Recombinant nematode anticoagulant protein c2, an inhibitor of tissue factor/factor VIIa, attenuates coagulation and the interleukin-10 response in human endotoxemia.

The tissue factor-factor (F)VIIa complex (TF/FVIIa) is responsible for the initiation of blood coagulation under both physiological and pathological conditions. Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent inhibitor of TF/FVIIa, mechanistically distinct from tissue factor pathway inhibitor. The first aim of this study was to elucidate the pharmacokinetics and pharmacodynamics of a single intravenous (i.v.) dose of rNAPc2. The second aim was to study its effect on endotoxin-induced coagulation and inflammation. Initially, rNAPc2 was administered to healthy volunteers in three different doses. There were no safety concerns and the pharmacokinetics were consistent with previous studies, in which rNAPc2 was administered subcutaneously. rNAPc2 elicited a dose-dependent reduction of the endogenous thrombin potential and a selective prolongation of prothrombin time. Subsequently, the effect on endotoxin-induced coagulation and inflammation was studied. The administration of rNAPc2 completely blocked the endotoxin-induced thrombin generation, as measured by plasma prothrombin fragment F1+2. The endotoxin-induced effect on fibrinolytic parameters such as plasmin-antiplasmin complexes and plasminogen activator inhibitor type 1 was not affected by rNAPc2. The administration of rNAPc2 attenuated the endotoxin-induced rise in interleukin (IL)-10, without affecting the rise in other cytokines. In conclusion, rNAPc2 is a potent inhibitor of TF/FVIIa, which was well tolerated and could safely be used intravenously in this Phase I study in healthy male volunteers. A single i.v. dose rNAPc2 completely blocked endotoxin-induced thrombin generation without affecting the fibrinolytic response. In addition, rNAPc2 attenuated the endotoxin-induced rise in IL-10, without affecting the rises in other cytokines.

Inhibition of the tissue factor pathway of coagulation by recombinant nematode anticoagulant protein c2 during elective coronary stent implantation.

BACKGROUND: Exposure of tissue factor (TF) to the circulation during coronary stent implantation initiates coagulation activation and may contribute to the risk of thrombotic complications. In this study, we investigated whether inhibition of TF-factor VIIa by recombinant Nematode Anti-coagulant Protein c2 (rNAPc2) is able to suppress haemostatic and inflammatory activity in patients undergoing elective intracoronary stenting. METHODS: In a randomised, double-blind design, 102 patients received either placebo or rNAPc2 (biological half-life >50 hours) at doses of 3.5, 5.0, 7.5 and 10.0 µg/kg as a single subcutaneous administration two to six hours before angioplasty. All patients also received aspirin, clopidogrel and unfractionated heparin (activated clotting time >250 seconds during angioplasty). Serial blood samples were collected before and after the intervention. RESULTS: At 30 hours after stenting, all rNAPc2 treatment groups but not the placebo group demonstrated a reduction from baseline of prothrombin fragment F1+2 and D-dimer plasma levels (to 23 and 12% below baseline values at the highest dose, respectively), which were significantly lower in three rNAPc2 groups compared with placebo (p≤0.03). TF plasma levels were initially reduced in all rNAPc2 groups and returned to baseline values 18 hours after stent implantation. These three markers all increased to above baseline values in the placebo group. Levels of P-selectin, antithrombin III and interleukin-8 were not or only slightly affected by the intervention or by rNAPc2, whereas a significant 2.8 to 4.1 fold increase of C-reactive protein plasma levels was found in all patient groups after the procedure. CONCLUSION: In contrast to the inflammatory response, coagulation activation after elective coronary stent implantation, which is observed in spite of the use of multiple antithrombotic drugs, can be attenuated by inhibition of the TF-factor VIIa complex using rNAPc2. Inhibition of the TF-mediated pathway of coagulation may be an important target to prevent thrombotic complications after coronary stenting.

Genetic variation in coagulation and fibrinolytic proteins and their relation with acute myocardial infarction: a systematic review.

BACKGROUND: It is pathophysiologically conceivable that genetic variations in coagulation and fibrinolytic proteins are associated with the risk of myocardial infarction. Methods and Results- We performed a literature search to identify published case-control studies correlating the factor V Leiden or prothrombin G20210A mutations or fibrinogen G-455A or plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphisms with the risk of myocardial infarction. Studies were included only if they used solid diagnostic criteria and complied with published methodological criteria. A common OR with corresponding 95% CI was calculated for the risk of myocardial infarction in a fixed-effect model according to Mantel-Haenszel. The factor V Leiden and prothrombin G20201A mutations did not significantly correlate with myocardial infarction (OR 1.26, 95% CI 0.94 to 1.67, P=0.12 and OR 0.89, 95% CI 0.59 to 1.35, P=0.6, respectively). Inclusion of the studies that investigated young patients (<55 years) made the association significant for factor V Leiden (OR 1.29, 95% CI 1.03 to 1.61, P=0.02). Homozygosity for the fibrinogen -455A allele was significantly associated with a decreased risk of myocardial infarction (OR 0.66, 95% CI 0.44 to 0.99, P=0.04), whereas the PAI-1 4G4G genotype was significantly associated with increased risk (OR 1.20, 95% CI 1.04 to 1.39, P=0.04). CONCLUSIONS: Associations between these genetic variations and myocardial infarction were weak or absent. In the absence of clinical implications, our results indicate that screening of patients with myocardial infarction for these genetic variations is not warranted.

