Thromboinflammation is a complex pathology associated with inflammation and coagulation. In cases of cardiovascular disease, in particular ischemia-reperfusion injury, thromboinflammation is a common complication. Increased understanding of thromboinflammation depends on an improved concept of the mechanisms of cells and proteins at the axis of coagulation and inflammation. Among these elements are activated protein C and platelets. This review summarizes the complex interactions of activated protein C and platelets regulating thromboinflammation in cardiovascular disease. By unraveling the pathways of platelets and APC in the inflammatory and coagulation cascades, this review summarizes the role of these vital mediators in the development and perpetuation of heart disease and the thromboinflammation-driven complications of cardiovascular disease. Furthermore, this review emphasizes the significance of the counteracting effects of platelets and APC and their combined role in disease states.

UNASSIGNED: Venous thromboembolic events have been reported in persons with hemophilia A who received emicizumab and activated prothrombin complex concentrate (APCC) concomitantly, but the relevant mechanism(s) remains unclear. We speculated that activated protein C (APC) and antithrombin (AT) resistance might be associated with these adverse events.
UNASSIGNED: To investigate APC and AT resistance in factor (F)VIII-deficient (FVIIIdef) plasma in the presence of emicizumab and APCC.
UNASSIGNED: In pooled normal plasma or FVIIIdef plasma samples mixed with emicizumab (50 μg/mL) and FVIII-bypassing agents, including recombinant FVIIa (2.2 μg/mL), APCC (1.3 IU/mL), or plasma-derived FVIIa/FX (1.5 μg/mL), the suppression effect of AT (0-2.4 μM) and APC (0-16 nM) was assessed by tissue factor-triggered thrombin generation assay. The APC effects in FVIIIdef plasma with the copresence of emicizumab, FII (1.3 μM), and/or FIXa (280 pM) were also examined.
UNASSIGNED: The AT resistance in emicizumab and each bypassing agent was not observed. Moreover, APC dose-dependent suppression effect was observed in pooled normal plasma or FVIIIdef plasma mixed with emicizumab and recombinant FVIIa or plasma-derived FVIIa/FX. However, APC-catalyzed inactivation had little effect on thrombin generation assay potential in FVIIIdef plasma spiked with emicizumab and APCC. The addition of FIXa to emicizumab in FVIIIdef plasma could lead to partial APC resistance. Furthermore, FVIIIdef plasma spiked with emicizumab, FIXa, and FII was markedly resistant to APC-mediated inactivation.
UNASSIGNED: FII and FIXa in APCCs were key clotting factors for APC resistance in FVIIIdef plasma supplemented with emicizumab and APCCs. The APC resistance in persons with hemophilia A receiving emicizumab and APCC may contribute to venous thromboembolic events.

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BACKGROUND: Inherited antithrombin, protein C, and protein S deficiency increase the risk of venous thromboembolism. The presence of defects can be identified by clinical laboratory assays. In most Chinese clinical laboratories, the screening tests for antithrombin, protein C, and protein S deficiency are their activity assays. Ensuring appropriate pre-analytical storage conditions for activity tests is essential. This study aimed to assess the effects of storage conditions on antithrombin, protein C, and protein S activity in frozen plasma.
METHODS: We collected the remaining plasma of 29 patients. The baseline of antithrombin, protein C, and protein S activity values were tested within 4 h. Then, each sample was sub-packaged into 4 EP tubes, and was stored at -20 °C for 3 days, -20 °C for 7 days, -80 °C for 3 days, and - 80 °C for 7 days, respectively. After thawing, samples were tested by two systems.
RESULTS: The percentage deviation of antithrombin and protein C activity assay was<10% compared with the initial values. Protein S activity showed a significant reduction in frozen plasma, with a deviation > 10%. Some samples, initially within the normal range, were classified as abnormal after freezing storage.
CONCLUSIONS: Our study indicated that antithrombin and protein C remain stable when stored at -20 °C or -80 °C in a week. We argued that Protein S activity is not stable in frozen plasma. The use of frozen-thawed plasma for PS activity assay may result in overdiagnosis of protein S deficiency.

