Protein C Research Articles

Selective inhibition of activated protein C anticoagulant activity protects against hemophilic arthropathy in mice

AbstractRecurrent spontaneous or trauma-related bleeding into joints in hemophilia leads to hemophilic arthropathy (HA), a debilitating joint disease. Treatment of HA consists of preventing joint bleeding by clotting factor replacement, and in extreme cases, orthopedic surgery. We recently showed that administration of endothelial cell protein C receptor (EPCR) blocking monoclonal antibodies (mAb) markedly reduced the severity of HA in factor VIII (FVIII)−/− mice. EPCR blocking inhibits activated protein C (APC) generation and EPCR-dependent APC signaling. The present study was aimed to define the role of inhibition of APC anticoagulant activity, APC signaling, or both in suppressing HA. FVIII−/− mice were treated with a single dose of isotype control mAb, MPC1609 mAb, that inhibits anticoagulant, and signaling properties of APC, or MAPC1591 mAb that only blocks the anticoagulant activity of APC. Joint bleeding was induced by needle puncture injury. HA was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections for synovial hypertrophy, macrophage infiltration, neoangiogenesis, cartilage degeneration, and chondrocyte apoptosis. No significant differences were observed between MPC1609 and MAPC1591 in inhibiting APC anticoagulant activity in vitro and equally effective in correcting acute bleeding induced by the saphenous vein incision in FVIII−/− mice. Administration of MAPC1591, and not MPC1609, markedly reduced the severity of HA. MAPC1591 inhibited joint bleed–induced inflammatory cytokine interleukin-6 expression and vascular leakage in joints, whereas MPC1609 had no significant effect. Our data show that an mAb that selectively inhibits APC's anticoagulant activity without compromising its cytoprotective signaling offers a therapeutic potential alternative to treat HA.

An evaluation of circulating activated TAFI in septic DIC: a case series and review of the literature

BackgroundAdministration of recombinant human soluble thrombomodulin (rTM) is often used in Japan to treat septic disseminated intravascular coagulation (DIC). Thrombin-activatable fibrinolysis inhibitor (TAFI) is a fibrinolysis inhibitor activated by the thrombin-thrombomodulin complex, however, it is unknown whether circulating activated TAFI is increased after rTM administration in patients with DIC. Furthermore, the relationship between TAFI activation and the prognosis of septic DIC is not defined yet.Case presentationWe report a series of 8 patient’s TAFI activation with septic DIC treated by rTM. We sought to investigate the effect of rTM on TAFI activation and the association of plasma activated TAFI (TAFIa/ai) levels with the prognosis of septic DIC.Using plasma samples from clinical studies conducted from May 2016–March 2017 on eight patients with septic DIC at Kagoshima University Hospital, we measured plasma levels of total TAFI, TAFIa/ai, thrombin-antithrombin complex (TAT), prothrombin fragment 1 + 2 (F1 + 2), soluble fibrin (SF), antithrombin (AT), protein C (PC), protein S (PS), and plasminogen activator inhibitor-1 (PAI-1) before and after intravenous rTM administration. Then, we evaluated the relationship of these marker levels to prognosis. The thrombin-rTM complex activated TAFI in vitro in plasma from a healthy volunteer. However, TAFIa/ai levels did not significantly increase over baseline in the septic DIC patients after intravenous rTM administration. Baseline TAFIa/ai levels in non-survivors were significantly higher than those in survivors.ConclusionsPlasma TAFIa/ai did not increase with rTM administration. Elevated baseline TAFIa/ai concentration may be a negative prognostic indicator in septic DIC. Larger studies are needed to confirm the in vivo effect of rTM on TAFI activation.

Classic Thrombophilias and Thrombotic Risk Among Middle-Aged and Older Adults: A Population-Based Cohort Study.

