Host DNases prevent vascular occlusion by neutrophil extracellular traps.

Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

Constructing protein-protein interaction network of hypertension with blood stasis syndrome via digital gene expression sequencing and database mining.

OBJECTIVE: To construct a protein-protein interaction (PPI) network in hypertension patients with blood-stasis syndrome (BSS) by using digital gene expression (DGE) sequencing and database mining techniques. METHODS: DGE analysis based on the Solexa Genome Analyzer platform was performed on vascular endothelial cells incubated with serum of hypertension patients with BSS. The differentially expressed genes were filtered by comparing the expression levels between the different experimental groups. Then functional categories and enriched pathways of the unique genes for BSS were analyzed using Database for Annotation, Visualization and Integrated Discovery (DAVID) to select those in the enrichment pathways. Interologous Interaction Database (I2D) was used to construct PPI networks with the selected genes for hypertension patients with BSS. The potential candidate genes related to BSS were identified by comparing the number of relationships among genes. Confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), gene ontology (GO) analysis was used to infer the functional annotations of the potential candidate genes for BSS. RESULTS: With gene enrichment analysis using DAVID, a list of 58 genes was chosen from the unique genes. The selected 58 genes were analyzed using I2D, and a PPI network was constructed. Based on the network analysis results, candidate genes for BSS were identified: DDIT3, JUN, HSPA8, NFIL3, HSPA5, HIST2H2BE, H3F3B, CEBPB, SAT1 and GADD45A. Verified through qRT-PCR and analyzed by GO, the functional annotations of the potential candidate genes were explored. CONCLUSION: Compared with previous methodologies reported in the literature, the present DGE analysis and data mining method have shown a great improvement in analyzing BSS.

Hemostatic disorders in a JAK2V617F-driven mouse model of myeloproliferative neoplasm.

Thrombosis is common in patients suffering from myeloproliferative neoplasm (MPN), whereas bleeding is less frequent. JAK2(V617F), the main mutation involved in MPN, is considered as a risk factor for thrombosis, although the direct link between the mutation and hemostatic disorders is not strictly established. We investigated this question using conditional JAK2(V617F) knock-in mice with constitutive and inducible expression of JAK2(V617F) in hematopoietic cells, which develop a polycythemia vera (PV)-like disorder evolving into myelofibrosis. In vitro, thrombosis was markedly impaired with an 80% decrease in platelet-covered surface, when JAK2(V617F) blood was perfused at arterial shear over collagen. JAK2(V617F) platelets presented only a moderate glycoprotein (GP) VI deficiency not responsible for the defective platelet accumulation. In contrast, a decreased proportion of high-molecular-weight von Willebrand factor multimers could reduce platelet adhesion. Accordingly, the tail bleeding time was prolonged. In the FeCl3-induced thrombosis model, platelet aggregates formed rapidly but were highly unstable. Interestingly, vessels were considerably dilated. Thus, mice developing PV secondary to constitutive JAK2(V617F) expression exhibit a bleeding tendency combined with the accelerated formation of unstable clots, reminiscent of observations made in patients. Hemostatic defects were not concomitant with the induction of JAK2(V617F) expression, suggesting they were not directly caused by the mutation but were rather the consequence of perturbations in blood and vessel homeostasis.

[Association of hemostatic disorders in diabetic patients with methylenetetrahydrofolate reductase (C677T) and coagulation factors II (G20210A) and V (G1691A) gene polymorphism].

The purpose of the investigation was to study an association of hemostatic disorders in diabetic patients with methylenetetrahydrofolate reductase (MTHFR) (C677T) and coagulation factors II (G20210A) and V (G1691A) gene polymorphism. The investigators examined 90 patients with diabetic nephropathy complicating types 1 and 2 diabetes in 54 and 36 cases, respectively. A control group comprised 100 healthy individuals. A polymerase chain reaction was used to diagnose single-nucleotide substitution of C6777T in the MTHFR gene, a point mutation in the coagulation factor V (FV) gene, and a factor II (FII) G20210A gene mutation in the coagulation factor II (FII) gene. The parameters of platelet and coagulation hemostasis were analyzed. Gene mutations (C677T in the MTHFR gene, G1691A in the FV gene and G20210A in FII gene) are encountered in diabetic patients more frequently than those in healthy individuals. The mutations are associated with increased blood coagulation potential and platelet hyperactivation.

