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.

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.

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.

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.

Assessment of markers of thrombin generation in patients with acute myocardial infarction complicated by ventricular fibrillation.

BACKGROUND: In most cases, sudden cardiac death is triggered by ischemia-related ventricular tachyarrhythmias and accounts for 50% of deaths from cardiovascular disease in developed countries. Chronic elevation of indicators of coagulation activation has been found in patients with coronary heart disease, but a role of coagulation activation as a potential risk factor for ventricular fibrillation (VF) during acute myocardial infarction (MI) has not been investigated. METHODS: We enrolled 50 patients with a history of MI, of whom 26 presented with VF in the acute phase of myocardial ischemia; 24 patients had an acute MI without ventricular tachyarrhythmias. Levels of thrombin-antithrombin complexes (TAT), prothrombin fragment F1 + 2 (F1 + 2), fibrinopeptide A (FPA), plasmin-antiplasmin complexes (PAP), protein C, antithrombin, activated partial thromboplastin time (aPTT), thromboplastin time, D-Dimer, fibrinogen, and high-sensitivity C-reactive protein (hs-CRP) were measured in plasma samples of all patients. Blood collection was obtained sequentially in two separate settings. Patients were studied at a median of 351 days after the acute coronary event. RESULTS: Higher levels of TAT complexes (13.4 +/- 22.2 vs. 3.03 +/- 4.3 microg/l; p = 0.02), FPA (79.7 +/- 132.3 vs. 24.04 +/- 41.3 ng/ml; p = 0.04), and F1+2 (1.89 +/- 1.3 vs. 1.16 +/- 0.5 nmol/l; p = 0.01) were observed in patients with VF compared with patients without ventricular tachyarrhythmias during the acute phase of MI. D-Dimer levels displayed a trend without reaching statistical significance (0.69 +/- 0.48 vs. 0.48 +/- 0.24 mg/l; p = 0.06). No differences were found in hs-CRP (3.25 +/- 4.5 vs. 4.4 +/- 8.8 mg/l; p = 0.5) and fibrinogen (2.8 +/- 0.9 vs. 2.7 +/- 0.9 g/l; p = 0.6) measurements. Repeat assessment of markers of coagulation activation at a median of 847 days revealed a highly significant decrease in patients with VF. CONCLUSIONS: Markers of thrombin generation are transiently increased in patients with VF during the acute phase of MI. These findings have implications for risk assessment and genetic screening of patients prone to VF during acute myocardial ischemia.

Protein C and protein S assessment in hospital laboratories: which strategy and what role for DNA sequencing?

This paper presents a critical assessment of protein C (PC) and protein S (PS) functional and immunological approaches with regard to DNA sequencing in a large hospital recruitment for thrombosis exploration in more than 1700 consecutive patients. After examination of clinical status and PC and PS phenotype, a genotypic study was implemented for 17 PC-deficient and 28 PS-deficient patients (activity < 70%). Sixty-five percent of the genotyped PC-deficient patients were found to have heterozygous mutations. Among the < 70% values, decreases in PC activity without gene mutation were always slight (mean value 64 +/- 7%) while patients presenting a PC gene mutation had a mean 50 +/- 17% activity (P < 0.05). Among the eight PC mutations found, only one has previously been described. A novel mutation in the promoter region (-1522), located in the HNF-1 site and associated with the Y226H heterozygous mutation, was found in a 9-month-old girl with 4% PC activity. Determination of PS functional activity was considerably improved by contemporaneous measurement of calibration and samples in a single step. Only 50% of the genotyped PS-deficient patients demonstrated heterozygous alterations of the gene. The benefit of sequencing to identify putative causal mutations was only 39% in PS-deficient women, while it was 90% in men. Among the nine PS mutations found, six have not yet been published. In the present paper, we explain our methodological choices and diagnostic strategy.

Hypercoagulability: clinical assessment and treatment.

This review emphasizes pathophysiology, clinical features, assessment, and therapy for hypercoagulability. Risk factors that further increase clotting include obesity, recent surgery, pregnancy, and cancer. Clinical examples of coagulation abnormalities may occur from single or multiple abnormalities and include both inherited and acquired defects. Laboratory testing undertaken at the time of acute thrombosis is often inaccurate or difficult to interpret. Individuals are best tested when they are not taking anticoagulants. Treatment of patients with either inherited or acquired abnormalities usually requires heparin compounds followed by warfarin, but the length of therapy has not yet been settled. Asymptomatic individuals with underlying hypercoagulability may not require treatment except in clot-promoting situations such as trauma, pregnancy, recent surgery, or use of venous access devices. The detection of one abnormality may no longer suffice because multiple defects can be found frequently. In patients with clotting and underlying risk factors, such as malignancy, pregnancy, estrogens, or surgery, an assessment for hypercoagulability should be considered.

[Significance of the activity of indicators of the fibrinolytic system on assessment of the hemostatic state]. / Znachymist' deiakykh pokaznykiv fibrynolitychnoï systemy v otsintsi stanu hemostazu.

Complex analysis of pregnant women haemostasis system before and after caesarian section allowed find the coagulation system activation. It was shown the thrombotic markers accumulation, AT III and protein C levels decrease. Also change of ratio fibrinolytic system components was exposed. Definition of t-PA and PAI-1 activities can be used as prognostic markers of the fibrinolytic chain haemostasis system disfunction.