BackgroundFive classic thrombophilias have been recognized: factor V Leiden (rs6025), the prothrombin G20210A variant (rs1799963), and protein C, protein S, and antithrombin deficiencies. This study aimed to determine the thrombotic risk of classic thrombophilias in a cohort of middle‐aged and older adults.Methods and ResultsFactor V Leiden, prothrombin G20210A and protein‐coding variants in the PROC (protein C), PROS1 (protein S), and SERPINC1 (antithrombin) anticoagulant genes were determined in 29 387 subjects (born 1923–1950, 60% women) who participated in the Malmö Diet and Cancer study (1991–1996). The Human Gene Mutation Database was used to define 68 disease‐causing mutations. Patients were followed up from baseline until the first event of venous thromboembolism (VTE), death, or Dec 31, 2018. Carriership (n=908, 3.1%) for disease‐causing mutations in the PROC, PROS1, and SERPINC1 genes was associated with incident VTE: Hazard ratio (HR) was 1.6 (95% CI, 1.3–1.9). Variants not in Human Gene Mutation Database were not linked to VTE (HR, 1.1; 95% CI, 0.8–1.5). Heterozygosity for rs6025 and rs1799963 was associated with incident VTE: HR, 1.8 (95% CI, 1.6–2.0) and HR, 1.6 (95% CI, 1.3–2.0), respectively. The HR for carrying 1 classical thrombophilia variant was 1.7 (95% CI, 1.6–1.9). HR was 3.9 (95% CI, 3.1–5.0) for carriers of ≥2 thrombophilia variants.ConclusionsThe 5 classic thrombophilias are associated with a dose‐graded risk of VTE in middle‐aged and older adults. Disease‐causing variants in the PROC, PROS1, and SERPINC1 genes were more common than the rs1799963 variant but the conferred genetic risk was comparable with the rs6025 and rs1799963 variants.

Reference interval of antithrombin, protein C, and protein S activities in healthy adults in Iran, the effect of age, sex, oral contraceptive intake, and menopause.

Antithrombin (AT), protein C (PC), and protein S (PS) are natural anticoagulant proteins that deficiency in each of them is associated with an increased risk of venous thromboembolism.The overlapping of plasma levels of AT, PC, and PS between healthy individuals and heterozygote carriers poses significant challenges in precise diagnosis. This study aimed to evaluate the effect of most influencing variables on plasma levels of these proteins and propose specific reference intervals to improve the interpretation of the laboratory results. This study was conducted on 1464 individuals who were referred to Massoud medical laboratory, Tehran, Iran, from 2019 to 2020. AT and PC were measured through chromogenic assay and PS plasma level with the clot-based assay. A multivariable linear regression model was performed to evaluate the effect of sex, age, oral contraceptive (OCP) intake, and menopause state. Normal deviate z value was used for different subgroups to justify the need for a separate reference interval. 1200 verified healthy individuals (434males and 766 females), aged between 18 and 69years were included in the study. The mean±SD age of the participants was 39.78±11.79years. The age-related effects for AT were found in men. In females, increasing age was associated with a rise in AT, PC, and PS plasma levels. No sex difference was found in AT plasma level. OCP-taking is associated with a decrease in AT and an increase in PC plasma levels. This is the largest study ever conducted on healthy individuals in the Iranian population, using specific reference interval results in accurate diagnosis of true AT, PC, and PS deficiency.

Prevention and Early Detection of Acute Kidney Injury in Intensive Care Unit: A Systematic Review

Acute Kidney Injury (AKI) is often associated with Intensive Care Unit (ICU) admission and results in a higher mortality rate around the world. The AKI cases have been rising for the past decade alongside the mortality rate. Literature has shown that early detection helps in overcoming the problem. This systematic review aims to identify the identify the relevant literature and to summarize the prevention and early detection of AKI in the ICU setting to reduce the morbidity and mortality rate of patients. A total of 20 articles identified in this review. This systematic review also is in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Relevant studies were identified based on the inclusion criteria with language filters but were limited to articles published from the year 2010 to 2020. Two reviewers independently conducted the quality assessment, data extraction, and analysis on all included studies. The result indicates there are several biomarkers which are helpful in detecting AKI such as Neutrophil Gelatinase-Associated Lipocalin (NGAL), Interleukin-18 (IL-18), Tissue Inhibitor of Metalloproteinase 2 (TIMP-2), Insulin-Like Growth Factor Binding Protein 7 (IGFBP-7), Protein C (PC), and soluble thrombomodulin (sTM). This review also addressed nursing responsibilities for early detection and prevention of AKI. Thus, severe cases of AKI and mortality cases associated with AKI can be minimized with better predictive and proper management.

