Protein C Research Articles

Thrombosis in Young Women on Ocps Likely Is Caused By an Unmasking Effect of Estrogens on Underlying Thrombophilias

Background: Estrogen-containing oral contraceptives (OCPs) increase the risk for thrombosis 3-4 fold in otherwise healthy young women, although the absolute thrombosis rate in young OCP users is only 0.05% per year (Trenor CC, Pediatrics. 2011;127: 347). In our prospective cohort database 70% of lower extremity venous thromboembolism in adolescent girls were associated with OCP use, usually of recent onset (<6months). OCPs are associated with acquired resistance to activated protein C (APC), an important thrombin regulatory pathway, with an increase in fibrinolysis (Douxfils J, Contraception, 2020;102:396). A causative role for estrogens in thrombus formation in young women is not completely understood. Aims: This study evaluated thrombin generation, thrombin reduction by APC (activated protein C resistance, APCR), fibrinolysis and thrombophilia in healthy young males, healthy young females who were using or not using estrogen hormone therapy (i.e. oral contraceptive pills/OCPs) and young females with thrombosis who were using or not using OCPs at the time of thrombus presentation. The goal of this study was to measure the effects of OCPs on laboratory assays of coagulation and fibrinolysis to reveal potential mechanisms in increased thrombosis risk. Methods: This was an analysis of a consented, prospective cohort study (Thrombo-PICS). Healthy controls included men and women with no personal or family history of bleeding or clotting who were using or not using OCPs and on no other relevant medications. Study groups included females who presented with thrombosis with no OCP use, or OCP use for less than six months versus six or more months. All participants with a history of thrombosis were studied three or more months following the onset of thrombosis and no longer on OCPs. Laboratory assays performed included calibrated automated thrombography (CAT) with and without thrombomodulin (CAT TM) to determine thrombin generation (endogenous thrombin potential, ETP) and thrombin reduction with APC (% ETP reduction, APCR) respectively, and the euglobulin clot lysis time (ELT) to model fibrinolysis. Factors known to cause APCR and to be associated with thrombosis were also assayed, including factor V Leiden (FVL), the prothrombin gene mutation (PTM), protein C, protein S, lipoprotein(a), and the lupus anticoagulant (LA). Overall results were presented using descriptive statistics. Chi-Squared tests and paired t-tests were used to make paired comparisons. Parametric and non-parametric paired comparisons were used to evaluate group differences in ETP, % ETP reduction, ELT and presence thrombophilic traits relative to estrogen use and history of thrombosis. Results: Table 1 and Figure 1 display results. All groups of females with a history of thrombosis had decreased mean %ETP reduction compared to healthy females not on OCPs (p=0.02), with the lowest values occurring in females with thrombosis following less than six months of OCP use. There was also a lower prevalence of abnormal CAT TM values amongst healthy controls not on OCPs as compared to thrombosis patients not on OCPs (19 vs 51%, p=0.005).Healthy females on OCPs showed an increased ETP (p=0.002) and a marked reduction in ELT (p<0.001) as compared to all other groups. Females with a history of thrombosis, whether associated with OCPs or not, were similar to healthy females not using OCPs in regard to ETP and ELT. Furthermore, females who developed thrombosis on OCPs were more likely to have a thrombophilic trait than healthy controls on OCPs (48 vs 4%, p<0.001). Healthy men were found to have a greater %ETP reduction than healthy women (p=0.04), while having a lower ETP than healthy women (p=0.007). There were no differences in ELTs between healthy male and female controls (p=0.39). Discussion: OCPs were associated with increased thrombin generation, increased APCR and accelerated fibrinolysis in healthy female controls. The lower ELT in healthy controls using OCPs suggests that increased fibrinolysis may partially balance acquired APCR from estrogen use. We were unable to test fibrinolysis in females on OCPs prior to the onset of thrombosis. Females with thrombosis onset within six months of starting OCPs showed the highest prevalence of thrombophilic traits known to cause APCR. These data suggest that the APCR effect of OCPs potentially augments the prothrombotic effect of underlying thrombophilias in the promotion of clinical thrombosis.