[Thrombocyte receptors: current views and therapeutic options]. / Trombocytenreceptoren: huidige inzichten en behandelingsmogelijkheden.

In the action of thrombocytes during stemming of a bleeding after damage to a blood vessel, receptors on the thrombocyte membrane play an important part. Adhesion of platelets takes place via specific binding of receptors; the main binding is that of glycoprotein (Gp) Ib to Von Willebrand factor which is synthetized by endothelial cells. Activation of thrombocytes is stimulated by adhesion and by agonists. Weak agonists, through production of thromboxane A2 and release of agonists from granules cause a self-fortifying process of thrombocyte stimulation; strong agonists (like thrombin) lead also to activation of Gp IIb/IIIa receptors. Aggregation of thrombocytes occurs after activation of Gp IIb/IIIa receptors. During stimulation, a change of shape occurs which enables binding to suitable plasma proteins of which the main one is fibrinogen. Knowledge of thrombocyte receptors enhances the insight into the prognosis and efficacy of certain treatments in diseases in which platelet aggregation is pivotal. Of the six categories of antiplatelet drugs, antagonists of Gp IIb/IIIa receptors are the most potent. In clinical trials good results have been obtained in patients with coronary disease of the intravenously administered form added to acetylsalicylic acid.

Low-oxygen stress and water deficit induce cytosolic pyruvate orthophosphate dikinase (PPDK) expression in roots of rice, a C3 plant.

Pyruvate orthophosphate dikinase (PPDK) is known for its role in C4 photosynthesis but has no established function in C3 plants. Abscisic acid, PEG and submergence were found to markedly induce a protein of about 97 kDa, identified by microsequencing as PPDK, in rice roots (C3). The rice genome was found to contain two ppdk loci, osppdka and osppdkb. We isolated osppdka cDNA, which encodes a cytosolic rice PPDK isoform of 96.6 kDa, that corresponded to the ABA-induced protein from roots. Western blot analysis showed a PPDK induction in roots of rice seedlings during gradual drying, cold, high salt and mannitol treatment, indicating a water deficit response. PPDK was also induced in the roots and sheath of submerged rice seedlings, and in etiolated rice seedlings exposed to an oxygen-free N2 atmosphere, which indicated a low-oxygen stress response. None of the stress treatments induced PPDK protein accumulation in the lamina of green rice seedlings. Ppdk transcripts were found to accumulate in roots of submerged seedlings, concomitant with the induction of alcohol dehydrogenase 1. Low-oxygen stress triggered an increase in PPDK activity in roots and etiolated rice seedlings, accompanied by increases in phosphoenolpyruvate carboxylase and malate dehydrogenase activities. The results indicate that cytosolic PPDK is involved in a metabolic response to water deficit and low-oxygen stress in rice, an anoxia-tolerant species.

A semi-quantitative ELISA for detection of Trypanosoma brucei gambiense specific antibodies in serum and cerebrospinal fluid of sleeping sickness patients.

A semi-quantitative ELISA, using variable surface glycoprotein of T.b. gambiense as antigen, was developed for the detection of antibodies of different immunoglobulin isotypes in serum and cerebrospinal fluid of sleeping sickness patients. Using the assay, the antibody profiles of paired serum and cerebrospinal fluid samples of 28 patients have been studied. Total concentrations of various Ig isotypes were determined as well. In serum and cerebrospinal fluid a drastic increase in IgG, basically IgG1, as well as in IgM levels was observed. The concentration of IgA remained relatively normal. The antitrypanosomal antibodies detected in serum and cerebrospinal fluid were mainly of the IgG (IgG1 and IgG3) and IgM isotypes. Measurement of immunoglobulin and trypanosome specific antibody concentrations in serum and CSF allows calculation of intrathecal antibody synthesis and is a possible tool for determining the clinical stage of sleeping sickness.

A group 3 LEA cDNA of rice, responsive to abscisic acid, but not to jasmonic acid, shows variety-specific differences in salt stress response.

A cDNA clone oslea3, encoding a group 3 late-embryogenesis abundant (LEA) protein was isolated from roots of rice seedlings (Oryza sativa L.). The encoded OSLEA3 protein has previously been found to accumulate to higher levels in roots of two salt-tolerant compared to a salt-sensitive rice variety in response to abscisic acid (ABA) [Moons et al., 1995. Plant Physiol. 107, 177-186]. The OSLEA3 protein (Mr 20.5, pI 6.5) characteristically contains ten imperfect 11-mer amino acid repeats. Exogenous application of ABA and exposure to salt shock (150 mM NaCl) rapidly induces a de novo, abundant oslea3 transcript accumulation in seedling roots, whereas application of jasmonic acid (9 microM) does not induce oslea3 expression. The stress-induced oslea3 transcript gradually declined upon prolonged salt shock, as wilting-induced damage became irreversible. oslea3 expression was compared for the salt-tolerant variety Pokkali and the salt-sensitive cultivar Taichung N1. Higher maximal mRNA levels were found in roots of the tolerant variety, also declining less rapidly upon sustained salt shock, concomitant with a delayed drop in shoot water content. DNA blot analysis indicated the existence of a small oslea3 gene family in rice with an equal gene number in both ecotypes. The results suggest that a differential regulation of oslea3 expression is an aspect of the varietal differences in salt stress tolerance.