Differential impact of tamoxifen and aromatase inhibitors on thrombin generation: the prospective HEMOBREAST cohort

AbstractTamoxifen and aromatase inhibitors (AIs) are potent antitumoral agents against breast cancer. Tamoxifen increases the risk of venous thromboembolism (VTE), but the influence of AIs on the risk of VTE remains unclear. To inform clinical decisions, we evaluated associations of tamoxifen or AIs with changes of surrogate hemostatic biomarkers. This prospective cohort included 107 women with localized breast cancer starting tamoxifen (n = 42) or an AI (n = 65). Thrombin generation (CAT) its sensitivity to thrombomodulin (TM) or activated protein C (APC), and specific coagulation parameters, were measured before and 10-16 weeks after initiation of treatmen Compared with baseline, endogenous thrombin potential and thrombin peak increased in tamoxifen users (+86 nM × min; 95% confidence interval [CI], 30-142; and +33 nM; 95% CI, 21-45) but not in AI users (n = 65; +44 nM × min; 95% CI, −4 to 93; and +7 nM; 95% CI, −3 to 17). Normalized TM sensitivity ratios increased with tamoxifen (+0.26; 95% CI, 0.19-0.33y) but not with AI (+0.02; 95% CI, −0.03 to 0.07). Plasma levels of fibrinogen, antithrombin, protein C, and Tissue Factor Pathway Inhibitor decreased, and free protein S increased with tamoxifen but not with AIs. The observed shift toward increased coagulability associated with tamoxifen is in line with its known increased risk of VTE. In contrast, AIs do not appear to impact hemostasis, suggesting a lack of associated VTE risk. The trial was registered at www.clinicaltrials.gov as #NCT03381963.

Activity of anticoagulant proteins on the polymer-coated and heparin-coated membranes in an extracorporeal circulation circuit.

We performed in vitro experiments using whole human blood without anticoagulants to clarify the activity of anticoagulant proteins on membranes coated with acrylate-copolymer (ACP) with a hydrophilic blood-contacting layer compared to those coated by immobilizing heparin (IHP) in extracorporeal circulation. Whole human blood from healthy volunteers was recirculated in two types of experimental circuits with an ACP-coated reservoir and tubes and an ACP-coated or IHP-coated membrane. To compare the fluctuation of anticoagulant proteins, the circuit pressure at the inlet and outlet of the membrane was measured every 5min; antithrombin antigen (ATQ), antithrombin activity, protein-C quantitation (PCQ), protein-C activity, protein-S free antigen (PSQ), and protein-S activity were measured at 0, 30, 60, 120, and 180min in each experiment (n = 5). The time taken to achieve high circuit pressure (> 300mmHg) at the inlet of the membrane was significantly shorter in the ACP-coated membrane circuit (28 ± 2.7min) than in the IHP-coated membrane circuit (54 ± 24min); however, the ATQ, PCQ, and PSQ at 180min of recirculation were significantly higher in the former than in the latter (all p < .05). ACP-coated membranes can prevent the consumption of anticoagulant proteins but cannot delay circuit thrombogenicity compared to IHP-coated membranes. Considering patient care during the post-extracorporeal circulation period, the use of ACP coating, which can preserve anticoagulant protein, is better in extracorporeal circulation circuits.

Effects of direct oral anticoagulants dabigatran and rivaroxaban on the blood coagulation function in rabbits.

The aim of this study was to explore the effects of dabigatran and rivaroxaban on the activities of various coagulation factors. To achieve that, 60 rabbits were randomly divided into experimental groups that received different doses of dabigatran or rivaroxaban. The effects of dabigatran and rivaroxaban on the activities of FII, FV, FVIII, FX, and activated protein C (APC) were analyzed. In the dabigatran groups, activated partial thromboplastin time and thromboplastin time (TT) were prolonged after drug administration, and the activities of FII, FV, FVIII, and FX were inhibited as the drug concentration increased. Low doses of dabigatran inhibited APC activity. In the rivaroxaban groups, APTT and TT were not significantly prolonged after drug administration. In contrast, the high-dose rivaroxaban group exhibited prolonged PT, and the degree of inhibition of the activities of FII, FV, FVIII, and FX increased as the drug concentration increased. Rivaroxaban had no significant effect on APC activity regardless of dosage. As the drug concentration increased, both NOACs had more significant inhibitory effects on the activities of FII, FV, FVIII, and FX. Low concentrations of dabigatran generated an inhibitory effect on APC activity, while high concentrations of dabigatran had no significant effect. Rivaroxaban had no significant effect on APC activity.

