Protein C Research Articles

Abstract 4136847: Administration of the Recombinant Activated Protein C Rescues the Cardiac Vulnerability to Ischemic Insults in Aging through Modulating Inflammatory Response during Ischemia and Reperfusion

Introduction: Activated protein C (APC) is an endogenous vitamin-K-dependent serine protease and exhibits therapeutic potential for ischemic heart disease. The APC derivatives with anticoagulant and/or cytoprotective properties were produced through chemical engineering to characterize the functional domain of APC for limiting ischemia/reperfusion (I/R) injury. Hypothesis: The thromboinflammatory regulation by APC ameliorates ischemic insults caused by I/R through the receptor EPCR/PAR1. Methods: Young (3 months)/aged (24 months) wild type C57BL/6J mice and PAR1 R46Q/R46Q (block cleavage by APC) knock in mutant (3 months, C57BL/6J) mice were subjected to ligation of left anterior descending coronary artery with 45 min of ischemia. 5 min prior to reperfusion, wild type APC, cytoprotective selective APC-2Cys, or anticoagulant selective APC-E170A (0.2 μg/kg i.v.) was administered followed by 24 hr of reperfusion. The left ventricles of hearts were used for immunoblotting and flow cytometry analysis. Results: APC and APC-2Cys but not APC-E170A reduced I/R-induced myocardial infarction. Echocardiography showed that APC and APC-2Cys but not APC-E170A can rescue systolic dysfunction by I/R in young/aged WT but not in PAR1 R46Q/R46Q mice. Biochemical analysis showed that administration of APC and APC-2Cys inhibited activation of inflammation signaling c-Jun N-terminal protein kinase (JNK) and NF-κB by I/R and reduced mRNA levels of the proinflammatory cytokines TNFα and IL-6 in young/aged WT but not in PAR1 R46Q/R46Q hearts. The plasma cytokine array demonstrated that APC and APC2-Cys reduced potency of pro-inflammatory cytokines and apoptotic signaling cytokines during I/R. Moreover, I/R-triggered inflammasome NLRP3 was reduced by APC and APC-2Cys in young/aged WT but not in PAR1 R46Q/R46Q hearts. The flow cytometry showed that APC and APC-2Cys increased lymphocyte and decreased myeloid cell abundance and increased the recruitment of inflammation-mediating CD8a + T-cells during I/R in young/aged WT but not PAR1 R46Q/R46Q hearts, indicating that the APC/EPCR/PAR1 axis mediates APC's immune regulation under I/R. Moreover, APC and APC-2Cys reduced I/R-triggered macrophage abundance in young/aged WT hearts, although the proportion of M1 and M2 macrophages was unchanged. Conclusions: The cytoprotective domain of APC is critical for its cardioprotection against ischemic insults. EPCR/PAR1 receptor complex mediates APC's immune regulation in aging during I/R conditions.

Cure of Congenital Purpura Fulminans via Expression of Engineered Protein C Through Neonatal Genome Editing in Mice.

PC (protein C) is a plasma anticoagulant encoded by PROC; mutation in both PROC alleles results in neonatal purpura fulminans-a fatal systemic thrombotic disorder. In the present study, we aimed to develop a genome editing treatment to cure congenital PC deficiency. We generated an engineered APC (activated PC) to insert a furin-cleaving peptide sequence between light and heavy chains. The engineered PC was expressed in the liver of mice using an adeno-associated virus vector or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated genome editing using an adeno-associated virus vector in vivo. The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of PS (protein S) in vitro. The adeno-associated virus vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation FV (factor V) in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6J mice. The insertion of EGFP sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using adeno-associated virus vector resulted in the expression of EGFP in 7% of liver cells, mainly via homology-directed repair, in mice. Finally, we succeeded in improving the survival of PC-deficient mice by expressing the engineered PC via neonatal genome editing in vivo. These results suggest that the expression of engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency.

Prevalence and Risk Factors of Pulmonary Embolism in COPD Patients Complicated with Secondary Polycythemia.