Clinical Consequences of Rare Germline Variation in Genes Associated with Coagulation across 451,958 Individuals

Introduction Despite deep biological insights over the preceding decades, our ability to assess for inherited hypercoagulable states remains limited. In light of these challenges and the increasing relevance of precision medicine and next-generation sequencing (NGS) to clinical practice, the identification of rare germline variants in coagulation-focused NGS panels is increasingly becoming accepted practice. However, the discovery of variants of unknown significance (VUS) remains a common clinical conundrum in molecular testing, particularly because evidence for the thrombosis risk associated with many genes derives from small, uncontrolled studies, case reports, and pedigree analysis. Large-scale evaluation of the clinical consequences associated with loss of function in individual coagulation-related genes would contribute to the clinical assessment of patients presenting with heterozygous VUS in poorly characterized genes. Objectives Determine the risk of four thrombosis phenotypes associated with rare, germline loss-of-function variants in a major clinical coagulation-focused NGS panel. Methods The UK Biobank (UKBB) contains paired clinical and whole exome sequencing data for over 450,000 participants. We identified rare (minor allele frequency <0.1%) germline variants that were predicted in silico to alter protein activity (functional impact score ≥0.8) within the 54 evaluable genes of the Yale NGS coagulation panel. For each gene, parallel multivariable Firth's logistic regression models were generated to assess the association between rare qualifying variants and four thrombotic phenotypes as well as coagulopathic bleeding in a gene-level collapsing analysis (N=270 independent models). We adjusted for age, sex, ancestry, and sequencing batch. Phenotypes of interest included venous thromboembolism (VTE), myocardial infarction (MI), stroke (CVA), peripheral artery disease (PAD), and coagulopathic bleeding. The Bonferroni corrected p-value for each phenotype was 0.001. We validated functional impact score predictions in seven secreted proteins measured in a subset of 47,923 UKBB participants who underwent plasma proteomic assessment (Olink Explore 1536 panel). Results A total of 451,958 individuals in the UKBB with whole exome sequencing data were evaluated. Among the 54 genes in the Yale NGS coagulation panel, there were 18,975 qualifying variants (98.4% heterozygous). Function-altering variants in 4 of 54 genes were associated with a statistically significant increased risk of thrombosis after adjusting for multiple comparisons: PROC (OR = 5.7, 95% CI: 3.2 - 9.6, p = 1.6x10 -7), SERPINC1 (OR = 5.0, 95% CI: 2.7 - 8.7, p = 4.0x10 -6), PROS1 (OR = 3.2, 95% CI: 2.0 - 4.8, p = 6.0x10 -6), and STAB2 (OR = 1.4, 95% CI: 1.2 - 1.7, p = 5.3x10 -4) (Figure 1). Nominal significance was achieved for 7 genes in the VTE phenotype, 1 gene in CVA, 3 genes in MI, 5 genes in PAD, and 0 genes in coagulopathic bleeding. Variants in a total of 37 genes did not appear to be significantly associated with any of the evaluated phenotypes. To assess the reliability of variant functional impact scores derived in silico, plasma levels for 13 of the 53 coagulation genes were analyzed. Of these, 8 were identified as secreted proteins amenable to assessment by plasma levels, including ADAMTS13 ( ADAMTS13), factor IX ( F9), factor VII ( F7), tissue plasminogen activator ( PLAT), urokinase ( PLAU), protein C ( PROC), plasminogen activator inhibitor 1 ( SERPINE1), and von Willebrand factor ( VWF). For these proteins, average plasma levels consistently demonstrated a negative correlation with functional impact score (Pearson r = -0.98, P=0.0009), indicating that our in silico assessments in this context are broadly reliable (Figure 2). Conclusion Within the commonly encountered clinical context of heterozygous loss of function, most genes contained in coagulation-focused NGS panels likely have fairly weak effects on disease risk that are limited to venous thrombosis. However, our findings do not rule out the possibility of more profound effects from homozygous or compound heterozygous loss of function in these same genes. By defining the clinical consequence of heterozygous loss of function in specific coagulation-related genes across a large population, our findings may help inform the interpretation of molecular testing results in which patients are found to have a VUS.