Reference intervals for coagulation parameters in non-pregnant and pregnant women

Established reference intervals (RIs) of coagulation parameters generally based on the general population are not applicable to specific women. In order to accurately evaluate the coagulation status of non-pregnant women and pregnant women, specific RIs should be established. Our study recruited 465 non-pregnant women and 1972 pregnant women aged 20–45 years. Eight tests including antithrombin (AT), protein C (PC), free protein S (fPS), lupus anticoagulant (LA), D-dimer, fibrin/fibrinogen degradation products (FDP), coagulation factor VII (FVII), and factor VIII (FVIII) were performed on ACL TOP automated coagulation instrument. The RIs for these tests were established in non-pregnant and pregnant women at different gestational weeks. Compared to the non-pregnant group, the medians of AT and fPS were lower, while the medians of PC, LA normalized ratio, D-dimer, FDP, FVII, and FVIII were higher. During pregnancy, AT and fPS activity showed a decreasing trend, with the increase of gestational age. PC activity, LA normalized ratio, D-dimer concentrations, FDP concentrations, FVII, and FVIII activity presented an increasing trend, with the increase of gestational age. The non-pregnant women-specific RIs and the gestational age-specific RIs of AT, PC, fPS, LA normalized ratio, D-dimer, FDP, FVII, and FVIII needed to be established for accurate clinical diagnoses.

The prognostic utility of protein C as a biomarker for adult sepsis: a systematic review and meta-analysis

BackgroundSepsis, the dysregulated host response to infection, triggers abnormal pro-coagulant and pro-inflammatory host responses. Limitations in early disease intervention highlight the need for effective diagnostic and prognostic biomarkers. Protein C’s role as an anticoagulant and anti-inflammatory molecule makes it an appealing target for sepsis biomarker studies. This meta-analysis aims to assess the diagnostic and prognostic value of protein C (PC) as a biomarker for adult sepsis.MethodsWe searched MEDLINE, PubMed, EMBASE, CINAHL and Cochrane Library from database inception to September 12, 2021. We included prospective observational studies of (1) adult patients (> 17) with sepsis or suspicion of sepsis that; (2) measured PC levels with 24 h of study admission with; and (3) the goal of examining PC as a diagnostic or prognostic biomarker. Two authors screened articles and conducted risk of bias (RoB) assessment, using the Quality in Prognosis Studies (QUIPS) and the Quality Assessment in Diagnostic Studies-2 (QUADAS-2) tools. If sufficient data were available, meta-analysis was conducted to estimate the standardized mean difference (SMD) between patient populations.ResultsTwelve studies were included, and 8 were synthesized for meta-analysis. Pooled analysis demonstrated moderate certainty of evidence that PC levels were less reduced in sepsis survivors compared to non-survivors (6 studies, 741 patients, SMD = 0.52, 95% CI 0.24–0.81, p = 0.0003, I2 = 55%), and low certainty of evidence that PC levels were less reduced in septic patients without disseminated intravascular coagulation (DIC) compared to those with DIC (3 studies, 644 patients, SMD = 0.97, 95% CI 0.62–1.32, p < 0.00001, I2 = 67%). PC could not be evaluated as a diagnostic tool due to heterogeneous control populations between studies.Conclusion and relevanceOur review demonstrates that PC levels were significantly higher in sepsis survivors compared to non-survivors and patients with sepsis but not disseminated intravascular coagulation (DIC). Our evaluation is limited by high RoB in included studies and poor reporting of the sensitivity and specificity of PC as a sepsis biomarker. Future studies are needed to determine the sensitivity and specificity of PC to identify its clinical significance as a biomarker for early sepsis recognition.Trial Registration PROSPERO registration number: CRD42021229786. The study protocol was published in BMJ Open.

