The presence of residual activated coagulation factor XI (FXIa) in some commercial intravenous immunoglobulin (IVIG) products has been identified as the root cause of a small number of thromboembolic events in patients who had received such therapy. Our objectives here were to design and evaluate the manufacturing process of GC5107, a 10% glycine-stabilized IVIG product, for its capacity to remove FXIa. The manufacturing process included a cation exchange chromatography (CEX) step, which employs a resin that binds immunoglobulin G (IgG) with high capacity. Procoagulant activity was assessed using Western blot analysis, enzyme-linked immunosorbent assay, thrombin generation assay, chromogenic FXIa assay, and non-activated partial thromboplastin time (NaPTT) assay. A spiking study in which large quantities of FXIa were added to samples before CEX chromatography was used to examine the robustness of the process to remove FXIa. Western blot and ELISA analyses demonstrated that residual FXIa remained in the intermediate manufacturing products until after CEX chromatography, when it was reduced to undetectable levels. The spiking study demonstrated that CEX chromatography removed >99% of FXI protein and reduced FXI activity to below detection limits, even in samples containing 158-fold greater FXIa levels than that of normal samples. Procoagulant activity in 9 consecutive lots of GC5107 was reduced to below the detection limits of the thrombin generation and chromogenic FXIa assays (<1.56 IU/ml and <0.16 IU/ml, respectively). The NaPTT of >250 s in all 9 lots indicated very low levels of procoagulant activity. We demonstrate that a novel 10% IVIG manufacturing process including CEX chromatography is a robust means of removing FXIa from the final preparation.

UNASSIGNED: Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis, and COVID-19. EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g.
UNASSIGNED: In this study, we analyzed the TF activity of 2 EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer, or COVID-19.
UNASSIGNED: EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer, or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured.
UNASSIGNED: LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in 2 patients with sepsis who had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs.
UNASSIGNED: We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

Southeast Asian ovalocytosis (SAO) is characterized by the misfolding of band 3 protein in red blood cells (RBC). The abnormal structure of the band 3 protein results in dysmorphic RBC and related functions. Previous data showed that in vitro storage under hypothermic conditions alters band 3 protein structure and function. Microvesiculation includes shedding of RBC membranes, called RBC-derived microparticles/extracellular vesicles (RMP/EVs), and storage lesions. Unfortunately, there is no evidence of RBC microvesiculation under in vitro storage conditions in heterozygous SAO individuals. This study determined the generation of REVs and procoagulant activity during the storage of SAO blood samples in southern Thailand. Venous blood was collected from eight SAO and seven healthy individuals, preserved in citrate phosphate dextrose-adenine 1 (CPDA-1) at 4 °C for 35 days. The absolute numbers of REVs and PS-expressing RBCs were analyzed using flow cytometry. The procoagulant activity of the produced extracellular vesicles was determined by a clotting time assay. The results showed a significant increase in the number of REVs and PS-expressing RBCs in the SAO blood samples. Significantly correlated PS externalization and procoagulant activity were observed in the SAO blood samples. These lines of evidence indicate that the abnormality of the Band 3 protein is possibly involved in aberrant microvesiculation, exerting procoagulant activity in vitro. Increased pools of REV production and abnormal storage lesions in SAO blood samples should be a concern. Notably, the mechanisms underlying membrane vesiculation depend on the extent of blood cell storage under hypothermic conditions.

Epstein-Barr virus (EBV)-positive T- or NK-cell neoplasms show progressive systemic inflammation and abnormal blood coagulation causing hemophagocytic lymphohistiocytosis (HLH). It was reported that inflammatory cytokines were produced and secreted by EBV-positive neoplastic T- or NK-cells. These cytokines can induce the differentiation of monocytes into macrophages leading to HLH. To clarify which products of EBV-positive neoplastic T- or NK-cells have effects on monocytes, we performed a co-culture assay of monocytes with the supernatants of EBV-positive T- or NK-cell lines. The expression of differentiation markers, the phagocytosis ability, and the mRNA expression of the inflammatory cytokines of THP-1, a monocytic cell line, clearly increased after culturing with the supernatants from EBV-NK-cell lines. Co-culturing with the supernatants promoted the expression of CD80 and CD206 as well as M1 and M2 macrophage markers in human monocytes. Co-culturing with the supernatants of EBV-NK-cell lines significantly enhanced the procoagulant activity and the tissue factor expression of monocytes. Interferon (IFN)-γ was elevated extremely not only in the supernatant of EBV-NK-cell lines but also in the plasma of EBV-positive NK-cell neoplasms patients accompanying HLH. Finally, we confirmed that IFN-γ directly enhanced the differentiation into M1-like macrophages and the procoagulant activity of monocytes. Our findings suggest that IFN-γ may potentially serve as a therapeutic target to regulate HLH in EBV-positive NK-cell neoplasms.