SARS-CoV-2 can induce vascular dysfunction and thrombotic events in patients with severe COVID-19; however, the cellular and molecular mechanisms behind these effects remain largely unknown. In this study, we used a combination of experimental and in silico approaches to investigate the role of PC in vascular and thrombotic events in COVID-19. Single-cell RNA-sequencing data from patients with COVID-19 and healthy subjects were obtained from the publicly available Gene Expression Omnibus (GEO) repository. In addition, HUVECs were treated with inactive protein C before exposure to SARS-CoV-2 infection or a severe COVID-19 serum. An RT-qPCR array containing 84 related genes was used, and the candidate genes obtained were evaluated. Activated protein C levels were measured using an ELISA kit. We identified at the single-cell level the expression of several pro-inflammatory and pro-coagulation genes in endothelial cells from the patients with COVID-19. Furthermore, we demonstrated that exposure to SARS-CoV-2 promoted transcriptional changes in HUVECs that were partly reversed by the activated protein C pretreatment. We also observed that the serum of severe COVID-19 had a significant amount of activated protein C that could protect endothelial cells from serum-induced activation. In conclusion, activated protein C protects endothelial cells from pro-inflammatory and pro-coagulant effects during exposure to the SARS-CoV-2 virus.

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BACKGROUND:  Migraine is associated with several genetic or acquired comorbidities. Studies conducted in recent years emphasize that the frequency of thrombophilia is high in migraine, especially migraine with aura (MA). Similarly, the presence of white matter lesions (WMLs) on brain magnetic resonance imaging (MRI) scans has been associated with migraine for many years.
OBJECTIVE:  Based on the knowledge that both WMLs and thrombophilia variants are frequently observed in MA, we aimed to investigate whether there is a relationship between genetic thrombophilia and the presence of WMLs in these patients.
METHODS:  The levels of proteins S and C, antithrombin III activities, activated protein C (APC) resistance, antiphospholipid immunoglobulin G/immunoglobulin M (IgG/IgM) and anticardiolipin IgG/IgM antibodies were investigated in 66 MA patients between the ages of 18 and 49 years who presented no cardiovascular risk factors. The presence of WMLs and the Fazekas grade was determined from the brain magnetic resonance imaging (MRI) scans\' T2-weighted and fluid-attenuated inversion recovery (FLAIR) sequence taken from the patients. The rates of WMLs were compared in patients with and without thrombophilia.
RESULTS:  Thrombophilia was detected in 34.8% of the patients, and 27.3% were determined to have WMLs in brain MRI scans. The WMLs were detected in 23.3% of the patients without thrombophilia, in 34.8% of those with thrombophilia, and in 50% of the subjects with multiple thrombophilia disorders. Among the thrombophilia disorders, only APC resistance was significantly more common in patients with WMLs.
CONCLUSIONS:  The results of the present study showed that thrombophilia may be a mechanism that should be investigated in the etiology of increased WMLs in MA.
BACKGROUND:  La migraña se asocia con una serie de comorbilidades genéticas o adquiridas. Los estudios realizados en los últimos años destacan que la frecuencia de trombofilia es elevada en la migraña, especialmente en la migraña con aura (MA). De manera similar, la presencia de lesiones de la sustancia blanca (LSB) en las imágenes por resonancia magnética (RM) del cerebro se ha asociado con la migraña hace muchos años.
OBJECTIVE:  Con base en la información de que se suelen observar tanto LSB como variantes de la trombofilia en MA, nuestro objetivo fue investigar si existe una relación entre la trombofilia genética y la presencia de LSB en estos pacientes. MéTODOS:  Se investigaron los niveles de proteína S y de proteína C, actividades de antitrombina III, resistencia a la proteína C activada (PCA), anticuerpos antifosfolípidos inmunoglobulina G/inmunoglobulina M (IgG/IgM) y anticuerpos anticardiolipina IgG/IgM en 66 pacientes con MA entre 18 y 49 años que no presentaban factores de riesgo cardiovascular. Se determinaron la presencia de LSB y el grado de Fazekas a partir de imágenes por RM del cerebro en la secuencia ponderada en T2 y recuperación de la inversión atenuada de fluido (fluid-attenuated inversion recovery, FLAIR, en inglés) obtenidas de los pacientes. Se compararon las tasas de LSB en pacientes con y sin trombofilia.
RESULTS:  Se detectó trombofilia en el 34,8% de los pacientes y LSB en el 27,3%. Las LSB estuvieron presentes en el 23,3% de los pacientes sin trombofilia, en el 34,8% de los que tenían trombofilia, y en el 50% de los que tenían múltiples trastornos trombofílicos. La resistencia a la PCA fue significativamente más común en aquellos pacientes con LSB. CONCLUSIóN:  Los resultados del presente estudio mostraron que la trombofilia puede ser un mecanismo que debe investigarse en la etiología del aumento de LSB en MA.

