Systematic Assessment of Protein C-Termini Mutated in Human Disorders.

All proteins have a carboxyl terminus, and we previously summarized eight mutations in binding and trafficking sequence determinants in the C-terminus that, when disrupted, cause human diseases. These sequence elements for binding and trafficking sites, as well as post-translational modifications (PTMs), are called minimotifs or short linear motifs. We wanted to determine how frequently mutations in minimotifs in the C-terminus cause disease. We searched specifically for PTMs because mutation of a modified amino acid almost always changes the chemistry of the side chain and can be interpreted as loss-of-function. We analyzed data from ClinVar for disease variants, Minimotif Miner and the C-terminome for PTMs, and RefSeq for protein sequences, yielding 20 such potential disease-causing variants. After additional screening, they include six with a previously reported PTM disruption mechanism and nine with new hypotheses for mutated minimotifs in C-termini that may cause disease. These mutations were generally for different genes, with four different PTM types and several different diseases. Our study helps to identify new molecular mechanisms for nine separate variants that cause disease, and this type of analysis could be extended as databases grow and to binding and trafficking motifs. We conclude that mutated motifs in C-termini are an infrequent cause of disease.

Assessment of Antithrombin III and Protein C in Saudi Myocardial Infarction Patients.

BACKGROUND: Myocardial infarction (MI) or acute myocardial infarction (AMI), commonly referred to as a heart attack, happens when the blood flow to part of the heart stops, causing damage to the heart muscle. Chest pain or discomfort that may flow into the shoulder, arm, back, neck, or jaw is the most common symptom. Most MIs occur due to coronary artery disease. High blood pressure, smoking, diabetes, lack of exercise, obesity, high blood pressure, poor diet, excessive alcohol use, etc. are risk factors. Antithrombin III (AT III) is a glycoprotein produced by the liver and consists of 432 amino acids. Protein C, also referred to as autoprothrombin IIA and factor XIV of blood coagulation, is a zymogen. In regulating anticoagulation, inflammation, cell death, and maintaining the permeability of blood vessel walls in humans and other animals, the activated form of protein C plays an important role. METHODS: A case control study was conducted in Saudi Arabia to determine the levels of AT III and protein C in Saudi MI patients. Samples (n = 150) from MI patients as well as healthy controls (n = 50) were collected (2.5 mL of venous blood for sandwich ELISA). RESULTS: This study showed that the mean AT III and protein C levels were within normal levels in patients (86 ± 19.63 and 76.20 ± 30.64, respectively). A comparison of mean AT III and protein C levels in patient and control groups showed no significant difference (p-value = 0.26, 0.2, and 0.19, respectively). The results also showed that some of the samples had low levels of AT III (8.7%) and protein C (11.3%). CONCLUSIONS: A deficiency of AT III and protein C were not strong significant risk factors for myocardial infarction.

A multi-laboratory assessment of congenital thrombophilia assays performed on the ACL TOP 50 family for harmonisation of thrombophilia testing in a large laboratory network.

OBJECTIVES: Thrombophilia testing is commonly performed within hemostasis laboratories, and the ACL TOP 50 family of instruments represent a new 'single platform' of hemostasis instrumentation. The study objective was to evaluate these instruments and manufacturer reagents for utility of congenital thrombophilia assays. METHODS: Comparative evaluations of various congenital thrombophilia assays (protein C [PC], protein S [PS], antithrombin [AT], activated protein C resistance [APCR]) using newly installed ACL TOPs 550 and 750 as well as comparative assessments with existing, predominantly STAGO, instrumentation and reagents. Verification of manufacturer assay normal reference ranges (NRRs). RESULTS: HemosIL PC and free PS assays showed good comparability with existing Stago methods (R>0.9) and could be considered as verified as fit for purpose. HemosIL AT showed high relative bias with samples from patients on direct anti-Xa agents, compromising utility. Manufacturer NRRs for PC, PS and AT were verified with minor variance. Given the interference with direct anti-Xa agents, an alternate assay (Hyphen) was evaluated for AT, and the NRR also verified. The HemosIL Factor V Leiden (APC Resistance V) evidenced relatively poor performance compared to existing assays, and could not be adopted for use in our network. CONCLUSIONS: This evaluation of HemosIL reagents on ACL TOP 50 family instruments identified overall acceptable performance of only two (PC, free PS) of four thrombophilia assays, requiring use of third-party reagents on ACL instruments for the other two assays (AT, APCR).