Expanding biomedical application of anatase titanium dioxide (TiO2) nanoparticles (NPs) is raising the public concern on its potential health hazards. Here, we demonstrated that TiO2 NPs can increase phosphatidylserine (PS) exposure and procoagulant activity of red blood cells (RBCs), which may contribute to thrombosis.
We conducted in vitro studies using RBCs freshly isolated from healthy male volunteers. TiO2 NPs exposure (≦ 25 μg/mL) induced PS exposure and microvesicles (MV) generation accompanied by morphological changes of RBCs. While ROS generation was not observed following the exposure to TiO2 NPs, intracellular calcium increased and caspase-3 was activated, which up-regulated scramblase activity, leading to PS exposure. RBCs exposed to TiO2 NPs could increase procoagulant activity as measured by accelerated thrombin generation, and enhancement of RBC-endothelial cells adhesion and RBC-RBC aggregation. Confirming the procoagulant activation of RBC in vitro, exposure to TiO2 NPs (2 mg/kg intravenously injection) in rats increased thrombus formation in the venous thrombosis model.
Collectively, these results suggest that anatase TiO2 NPs may harbor prothrombotic risks by promoting the procoagulant activity of RBCs, which needs attention for its biomedical application.

Curcumin is a natural bioactive component derived from the turmeric plant Curcuma longa, which exhibits a range of beneficial activities on human cells. Previously, an inhibitory effect of curcumin on platelets was demonstrated. However, it is unknown whether this inhibitory effect is due to platelet apoptosis or procoagulant platelet formation. In this study, curcumin did not activate caspase 3-dependent apoptosis of human platelets, but rather induced the formation of procoagulant platelets. Interestingly, curcumin at low concentration (5 µM) potentiated, and at high concentration (50 µM) inhibited ABT-737-induced platelet apoptosis, which was accompanied by inhibition of ABT-737-mediated thrombin generation. Platelet viability was not affected by curcumin at low concentration and was reduced by 17% at high concentration. Furthermore, curcumin-induced autophagy in human platelets via increased translocation of LC3I to LC3II, which was associated with activation of adenosine monophosphate (AMP) kinase and inhibition of protein kinase B activity. Because curcumin inhibits P-glycoprotein (P-gp) in cancer cells and contributes to overcoming multidrug resistance, we showed that curcumin similarly inhibited platelet P-gp activity. Our results revealed that the platelet inhibitory effect of curcumin is mediated by complex processes, including procoagulant platelet formation. Thus, curcumin may protect against or enhance caspase-dependent apoptosis in platelets under certain conditions.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) patients are at a high risk of developing venous thromboembolism, with a high rate of morbidity and mortality. The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in patients with NASH remains unclear. Our study aimed to investigate the formation of NETs in NASH patients stimulated by specific pro-inflammatory factors. Moreover, we evaluated the pivotal role of NETs in the induction of hypercoagulability in NASH and the interaction between NETs and endothelial injury.
METHODS: The levels of the NETs biomarkers were evaluated in the plasma samples of 27 NASH patients and 18 healthy subjects. The formation of NETs was visualized using immunofluorescence microscopy. The PCA of the NETs was assessed using coagulation time, purified coagulation complex, and fibrin formation assays. Confocal microscopy was further used to evaluate the interactions between the NETs and HUVECs.
RESULTS: The levels of NETs markers in the plasma of NASH patients were significantly higher than healthy controls. NETs derived from NASH enhanced thrombin and fibrin formation and significantly reduced CT (p<0.05). The mixture of IL-6 and TNF-α triggered the NETs release in the plasma rather than them alone. Additionally, the NETs exerted cytotoxic effects on the endothelial cells, converting them to a procoagulant and pro-inflammatory phenotype, and DNase I could reverse these effects.
CONCLUSIONS: Our results revealed the primary role of NETs in promoting the hypercoagulable state in NASH patients. Methods that prevent the formation of NETs may be a novel approach for the prevention and treatment of NASH.