Influence of Activated Protein C Resistance on Short-Term Hospital Outcomes Following Admission for Acute Ischemic Stroke in the United States

Introduction: Activated protein C (APC) plays a crucial role in breaking down factor Va and VIIIa, preventing excessive blood clotting. APC resistance occurs when protein C is unable to degrade these factors, leading to a higher risk of thrombosis. This condition can be either hereditary or acquired, with the most common cause being the factor V Leiden mutation. While previous studies have indicated that APC resistance increases the likelihood of acute ischemic stroke (AIS), their specific outcomes have not been extensively studied. Our research aims to bridge this gap via a national study. Methods: We used hospital records via the National Inpatient Sample (NIS) for our study. It consists of de-identified in-patient data, released on an annual basis by HCUP, AHRQ, and collaborating hospitals. Patients admitted with a primary diagnosis of AIS were recruited. We excluded all hospitalizations of ages <18 years. The impact of APCR status on outcomes and in-hospital complications such as acute kidney injury (AKI), myocardial infarction (MI), thrombolysis, mechanical thrombectomy (MT), vasopressor use, seizures, unfavorable discharge (discharge to another center such as short-term hospital, skilled nursing facility, intermediate care facility, and another type of facility) was studied using multivariable regression analysis, while adjusting for the impact of several potential patient and hospital characteristics. Results: Our selection criteria identified 2631890 admissions for AIS between 2016-2020, including 5910 (0.2%) patients also reporting a diagnosis of APCR (Table 1). In addition, APCR patients were younger (mean age 60.1 vs. 70.1 years, p<0.01) with a lower mean Charlson Comorbidity Index (CCI) score (3.50 vs. 3.87, p<0.01), and showed higher odds of a history of prior stroke (19.3% vs. 15.4%, aOR 1.482, 95% CI 1.387-1.584, p<0.01). While both groups were primarily covered by Medicare (65.0% in non-APCR group and 47.3% in APCR patients), a higher proportion of patients with APCR were also covered by private insurance (33.0% vs 19%, p<0.01). The APCR group also showed a higher prevalence of Whites (85.9% vs. 68.2%, p<0.01). Most cases in both cohorts were treated in Urban teaching centers. While higher prevalences of smoking(43.2% vs. 39.8%, p<0.01), obesity (20.3% vs. 14.5%, p<0.01), and drug abuse (4.4% vs. 2.8%, p<0.01) were seen in APCR patients, they also expressed fewer cases of cirrhosis (0.3% vs. 0.7%, p<0.01), diabetes (30.6% vs 39.2%, p<0.01), hypertension (51.9% vs 58.2%, p<0.01), chronic kidney disease (13.7% vs 18.3%, p<0.01), atrial fibrillation (18.8% vs 24.9%, p<0.01), and lipid disorder (53.8% vs 60.3%, p<0.01). After careful adjustments of confounders, APCR patients showed a higher use of mechanical thrombectomy (aOR 1.523, 95% CI 1.374-1.689, p<0.01) and vasopressor use (aOR 1.877, 95% CI 1.435-2.455, p<0.01) and also reported higher odds of seizures (aOR 1.413, 95% CI 1.265-1.578, p<0.01) and unfavorable discharge (aOR 1.065, 95% CI 1.006-1.128, p=0.03). However, lower odds of acute kidney injury (aOR 0.87, 95% CI 0.794-0.952, p<0.01) and death(aOR 0.708, 95% CI 0.597-0.838, p<0.01) were noted. No differences in events of MI (aOR 1.161, 95% CI 0.994-1.358, p=0.06) and thrombolysis (aOR 0.94, 95% CI 0.863-1.024, p=0.159) use were seen between the two groups. Conclusion: The study reported higher short-term outcomes, including increased use of mechanical thrombectomy and vasopressors and higher odds of seizures and unfavorable discharge in APCR patients with acute stroke. However, no similar increased odds of in-patient death, acute kidney injury, thrombolysis, and myocardial infarction were noted. While the impact of APCR on stroke recurrence has been highly debated in prior studies, our analysis provides a novel update showing an increased risk of subsequent stroke in patients with a history of stroke. Encouraging more extensive studies in a clinical setting, with larger groups of people and more extended observation periods, can provide a clearer understanding of the situation and thus improve their long-term outcomes.

Serpin-PC in Persons with Severe Hemophilia (PwH): Updated Results from a Multicenter Multi-Part, First-in-Human Study