This study aimed to establish the prevalence of pulmonary embolism (PE) in chronic obstructive pulmonary disease (COPD) patients with secondary polycythemia (SP) and explore the risk factors for PE in COPD patients with SP. We analyzed the prevalence of PE among COPD patients with SP who were hospitalized at Qinghai Provincial People's Hospital between January 2015 and December 2020. From January 2021 to January 2024, we enrolled patients into three groups (COPD+SP+PE, COPD+SP, and control) and performed laboratory measurements, biomarkers, echocardiography, and pulmonary function tests. Patients in the COPD+SP group received clinical treatment, and biomarkers were measured again seven days after treatment. The prevalence of PE in patients with COPD SP was 5.21%. We found that COPD+SP+PE group had significantly higher levels of erythrocyte distribution width (RDW), platelet volume distribution width (PDW), mean platelet volume (MPV), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), monocyte to large platelet ratio (MLPR), 5-hydroxytryptamine (5-HT), activated protein C (APC), urokinase-type plasminogen activator (u-PA), thrombomodulin (TM), interleukin-38 (IL-38), tissue factor (TF), and fractalkine (FKN) in contrast to COPD+SP group. Biomarkers, such as FKN, β-thromboglobulin (β-TG), APC, u-PA, TM, TF, and IL-38, were risk factors for COPD patients with SP who are complicated by PE. Clinical treatment significantly reduced the levels of β-TG, IL-38, APC, endothelin-1 (ET-1), u-PA, FKN, TM, 5-HT, and neutrophil extracellular traps (NETs) in patients with COPD+SP. PE incidence was significantly higher in patients with COPD and SP. In COPD patients with SP, routine joint detection of blood and cardiac markers, blood gas analysis, and pulmonary function tests can help to identify patients with PE. APC, u-PA, TF, FKN, TM, and IL-38 are risk factors for PE in patients with COPD and SP, and clinical treatment can effectively reduce this risk.

A Novel Murine Model Enabling rAAV8-PC Gene Therapy for Severe Protein C Deficiency.

Severe protein C deficiency (SPCD) is a rare inherited thrombotic disease associated with high morbidity and mortality. In the current study, we established a viable murine model of SPCD, enabling preclinical gene therapy studies. By creating SPCD mice with severe hemophilia A (PROC-/-/F8-), the multi-month survival of SPCD mice enabled the exploration of recombinant adeno-associated viral vector-PC (rAAV8-PC) gene therapy (GT). rAAV8- PC (1012 vg/kg of AAV8-PC) was injected via the tail vein into 6-8-week-old PROC-/-/F8- mice. Their plasma PC antigen levels (median of 714 ng/mL, range 166-2488 ng/mL) and activity (303.5 ± 59%) significantly increased to the normal range after GT compared to untreated control animals. PC's presence in the liver after GT was also confirmed by immunofluorescence staining. Our translational research results provide the first proof of concept that an infusion of rAAV8-PC increases PC antigen and activity in mice and may contribute to future GT in SPCD. Further basic research of SPCD mice with prolonged survival due to the rebalancing of this disorder using severe hemophilia A may provide essential data regarding PC's contribution to specific tissues' development, local PC generation, and its regulation in inflammatory conditions.

Unraveling the Molecular Pathogenesis of Protein C Deficiency-Associated VTE: Insights from Protein C Mutations C238G and R189W in Thai Patients.

Protein C (PC) deficiency is a well-established risk factor for thromboembolism (TE), commonly manifesting in pediatric patients. This study aimed to elucidate the pathogenic mechanisms of two novel PC mutations, C238G and R189W, identified in Thai children with both venous and arterial TE. The effects of wild-type (WT), C238G, and R189W PC variants were investigated through transient transfection of HEK293T cells. PC secretion levels were measured, and immunofluorescence analysis was performed to assess intracellular localization. ER stress-related gene expression and UPR activation were evaluated. Structural analysis was conducted to explore the significance of the C238 and R189W residue in PC functionality. The C238G mutation led to a severe 95% reduction in PC secretion, while R189W showed a 30% decrease compared with WT. Immunofluorescence revealed that C238G-PC was predominantly retained in the ER, indicating protein misfolding. C238G-expressing cells exhibited significant upregulation of ER stress-related genes and UPR activation. In contrast, R189W resulted in only a modest increase in UPR gene expression, suggesting a less pronounced impact on protein folding and secretion. Structural analysis demonstrated the critical role of the C238 residue in maintaining PC's disulfide bond and overall conformation. This study reveals distinct molecular mechanisms by which the C238G and R189W mutations contribute to PC deficiency and increased thrombotic risk. The findings emphasize the essential role of the C238 residue in preserving PC structure and secretion, enhancing the understanding of PC deficiency-associated TE in pediatric patients.

