The role of autolysis loop in determining the specificity of coagulation proteases

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an ~500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.

Proteína C ativada no tratamento de recém-nascido com sepse, choque e disfunção de múltiplos órgãos e sistemas: relato de caso e revisão de literatura / Activated C protein in the treatment of a newborn with sepsis, shock and multiple organ dysfunction systems: case report and literature review

JUSTIFICATIVA E OBJETIVOS: A sepse grave representa a síndrome de resposta inflamatória sistêmica resultante de uma infecção, na presença de disfunção cardiovascular, síndrome do desconforto respiratório agudo ou duas ou mais disfunções orgânicas. Embora a mortalidade atribuída à sepse em crianças tenha sido reduzida de maneira significativa nas últimas décadas, a incidência de óbitos em recém-nascidos permanece elevada (20 por cento a 40 por cento), a despeito dos avanços em cuidados intensivos. O objetivo deste estudo foi descrever o caso de um recém-nascido com sepse, choque e disfunção de múltiplos órgãos e sistemas (DMOS) que se beneficiou do uso da proteína C ativada. RELATO DO CASO: Recém-nascido prematuro, do sexo masculino, nascido de cesariana em decorrência de ruptura prematura de membranas e sofrimento fetal agudo. Internado na UTI-Neonatal por insuficiência respiratória aguda secundária à pneumonia intra-útero. Recebeu assistência ventilatória, surfactante pulmonar exógeno e antibioticoterapia precocemente, evoluindo, no entanto, com hipertensão pulmonar persistente e choque. Houve difícil controle do quadro infeccioso, a despeito de ajustes no esquema de antibioticoterapia, evoluindo com DMOS. No 28º dia, foi iniciado o uso da proteína C ativada. O paciente evoluiu favoravelmente à medicação, com resolução das disfunções orgânicas e ausência de sangramentos. CONCLUSÕES: A proteína C ativada não pode ser prescrita de maneira rotineira no tratamento de recém-nascidos com sepse grave. No caso relatado, no entanto, acredita-se que ela tenha contribuído para a resolução das disfunções orgânicas apresentadas pelo paciente.

BACKGROUND AND OBJECTIVES: Severe sepsis represents the systemic inflammatory response resulting from an infection, associated with one of the following: cardiovascular organ dysfunction, acute respiratory distress syndrome or two or more organ dysfunctions. Although the mortality rate from sepsis in children has steadily decreased in the last decades, the mortality rate in newborns remains high (20 percent to 40 percent) despite the development in intensive care. The authors describe a newborn who suffered from sepsis, shock and multiple organ dysfunction syndromes (MODS) that recovered after the administration of activated C protein. CASE REPORT: A premature newborn underwent cesarean section because of a premature rupture of membranes and acute fetal distress. The newborn developed acute respiratory distress due to intrauterine pneumonia and was taken to the Neonatal Intensive Care Unit. The patient was given mechanical ventilation, exogenous pulmonary surfactant and antibiotics early in the treatment. Nevertheless, he developed persistent pulmonary hypertension and shock. The control of the infection was difficult, despite the adjustment of the antibiotics, resulting in the development of MODS. On the 28th day, activated C protein was given to the patient. The administration of the drug was successful and the patient recovered from the organ dysfunction without bleeding. CONCLUSIONS: The activated C protein can't be recommended as a routine in the treatment of newborns with severe sepsis. However, in this case, it contributed to the recovery of the organ dysfunctions presented by the patient.

Combination of thrombophilia markers in acute myocardial infarction of the young.

BACKGROUND: The pathogenesis of arterial thrombotic disease involves multiple genetic and environmental factors related to atherosclerosis and thrombosis. But, there have been very few studies in India which have investigated some of the thrombophilia markers. AIM: To look for combined thrombophilia in MI patients. SETTINGS AND DESIGN: One hundred twenty patients of myocardial infarction (age below 40 yrs.) were recruited 8-10 weeks after stabilization. Hundred age and sex-matched healthy controls were also recruited in the present study. METHODS AND MATERIAL: Following thrombophilia markers were screened in these patients--plasma fibrinogen, protein C, protein S, antithrombin III, factor V Leiden, PT G20210A polymorphism, MTHFR C677T, homocysteine, fibrinogen b448 Arg/Lys polymorphism and CBS T833C mutation. STATISTICAL ANALYSIS: Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 10.0, SPSS Inc., Chicago, USA. RESULTS AND CONCLUSION: Elevated fibrinogen levels, homocysteine (p< 0.001 and homocysteine with odds ratio 6.26) and factor V Leiden (p=0.038) were independently associated with MI in our patients. A total of 37 patients (42.5%) had the presence of more than one thrombophilia markers in combination. Out of these, 10 had the presence of three markers in combination and 1 had five thrombophilia markers in combination. Only 2 controls had prothrombotic markers in combination. Combined prothrombotic risk factors were significant in cases in comparison to controls (p< 0.001). Further larger studies on a nationwide basis recruiting a large number of young MI patients should be done to substantiate these findings.