Introduction SerpinPC is an investigational serine protease inhibitor (SERPIN) engineered to specifically inhibit Activated Protein C (APC). The previously presented data from the completed parts of AP-0101 showed that administration of SerpinPC reduced bleeding in persons with severe hemophilia with no observations of unexplained chronic elevation in D-dimer. We will now present all results up to the end of Part 5 by which time we anticipate the median continuous exposure will be more than 3 years. Methods AP-0101 is an ongoing first-in-human open-label multicenter study utilizing an adaptive design to investigate the safety, tolerability, pharmacokinetics and efficacy of SerpinPC in subjects with severe hemophilia A and B. Part 1a was a Single Ascending Dose Study of SerpinPC in 15 healthy male volunteers and 12 males with severe hemophilia. Part 2 enrolled 23 males with severe hemophilia (19 hemophilia A and 4 hemophilia B), who were not on replacement factor prophylaxis, to receive SerpinPC at 0.3, 0.6 or 1.2 mg/kg, administered as a subcutaneous (SC) injection once every 4 weeks over a 24-week period (6 total doses). In Part 3, subjects who completed Part 2 received a flat dose of 60 mg of SerpinPC once every 4 weeks for 48 weeks. Part 4 was a further extension in which subjects who completed Part 3 received 1.2 mg/kg of SerpinPC once every 2 weeks for 24 weeks. Part 5 was a further extension in which subjects who completed Part 4 continued to receive 1.2 mg/kg of SerpinPC once every 2 weeks for 52 weeks. Results Annualized bleed rates, safety and tolerability for Part 5 will be available and a complete summary of all results to the end of Part 5 will be presented, including available pharmacokinetic and anti-drug antibody data.

Thrombophilia Screening in Autoimmune Hemolytic Anemia: A Single-Center Cross-Sectional Study

Background Autoimmune hemolytic anemia (AIHA) is characterized by an increased rate of thrombosis, whose predictors are not completely clarified, although an association with active hemolysis (i.e., LDH >1.5 x ULN), anemia severity and previous splenectomy has been reported. Moreover, antithrombotic prophylaxis and its duration are not standardized in this patient-population. The value of thrombophilia screening, including congenital and acquired abnormalities, has not been established yet. Aim To investigate the prevalence of inherited and acquired thrombophilia abnormalities in a cohort of AIHA patients with or without thrombosis. Methods AIHA patients followed from January 2019 until September 2022 at a tertiary hematologic center in Milan, have been systematically tested for Factor V Leiden (FVL) and prothrombin (FII) G20210A mutation, protein C (PC), protein S (PS), antithrombin (AT) levels, and the presence of antiphospholipid antibodies (APLA, i.e., lupus anticoagulant, LAC, anti-cardiolipin and anti-b2-glycoprotein-1). Patients' hematological characteristics, additional thrombotic risk factors, history of venous/arterial thrombosis before and after AIHA diagnosis, and anticoagulant and/or antiplatelet therapies were retrospectively recorded. Results As shown in table 1, 59% of 92 enrolled patients were female with a median age at diagnosis of 70.5 years (range 24-89), and were classified as warm AIHA (50%), cold agglutinin disease (31.5%), mixed/atypical AIHA (9.8%), and Evans syndrome (association of AIHA and other immune cytopenia, 8.7%). Thrombophilia screening resulted positive in 19 patients (20.7%) including FVL (1.1%), F II (1.1%), APLA (10.8%), PC deficiency (1.1%), PS deficiency (2.2%), AT deficiency (2.2%), and more than one abnormality (2.2%). Additional risk factors considered were thrombosis prior to AIHA diagnosis, reported in 6 patients (7%, 3 venous and 3 arterial), and associated conditions (29%), including autoimmune disease (N=17), lymphoproliferative disease (N=7), myeloproliferative neoplasm (N=1), and paroxysmal nocturnal hemoglobinuria (N=1). Furthermore, 97% of subjects presented at least one risk factor (i.e., diabetes, obesity, bed rest, etc.) in addition to AIHA. Twenty-seven patients (28%) received anticoagulant prophylaxis after AIHA diagnosis, mainly short-term for hemolysis (42%) or surgery of lower limb fractures (37%), and long-term for atrial fibrillation in 24%; 22% of patients were on antiplatelet prophylaxis, and 4 patients had been splenectomized. Since AIHA diagnosis, 26 patients (28%) experienced thrombosis (77% venous, 23% arterial), with 33 total events (3 patients had > 1 event). Median hemoglobin and LDH at thrombosis were 8.6 g/dL (2-11.7), and 374 U/L (129-1873) respectively, indicating active hemolysis. No relationship was found with inherited thrombophilia abnormalities (3 patients, 8% experienced thrombosis vs 7, 10.5% who did not), whilst a significant association was noted with APLA positivity (p<0.05). Two patients with APLA experienced more than 1 event, one had 1 venous and 1 arterial event. Conclusions Along with disease activity and Evans syndrome, positivity for APLA appears the main predictor of thrombosis in AIHA and is therefore worth of testing, whilst extensive thrombophilia screening encompassing inherited abnormalities appears less useful.