Thrombomodulin (p.Cys537Stop) is released from cells by an unusual membrane insertion/leakage mechanism

AbstractExpression of the thrombomodulin (TM) variant c.1611C>A (p.Cys537Stop) leads to the synthesis of a protein with no cytoplasmic tail and a transmembrane domain shortened by 3 amino acids (TM536). However, little is known regarding the release mechanism and properties of TM536. Using umbilical vein endothelial cells and peripheral blood-derived endothelial colony-forming cells from a heterozygous carrier of the TM536 variant as well as overexpression cell models, we demonstrated that TM536 is released from cells by an unusual mechanism. First, TM536 is inserted into the endoplasmic reticulum (ER) membrane, then, because of the low hydrophobicity of its intramembrane domain, it escapes from it and follows the conventional secretory pathway to be released into the extracellular compartment without the involvement of proteolysis. This particular secretion mechanism yields a soluble TM536, which is poorly modified by chondroitin sulfate glycosaminoglycan compared with conventionally secreted soluble forms of TM, and therefore has a suboptimal capacity to mediate thrombin-dependent activation of protein C (PC). We also showed that TM536 cellular trafficking was altered, with retention in the early secretory pathway and increased sensitivity to ER-associated degradation. As expected, activation of ER-associated degradation increased TM536 degradation and reduced its release. The expression of TM536 at the cell surface was low, and its distribution in lipid raft-like membrane microdomains was altered, resulting in low thrombin-dependent PC activation on the cell surface.

Decreased Protein C Pathway Activity in COVID-19 Compared to Non-COVID Sepsis: An Observational and Comparative Cohort Study.

Sepsis-associated coagulopathy increases risk of mortality. Impairment of the anticoagulant protein C (PC) pathway may contribute to the thrombotic phenotype in coronavirus disease 2019 (COVID-19) sepsis. This study assessed the functionality of this pathway in COVID-19 and non-COVID sepsis by measuring its key enzymes, thrombin and activated PC (APC). The study population included 30 patients with COVID-19, 47 patients with non-COVID sepsis, and 40 healthy controls. In healthy controls, coagulation activation and subsequent APC formation was induced by 15 µg/kg recombinant activated factor VII one hour before blood sampling. APC and thrombin in plasma were measured using oligonucleotide-based enzyme capture assays. The indirect thrombin markers prothrombin-fragment 1+2 (F1+2) and thrombin-antithrombin complex (TAT) were also measured. Compared with stimulated healthy controls, median thrombin, F1+2, and TAT levels were higher in patients with COVID-19 (up to 6-fold, p < 2 × 10-6) and non-COVID sepsis (up to 4.7-fold, p < 0.010). APC levels were 2.4-fold higher in patients with COVID-19 (7.44 pmol/L, p = 0.011) and 3.4-fold higher in non-COVID sepsis patients (10.45 pmol/L, p = 2 × 10-4) than in controls (3.08 pmol/L). Thrombin markers and APC showed correlation in both COVID-19 (r = 0.364-0.661) and non-COVID sepsis patients (r = 0.535-0.711). After adjustment for PC levels, median APC/thrombin, APC/F1+2, and APC/TAT ratios were 2-fold (p = 0.036), 6-fold (p = 3 × 10-7) and 3-fold (p = 8 × 10-4) lower in the COVID-19 group than in the non-COVID sepsis group, and the latter two were also lower in the COVID-19 group than in stimulated healthy controls. In conclusion, it was found that a comparatively lower anticoagulant APC response in COVID-19 patients as compared to non-COVID sepsis patients, potentially linked to endothelial dysfunction, contributes to the prothrombotic phenotype of COVID-19 sepsis.