Patients affected by the rare Glanzmann thrombasthenia (GT) suffer from defective or low levels of the platelet-associated glycoprotein (GP) IIb/IIIa, which acts as a fibrinogen receptor, and have therefore an impaired ability to aggregate platelets. Because the procoagulant activity is a dichotomous facet of platelet activation, diverging from the aggregation endpoint, we were interested in characterizing the ability to generate procoagulant platelets in GT patients. Therefore, we investigated, by flow cytometry analysis, platelet functions in three GT patients as well as their ability to generate procoagulant collagen-and-thrombin (COAT) platelets upon combined activation with convulxin-plus-thrombin. In addition, we further characterized intracellular ion fluxes during the procoagulant response, using specific probes to monitor by flow cytometry kinetics of cytosolic calcium, sodium, and potassium ion fluxes. GT patients generated higher percentages of procoagulant COAT platelets compared to healthy donors. Moreover, they were able to mobilize higher levels of cytosolic calcium following convulxin-plus-thrombin activation, which is congruent with the greater procoagulant activity. Further investigations will dissect the role of GPIIb/IIIa outside-in signalling possibly implicated in the regulation of platelet procoagulant activity.

Patients with obstructive jaundice (OJ) are considered to be prothrombotic with increased risk of thromboembolism complications. The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) and thrombosis risk in patients with OJ is unclear. In this study, we investigated NETs formation in OJ patients and the role of elevated unconjugated bilirubin (UCB) in inducing NETs, resulting in enhanced PCA and endothelial injury.
NETs of OJ patients and healthy controls were measured. NETs PCA was assessed via coagulation time (CT), fibrin formation and purified coagulation complex production assays. Visualization of NETs and mitochondrial reactive oxygen species (MitoROS) were performed with a fluorescence microscope. We further used confocal microscopy to quantify the exposure of phosphatidylserine (PS), fibrin strands and FVa/Xa on Human umbilical vein endothelial cells (HUVECs).
Assessment of NETs components levels revealed greater NETs production in OJ patients than in healthy controls. Importantly, OJ-NETs were responsible for enhanced PCA. UCB induced NETs formation via MitoROS accumulation and mitochondrial mobilization. HUVECs cocultured with OJ NETs lost their cell-cell junctions and consequently converted to a procoagulant phenotype. The PCA was attenuated by using DNase I alone or in combination with lactadherin.
Our results suggest that UCB-induced NETs play a prominent role in promoting the hypercoagulable and prothrombotic state in OJ patients. The increased MitoROS accumulation in neutrophils initiated NETosis. NETs are promising targets for indicating or improving coagulation disorders in OJ patients.

Traumatic brain injury (TBI) could highly induce coagulopathy through breaking the dynamic balance between coagulation and fibrinolysis systems, which may be a major contributor to the progressive secondary injury cascade that occurs after TBI. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibition is reported to exert neuroprotection in TBI, making it a potential regulatory target involved in TBI-induced coagulation disorder. PTEN level is controlled in a major way by E3 ligase-mediated degradation through the ubiquitin-proteasome system. The C terminus of Hsc70-interacting protein (CHIP) has been shown to regulate proteasomal degradation and ubiquitination level of PTEN. In the present study, CHIP was overexpressed and knocked down in mouse brain microvascular endothelial cells (bEnd.3) and tissues during the early phase of TBI. In vitro cell proliferation, cell apoptosis, migration capacity, and invasion capacity were determined. The changes of procoagulant and apoptosis molecules after TBI were also detected as well as the micrangium density and blood-brain barrier permeability after in vivo TBI. In vitro results demonstrated that CHIP overexpression facilitated bEnd.3 cell proliferation, migration, and invasion and downregulated cell apoptosis and the expressions of procoagulant molecules through promoting PTEN ubiquitination in a simulated TBI model with stretch-induced injury treatment. In vivo experiments also demonstrated that CHIP overexpression suppressed post-TBI apoptosis and procoagulant protein expressions, as well as increased microvessel density, reduced hemorrhagic injury, and blood-brain barrier permeability. These findings suggested that the upregulation of CHIP may attenuate apoptosis and procoagulant activity, facilitate brain repair, and thus exerts neuroprotective effects in TBI.