Levels of Some Blood Clotting Factors in COVID-19 Patients: Comparative Study Before and After Pfizer- BioNTech Vaccinations

One of the health issues that a coronavirus can induce is blood clotting. Coronavirus can be prevented in a number of ways. Vaccination is one of the critical methods for preventing illness or lessening its impact. This study seeks to estimate a few blood coagulation variables. 147 samples were collected from the Baghdad Governorate in the autumn of 2021. The samples were split into three groups: COVID-19 patients, healthy individuals before and after receiving the (Pfizer-BioNTech) vaccine, and healthy individuals only. Prothrombin Time (PT), Partial Thromboplastin Time (PTT), Protein C (PTN-C), Protein S (PTN-S), and International Normalized Ratio (INR) for 49 samples were measured and computed for each group. The results have shown that the PTN-C and PTN-S concentrations were significantly decreased in the COVID-19 patients compared to unvaccinated healthy individuals. While INR of COVID-19 patients showed a highly significant increase when compared with unvaccinated healthy individuals. At the same time, there was no significant difference for each PT and PTT between these studied groups. The parameters exhibit identical findings when COVID-19 patients are compared to those who have had vaccinations, with the exception of PT, which reveals a highly significant rise in COVID-19 patients when compared to those who have received vaccinations. Conclusion: COVID-19 causes blood clots and may be recognized by a decrease in PTN-C and PTN-S content.

Activation of intravascular blood coagulation in the early postoperative period in reconstructive abdominoplasty

Purpose of the study: to determine the activity of intravascular blood coagulation during the abdominoplasty and justify pharmacological thromboprophylaxis.Material and Methods. An open prospective study was conducted on a group of 100 patients undergoing abdominoplasty. The risk of venous thromboembolism (VTE) before the surgery was assessed using the Caprini risk assessment model in 2010. Prothrombin (PT) and fibrinogen (FG) levels, soluble fibrin-monomer complexes (SFMC), euglobulin clot lysis time (ECLT), antithrombin III (AT-III) level, and protein C (PC) level were determined before and after the surgery.Results. Using the Caprini risk assessment model, patients were categorized by VTE risk as follows: moderate risk – 64% and high risk – 36%. After the surgery, an increase in PT by 12.3% and 15.5% on days 3 and 5 was observed (p < 0.01); an increase in FG by 0.49; 2.34 and 2.04 g/L on days 1, 3, and 5 was observed (p < 0.01); an increase in SFMC at all postoperative stages by 4.7; 8.2; 10.8 and 10.0 times (p < 0.01); an increase in ECLT by 2.8; 9.7, and 6.3 minutes on days 1, 3, and 5 (p < 0.001); and a decrease in AT-III and PC levels immediately after the operation by 18.8% and 8.9%, on day 1 by 14.2% and 11.2%, and on day 3 by 8.8% and 6.3% (p < 0.01).Conclusion. All patients undergoing abdominoplasty require pharmacological thromboprophylaxis due to moderate and high risk of venous thromboembolic complications. Abdominoplasty in the early postoperative period is accompanied by activation of plasma coagulation, inhibition of fibrinolytic system activity, and reduced activity of natural anticoagulants, which justifies the need for pharmacological thromboprophylaxis for at least 5 days.

Assessment of plasma hemostasis system in the early postoperative period in patients after abdominoplasty