Coagulation aberrations in adult patients with acute leukemia: A single center experience

Background and objective: Acute leukemia (AL) patients are at high risk for bleeding and thrombotic events due to the disruption of the normal hemostatic system associated with this disease. As a result of the lack of data on coagulopathy in AL patients in our locality, we conducted this study to assess the hemostatic parameters in patients with newly diagnosed AL at the time of presentation. Methods: This prospective cross-sectional study included 59 patients with de novo AL, along with 35healthy control subjects. The study was conducted at Nanakali Hemato-Oncology Center between September 2021 and May 2022. Coagulation markers, including prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen levels (FBG), D-dimer (D-Di), antithrombin (AT), protein C (PC), and protein S (PS) were measured for each patient and control. Results: The median age of the patients was 41 years, with a male to female ratio of 1.1:1. Thirty-eight (64.4%) patients had acute myeloid leukemia (AML) and 21 (35.6%) patients had acute lymphoblastic leukemia (ALL). D-Dilevels were significantly raised in both AML and ALL compared to controls. PT was significantly higher, and PC and AT were significantly lower in AML compared to ALL or control groups. The Bleeding incidence in AML was significantly higher than in ALL (65.8% versus 23.8%). Thrombosis occurred in two (3.4%) patients. Conclusion: Coagulation abnormalities were common in AL. The hemostatic derangement and bleeding at the time of presentation were more pronounced in patients with AML than in ALL.

Specific DNA aptamer-immobilized cryogel membranes as novel bioaffinity supports and their potential for the purification of activated protein C.

Activated protein C (APC), a serine protease produced from zymogen protein C (PC), is the key enzyme of the protein C pathway. APC has anticoagulant, anti-inflammatory, and cytoprotective features. APC has recently been shown to significantly reduce coagulation as well as mortality in patients with severe sepsis. Herein, we aimed to develop an affinity support material that allows the purification of plasma APC for the first time. In this research, a novel APC-specific DNA aptamer-based poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) (poly(HEMA-GMA/DNA-Apt)) macroporous cryogel membrane at different molar ratios was prepared using affinity binding method and their potential for purification and identification of APC was investigated. The DNA aptamer-immobilized cryogels were characterized to examine their structural and morphological properties. The effect of pH, initial concentration, temperature, ionic strength difference, and flow rate changes was examined. Selectivity studies were performed in the presence of APC and competitive proteins, and cryogel support materials were shown to have a very high affinity for APC. Adsorption capacity was found to be 89.02 mg/g. Finally, NaCl revealed efficiency for APC desorption and the reuse of cryogels was successfully tested for ten cycles.

Venous thromboembolism risk in adults with hereditary thrombophilia: a systematic review and meta-analysis.

This systematic review and meta-analysis assesses venous thromboembolism (VTE) risk in adults with hereditary thrombophilia, including Factor V Leiden (FVL) mutation, prothrombin G20210A (FII) mutation, compound heterozygosity, protein C (PC), protein S (PS), and antithrombin (AT) deficiency. Eligibility criteria included studies suitable for quantitative synthesis with extractable information on VTE risk in adults (> 15 years). There were no restrictions on VTE type, location, or occurrence. Two authors reviewed all studies and extracted data from 107 publications, encompassing 107,130 individuals (21,560 experiencing VTE). We used a random effects model and calculated odds ratios (ORs) with 95% confidence intervals (CIs). The highest risk was associated with homozygous FVL (OR 5.58, 95% CI 4.61-6.74), homozygous FII (OR 5.16, 95% CI 3.12-8.52), and compound heterozygosity (OR 4.64, 95% CI 2.25-9.58). In contrast, VTE risk was lowest for FVL heterozygosity (OR 2.97, 95% CI 2.41-3.67) and FII heterozygosity (OR 2.21, 95% CI 1.70-2.87), whereas PC (OR 3.23, 95% CI 2.05-5.08), PS (OR 3.01, 95% CI 2.26-4.02), and AT deficiency (OR 4.01, 95% CI 2.50-6.44) demonstrated an intermediate VTE risk. These results highlight an increased risk of venous thromboembolism in adults with hereditary thrombophilia. However, the risk for patients with PC, PS, and AT deficiency appears to be lower than previously stated, likely due to varying thrombogeneity of the underlying genetic mutations. Further research addressing this aspect of VTE risk in hereditary thrombophilia is imperative to improve patient management. TRIAL REGISTRATION: PROSPERO registration number CRD42022376757.