Stability of coagulation parameters in plasma samples at room temperature after one freeze/thaw cycle.

Sample freezing is a part of routine laboratory tasks because some coagulation parameters are analysed in batches to optimize reagent consumption. The coagulation parameter stability in fresh and frozen samples has been described, but data are scarcer after thawing. This study objective was to determine the stability of the main coagulation parameters (from blood withdrawn on siliconized CTAD tubes and double-centrifuged) after one freeze/thaw cycle to generate procedures for appropriate handling, storage and testing. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, D-dimers, clotting factors (F), protein C, protein S, antithrombin, lupus anticoagulant (LA)-sensitive aPTT and diluted-Russel's viper venom time (dRVVT) were assessed in 60 plasma samples (n=30, normal range and n=30, outside the normal range). Thirty samples from anticoagulated patients [unfractionated heparin (UFH), low-molecular weight heparin (LMWH), apixaban or rivaroxaban] were assessed using specific anticoagulant assays. Frozen samples were thawed, and assays were performed at 15min, 2, 4 and 6h after thawing. The coagulation parameter stability was assessed with the method of rejection limit. After thawing, aPTT, PT, fibrinogen, D-dimers, FII, FV, FX, FIX, FXI, FXII, PC and UFH anti-Xa activity remained stable for at least 6h, FVII for 5h, PS, AT, dRVVT screen assay and LMWH anti-Xa activity for 4h, and LA-sensitive aPTT and apixaban-specific anti-Xa activity for 3h. FVIII, dRVVT confirm assay and rivaroxaban specific anti-Xa activity were stable for 2h. These results suggest that sample stability for some haemostasis assays is limited after thawing.

Activated Protein C Protects against Murine Contact Dermatitis by Suppressing Protease-Activated Receptor 2.

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with excessive inflammation and defective skin barrier function. Activated protein C (APC) is a natural anticoagulant with anti-inflammatory and barrier protective functions. However, the effect of APC on AD and its engagement with protease activated receptor (PAR)1 and PAR2 are unknown. Methods: Contact hypersensitivity (CHS), a model for human AD, was induced in PAR1 knockout (KO), PAR2KO and matched wild type (WT) mice using 2,4-dinitrofluorobenzene (DNFB). Recombinant human APC was administered into these mice as preventative or therapeutic treatment. The effect of APC and PAR1KO or PARKO on CHS was assessed via measurement of ear thickness, skin histologic changes, inflammatory cytokine levels, Th cell phenotypes and keratinocyte function. Results: Compared to WT, PAR2KO but not PAR1KO mice displayed less severe CHS when assessed by ear thickness; PAR1KO CHS skin had less mast cells, lower levels of IFN-γ, IL-4, IL-17 and IL-22, and higher levels of IL-1β, IL-6 and TGF-β1, whereas PAR2KO CHS skin only contained lower levels of IL-22 and IgE. Both PAR1KO and PAR2KO spleen cells had less Th1/Th17/Th22/Treg cells. In normal skin, PAR1 was present at the stratum granulosum and spinosum, whereas PAR2 at the upper layers of the epidermis. In CHS, however, the expression of PAR1 and PAR2 were increased and spread to the whole epidermis. In vitro, compared to WT cells, PAR1KO keratinocytes grew much slower, had a lower survival rate and higher para permeability, while PAR2KO cells grew faster, were resistant to apoptosis and para permeability. APC inhibited CHS as a therapeutic but not as a preventative treatment only in WT and PAR1KO mice. APC therapy reduced skin inflammation, suppressed epidermal PAR2 expression, promoted keratinocyte growth, survival, and barrier function in both WT and PAR1KO cells, but not in PAR2KO cells. Conclusions: APC therapy can mitigate CHS. Although APC acts through both PAR1 and PAR2 to regulate Th and mast cells, suppression of clinical disease in mice is achieved mainly via inhibition of PAR2 alone. Thus, APC may confer broad therapeutic benefits as a disease-modifying treatment for AD.