Two Consecutive Invasive Surgeries Utilizing Zymogen Protein C (ZPC) That Enhanced Patient Safety and Reduced Costs.

This case report describes a major surgical procedure for a protein C-deficient, hypercoagulable patient who underwent two back-to-back invasive surgeries, hip replacement, and spinal stenosis correction. The patient, an 84-year-old male with a history of deep vein thromboses (DVT) and pulmonary emboli (PE), was treated pre-, peri-, and postoperatively with zymogen protein C (ZPC-Baxter, International) and recovered without clotting or increased bleeding. During the procedure, the patient was not administered any other anticoagulants. There have now been several case reports on different patients with unrelated teams in various locations worldwide using zymogen protein C during surgical procedures. Thus, this procedure is becoming a viable choice for patients with a high probability of clotting during and after invasive surgery. This case focuses on accomplishing safer surgery and reducing costs, by using less ZPC while accomplishing two surgeries in one procedure. As a result, this procedure might be useful for many medical situations where acquired protein C deficiency could be a problem (e.g., sepsis, pregnancy, etc.). This approach may have greater application to medical conditions other than protein C deficiency, where clotting and inflammation can become issues.

Identifying Genetic and Biological Determinants of Race-Ethnic Disparities in Stroke in the United States.

In the United States, causes of racial differences in stroke and its risk factors remain only partly understood, and there is a long-standing disparity in stroke incidence and mortality impacting Black Americans. Only half of the excess risk of stroke in the United States Black population is explained by traditional risk factors, suggesting potential effects of other factors including genetic and biological characteristics. Here, we nonsystematically reviewed candidate laboratory biomarkers for stroke and their relationships to racial disparities in stroke. Current evidence indicates that IL-6 (interleukin-6), a proinflammatory cytokine, mediates racial disparities in stroke through its association with traditional risk factors. Only one reviewed biomarker, Lp(a) (lipoprotein[a]), is a race-specific risk factor for stroke. Lp(a) is highly genetically determined and levels are substantially higher in Black than White people; clinical and pharmaceutical ramifications for stroke prevention remain uncertain. Other studied stroke risk biomarkers did not explain racial differences in stroke. More research on Lp(a) and other biological and genetic risk factors is needed to understand and mitigate racial disparities in stroke.

Oral Contraceptives and Venous Thromboembolism: Focus on Testing that May Enable Prediction and Assessment of the Risk.

Combined oral contraceptives (COCs) induce several changes in the levels of coagulation factors. The levels of procoagulant factors are often increased, while levels of anticoagulant factors are decreased. Fibrinolysis is also affected, even if the effect seems to be more counterbalanced by opposite regulation of profibrinolytic and antifibrinolytic factors. These effects on hemostasis are more pronounced with third- or fourth-generation COC compared with second-generation COC. Venous thromboembolism (VTE) risk increases when multiple risk factors, including genetic and environmental, are present simultaneously. COC use causes changes in coagulation that modify the prothrombotic state induced by preexisting hemostatic alterations in a supra-additive manner. Therefore, testing appears to be of importance not only before implementing COC but also to monitor any potential thrombogenicity induced by COC therapy. Inherited genetic factors, such as factor V Leiden, G20210A prothrombin mutation, antithrombin, protein C or protein S deficiencies, non-O blood group, as well as CYP2C9*2 and the rs4379368 mutations, have all been identified as genetic predictive risk factors of VTE in women. Nevertheless, the screening of these genetic biomarkers is not capable of assessing the phenotypic expression of the risk. This review will focus on the different options for screening the thrombogenic status in this population. Specific attention will be given to the endogenous thrombin potential-based activated protein C resistance, a test aiming at assessing the thrombogenicity induced by hormonal therapies and inherited or acquired thrombophilia.