Introduction Venous thromboembolic complications after abdominoplasty are associated with increased intraabdominal pressure and altered venous return without taking into account possible changes in the activity of the blood coagulation system.Purpose of work was to evaluate the state of the plasma hemostasis system in the early postoperative period in patients after abdominoplasty.Materials and methods The study included 100 patients who underwent aesthetic abdominoplasty. Prothrombin (PT), fibrinogen (FG), activated partial thromboplastin time (APTT), soluble fibrin-monomer complexes (SFMC), euglobulin clot lysis (ECL), antithrombin-III (AT-III), and protein C (PrC) levels were evaluated before surgery, immediately after surgery, on the first, third and fifth days after surgery. Statistical data analysis was performed using SPSS 16, Excel 2010 for Windows, using the Kruskal – Wallis criterion.Results Coagulation hemostasis was characterized by a decrease in PT immediately after the surgery and on the first day by 13.9 and 5.3, and an increase in PT on the third and fifth days by 12.3 and 15.5 (p < 0.001), decrease in FG on the first, third and fifth days by 0.49 g/L, 2.34 g/L, and 2.04 g/L, respectively (p < 0.001). SFMC increased at all postoperative stages by 4.7, 8.2, 10.8, and 10.0 times (p < 0.001). Inhibition of XII-dependent fibrinolysis was characterized by an increase in ECL time on the first, third and fifth days by 2.8, 9.7, and 6.3 minutes, respectively (p < 0.001). AT-III and PrC levels decreased immediately after the surgery by 18.8 and 8.9, on the first day by 14.2 and 11.2, and on the third day by 8.8 and 6.3 respectively (p < 0.001).Discussion Activation of blood coagulation processes with imbalance of coagulation, anticoagulation, and fibrinolytic systems create conditions for venous thrombosis formation after abdominoplasty, which substantiates the necessity of VTE pharmacoloprophylaxis for at least five days.Conclusion In the early postoperative period abdominoplasty is accompanied by activation of plasma coagulation, inhibition of fibrinolytic system activity and reduction of natural anticoagulant activity.

Influence of emicizumab on protein C-mediated clotting regulation

Emicizumab, a FVIII-mimetic bispecific antibody, is insensitive to degradation by activated protein C (APC) and may thus induce a procoagulant state. We investigated the effect of emicizumab on protein C-mediated inhibition of coagulation under in vitro conditions mimicking physiological and pathological clotting activation. Thrombin generation (TG) in tissue factor-triggered hemophilic plasma containing emicizumab (50 μg/mL) was inhibited by APC or thrombomodulin in a concentration-dependent manner, and to a similar extent as in plasma added with FVIII (Kovaltry, 1 IU/mL). However, when clotting was activated via the intrinsic pathway, emicizumab-plasma displayed resistance to APC, manifested by a barely detectable prolongation of the lag time of TG, and by the lack of inhibition of FXa generation. Moreover, in contact-activated plasma added with APC, the generation of a second wave of thrombin, following prothrombin replenishment, was much greater in emicizumab-plasma than in Kovaltry-plasma, suggesting that the insensitivity of emicizumab to APC may translate in greater thrombin formation. Considering the major role played by the contact system in the thrombotic process, hemophilia A patients under emicizumab treatment might be at increased thrombotic risk.

Unraveling the Molecular Mechanisms of Activated Protein C (APC) in Mitigating Reperfusion Injury and Cardiac Ischemia: a Promising Avenue for Novel Therapeutic Interventions.

Ischemic heart disease, which results from plaque formation in the coronary arteries, hinders the flow of oxygenated blood to the heart, leading to ischemia. Reperfusion injury remains a significant challenge for researchers, and the mechanisms underlying myocardial ischemia-reperfusion injury (MIRI) are not entirely understood. The review directs future research into potential targets in clinical treatment based on our present understanding of the pathophysiological mechanisms of MIRI. The study provides insights into the mechanisms underlying MIRI and offers direction for future research in this area. The use of targeted therapies may hold promise in improving cardiac function in the elderly and minimizing the adverse effects of revascularization therapies. The purpose of this review is to analyze the role of activated protein C (APC) in the pathogenesis of ischemic heart disease, heart failure, and myocardial ischemia-reperfusion injury, and discuss the potential of APC-based therapeutics.

Activated protein C in epilepsy pathophysiology.

Epilepsy is one of the most common neurologic disorders that is characterized by recurrent seizures, and depending on the type of seizure, it could lead to a severe outcome. Epilepsy's mechanism of development is not fully understood yet, but some of the common features of the disease are blood-brain barrier disruption, microglia activation, and neuroinflammation. Those are also targets of activated protein C (APC). In fact, by downregulating thrombin, known as a pro-inflammatory, APC acts as an anti-inflammatory. APC is also an anti-apoptotic protein, instance by blocking p53-mediated apoptosis. APC's neuroprotective effect could prevent blood-brain barrier dysfunction by acting on endothelial cells. Furthermore, through the downregulation of proapoptotic, and proinflammatory genes, APC's neuroprotection could reduce the effect or prevent epilepsy pathogenesis. APC's activity acts on blood-brain barrier disruption, inflammation, and apoptosis and causes neurogenesis, all hallmarks that could potentially treat or prevent epilepsy. Here we review both Activated Protein C and epilepsy mechanism, function, and the possible association between them.