Clinical and genetic features of Chinese pediatric patients with severe congenital protein C deficiency who first presented with purpura fulminans: A case series study and literature review

IntroductionPurpura fulminans (PF) is a hematological emergency that can be caused by severe congenital protein C (PC) deficiency. It has been rarely reported in the Chinese population. We aimed to characterize the clinical and genetic features of Chinese pediatric patients with severe congenital PC deficiency who first presented with PF. Materials and methodsTwelve pediatric patients were diagnosed with severe congenital PC deficiency with PF, which was diagnosed based on our hospital records and previous reports from 1988 to July 2021 in China. We evaluated the clinical and genetic features of these patients. ResultsNine patients (9/12, 75%) had onsets that were observed within the first 48 h after birth. Six patients had a family history of thromboembolism. There was no consanguinity. Other symptoms were intracranial thrombosis or hemorrhage (4, 33.3%), ocular lesions (2, 16.7%), gastrointestinal hemorrhage (2, 16.7%) and kidney infarction before birth (1, 8.3%). All but one of the patients (one case not detected) had a plasma PC activity of <10%. The genetic study indicated that in the eight patients with inherited PC deficiency, two were homozygous, five were compound heterozygous and one was heterozygous for PC deficiency. ConclusionThis is the first and largest case series of Chinese pediatric patients with severe congenital PC deficiency who first presented with PF. It has been shown that treatment with both fresh frozen plasma and anticoagulants is recommended when PC concentrate is not easily available, especially in developing countries.

Clinical phenotype and gene mutation analysis of 12 patients with hereditary protein C deficiency in different families

目的探讨来自不同家系12例遗传性蛋白C（PC）缺陷症先证者的基因突变类型与临床特征。方法采用发色底物法检测血浆PC活性，酶联免疫吸附法检测PC抗原含量。采用PCR直接测序法分析先证者PROC基因9个外显子及其侧翼序列，对发现的疑似突变用反向（缺失突变用克隆）测序予以验证。结果12例先证者的PC活性均明显下降（18％～55％），其中10例先证者的PC抗原水平显著降低（13％～58％）。共发现11种PROC基因突变，其中c.383G>A（p.Gly128Asp）、c.997G>A（p.Ala291Thr）、c.1318C>T（p.Arg398Cys）和c.532G>C（p.Leu278Pro）4种杂合突变为首次发现；6种突变发生在丝氨酸蛋白酶结构域、4种发生在表皮生长因子同源区域（EGF）、1种突变在EGF和丝氨酸蛋白酶结构域之间的激活肽区域；缺失突变（p.Met364Trp fsX15和p.Lys192del）2种，其余为错义突变。有3例无亲缘关系的先证者检出p.Phe181Val和p.Arg189Trp纯合或杂合突变。所有基因突变可能来自先证者的父亲和（或）母亲，其中2个家系存在近亲婚配。先证者有9例出现静脉血栓形成、2例有不良妊娠表现、1例出现紫癜。结论PROC基因缺陷导致的PC缺陷症患者易发生静脉血栓形成，尤其当同时存在其他易栓因素时。

Protein C system in preterm babies with chronic lung disease: Prospective study.

Chronic lung disease (CLD) is a major neonatal pulmonary disorder associated with inflammation. Recent studies have shown that protein C anticoagulant pathways, such as those for protein C (PC), protein S (PS), and thrombomodulin (TM), could be useful indices for reflecting pulmonary injury. However, the involvement of these factors in preterm infants with very low birthweight (VLBW) who have developed CLD remains to be investigated. Here, we investigated whether PC pathway-related factors could predict the development of CLD in preterm infants with VLBW. We collected plasma samples from 26 preterm infants with VLBW (13 each from those with and without CLD) at the time of birth and measured TM, PC, and PS levels in their plasmas. We analyzed prospectively the relationship between these factors in infants with and without CLD. There were significant differences in gestational age, birthweight, Apgar score (5 min), and duration of mechanical ventilation between the CLD and non-CLD groups. No significant differences in the PC and PS levels at birth were observed between the two groups, whereas the TM levels in the CLD group were significantly higher than those in the non-CLD group (P = 0.013). The TM levels correlated with gestational age and duration of mechanical ventilation. However, covariance analysis demonstrated that gestational age was significantly associated with TM levels, and consequently, development of CLD was not associated with TM level at birth. Thrombomodulin, PC, and PS levels at birth could not predict the development of CLD in preterm infants with VLBW.