Data on the pathophysiological mechanisms of hemostatic alterations in the thrombotic events that occur during Ramadan intermittent fasting (RIF), particularly in the natural coagulation inhibitors, are very limited. Thus, our objective was to evaluate the effect of RIF on the natural anticoagulants level, antithrombin, protein C, and total and free protein S (PS) in healthy participants. Participants were divided into two groups. Group I consisted of 29 healthy fasting participants whose blood samples were taken after 20 days of fasting. Group II included 40 healthy non-fasting participants whose blood samples were taken 2-4 weeks before the month of Ramadan. Coagulation screening tests including prothrombin time (PT), activated partial thromboplastin time (APTT) and plasma fibrinogen level, natural anticoagulants; antithrombin, protein C, free and total PS and C4 binding protein (C4BP) levels were evaluated in the two groups. High levels of total and free PS without change in antithrombin, protein C, and C4BP levels were noted in the fasting group as compared with non-fasting ones (p < 0.05). PT and APTT showed no difference between the two groups. However, the fibrinogen level was higher in the fasting group. In conclusion, RIF was found to be associated with improved anticoagulant activity in healthy participants, which may provide temporal physiological protection against the development of thrombosis in healthy fasting people.

Protein C (PC), a vitamin K-dependent serine protease zymogen in plasma, can be activated by thrombin-thrombomodulin(TM) complex, resulting in the formation of activated protein C (APC). APC functions to downregulate thrombin generation by inactivating active coagulation factors V(FVa) and VIII(FVIIIa). Deficiency in PC increases the risk of venous thromboembolism (VTE). We have identified two unrelated VTE patients with the same heterozygous mutation (c.1384 T > C, p.Ter462GlnextTer17) in PROC. To comprehend the role of this mutation in VTE development, we expressed recombinant PC-Ter462GlnextTer17 in mammalian cells and evaluated its characteristics using established coagulation assay systems. Functional studies revealed a significant impairment in the activation of the mutant by thrombin or thrombin-TM complex. Furthermore, APC-Ter462GlnextTer17 demonstrated diminished hydrolytic activity towards the chromogenic substrate S2366. APTT and FVa degradation assays showed that both the anticoagulant activity of the mutant protein was markedly impaired, regardless of whether protein S was present or absent. These results were further supported by a thrombin generation assay conducted using purified and plasma-based systems. In conclusion, the Ter462GlnextTer17 mutation introduces a novel tail at the C-terminus of PC, leading to impaired activity in both PC zymogen activation and APC\'s anticoagulant function. This impairment contributes to thrombosis in individuals carrying this heterozygous mutation and represents a genetic risk factor for VTE.