Factor VIII, Protein C and Cardiovascular Disease Risk: The REasons for Geographic and Racial Differences in Stroke Study (REGARDS).

BACKGROUND: Haemostatic balance represented by low protein C (PC) and elevated factor VIII (FVIII) has been inconsistently associated with stroke and coronary heart disease (CHD) risk. OBJECTIVE: This article assesses whether an elevated FVIII and a low PC would increase cardiovascular risk more than either individually. PATIENTS AND METHODS: REGARDS recruited 30,239 black and white U.S. participants aged ≥ 45 years between 2003 and 2007. FVIII and PC were measured in a case-cohort sample of 646 stroke, 654 CHD, and a 1,104-person random sample with follow-up for approximately 4.5 years. Hazard ratios (HRs) were estimated using Cox models adjusted for demographic and cardiovascular risk factors. RESULTS: Elevated FVIII (per standard deviation [SD] increase) was associated with increased risk of both stroke (HR, 1.26; 95% confidence interval [CI], 1.08, 1.46) and CHD (HR, 1.52; 95% CI, 1.29, 1.79), while there was no association of PC per SD decrease. For PC, there was a trend towards increased cardiovascular disease risk in the lowest values (bottom 5%). For stroke, there was no interaction between FVIII and low PC (pinteraction = 0.55). For CHD, the adjusted HR of FVIII per SD increase was significantly greater with PC in the bottom 5% (HR, 3.59; 95% CI, 1.39, 8.29) than PC in the upper 95% (HR, 1.45; 95% CI, 1.23, 1.71; pinteraction = 0.07). CONCLUSION: Higher FVIII was associated with both CHD and stroke risk and the risk potentiated by low PC for CHD. Findings demonstrate that risks for cardiovascular diseases conferred by adverse levels of haemostasis biomarkers may be augmented by levels of other biomarkers.

Assessment of Hereditary Thrombophilia: Performance of Protein C (PC) Testing.

Protein C (PC) is a plasma Vitamin K-dependent pro-enzyme protein that is synthesized in the liver. Upon activation, PC regulates the coagulation process by neutralizing the procoagulant activities of factors V and VIII in the presence of the cofactor Protein S. PC is a major regulator of the coagulation process. The clotting based Protein C assay, the protocol described in this chapter, quantitates the amount of functional PC present in the specimen in a proportional fashion based on the prolongation of the Activated Partial Thromboplastin Time (APTT). Other methods for assessing PC are also available, including chromogenic and antigenic assays, but protocols for these assays are not provided.

A Compelling Case for the Use of Perioperative Zymogen Protein C for Increased Patient Safety.

It is imperative to maintain normal blood flow to provide adequate oxygen supply to specific organs and cells, as well as for the removal of metabolic byproducts. Therefore, any situation that results in blood clotting can injure or kill living tissues. In this paper, we describe a case where a protein C deficient subject who would, by all medical indicators, be at 100 % risk of experiencing thrombophlebitis, deep vein thrombosis, and or lung emboli, is able to escape all pathologies by using perioperative zymogen protein C (ZPC). This protein C deficient patient has a long history of blood clotting, particularly from surgical procedures. The patient is 81 years old and first experienced clotting due to hernia surgery in 1964, when he was hospitalized for 16 days post-surgery with life threatening complications. It was later determined in 1980, after many episodes, that the patient had hereditary protein C deficiency at the 38 % level. In his hernia surgery, perioperative ZPC was used along with accepted anticoagulation procedures with no blood clots or other related side effects occurring. This procedure can greatly benefit protein C deficient patients, and could potentially find use for non-PC deficient patients in surgeries and a variety of other medical treatments. This particular case helps to validate the importance of ZPC in effecting safer surgery in high-risk patients. It also supports the mechanism of ZPC acting as an anticoagulant without causing bleeding. Most importantly, each clinical case study represents a unique combination of surgeon, hematologist, medical staff, and patient functioning as a coordinated team. In this case, smaller amounts of very expensive ZPC achieved safe and efficacious results, which is hugely important for future clinical applications when considering the production cost of ZPC. More studies must be done to establish minimum dosing while achieving safe and efficacious outcomes.