Significance of altered anticoagulant proteins and D-dimer in cirrhotic portal vein thrombosis: relation to the degree of liver dysfunction.

Portal vein thrombosis (PVT) is a well-known consequence of cirrhosis. Its pathophysiology is complex, with possible downstream hepatic decompensation. This study was conducted to describe the changes of protein C (PC), protein S (PS) and D-dimer blood levels associated with PVT formation in cirrhosis and the relation to the degree of liver dysfunction. This was a case-control study that included 50 cirrhotic patients who presented with acute de novo non-malignant PVT and 50 cirrhotic patients without PVT as a control group. The severity of liver disease was classified as per the Child-Turcotte-Pugh (CTP) score. Doppler ultrasonography identified acute portal vein occlusion, and dynamic contrast-enhanced computed tomography confirmed the extent and nature of PVT. Blood PC, PS and D-dimer levels were measured using enzyme-linked immunosorbent assay. PC and PS levels were significantly lower, and the D-dimer level was significantly higher, in cirrhotic patients with PVT compared to the control group. PC and PS levels were significantly decreased in patients with higher CTP score of both groups. The D-dimer level did not vary significantly with the degree of liver dysfunction in patients of either group. PC, PS and D-dimer at the cut-off points of ≤ 77 IU/dl, ≤ 63 IU/dl, and > 300 ng/ml, respectively, significantly suggested PVT occurrence. Alteration of the anticoagulant proteins and D-dimer contributed to PVT formation in cirrhotic patients and could help stratify the degree of liver dysfunction. Blood level of these hemostatic proteins could be incorporated into a probability score for early diagnosis and treatment of PVT in cirrhosis.

Comparative Effectiveness of Peg-Asparaginase and L-Asparaginase on Coagulation Markers Among Pediatric Patients with Acute Lymphoblastic Leukemia.

Asparaginase (ASP), a chemotherapy component in the acute lymphoblastic leukemia (ALL) treatment, could impair normal coagulation state. Usually, a decline in the levels of several coagulation factors occurs which ultimately could lead to thrombotic events and abnormal coagulation tests. In this study, we aimed to compare the effects of two different subtypes of ASP, pegylated asparaginase (PEG-ASP) and L-asparaginase (L-ASP) on coagulation markers and test among 40 pediatric patients with ALL. In this cohort study a total of 40 pediatric patients with newly diagnosed ALL were enrolled and divided into two groups by simple randomization. In group A, 20 patients received PEG-ASP while in group B, 20 patients received L-ASP during the induction treatment. Coagulation markers included prothrombin time (PT), partial thrombin time (PTT), protein-C (Pr-C), protein-S (Pr-S), and antithrombin III (ATIII) and were assessed before start and after of induction chemotherapy. Coagulation profile including PT, PTT, INR, Pr-C, Pr-S, and ATIII before start of treatment were not statistically significant between the two groups. Anticoagulant factors decreased significantly after consuming both drugs. Tests for PT and INR of those who took L-ASP decreased significantly. Overall, when comparing the changes of the six studied factors, ATIII and Pr-C were the significant factors which were different between groups. ASP has a negative effect on anticoagulant factors including (ATIII, Pr-C, Pr-S). Additionally, the negative effect of L-ASP on anticoagulant factors was more prominent than PEG-ASP. Therefore, the risk of thrombosis probably was negligible in PEG-ASP in comparison with L-ASP.

Protein S: a Central Regulator of Blood Coagulation.