Avaliaçäo de coagulaçäo, fibrinólise e proteína C em pacientes de risco e com doenças coronarianas / Evaluation of coagulation, fibrinolysis and protein C in risk patients and patients presenting coronarian diseases

A hemostasia é resultante do equilíbrio entre pró-coagulantes e anticoagulantes, envolvendo vasos, plaquetas, proteínas da coagulaçäo e da fibrinólise e anticoagulantes naturais. Todos estes componentes estäo inter-relacionados, constituindo os sistemas de coagulaçäo, anticoagulaçäo e fibrinólise. Muitos fatores, genéticos ou adquiridos, podem contribuir para romper este equilíbrio, levando a estados de hipo ou hipercoagulabilidade. Em doenças coronarianas como a angina e o infarto, há uma maior ativaçäo das plaquetas e das proteínas da coagulaçäo, favorecendo a formaçäo de trombos. Na tentativa de restaurar a hemostasia, ocorre a intervençäo do sistema fibrinolítico, o qual promove a lise do coágulo e desobstrui o vaso. Neste trabalho foram avaliados os mecanismos da coagulaçäo e da fibrinólise e a proteína C, um anticoagulante natural. Foram estudados 20 pacientes com doenças coronarianas, notadamente angina de peito (n = 8) e infarto agudo do miocárdio (n = 12), além de pacientes potencialmente em risco de desenvolver doença cardiovascular (n = 17). O grupo infarto foi pareado com indivíduos sadios do ponto de vista clinicolaboratorial (grupo-controle, n = 12). Os resultados revelaram uma diferença significativa nos níveis de fibrinogênio nos grupos de angina e infarto quando comparados ao grupo-controle. Níveis de proteína C ativada também mostraram diferença significativa entre os grupos de risco e infarto. Os demais parâmetros hemostáticos avaliados näo diferiram significativamente entre os grupos estudados, porém foi observada uma tendência à hipercoagulabilidade nos grupos de pacientes quando comparados ao grupo-controle

Haemostatic disorders in nonsplenectomized and splenectomized thalassaemic children

A group of 40 thalassaemic patients [20 splenectomized and 20 nonsplenectomized] from the Haematology Unit of Tanta University Hospital [age range: 3-14 years] were studied to identify the mechanisms by which haemorrhagic and thrombotic complications occur in thalassaemic patients. The patients' levels of protein C, antithrombin III and in vitro platelet aggregation in response to collagen were compared with those of 20 controls. The study suggests that thrombocytosis, increased platelet aggregation and decreased natural coagulation inhibitors [protein C and antithrombin III] in splenectomized thalassaemic children may be significant in thrombotic complications in such patients. Defective platelet aggregation and prothrombin activity in nonsplenectomized children may also give rise to haemorrhagic tendencies

Resistencia a la proteína C activada / Resistence to protein C activation

La trombosis es la causa más frecuente de muerte en el mundo occidental, en EUA por tromboembolismo pulmonar, trombosis coronaria y accidentes cerebrovasculares mueren 1.990.000/año. Aparecen 398.000 trombosis venosas profundas en paciente/año y abortos o pérdidas fetales por trombosis de la placenta. El sistema inhibidor de la coagulación está compuesto por la Antitrombina III(ATIII) cofactor II de la Heparina (CIIIHep), inhibudor de la vía del factor tisular (TFPI), la vía fibronolítica y el sistema anticoagulante de la proteína C, que posee el mayor número de trastornos genéticos asociados con trombosis venosa (TV). La trombina activa a la proteína C; (proteína C activada) (PCA) con la finalidad de clivar a los cofactores VIIIa y la Va.La proteína c recibe la potenciación de la proteína S. El factor V (FV) sinérgicamente con la S como cofactor de la PCA en la degradación del VIIIa. La PCA produce un efecto anticoagulante medible por APTT. La falla de éste, es la resistencia a la proteína C activada (RPCA) común en la población caucasiana. Se hereda en forma autosómica dominante. Algunos portadores nunca desarrollan trombosis, en otros el cuadro es recurrente a tempbrana edad. Se agrava la situación por la coexistencia de otros factores de riesgo congénitos o adquiridos : ingestión de anticonceptivos, embarazo, cáncer, traumas y cirugías. La mutación se describió en el síndrome HELLP , y en la preeclampsia con un 22 por ciento de positividades. El estudio de la anomalía por el test RPCA es de utilidad clínica pero solo el estudio del factor V LEIDEN por PCR permite el conocimiento de la alteración genética. No hay pautas firmes sobre tratamiento. Si la trombosis es recurrentes, puede llegarse a la terapéutica profiláctica por vida

Resistência à proteína C ativada / Resistance to activeted protein C

O fator V da coagulaçäo, quando ativado, participa como um co-fator essencial na ativaçäo da pró-trombina pelo fator X ativado. O fator V ativado é inativado pela proteína C ativada, numa reaçäo potencializada pela proteína S. Uma mutaçäo no éxon 10 (Arg 506 Gln) do gene do fator V dß origem a uma molécula do fator V que näo é adequadamente inativada pela proteína C ativada, o que causa a chamada resistência à proteína C ativada.

Resistance to activated protein C (APC) in Indian patients of thrombophilia.

In a pilot study, 28 consecutive patients with thrombosis and 23 age and sex matched healthy normal controls were investigated with normalised APC sensitivity ratio (n-APC-SR) to know the defect exists and if so its prevalence in India. Six of the 28 (21.4%) thrombotic patients had n-APC-SR of < 0.76 (range 0.55 to 0.74) as compared to the normal control value (0.97 +/- 0.105). Our study shows APC resistance defect in a significant proportion (21.4%) of Indian patients with thrombotic disorders.

Protein C levels in ischaemic heart disease.

Protein C is a circulating glycoprotein with anticoagulant properties. Functional and immunological levels of protein C were determined in 34 cases of ischaemic heart disease and 12 healthy age-matched controls. The sensitive colorimetric assay was used to determine the functional levels and ELISA for antigenic levels. Mean protein C activity and antigenic levels were found to be elevated in these patients as compared to controls. Protein C levels in the three individual subgroups-acute myocardial infarction, previous myocardial infarction and chronic stable angina pectoris-were also raised as compared to controls. The elevation was significant in the case of the acute myocardial infarction group. These results further support the hypothesis that the body synthesises increased amounts of protein C in ischaemic heart disease to compensate for the hypercoagulable state that exists in this disorder, thus playing a protective role.