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.

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.

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.

OBJECTIVE: Many pieces of literature have reported that inherited and acquired thrombophilia might be a risk factor for recurrent implantation failure (RIF), however, most studies have only focused on RIF patients and not their male partners. We studied the possible association of paternal thrombophilia with RIF risk.
METHODS: Forty-two male partners aged 20-45 suffered from RIF compared with 42 males from couples with at least one successful pregnancy. All participants were investigated for thrombophilia markers.
RESULTS: The prevalence of coagulation Factor V activity was significantly higher in the case group (42.9%) than in the control group (16.7%) (p=0.008) (OR=3.75; 95% CI, 1.38, 10.12). The prevalence of protein C and protein S deficiencies in RIF patients were 4.8% and 2.4%, respectively, and 0% in the controls. The prevalence of antithrombin III (ATIII) deficiency was significantly higher in the case group (19%) than in the control group (2.4%) (p=0.01). None of MTHFR C677T and MTHFR A1298C were statistically significant between the two groups. Combined thrombophilia was 45.2% in the men of the RIF group when compared with the control, 14.2% (p=0.001) (OR = 4.95; 95% CI, 1.75-13.86).
CONCLUSIONS: Paternal thrombophilia may be related to recurrent implantation failure, so evaluation of this factor in RIF patients could be used to identify relevant risk groups and may help in the proper management of these cases to enhance the chance of implantation.

UNASSIGNED: Protein C (PC) is an anticoagulant that is encoded by the PROC gene. Validation for the function of PC was carried out in mouse models.
UNASSIGNED: In this study, autosomal recessive PC deficiency (PCD) was selected as the target, and the specific mutation site was chromosome 2 2q13-q14, PROC c.1198G>A (p.Gly400Ser) which targets G399S (GGT to AGC) in mouse models. To investigate the role of hereditary PC in mice models, we used CRISPR/Cas9 gene editing technology to create a mouse model with a genetic PCD mutation.
UNASSIGNED: The two F0 generation positive mice produced using the CRISPR/Cas9 gene editing technique were chimeras, and the mice in F1 and F2 generations were heterozygous. There was no phenotype of spontaneous bleeding or thrombosis in the heterozygous mice, but some of them were blind. Blood routine results showed no significant difference between the heterozygous mice and wild-type mice (P > 0.05). Prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) were prolonged in the heterozygous mice, while the level of fibrinogen content (FIB) decreased, suggesting secondary consumptive coagulation disease. The protein C activity of heterozygous mice was significantly lower than that of wild-type mice (P < 0.001), but there was no significant difference in protein C antigen levels (P > 0.05). H&E staining showed steatosis and hydrodegeneration in the liver of heterozygous mice. Necrosis and exfoliated epithelial cells could be observed in renal tubule lumen, forming cell or granular tubules. Hemosiderin deposition was found in the spleen along with splenic hemorrhage. Immunohistochemistry demonstrated significant fibrin deposition in the liver, spleen, and kidney of heterozygous mice.
UNASSIGNED: In this study, heterozygotes of the mouse model with a PC mutation were obtained. The function of PC was then validated in a mouse model through genotype, phenotype, and PC function analysis.

UNASSIGNED: β-thalassemia is a group of inherited blood disorders that affect the production of β-globin chains, leading to the reduction or absence of these chains. One of the complications observed in patients with β-thalassemia major (β-TM) is thrombosis, especially in those who receive frequent blood transfusions. This may be due to a decrease in the levels of the natural anticoagulants: protein C (PC), total protein S (PS), and antithrombin (AT).
UNASSIGNED: In this case-control study, patients with β-TM, who had received at least 20 packed cell transfusions during their lifetime, were included. Patients with other underlying diseases like bleeding or thrombotic disorders were excluded. Totally, 118 patients with β-TM and 120 healthy individuals were included.
UNASSIGNED: The mean level of PC and AT was significantly lower in patients with β-TM (48.2 ± 65.4 and 57.42 ± 13.6, respectively) compared to the control group (97.1 ± 21.46 and 81.79 ± 14.3, respectively), with P value of 0.001 and 0.01, respectively. Although the difference was not statistically significant (P = 0.1), a similar trend was observed for total PS (61.12 ± 21.12 for patients versus 72.2 ± 35.2 for the control group). Of note, the decrease in PC, AT, and total PS levels compared to the control group was 50.36%, 27.5%, and 15.34%, respectively.
UNASSIGNED: It seems that β-TM patients who receive prolonged blood transfusions frequently are at an increased risk of decreased in natural anticoagulants levels and therefore potentially are at risk of thrombosis.