Adoption and de-adoption of drotrecogin alfa for severe sepsis in the United States.

PURPOSE: Drotrecogin alfa was a landmark drug for treatment of severe sepsis, yet little is known about how it was adopted and de-adopted during its 10-year period of availability. METHODS: We used hospitalization data on fee-for-service Medicare beneficiaries from 2002 to 2011 to characterize trends in the use of drotrecogin alfa in the United States. RESULTS: Drotrecogin alfa use peaked at 5.87 per 1000 severe sepsis hospitalizations in 2003 and then steadily declined to 0.94 administrations per 1000 severe sepsis hospitalizations in 2010. Large teaching hospitals were more likely to use drotrecogin alfa than small, nonteaching hospitals. The addition of "add-on payments" to hospitals for using drotrecogin alfa in 2002 was associated with significantly increased use (P < .0001), and the withdrawal of those payments in 2004 was associated significantly decreased use (P < .0001). Neither the publication of international sepsis guidelines with favorable drotrecogin alfa recommendations (in 2004 and 2008) nor the publication of a clinical trial focused on drotrecogin alfa (in 2005) were associated with consistent changes use (P > .05). CONCLUSIONS: Drotrecogin alfa use declined over time, with marked changes in use associated with drug-specific financial incentives but not the publication of clinical practice guidelines or clinical trials.

Assessment of the relationships among coagulopathy, hyperfibrinolysis, plasma lactate, and protein C in dogs with spontaneous hemoperitoneum.

OBJECTIVE: To relate coagulation and fibrinolysis derangements to shock severity as reflected by plasma lactate concentrations in dogs with spontaneous hemoperitoneum (SHP) and determine the impact on transfusions. DESIGN: Prospective, observational, case-control study. SETTING: Three veterinary teaching hospitals. ANIMALS: Twenty-eight client-owned dogs with SHP and 28 breed- and age-matched control dogs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood samples for platelet counts, coagulation, and anticoagulant assays (prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin, and protein C, thromboelastography [TEG]), fibrinolysis testing (d-dimer and TEG lysis parameters with and without the addition of 50 U/mL of tissue plasminogen activator [TEG LY30 measured with the addition of 50 U/mL of tPA to the blood sample, LY3050 and TEG LY60 measured with the addition of 50 U/mL of tPA to the blood sample, LY6050 ; LY30 and LY60]), and plasma lactate as an indicator of severity of shock were collected from SHP dogs at the time of diagnosis. SHP dogs were hypocoagulable (prolonged prothrombin time and activated partial thromboplastin time, decreased TEG maximum amplitude) and hyperfibrinolytic (increased LY3050 and TEG LY6050 ) compared to controls. The severity of hypocoagulability was related to protein C activity, while the severity of hyperfibrinolysis was related to plasma lactate concentration. Among the 18 dogs discharged from the hospital, LY3050 was significantly associated with the dose of fresh frozen plasma administered, but none of the parameters were associated with the dose of red blood cells administered. CONCLUSIONS: Dogs with SHP have evidence of hypocoagulability, protein C deficiency, and hyperfibrinolysis. Parameters of hyperfibrinolysis were related to plasma lactate concentrations and volume of plasma transfused during hospitalization. These derangements resemble those found in people with acute coagulopathy of trauma and shock, and activation of protein C may be a common feature to both syndromes.

Human recombinant activated protein C-coated stent for the prevention of restenosis in porcine coronary arteries.

