Valeria Matus

Circulating platelet-derived microparticles in systemic lupus erythematosus. Association with increased thrombin generation and procoagulant state.

The risk for thrombosis is significantly increased in systemic lupus erythematosus (SLE), affecting both venous and arterial vessels. Activated platelets are known to participate in thrombus formation and growth. A general feature of activated cells is the shedding of microparticles (MP) which support coagulation by exposure of negatively charged phospholipids and possibly tissue factor (TF). In this work we characterized circulating MP in patients with SLE and their relationship with a procoagulant state. Thirty patients with SLE (aged 15-72 years, mean age 38 years) and 20 healthy controls (aged 22-54 years, mean age 34 years) were studied; patients fulfilled 4 revised criteria for SLE. The number and cellular source of circulating MP were determined by flow cytometry using double labeling with specific monoclonal antibodies and annexin V. Thrombin generation was measured as the endogenous thrombin potential (ETP) without the addition of exogenous phospholipids and TF; under these conditions the generation of thrombin depended directly on the number of MP present in plasma. Thrombin anti-thrombin (TAT) and plasmin-antiplasmin (PAP) complexes were measured by ELISA. Compared to the controls, circulating MP were significantly elevated in the patient group (1218 +/- 136 vs 653 +/- 74 x 10(3)/ml plasma, p: 0.0007). In both groups, most of these MP were of platelet origin (927 +/- 131 vs 517 +/- 72 x 10(3)/ml plasma, p:0.009 ). ETP was higher among patients as compared to the controls (804 +/- 64 vs 631 +/- 37 nM thrombin, p: 0.025). Plasma levels ofTAT in patients and controls were 3.4 +/- 0.8 and 1.4 +/- 0.5 microg/L, respectively (p:0.04), and of PAP complexes were 62.5 +/- 14 and 24.05 +/- 2.5 microg/ml, respectively (p: 0.014). The number of platelet-derived MP correlated significantly with thrombin generation (r: 0.42; p: 0.038) and TAT levels (r: 0.40; p: 0.035). We did not find an association of circulating MP with disease activity nor with the presence of antiphospholipid antibodies. The increased number of circulating platelet-derived microparticles and their association with high ETP and activation of the coagulation system suggest that these microparticles play an important role in the pathogenesis of the prothrombotic state in SLE patients.

Degradation of 2,4,6-trichlorophenol via chlorohydroxyquinol in Ralstonia eutropha JMP134 and JMP222.

Abbreviations: 2,4‐D: 2,4‐dichlorophenoxyacetate; 2,4,6‐TCP: 2,4,6‐trichlorophenol; HQ: hydroxy‐quinol; 6‐CHQ: 6‐chlorohydroxyquinol; 2,6‐DCBQ: 2,6‐dichlorobenzoquinone; 2,6‐DCHQ: 2,6‐dichlorohydroxyquinone; CMA: 2‐chloromaleylacetate; MA: maleylacetate; HQDO: hydroxyquinol dioxygenase; MAR: maleylacetate reductase.

Diagnosis of mild platelet function disorders. Reliability and usefulness of light transmission platelet aggregation and serotonin secretion assays

Light transmission platelet aggregation (PA), adapted to measure platelet secretion (PS), is the reference test for diagnosing platelet functional disorders (PFD). Problems with these assays include lack of standardisation, unknown reproducibility and lack of universally accepted diagnostic criteria. We addressed these issues in patients with inherited mucocutaneous bleeding (MCB). Normal and abnormal PA tests in 213 patients were reproducible in 93·3% and 90·4% of the cases, respectively. Mean intra‐subject coefficient of variation for PA with strong agonists were <9% and mean intra‐class correlation coefficient for weak agonists were >0·86 (P < 0·0001). Concomitant impaired PA with 10 μmol/l‐adrenaline and 4 μmol/l‐ADP was observed in 13·7% of the controls. This combination was not considered per se a criterion for PFD. PA with adrenaline ≥42% or irreversible aggregation with 4 μmol/l ADP had 93% and 95% Negative Predictive Value for diagnosing PFD, respectively. PA defects were consistently associated with abnormal PS. In contrast, 14·3% of patients with MCB had isolated PS. Thus, standardized PA/PS assays are highly reproducible and concordant in normal and patient populations. Normal PA with adrenaline and low ADP concentration robustly predict a normal PA. Simultaneous PA/PS assays enable the diagnosis of isolated PS defects. This study confirmed that hereditary PA–PS defects are highly prevalent.