UNASSIGNED: Sickle cell disease (SCD) is a hereditary hemoglobinopathy marked by hemolytic anemia and vaso-occlusive events (VOEs). Chronic endothelial activation, inflammation, and coagulation activation contribute to vascular congestion, VOEs, and end-organ damage. Coagulation proteases such as thrombin and activated protein C (APC) modulate inflammation and endothelial dysfunction by activating protease-activated receptor 1 (PAR1), a G-protein-coupled receptor. Thrombin cleaves PAR1 at Arg41, while APC cleaves PAR1 at Arg46, initiating either proinflammatory or cytoprotective signaling, respectively, a signaling conundrum known as biased agonism. Our prior research established the role of thrombin and PAR1 in vascular stasis in an SCD mouse model. However, the role of APC and APC-biased PAR1 signaling in thrombin generation, inflammation, and endothelial activation in SCD remains unexplored. Inhibition of APC in SCD mice increased thrombin generation, inflammation, and endothelial activation during both steady state and tumor necrosis factor α challenge. To dissect the individual contributions of thrombin-PAR1 and APC-PAR1 signaling, we used transgenic mice with point mutations at 2 PAR1 cleavage sites, ArgR41Gln (R41Q) imparting insensitivity to thrombin and Arg46Gln (R46Q) imparting insensitivity to APC. Sickle bone marrow chimeras expressing PAR1-R41Q exhibited reduced thrombo-inflammatory responses compared with wild type PAR1 or PAR1-R46Q mice. These findings highlight the potential benefit of reducing thrombin-dependent PAR1 activation while preserving APC-PAR1 signaling in SCD thromboinflammation. These results also suggest that pharmacological strategies promoting biased PAR1 signaling could effectively mitigate vascular complications associated with SCD.

UNASSIGNED: Recently, we have shown alterations in the anticoagulant response to recombinant activated factor VII (rFVIIa)-induced coagulation activation in patients with thrombophilia.
UNASSIGNED: This study aimed to extend this in vivo model to fibrinolysis biomarkers.
UNASSIGNED: This interventional in vivo study included 56 patients with thrombophilia and previous venous thromboembolism (VTE+), 38 without VTE (VTE-), and 35 healthy controls. Plasma levels of D-dimer, plasmin-α2-antiplasmin (PAP) complex, and plasminogen activator inhibitor-1 (PAI-1) were monitored for over 8 hours after rFVIIa infusion (15 μg/kg) along with thrombin markers and activated protein C (APC).
UNASSIGNED: Throughout cohorts, median PAP increased by 40% to 52% (P < 3.9 × 10-10) and PAI-1 decreased by 59% to 79% (P < 3.5 × 10-8). In contrast to thrombin-antithrombin (TAT) complex, which also increased temporarily (44% to 115%, P < 3.6 × 10-6), changes in PAP and PAI-1 did not reverse during the observation period. The area under the measurement-time curves (AUCs) of PAP and TAT, which are measures of plasmin and thrombin formation, respectively, were each greater in the VTE+ cohort than in healthy controls (median PAP-AUC = 0.48 vs 0.27 ng·h/L [P = .003], TAT-AUC = 0.12 vs 0.03 nmol·h/L [P = 2.5 × 10-4]) and were correlated with one another (r = 0.554). As evidenced by the respective AUCs, asymptomatic factor (F)V Leiden carriers showed less PAP formation (0.22 vs 0.41 ng·h/L, P = 9 × 10-4), more pronounced PAI-1 decline (0.10 vs 0.18 ng·h/L, P = .01), and increased APC formation (28.7 vs 15.4 pmol·h/L, P = .02) than those within the VTE+ group (n = 19 each).
UNASSIGNED: rFVIIa-induced thrombin formation is associated with fibrinolysis parameter changes outlasting the concomitant anticoagulant response. Both correlate with thrombosis history in FV Leiden and might help explain its variable clinical expressivity.