Activated protein C (APC), an endogenous protein, inhibits inflammation and thrombosis and interrupts the coagulation cascade. Here, we investigated the effect of human recombinant APC on the development of neointimal hyperplasia in porcine coronary arteries. Yukon Choice bare metal stents were coated with 2.6 µg APC/mm(2). Under general anesthesia, APC-coated and bare stents were implanted in the left anterior descending and circumflex coronary arteries of 10 domestic pigs. During the 4-week follow-up, animals were treated with dual antiplatelet therapy and neointimal hyperplasia was evaluated via histology. Scanning electron microscopy indicated successful but unequal coating of stents with APC; nearly complete drug release occurred within 4 h. Enzyme-linked immunosorbent assay revealed that intracoronary stent implantation rapidly increased the levels of monocyte chemoattractant protein-1, an effect that was inhibited by APC release from the coated stent. Fibrin deposition and adventitial inflammation were significantly decreased 1 month after implanting APC-coated stents versus bare stents, paralleled by significantly smaller neointimal area (0.98 ± 0.92 vs. 1.44 ± 0.91 mm(2), P = 0.028), higher lumen area (3.47 ± 0.94 vs. 3.06 ± 0.91 mm(2), P = 0.046), and lower stenosis area (22.2 ± 21.2% vs. 32.1 ± 20.1%, P = 0.034). Endothelialization was complete with APC-coated but not bare (90%) stents. P-selectin immunostaining revealed significantly fewer activated endothelial cells in the neointima in the APC group (4.6 ± 1.9 vs. 11.6 ± 4.1%, P < 0.001). Thus, short exposure of coronary arteries to APC reduced inflammatory responses, neointimal proliferation, and in-stent restenosis, offering a promising therapy to improve clinical outcomes of coronary stenting. However, coating stents with APC for prolonged, controlled drug release remains technically challenging.

Proteína C ultrasensible como factor independiente de riesgo en población con y sin antecedentes cardiovasculares / High sensitivity C protein as an independent risk factor in people with and without history of cardiovascular disease

Entre los nuevos biomarcadores del riesgo de eventos cardiovasculares (ECV), la proteína C reactiva detectada mediante técnicas de alta sensibilidad (PCRus) ha sido uno de los más evaluados. En esta revisión se exploró la evidencia existente sobre la utilidad de la PCRus como factor independiente del riesgo de eventos en sujetos sin antecedentes cardiovasculares, y como marcador pronóstico en sujetos con enfermedad cardiovascular crónica o aguda. Se realizó un overview (revisión de revisiones) con búsqueda en las principales bases bibliográficas suplementada por buscadores genéricos de Internet. En una primera etapa se detectaron revisiones sistemáticas, guías de práctica clínica, evaluaciones de tecnologías sanitarias y políticas de cobertura, y en una segunda etapa se identificaron estudios primarios publicados posteriormente a las fechas de búsqueda de las revisiones sistemáticas. Fueron recuperadas 774 citas, de las cuales se incluyeron 36 publicaciones que evaluaron el papel de la PCRus en poblaciones sanas o con antecedentes coronarios. Se encontró evidencia de alta calidad señalando a la PCRus, tanto como factor de riesgo en población general, como pronóstico en aquellos con ECV en todas las poblaciones evaluadas. Su mayor utilidad residió en sujetos sin historia de ECV y riesgo intermedio de eventos a 10 años, en donde la adición de la PCRus a modelos clásicos de estimación del riesgo de eventos mejora la estratificación del riesgo. No se identificó, en cambio, consenso sobre su utilidad clínica como marcador pronóstico en sujetos con enfermedad coronaria crónica o aguda.