Molecular mechanisms and clinical manifestations of hereditary hemorrhagic telangiectasia

IntroductionHereditary Hemorrhagic Telangiectasia (HHT) is charactered by telangiectasia and arteriovenous malformations (AVMs). Recurrent visceral and mucocutaneous bleeding is frequently reported among HHT patients, while data on the prevalence of thrombosis remains limited. This study aims to describe the clinical manifestations and molecular biological characteristics of HHT patients. MethodsWe conducted a retrospective study at Ruijin Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. A total of 24 HHT patients, observed between January 2019 and December 2023, were included. We recorded the biological, clinical, and therapeutic events, with particular attention to bleeding and thrombotic events. Gene mutation analysis and blood constituent measurements were performed. ResultsThe prevalence of bleeding among all HHT patients was 100 %, while thrombotic events were noted in 41.70 % of cases. Hepatic arteriovenous malformations (HAVMs) were identified in six patients, pulmonary arteriovenous malformations (PAVMs) in five patients, and cerebral arteriovenous malformations (CAVMs) in one patient. For patients with thrombosis, the discontinuation rates were 23.08 % for antiplatelet therapy and 33.33 % for anticoagulant therapy due to the increased risk of bleeding. Genetic mutations related to HHT were present in 16 patients, with ACVRL1 (activin A receptor-like type 1) mutations being the most frequent at 41.67 %, followed by ENG (endoglin) mutations at 20.83 %, and GDF2 (growth differentiation factor 2) mutations at 4.17 %. The incidence of PAVMs was 75.00 % in HHT1 patients with ENG mutations and 20 % in HHT2 patients with ACVRL1 mutations, while HAVMs occurred in 0 % and 40.00 % of these groups, respectively. Patients were divided into non-AVMs and AVMs groups. Compared to normal controls, von Willebrand factor (vWF) activity was significantly increased in all HHT patients (149.10 % vs. 90.65 %, P < 0.001). In the non-AVMs group, the median level of stromal cell-derived factor-1 (SDF-1) was significantly elevated (124.31 pg/mL vs. 2413.57 pg/mL, P < 0.05), while vWF antigen levels were markedly higher in the AVMs group (165.30 % vs. 130.60 %, P = 0.021). Further grouping of HHT patients based on bleeding and thrombosis phenotypes revealed that those with thrombosis had significantly higher median percentages of schistocytes (3.50 % vs. 0 %, P = 0.002), ferritin concentrations (318.50 μg/L vs. 115.50 μg/L, P = 0.001), and lactate dehydrogenase (LDH) levels (437 U/L vs. 105 U/L, P < 0.001). There were no significant differences in the activity of vWF, protein C (PC), protein S (PS), and factor VIII (FVIII) between the two groups. ConclusionThis study highlighted the complex relationship between arteriovenous malformations and genetic mutations in HHT patients. A comprehensive assessment of bleeding and thrombosis risks should be conducted for each HHT patient, additionally, further clinical studies are needed to explore the risk factors for thrombosis and anticoagulant-related bleeding in HHT.

Heterozygous Prothrombin Mutation-Associated Thrombophilia.