Prekallikrein deficiency in a dog.

Prekallikrein (PK) deficiency is an uncommon disorder in dogs. In this report, we describe a case of a dog that was referred for neurological defects and had a prolonged activated partial thromboplastin time (aPTT) and normal prothrombin time (PT) with no hemostatic defects. By using human PK-deficient plasma, the dog was diagnosed to have PK deficiency. The nucleotide sequence of normal canine PK cDNA was determined and compared with the genomic sequences of PK in the affected dog. The comparison revealed that the dog had a point mutation in exon 8 that leads to an amino acid substitution in the fourth apple domain of PK. This is the first report showing a point mutation of PK in a dog with PK deficiency.

Molecular basis of iron-loading disorders.

Iron-loading disorders (haemochromatosis) represent an important class of human diseases. Primary iron loading results from inherited disturbances in the mechanisms regulating intestinal iron absorption, such that excess iron is taken up from the diet. Body iron load can also be increased by repeated blood transfusions (secondary iron loading), usually as part of the treatment for various haematological disorders. In these syndromes, an element of enhanced iron absorption is also often involved. The central regulator of body iron trafficking is the liver-derived peptide hepcidin. Hepcidin limits iron entry into the plasma from macrophages, intestinal enterocytes and other cells by binding to the sole iron-export protein ferroportin, and facilitating its removal from the plasma membrane. Mutations in hepcidin or its upstream regulators (HFE, TFR2, HFE2 and BMP6) lead to reduced or absent hepcidin expression and a concomitant increase in iron absorption. Mutations in ferroportin that prevent hepcidin binding produce a similar result. Increased ineffective erythropoiesis, which often characterises erythrocyte disorders, also leads to reduced hepcidin expression and increased absorption. Recent advances in our understanding of hepcidin and body iron homeostasis provide the potential for a range of new diagnostic and therapeutic tools for haemochromatosis and related conditions.

Bleeding disorders in Lowe syndrome patients: evidence for a link between OCRL mutations and primary haemostasis disorders.

Lowe syndrome (LS) is a rare X-linked disorder caused by mutations in the oculocerebrorenal gene (OCRL), encoding OCRL, a phosphatidylinositol 5-phosphatase with a RhoGAP domain. An abnormal rate of haemorrhagic events was found in a retrospective clinical survey. Herein, we report the results of exploration of haemostasis in six LS patients. All patients had normal coagulation tests but prolonged closure times (CTs) in the PFA-100 system. Healthy donors' blood samples incubated with a RhoA kinase inhibitor had prolonged CTs. This suggests that an aberrant RhoA pathway in platelets contributes to CT prolongation and primary haemostasis disorders in LS.

Subclinical pulmonary haemorrhage causing a restrictive lung defect in three siblings with a unique urticarial vasculitis syndrome.

Three siblings with urticarial vasculitis syndrome (UVS) are described. All had restrictive lung function abnormalities caused by subclinical pulmonary haemorrhage. The latter was suspected after finding haemosiderin-laden macrophages and a friable bronchial mucosa during elective bronchopulmonary alveolar lavage (BAL). The chest radiographs were normal at presentation but after steroid withdrawal symptoms worsened, haemoglobin levels fell, and Case 1 developed acute pulmonary haemorrhage. This was documented by lung biopsy, which also revealed evidence of old haemorrhage and fibrosis. We concluded that these patients had a unique familial variant of UVS with a previously unreported restrictive lung disease due to subclinical pulmonary haemorrhage.

Vascular thrombohemorrhagic disorders: hereditary and acquired.

This review has summarized the more important diseases that may be accompanied by or lead to a disorder of hemostasis or thrombosis via alterations of the vasculature. It is to be stressed that the vascular component of hemostasis is often overlooked by clinicians caring for patients with disorders of hemostasis and thrombosis. It should be appreciated that the vasculature is intricately related to the coagulation protein system and to platelets when involved in thrombohemorrhagic diatheses. Although many vascular disorders may lead to hemorrhage or thrombosis, it must be appreciated that often it is impossible to discern between a primary vascular defect/damage and a defect that has been induced by platelet activation/dysfunction or procoagulant abnormalities.