Among the new cardiovascular event (CVE) risk biomarkers, C-reactive protein detected using high sensitive techniques (hs-CRP) has been one of the most commonly evaluated. In this review, the available evidence on the usefulness of hs-CRP was explored as an independent risk event factor in subjects with no cardiovascular history and as prognosis in case of chronic or acute cardiovascular condition. An overview (revision of revisions) was carried out searching in the main bibliographic databases and in other general Internet search engines. During the first stage, systematic reviews, clinical practice guidelines, health technology assessments and coverage policies were found and, during the second stage primary studies published after the systematic review search dates were added. Seven hundred and seventy four quotes were found, including 36 papers assessing the role of hs-CRP in healthy populations or with cardiovascular history. High quality evidence was found pointing out hs-CRP, both as risk factor in the general population and as prognostic factor in those with CVE, in all the populations assessed. It was most useful in subjects with a history of CVE and intermediate risk of events at 10 years; where adding hs-CRP to the classical models for event risk estimation improves risk staging. There was no consensus on its clinical usefulness as a prognostic marker in subjects with chronic or acute disease.

[High sensitivity C protein as an independent risk factor in people with and without history of cardiovascular disease]. / Proteína C ultrasensible como factor independiente de riesgo en población con y sin antecedentes cardiovasculares.

Among the new cardiovascular event (CVE) risk biomarkers, C-reactive protein detected using high sensitive techniques (hs-CRP) has been one of the most commonly evaluated. In this review, the available evidence on the usefulness of hs-CRP was explored as an independent risk event factor in subjects with no cardiovascular history and as prognosis in case of chronic or acute cardiovascular condition. An overview (revision of revisions) was carried out searching in the main bibliographic databases and in other general Internet search engines. During the first stage, systematic reviews, clinical practice guidelines, health technology assessments and coverage policies were found and, during the second stage primary studies published after the systematic review search dates were added. Seven hundred and seventy four quotes were found, including 36 papers assessing the role of hs-CRP in healthy populations or with cardiovascular history. High quality evidence was found pointing out hs-CRP, both as risk factor in the general population and as prognostic factor in those with CVE, in all the populations assessed. It was most useful in subjects with a history of CVE and intermediate risk of events at 10 years; where adding hs-CRP to the classical models for event risk estimation improves risk staging. There was no consensus on its clinical usefulness as a prognostic marker in subjects with chronic or acute disease.

[From the "note limitative" to the "registri AIFA"]. / Dalle note limitative ai registri AIFA.

In Italy, over the past 10 years, we have had a proliferation of registers associated with the reimbursement of drugs by the National Health Service. The regulatory path that made them grow comes from the so-called "note limitative" and treatment plans associated with the use of certain drugs. From these experiences different types of registries have been created that collect, at the time of prescription, information about the safety and appropriateness of use of medication where a benefit-risk profile in the general population is still not well defined. The critical analysis of some of these experiences can present positive and negative aspects associated with a regulatory reality now used in clinical practice nationwide.

Evaluating treatment effectiveness in patient subgroups: a comparison of propensity score methods with an automated matching approach.

Propensity score (Pscore) matching and inverse probability of treatment weighting (IPTW) can remove bias due to observed confounders, if the Pscore is correctly specified. Genetic Matching (GenMatch) matches on the Pscore and individual covariates using an automated search algorithm to balance covariates. This paper compares common ways of implementing Pscore matching and IPTW, with Genmatch for balancing time-constant baseline covariates}. The methods are considered when estimates of treatment effectiveness are required for patient subgroups, and the treatment allocation process differs by subgroup. We apply these methods in a prospective cohort study that estimates the effectiveness of Drotrecogin alfa activated, for subgroups of patients with severe sepsis. In a simulation study we compare the methods when the Pscore is correctly specified, and then misspecified by ignoring the subgroup-specific treatment allocation. The simulations also consider poor overlap in baseline covariates, and different sample sizes. In the case study, GenMatch reports better covariate balance than IPTW or Pscore matching. In the simulations with correctly specified Pscores, good overlap and reasonable sample sizes, all methods report minimal bias. When the Pscore is misspecified, GenMatch reports the least imbalance and bias. With small sample sizes, IPTW is the most efficient approach, but all methods report relatively high bias of treatment effects. This study shows that overall GenMatch achieves the best covariate balance for each subgroup, and is more robust to Pscore misspecification than common alternative Pscore approaches.