Venous thromboembolism (VTE) is predisposed by thrombotic mutations in patients with hereditary thrombophilia. Although prothrombin deficiencies caused by homozygous or compound heterozygous mutations are associated with bleeding diathesis, rare cases have shown a correlation between heterozygous prothrombin mutations and thrombosis. We surveyed genetic variants involved in thrombosis and hemostasis in 347 patients with unprovoked VTE or having a positive family history of thrombosis. For patients identified with heterozygous prothrombin mutations, we conducted family investigations and performed a thrombin generation test (TGT) to elucidate the thrombotic risk. Novel mutants were expressed and subjected to functional assays to clarify the underlying thrombotic mechanisms. Heterozygous prothrombin mutations were identified in 3.5% of patients (12/347), including three novel mutations Phe382Ser, Phe382Leu, and Asp597Tyr found in one patient each, as well as previously reported Arg541Trp mutation in four patients and Arg596Gln mutation in five patients. A total of 42 mutation carriers were identified within the 12 pedigrees, among whom 64.3% (27/42) had experienced thrombotic events. TGT results demonstrated hypercoagulability for carriers of the five mutations, with Arg596Gln showing the highest thrombin generation potential followed by Arg541Trp. The Phe382-associated mutations severely impaired thrombomodulin-binding ability of thrombin, resulting in obviously reduced protein C (PC) activation. The Asp597Tyr mutation exhibited a mild reduction in both inactivation by antithrombin and PC activation reactions. The presence of heterozygous prothrombin mutations represents a potential genetic predisposition for VTE. All thrombosis-associated mutations potentiate coagulation activity by either conferring antithrombin resistance and/or impairing PC pathway activity.

Investigation of the relationship between thrombophilic disorders and brain white matter lesions in migraine with aura.

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. 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. 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. 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. 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.

A novel factor V compound heterozygous mutation associated with thrombosis (Y1961C; FV-Kanazawa, together with 1982_1983del)

BackgroundFactor (F)V is pivotal in both procoagulant and anticoagulant mechanisms. The present report describes a novel F5 mutation in a FV-deficient patient (FV activity, 6 IU/dL; FV antigen, 32 IU/dL) complicated by recurrent deep vein thrombosis. The patient demonstrated activated protein C resistance (APCR) with compound heterozygous mutations consisting of FV-Y1961C (FVKanazawa) and FV-1982_1983del. ObjectivesTo clarify thrombotic mechanisms associated with this FV abnormality. Methods and ResultsLevels of FV-1982_1983del were below the detection sensitivity in our expression experiments using human embryonic kidney 293T cells, and analyses were targeted, therefore, on the FV-Y1961C mutation. Activated partial thromboplastin time–based clotting assays demonstrated that FV-Y1961C exhibited APCR and that the reduced activated protein C (APC) susceptibility in FVa-Y1961C resulted in a marked depression of APC-catalyzed inactivation with delayed cleavage at Arg506 and little cleavage at Arg306 with or without protein S. The APC cofactor activity of FV-Y1961C in APC-catalyzed FVIIIa inactivation promoted by Arg336 cleavage in FVIII was impaired. The binding affinity of FVa-Y1961C to phospholipid membranes was reduced in reactions involving APC/protein S–catalyzed inactivation and in prothrombinase activity. Furthermore, the addition of FVa-Y1961C to plasma failed to inhibit tissue factor–induced procoagulant function. These characteristics were similar to those of FV-W1920R (FVNara) and FV-A2086D (FVBesançon). ConclusionWe identified a compound heterozygous FV-Y1961C mutation in the C1 domain representing a novel FV mutation (FVKanazawa) resulting in not only APCR due to impaired FVa susceptibility and FV cofactor activity for APC function but also impaired inhibition of tissue factor–induced procoagulant function. These defects in anticoagulant function associated with FV in FV-Y1961C contributed to a prothrombotic state.

A Compact Differential Dynamic Microscopy-based Device (cDDM): An Approach Tool for Early Detection of Hypercoagulable State in Transfusion-Dependent-β-Thalassemia Patients.