Evaluation of prothrombotic risk of two PROC hotspot mutations (Arg189Trp and Lys193del) in Chinese population: a retrospective study

BackgroundR189W and K193del of protein C (PC) were hotspot mutations in Chinese population with venous thromboembolism (VTE), but almost two-thirds of patients with above mutations coexisting with other genetically or aquiredly prothrombotic risk factors. The aim of this study is to clarify the independent contributions of R189W or K193del to VTE risk.Methods490 unrelated patients with a personal history of VTE and 410 healthy participants were enrolled in this study. Data of their demographics, family history, genetic and acquired thrombosis risk factors were collected and statistically analyzed.ResultsPC R189W and K193del were identified in 3/410 (0.7%) and 7/410 (1.7%) healthy controls, and in 27/490 (5.5%) and 43/490 (8.8%) patients with VTE, respectively. Notably, about 70% of these mutant carriers combined with other genetic or acquired thrombophilic factors. After adjustment for age, gender, other inherited and acquired risk factors, we demonstrated that R189W and K193del were associated with 5.781-fold and 4.365-fold increased risk of VTE, respectively, which were significantly lower than the prothrombotic risk of anticoagulant deficiencies induced from rare mutations. Independent R189W or K193del mutation was not associated with earlier first-onset age as well as higher recurrent rate of VTE. However, combination of other genetic or acquired thrombophilic factors had supra-additive effects on those consequences. The more additional risk factors the patients had, the younger first-onset ages and higher risk of recurrence would be.ConclusionsAs the most frequent mutations for PC deficiency in Chinese population, both R189W and K193del mutations had limited independent contributions to VTE development compared with other rare mutations in PROC gene, but may act in concert with other genetic defects or acquired thrombotic risk factors to produce the final severe phenotype.

PB1109 Development and Evaluation of an Aptamer-Based Affinity Purification Method for Activated Protein C (APC)

PB1109 Development and Evaluation of an Aptamer-Based Affinity Purification Method for Activated Protein C (APC)

PB1059 Single Amino Acid Substitutions of an Antibody Specific for a Non-Active Site of Activated Protein C (APC) Yielded an Antibody Library Capable of Modulating and Uncoupling APC Pleiotropic Functions

PB1059 Single Amino Acid Substitutions of an Antibody Specific for a Non-Active Site of Activated Protein C (APC) Yielded an Antibody Library Capable of Modulating and Uncoupling APC Pleiotropic Functions

Expression of human thrombomodulin by GalTKO.hCD46 pigs modulates coagulation cascade activation by endothelial cells and during ex vivo lung perfusion with human blood.

Thrombomodulin is important for the production of activated protein C (APC), a molecule with significant regulatory roles in coagulation and inflammation. To address known molecular incompatibilities between pig thrombomodulin and human thrombin that affect the conversion of protein C into APC, GalTKO.hCD46 pigs have been genetically modified to express human thrombomodulin (hTBM). The aim of this study was to evaluate the impact of transgenic hTBM expression on the coagulation dysregulation that is observed in association with lung xenograft injury in an established lung perfusion model, with and without additional blockade of nonphysiologic interactions between pig vWF and human GPIb axis. Expression of hTBM was variable between pigs at the transcriptional and protein level. hTBM increased the activation of human protein C and inhibited thrombosis in an in vitro flow perfusion assay, confirming that the expressed protein was functional. Decreased platelet activation was observed during ex vivo perfusion of GalTKO.hCD46 lungs expressing hTBM and, in conjunction with transgenic hTBM, blockade of the platelet GPIb receptor further inhibited platelets and increased survival time. Altogether, our data indicate that expression of transgenic hTBM partially addresses coagulation pathway dysregulation associated with pig lung xenograft injury and, in combination with vWF-GP1b-directed strategies, is a promising approach to improve the outcomes of lung xenotransplantation.

Protein C and protein S deficiencies are associated with increased risk of deep vein thrombosis in pregnant women using oral contraceptives.