An adenine insertion in exon 6 of human GP6 generates a truncated protein associated with a bleeding disorder in four Chilean families

Glycoprotein VI (GPVI), 60–65 kDa, is a major collagen receptor on platelet membranes involved in adhesive and signaling responses. Mice lacking GPVI have impaired platelet response to collagen and defective primary adhesion and subsequent thrombus formation. Complete or partial deficiency of GPVI in humans is a rare condition presenting as a mild bleeding disorder. The defect in most of the reported patients is acquired and associated with other diseases. To date, only two patients have been characterized at the molecular level who carry different compound heterozygous mutations in the GP6 gene.

Degradation of environmental pollutants by Alcaligenes eutrophus JMP134 (pJP4)

In this work, the degradation of several environmental pollutants by Alcaligenes eutrophus JMP134 (pJP4) was studied. It has been reported that this strain grows on 2,4-dichlorophenoxyacetate, 3-chlorobenzoate, and phenol, and that it is resistant to Hg2+ toxicity. Except degradation of phenol, all these properties are encoded on pJP4. Although this bacterium was unable to grow on several chlorophenols or chloroguaiacols as sole carbon and energy source, it proliferated in 4-chlorophenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol. Degradation of 2,4,6-trichlorophenol (up to 0.4 mM) did not depend on the presence of pJP4, since the cured strain JMP222 also expressed this property. In chemostats fed with 2,4,6-trichlorophenol (0.4 mM) or phenol (1 mM) plus 2,4,5-trichlorophenol (0.05 mM), strain JMP134 was able to degrade 2,4,5-trichlorophenol. On the other hand, strain JMP134 grew at higher levels of HgCl2 (0.1 mM) or merbromine (1.5 mM) in rich medium, but growth was not detected in minimal medium cultures, containing either chlorinated or nonchlorinated compounds. The ability of strain JMP134 to survive and degrade chlorphenols in the presence of pulp bleaching effluent was assessed in aerated batch cultures. This biodegradable organic matter-and chlorophenol-containing effluent allowed the proliferation of the strain JMP134. When 2,4-dichlorophenoxyacetate or 2,4,6-trichlorophenol was added, complete degradation of these compounds was detected. The degradation was not affected by the presence of indigenous microorganisms or the amount of degradable material.

Human platelet interaction with E. coli O111 promotes tissue-factor-dependent procoagulant activity, involving Toll like receptor 4

Platelets have a major role in clotting activation and contribute to the innate immune response during systemic infections. Human platelets contain tissue factor (TF) and express functional Toll-like receptor 4 (TLR4). However, the role of TLR4 in triggering the procoagulant properties of platelets, upon challenge with bacteria, is yet unknown. Our hypothesis is that E. coli O111-TLR4 interaction activates platelets and elicits their procoagulant activity. We demonstrated that the strain, but not ultrapure LPS, increased surface P-selectin expression, platelet dependent TF procoagulant activity (TF-PCA) and prompted a faster thrombin generation (TG). Blockade of TLR4 resulted in decreased platelet activation, TF-PCA and TG, revealing the participation of this immune receptor on the procoagulant response of platelets. Our results provide a novel mechanism by which individuals with bacterial infections would have an increased incidence of blood clots. Furthermore, the identification of platelet TF and TLR4 as regulators of the effect of E. coli O111 might represent a novel therapeutic target to reduce the devastating consequences of the hemostatic disorder during sepsis.

Procarboxypeptidase U (TAFI) and the Thr325Ile proCPU polymorphism in patients with hereditary mucocutaneous hemorrhages.

BACKGROUND Patients with hereditary mucocutaneous bleeding are difficult to diagnose and many of them fulfill the category of bleeders of unknown cause (BUC). The pathogenic role of hyperfibrinolysis has received little attention, despite the successful use of antifibrinolytic drugs in treating many of these patients. Theoretically, decreased plasma procarboxypeptidase U (proCPU) levels or lower carboxypeptidase U (CPU) stability would result in higher fibrinolytic activity and bleeding tendency. METHODS We analyzed plasma proCPU and the distribution of the Thr325Ile proCPU polymorphism in 193 patients with mucocutaneous bleeding of whom 116 were bleeders of unknown cause (BUC), and in 143 healthy, age and sex-matched controls. RESULTS ProCPU concentration was higher in women than in men, increased with age, and was significantly correlated with clot lysis time, platelet count, APTT, and PT. However, proCPU levels were unexpectedly higher in patients than in controls (968+/-134 vs. 923+/-147 U/L, p=0.004). The allele distribution of the Thr325Ile proCPU polymorphism was similar in both groups, with a low percentage of homozygous Ile/Ile. CONCLUSIONS Our results indicate that the proCPU system is not of major importance in the bleeding pathogenesis of these patients. The higher proCPU levels in the patients may even modestly counteract the bleeding tendency.