β-Thalassemia especially transfusion-dependent thalassemia (TDT) associates with a hypercoagulable state, which is the main cause of thromboembolic events (TEE). Plasma viscosity and rheological parameters could be essential markers for determining hypercoagulable state in β-thalassemia patients. The traditional methods for measuring viscosity are often limited by large sample volumes and are impractical for routine clinical monitoring. The compact differential dynamic microscopy-based device (cDDM), an optical microscopy for quantitative rheological assessment, was developed and applied for prognosis of the hypercoagulable state in β-TDT with and without splenectomy. The device was performed plasma viscosity measurement using low plasma volume (8 μL) and revealed a value as modulus of complex viscosity |η(ω)| in 7 min. We also parallelly demonstrated the correlation of the viscosity and related-coagulable parameters: complete blood count, prothrombin time (PT), activated partial thromboplastin time (APTT), protein C (PC), protein S (PS), CD62P and CD63 expression, and platelet aggregation test. The thalassemia plasma exhibited a higher value of |η(ω)| than healthy plasma, which can represent a different viscoelastic property among the groups. Even all related-coagulable parameters indicated hypercoagulable state in both nonsplenectomies and splenectomies β-TDT patients when compared to control, only high platelet numbers significantly correlated to high plasma viscosity in the splenectomy group. However, the other coagulable parameters have shown a trend of positive relationship with high plasma viscosity in all β-1thalassemia TDT patients. The relative results suggested that our device would be an approach tool for early detection of hypercoagulable state in transfusion-dependent-β-thalassemia patients, which can help to prevent TEE and the critical consequent-complications.

Use of a fluoroscopy‐based robotic‐assisted total hip arthroplasty system resulted in greater improvements in hip‐specific outcome measures at one‐year compared to a CT‐based robotic‐assisted system

AbstractBackgroundThe purpose of this study was to compare one‐year patient reported outcome measures between a novel fluoroscopy‐based robotic‐assisted (FL‐RTHA) system and an existing computerised tomography‐based robotic assisted (CT‐RTHA) system.MethodsA review of 85 consecutive FL‐RTHA and 125 consecutive CT‐RTHA was conducted. Outcomes included one‐year post‐operative Veterans RAND‐12 (VR‐12) Physical (PCS)/Mental (MCS), Hip Disability and Osteoarthritis Outcome (HOOS) Pain/Physical Function (PS)/Joint replacement, and University of California Los Angeles (UCLA) Activity scores.ResultsThe FL‐RTHA cohort had lower pre‐operative VR‐12 PCS, HOOS Pain, HOOS‐PS, HOOS‐JR, and UCLA Activity scores compared with patients in the CT‐RTHA cohort. The FL‐RTHA cohort reported greater improvements in HOOS‐PS scores (−41.54 vs. −36.55; p = 0.028) than the CT‐RTHA cohort. Both cohorts experienced similar rates of major post‐operative complications, and had similar radiographic outcomes.ConclusionsUse of the fluoroscopy‐based robotic system resulted in greater improvements in HOOS‐PS in one‐year relative to the CT‐based robotic technique.

PC Gene Affects Milk Production Traits in Dairy Cattle.

In previous work, we found that PC was differentially expressed in cows at different lactation stages. Thus, we deemed that PC may be a candidate gene affecting milk production traits in dairy cattle. In this study, we found the polymorphisms of PC by resequencing and verified their genetic associations with milk production traits by using an animal model in a cattle population. In total, we detected six single-nucleotide polymorphisms (SNPs) in PC. The single marker association analysis showed that all SNPs were significantly associated with the five milk production traits (p < 0.05). Additionally, we predicted that allele G of 29:g.44965658 in the 5' regulatory region created binding sites for TF GATA1 and verified that this allele inhibited the transcriptional activity of PC by the dual-luciferase reporter assay. In conclusion, we proved that PC had a prominent genetic effect on milk production traits, and six SNPs with prominent genetic effects could be used as markers for genomic selection (GS) in dairy cattle, which is beneficial for accelerating the improvement in milk yield and quality in Chinese Holstein cows.

