous environments can interact with ZnO NPs, thereby affecting their aggregation, sedimentation and biotoxicity. Here, we systematically investigated the effects of humic acid (HA) and sodium alginate (SA) on the aggregation behavior of ZnO NPs and their biotoxicity to Daphnia magna. High concentrations (9.0 mg/L) of HA and SA accelerated the aggregation of ZnO NPs with maximum aggregation rates (ΔD/Δt) of 22.1 and 19.2 nm/min, respectively. Both HA and SA led to 31.2% and 30.1% decrease of ZnO NPs concentration compared with the control experiment. The results calculated by Derjaguin-Landau-Verwey-Overbeek theoretical formula were consistent with these of aggregation and sedimentation of ZnO NPs. Furthermore, excitation-emission-matrix fluorescence spectroscopy verified that the carboxylic groups of HA and SA have high complexation capacity with ZnO NPs. Daphnia magna was used to evaluate the biotoxicity of ZnO NPs, and the toxicity of ZnO NPs to Daphnia magna was alleviated as the HA concentration increased from 0 to 1.2 mg/L. Toxicity mitigation experiments confirmed that photocatalytic generation of reactive oxygen species was more toxic to Daphnia magna than dissolved Zn2+ in acute and chronic toxicity tests. Moreover, the attacks of active oxygen free radical damaged the antioxidant system of Daphnia magna. The information obtained will help us to improve the understanding of the impacts of ZnO NPs on freshwater ecosystems.