An abscisic-acid- and salt-stress-responsive rice cDNA from a novel plant gene family.

A novel cDNA clone osr40cl, encoding a abscisic acid (ABA)-responsive 40-kDa protein previously associated with salt tolerance (Moons et al. '1995' Plant Physiol 107: 177-186), was isolated from roots of rice seedlings (Oryza sativa L.). Exogenously applied ABA and salt shock induced a marked increase of the osr40cl transcript level in roots of seedlings whereas constant osr40cl mRNA levels were found in the shoot. The root-specific salinity-induced osr40cl mRNA accumulation was rapid and gradually declined upon prolonged salt shock. Plant growth regulators, signalling the wounding and the pathogen response, did not enhance osr40cl expression, indicating a salt- and osmotic-stress-specific response. The encoded OSR40cl protein was found to be hydrophilic, rich in histidine residues (6%) constituting putative metal-binding domains, and to consist of a duplicated domain of 151 amino acids (75% identical), that can form amphiphilic alpha-helical structures. The gene osr40cl belongs to a multigene family. Two osr40 genes were isolated, osr40g2 and osr40g3, tandemly arranged in an 8-kb region of the rice genome. Antisera raised against a conserved OSR40 peptide recognized different OSR40 proteins that accumulated in roots upon exposure to salt stress. The OSR40 protein family included 29-KDa proteins and two 40-kDa proteins, the latter most probably corresponding to OSR40cl and OSR40g2 with duplicated domain structures. The osr40g3 transcript encoded a single copy of the OSR40 domain and exhibited a shoot-specific expression. Results indicate that OSR40cl plays a role in the adaptive response of roots to an hyper-osmotic environment and belongs to a novel plant protein family that most probably has structural functions.

Antagonistic effects of abscisic acid and jasmonates on salt stress-inducible transcripts in rice roots.

Abscisic acid (ABA) and jasmonates have been implicated in responses to water deficit and wounding. We compared the molecular and physiological effects of jasmonic acid (JA) (< or = 10 microM), ABA, and salt stress in roots of rice. JA markedly induced a cationic peroxidase, two novel 32- and 28-kD proteins, acidic PR-1 and PR-10 pathogenesis-related proteins, and the salt stress-responsive SalT protein in roots. Most JA-responsive proteins (JIPs) from roots also accumulated when plants were subjected to salt stress. None of the JIPs accumulated when plants were treated with ABA. JA did not induce an ABA-responsive group 3 late-embryogenesis abundant (LEA) protein. Salt stress and ABA but not JA induced oslea3 transcript accumulation. By contrast, JA, ABA, and salt stress induced transcript accumulation of salT and osdrr, which encodes a rice PR-10 protein. However, ABA also negatively affected salT transcript accumulation, whereas JA negatively affected ABA-induced oslea3 transcript levels. Endogenous root ABA and methyl jasmonate levels showed a differential increase with the dose and the duration of salt stress. The results indicate that ABA and jasmonates antagonistically regulated the expression of salt stress-inducible proteins associated with water deficit or defense responses.

Molecular and physiological responses to abscisic acid and salts in roots of salt-sensitive and salt-tolerant Indica rice varieties.

The Indica rice (Oryza sativa L.) varieties Pokkali and Nona Bokra are well-known salt tolerance donors in classical breeding. In an attempt to understand the molecular basis of their tolerance, physiological and gene expression studies were initiated. The effect of abscisic acid (ABA) on total proteins in roots from 12-d-old seedlings of Pokkali, Nona Bokra, and the salt-sensitive cultivar Taichung N1 were analyzed on two-dimensional gels. The abundance of ABA-induced proteins was highest in the most tolerant variety, Pokkali. Three ABA-responsive proteins, present at different levels in roots from tolerant and sensitive varieties, were further characterized by partial amino acid analysis. A novel histidine-rich protein and two types of late embryogenesis abundant (LEA) proteins were identified. Protein immunoblotting revealed that the levels of dehydrins and group 3 LEA proteins were significantly higher in roots from tolerant compared with sensitive varieties. Endogenous ABA levels showed a transient increase in roots exposed to osmotic shock (150 mM NaCl). Peak ABA concentrations were 30-fold higher for Nona Bokra and 6-fold higher for Pokkali compared with Taichung N1. Both the salt-induced endogenous ABA levels and a greater molecular response of root tissue to ABA were associated with the varietal differences in tolerance.