COAGULATION AND ANTICOAGULATION PARAMETERS IN MULTIPLE SCLEROSIS PATIENTS WITH AND WITHOUT COVID-19

The aim. To investigate plasma levels of main coagulation and fibrinolytic factors in MS patients with and without COVID-19 history. Materials and methods. A total of 127 participants were enrolled in this study, including 97 MS patients and 30 healthy controls (HC). Patients with MS were divided into two groups: MS+Covid group (n=41) – patients with MS, who had a laboratory-verified diagnosis of COVID-19 in the past 3-6-month period and MS group (n=56) – patients with MS, who did not suffer from COVID-19 previously. Determination of plasma levels of prothrombin, plasminogen, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), protein C (PC), soluble thrombomodulin (TM) was performed by means of enzyme-linked immunosorbent assay. Spectrophotometric techniques were used to determine concentrations of fibrinogen, soluble fibrin monomeric complexes (SFMC) as well as plasminogen activity and inhibitory potential of α-2-antiplasmin. Results. The MS group was characterized by elevated levels of plasma prothrombin, fibrinogen, D-dimer, SFMC, soluble TM compared to HC, while PC concentration did not differ between MS and HC groups. Plasma plasminogen level as well as plasma level of the potential plasmin activity were significantly decreased in MS patients compared to HC group. The plasma tPA level was significantly reduced while plasma PAI-I level was significantly increased in MS patients compared to HC. Patients of MS group had an increased level of plasma α-2-antiplasmin activity compared with HC group. To note, most of studied parameters did not differ between two MS groups, except protein C, soluble thrombomodulin levels and plasma α-2-antiplasmin activity. Conclusions. The results of our study showed that MS patients have got altered hemostasis parameters; however, further study is necessary to find out the relationship between particular components of coagulation and fibrinolytic systems and pathophysiology of MS. Additionally, our findings demonstrated that a SARS-CoV-2 infection had a limited effect on hemostasis parameters in MS patients, causing changes in only a few parameters, including thrombomodulin and protein C levels as well as α-2-antiplasmin activity.

Activated protein C resistance in the copresence of emicizumab and activated prothrombin complex concentrates

BackgroundVenous 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. ObjectivesTo investigate APC and AT resistance in factor (F)VIII–deficient (FVIIIdef) plasma in the presence of emicizumab and APCC. MethodsIn 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. ResultsThe 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. ConclusionFII 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.

Limb Ischemic Necrosis Secondary to Microvascular Thrombosis: A Brief Historical Review.

Ischemic limb injury can be broadly classified into arterial (absent pulses) and venous/microvascular (detectable pulses); the latter can be divided into two overlapping disorders-venous limb gangrene (VLG) and symmetrical peripheral gangrene (SPG). Both VLG and SPG feature predominant acral (distal) extremity ischemic necrosis, although in some instances, concomitant nonacral ischemia/skin necrosis occurs. Historically, for coagulopathic disorders with prominent nonacral ischemic necrosis, clinician-scientists implicated depletion of natural anticoagulants, especially involving the protein C (PC) system. This historical review traces the recognition of natural anticoagulant depletion as a key feature of nonacral ischemic syndromes, such as classic warfarin-induced skin necrosis, neonatal purpura fulminans (PF), and meningococcemia-associated PF. However, only after several decades was it recognized that natural anticoagulant depletion is also a key feature of predominantly acral ischemic microthrombosis syndromes-VLG and SPG-even when accompanying nonacral thrombosis is not present. These acquired acral limb ischemic syndromes typically involve the triad of (a) disseminated intravascular coagulation, (b) natural anticoagulant depletion, and (c) a localizing explanation for microthrombosis occurring in one or more limbs, either deep vein thrombosis (helping to explain VLG) or circulatory shock (helping to explain SPG). In most cases of VLG or SPG there are one or more events that exacerbate natural anticoagulant depletion, such as warfarin therapy (e.g., warfarin-associated VLG complicating heparin-induced thrombocytopenia or cancer hypercoagulability) or acute ischemic hepatitis ("shock liver") as a proximate factor predisposing to severe depletion of hepatically synthesized natural anticoagulants (PC, antithrombin) in the setting of circulatory shock.