Oral contraceptives are commonly taken by women and are known to increase the risk of venous thromboembolism (VTE). The aim of this study was to investigate the association between oral contraceptive use and natural anticoagulants, that is, protein C (PC), protein S (PS), and antithrombin in pregnant women with deep vein thrombosis (DVT). This case-control study was conducted on 330 pregnant women, that is, cases 165 (who used oral contraceptives) and controls 165 (who did not use oral contraceptives). The levels of PC, PS, and antithrombin were measured and compared between the two groups. The use of different types of oral contraceptives and their association with DVT and PC and PS were also analyzed. The study found that women with DVT had significantly lower levels of PC and PS compared with controls ( P < 0.001). However, no significant difference was found in the levels of AT. Among the different types of oral contraceptives, first-generation progestin pills including Ethynodiol Diacetate, Norethindrone Acetate, Norethynodrel, and second-generation oral contraceptives (Lynestrenol, Levonorgestrel and Norgestrel) were not found to be associated with lower levels of PC and AT while Desogestrel, Norgestimate, and Gestodene (third-generation) were associated with lower levels of PS. This study suggests that the use of contraceptives, particularly those containing Desogestrel, Norgestimate, and Gestodene, may be associated with a higher risk of thrombosis because of the associated lower levels of PS. Monitoring anticoagulant levels is crucial in preventing DVT in this population.

Activated protein C signaling mediates neuroinflammation in seizure induced by pilocarpine

Epilepsy is one of the most common and oldest neurological disorders, characterized by periodic seizures that affect millions globally. Despite its long history, its pathophysiology is not fully understood. Additionally, the current treatment methods have their limitations. Finding a new alternative is necessary. Activated Protein C (APC) has been proven to have neurological protection in other neurological disorders; however, there is no study that focuses on the role of APC in seizures. We propose that APC's protective effect could be associated with seizures through inflammation and apoptosis regulation. The results demonstrated that APC's pathway proteins are involved in neuroprotection mechanisms in seizure-induced models by acting on certain inflammatory factors, such as NF-κB and apoptosis proteins.

Design and characterization of novel activated protein C variants for the proteolysis of cytotoxic extracellular histone H3

BackgroundExtracellular histone H3 is implicated in several pathologies including inflammation, cell death, and organ failure. Neutralization of histone H3 is a strategy that was shown beneficial in various diseases, such as rheumatoid arthritis, myocardial infarction, and sepsis. It was shown that activated protein C (APC) can cleave histone H3, which reduces histone cytotoxicity. However, due to the anticoagulant properties of APC, the use of APC is not optimal for the treatment of histone-mediated cytotoxicity, in view of its associated bleeding side effects. ObjectivesThis study aimed to investigate the detailed molecular interactions between human APC and human histone H3, and subsequently use molecular docking and molecular dynamics simulation methods to identify key interacting residues that mediate the interaction between APC and histone H3 and to generate novel optimized APC variants. MethodsAfter molecular simulations, the designed APC variants 3D2D-APC (Lys37-39Asp and Lys62-63Asp) and 3D2D2A-APC (Lys37-39Asp, Lys62-63Asp, and Arg74-75Ala) were recombinantly expressed and their abilities to function as anticoagulant, to bind histones, and to cleave histones were tested and correlated with their cytoprotective properties. ResultsCompared with wild type-APC, both the 3D2D-APC and 3D2D2A-APC variants showed a significantly decreased anticoagulant activity, increased binding to histone H3, and similar ability to proteolyze histone H3. ConclusionsOur data show that it is possible to rationally design APC variants that may be further developed into therapeutic biologicals to treat histone-mediated disease, by proteolytic reduction of histone-associated cytotoxic properties that do not induce an increased bleeding risk.

Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression.

Introduction: Dysfunction of the endothelium impairs its' protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses. Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs.

Activated protein C, protein S, and tissue factor pathway inhibitor cooperate to inhibit thrombin activation

IntroductionThrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life-threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). However, prothrombinase is resistant to either of these inhibitory systems in isolation. Materials and methodsWe hypothesized that these anticoagulants function best together, and tested this hypothesis using purified proteins and plasma-based systems. ResultsIn plasma, TFPIα had greater anticoagulant activity in the presence of APC and PS, maximum PS activity required both TFPIα and APC, and antibodies against TFPI and APC had an additive procoagulant effect, which was mimicked by an antibody against PS alone. In purified protein systems, TFPIα dose-dependently inhibited thrombin activation by prothrombinase, but only in the presence of APC, and this activity was enhanced by PS. Conversely, FXa protected FVa from cleavage by APC, even in the presence of PS, and TFPIα reversed this protection. However, prothrombinase assembled on platelets was still protected from inhibition, even in the presence of TFPIα, APC, and PS. ConclusionsWe propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. Platelets protect prothrombinase from inhibition, however, supporting a procoagulant environment within the clot.