Single and combined prothrombotic factors in patients with idiopathic venous thromboembolism: prevalence and risk assessment.

The inherited thrombophilias--deficiencies of protein C, protein S, and antithrombin III--and the prothrombotic polymorphisms factor V G1691A and factor II G20210A predispose patients toward venous thromboembolism (VTE). The aim of this study was to determine the prevalence of single and combined prothrombotic factors in patients with idiopathic VTE and to estimate the associated risks. The study group consisted of 162 patients referred for work-up of thrombophilia after documented VTE. The controls were 336 consecutively admitted patients. In all subjects factor V G1691A, factor II G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T were analyzed by specific polymerase chain reactions and restriction enzymes. Activities of antithrombin III and protein C, free protein S antigen, and lupus anticoagulant were determined in a subset of 109 patients who were not receiving oral anticoagulants. The prevalences of heterozygotes and homozygotes for factor V G1691A and factor II G20210A among patients and controls were 40.1% versus 3.9% and 18.5% versus 5.4%, respectively (P=0.0001). The prevalence of homozygotes for MTHFR C677T in patients was 22.8% and in controls, 14.3% (P=0.025). Heterozygous and homozygous factor V G1691A, factor II G20210A, and homozygous MTHFR C677T were found to be independent risk factors for VTE, with odds ratios of 16.3, 3.6, and 2.1, respectively. Two or more polymorphisms were detected in 27 of 162 patients (16.7%) and in 3 of 336 controls (0.9%). Logistic regression analysis disclosed odds ratios of 58.6 (confidence interval [CI], 22.1 to 155.2) for joint occurrence of factor V and factor II polymorphisms, of 35.0 (CI, 14.5 to 84.7) for factor V and MTHFR polymorphisms, and of 7.7 (CI, 3.0 to 19.6) for factor II and MTHFR polymorphisms. Among 109 patients in whom a complete thrombophilic work-up was performed, 74% had at least 1 underlying defect. These data indicate that in most patients referred for evaluation of thrombophilia due to idiopathic VTE, 1 or more underlying genetic predispositions were discernible. The presence of >1 of the prothrombotic polymorphisms was associated with a substantial risk of VTE.

Assessment of activated protein C resistance using a new and rapid venom-based test: STA Staclot APC-R.

Activated protein C (APC) resistance is related to a single point mutation in the factor V gene (FV:Q506) and appears to be the most common inherited risk factor for venous thromboembolism. A reliable screening test is therefore useful. We aimed to evaluate a new APC resistance test, on the basis of the procoagulant activity present in one snake venom of a crotalidae family: STA Staclot APC-R. We studied 36 consecutive patients with an acute deep venous thrombosis (DVT) confirmed by compression ultrasonography and carrying the FV:Q506 allele, assessed by DNA analysis, 103 of their family members and 35 consecutive patients with a proven DVT but who did not carry the FV:Q506 allele. Blood samples were collected within 24 h of admission for the DVT cases and on the day of medical registration for the family members. Tests were performed blind. The STA Staclot APC-R test, using a cut-off value of 0.80, had an overall sensitivity of 100% (95% CI, 95-100) and a specificity of 98.8% (95% CI, 92.0-99.6). An acute thrombosis process did not influence the performance of the test. We conclude that this test is easy and rapid to perform in every day practice and fulfills the criteria for a screening test.

Structure-function assessment of the role of the helical stack domain in the properties of human recombinant protein C and activated protein C.

The role of the helical stack (HS) in defining the properties of human recombinant (r) protein C (PC) and activated protein C (APC) was assessed. To do so, several mutations were made in this region of the molecule and their effects on the proteins examined. Substitution of the entire HS of PC (residues 38-46) by that of human coagulation factor (f) IX (residues 39-47), yielding r-[HSIX]PC, did not result in any substantial changes in the gamma-carboxyglutamic acid domain (GD)-related Ca(2+)-dependent properties of PC or APC, suggesting that the conformation of the HS may play a more dominant role in these Ca(2+)-dependent properties than do the specific amino acids that differ between these two HS regions. On the other hand, the catalytic efficiency of activation of r-[HSIX]PC by the thrombin/thrombomodulin complex was reduced to approximately one-third of that of wtr-PC, a result that demonstrates a specific role for the HS of PC in this activation process. Another mutation, [Ser42-->Pro], was generated in the HS region of r-PC, providing r-[S42P]PC, a change that according to the empirical algorithm based on the Chou-Fasman secondary structure rules, would disrupt the alpha-helical conformation of the HS. The anticoagulant activity of the corresponding r-[S42P]APC was found to be approximately 35% of that of wtr-APC. Because of the lack of any notable effects of this mutation on other GD-related Ca(2+)-dependent properties of r-PC and r-APC, the basis of this anticoagulant activity loss may be due to its nonmaximal alignment with substrate on the PL surface. The results of this study indicate that the role of the HS of r-PC and r-APC is to provide a region of the protein that is needed to assure optimal alignment on the PL or cell surface of the active site of the enzyme with that of the cleavage sites of the substrates, perhaps by functioning as a scaffold for separation of the active site of APC from the PL surface.