Protein S is a central regulator of coagulation as it critically participates in down-regulation of both extrinsic and intrinsic pathways of the coagulation cascade. In this review, we aim to provide an update on protein S and its anticoagulant functions as a central hemostatic regulator. Electronic databases including, Google, Google Scholar, PMC, PubMed, Science Direct, and Scopus were rigorously searched using the terms protein S, hemostasis, natural anticoagulants, regulators of coagulation, and coagulation inhibitors for the completion of this descriptive review. Literature review shows that protein S is a potent cofactor for activated protein C (APC) in the regulation of the intrinsic pathway and a cofactor for tissue factor pathway inhibitor (TFPI) in the regulation of the extrinsic pathway. The strong association between protein S deficiency either hereditary or acquired and increased risk for venous thrombosis indicates the important and central role of protein S in controlling the initiation and propagation phase of coagulation cascade and that protein S is an important determinant for optimal activity of both APC and TFPI in coagulation regulation. Available evidence suggests that the role of protein S in the down-regulation of blood coagulation is mainly mediated through its high affinity binding to negatively charged phospholipid surfaces. This high affinity binding to negatively charged phospholipids helps bring the anticoagulant proteins to the membranes, resulting in efficient and targeted regulation of coagulation. In the shade of current COVID-19 pandemic, protein S deficiency has been found to be a leading cause of thrombotic complications associated with COVID-19.

Influence of DOACS and DOAC-REMOVE® on coagulation assays during thrombophilia testing in DOAC-treated patients

Background/Aim. Direct oral anticoagulants (DOACs) administration significantly interferes with coagulation as-says. The aim of the study was to evaluate the effect of DOACs and DOAC-Remove? on coagulation assays during thrombophilia testing. Methods. The study was carried out from January 2019 to the end of June 2020. It included 30 DOAC-treated patients, 14 females and 16 males aged 23 to 63 (median age 47.6 years), tested for thrombophilia due to venous thromboembolism (VTE). Thrombophilia testing was performed using DOAC-Remove? tablets (activated charcoal). The results before and after DOAC-Remove? were compared. Results. Positive lupus anticoagulant (LA) results were observed in 20% apixaban, 100% dabigatran, and 70% rivaroxaban-treated patients, while in samples after DOAC-Remove?, the LA positivity was observed only in one from the apix-aban group. Before DOAC-Remove?, the activated protein C (APC) resistance (APC-R) was measurable in 40% dabigatran and 80% rivaroxaban-treated patients, while, after using DOAC-Remove?, the APC-R was measurable in all cases. Comparing the results obtained from the samples before and after DOAC-Remove?, a difference was noted in relation to all dilute Russell?s viper venom time (dRVVT) coagulation tests, except for the dRVVT ratio in the apixaban group. Clot-based methods for detecting the APC resistance were significantly affected by dabigatran and less by rivaroxaban. Conclusion. DOACs were practically inactivated after the addition of the DOAC-Remove?, which made it possible to perform analyses for the LA and APC-R testing freely and obtain relevant results.

Clinical manifestations and gene analysis of 18 cases of hereditary protein S deficiency

目的分析18例遗传性蛋白S（PS）缺乏症患者的临床表现及分子致病机制。方法对2016年7月至2019年2月就诊于中国医学科学院血液病医院的18例PS缺乏症患者进行回顾性分析，应用凝固法测定PS活性、应用发色底物法测定蛋白C（PC）和抗凝血酶（AT）活性并进行初步诊断，使用高通量测序（HTS）筛查凝血疾病相关基因变异并进行Sanger测序验证；使用Swiss-model软件进行三维结构分析。结果18例患者中男15例，女3例，中位年龄37（14～62）岁。均有深静脉血栓栓塞病史，PS活性为12.5～48.2 U/dl。所有患者均检出PROS1基因变异，其中5个无义突变（c.134_162del/p.Leu45*、c.847G>T/p.Glu283*、c.995_996delAT/p.Tyr332*、c.1359G>A/p.Trp453*、c.1474C>T/p.Gln492*）、2个移码突变（c.1460delG/p.Gla487Valfs*9、c.1747_1750delAATC/p.Asn583Wfs*9）和1个大片段缺失（外显子9 缺失）为首次报道。此外，1例妊娠期深静脉血栓形成女性患者的PS活性为55.2 U/dl，并且检出PROC基因c.565C>T/p.Arg189Trp突变。结论该研究新发现的基因突变丰富了与遗传性PS缺乏症相关的PROS1基因突变谱。