Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo.

Platelets play a central role in thrombosis, hemostasis, and inflammation. We show that activated platelets release inorganic polyphosphate (polyP), a polymer of 60-100 phosphate residues that directly bound to and activated the plasma protease factor XII. PolyP-driven factor XII activation triggered release of the inflammatory mediator bradykinin by plasma kallikrein-mediated kininogen processing. PolyP increased vascular permeability and induced fluid extravasation in skin microvessels of mice. Mice deficient in factor XII or bradykinin receptors were resistant to polyP-induced leakage. PolyP initiated clotting of plasma via the contact pathway. Ablation of intrinsic coagulation pathway proteases factor XII and factor XI protected mice from polyP-triggered lethal pulmonary embolism. Targeting polyP with phosphatases interfered with procoagulant activity of activated platelets and blocked platelet-induced thrombosis in mice. Addition of polyP restored defective plasma clotting of Hermansky-Pudlak Syndrome patients, who lack platelet polyP. The data identify polyP as a new class of mediator having fundamental roles in platelet-driven proinflammatory and procoagulant disorders.

Factor XIIa inhibitor recombinant human albumin Infestin-4 abolishes occlusive arterial thrombus formation without affecting bleeding.

BACKGROUND: Blood coagulation is a tightly regulated process of sequentially activated serine proteases culminating in fibrin formation, which is critical for limiting posttraumatic blood loss but also may contribute to acute thrombotic diseases, most notably myocardial infarction and stroke. Recent studies with factor XII-deficient mice revealed that the factor XII-induced intrinsic coagulation pathway is essential for pathological thrombus formation but dispensable for hemostasis. Consequently, these findings led to the hypothesis that factor XII could be a promising pharmacological target for safe antithrombotic therapy. METHODS AND RESULTS: The complementary DNA of the previously described factor XIIa inhibitor Infestin-4, cloned from the midgut of Triatoma infestans, was fused to recombinant human albumin (rHA) and analyzed in vitro. The resulting protein rHA-Infestin-4 specifically inhibits factor XIIa and causes prolonged activated partial thromboplastin time in human, mouse, and rat plasma. To assess its inhibitory potency in vivo, mice and rats were injected with rHA-Infestin-4 and challenged in pathological thrombus formation models. In addition, bleeding assays were performed. rHA-Infestin-4 completely abolished occlusive arterial thrombus formation in mice and rats while leaving hemostasis fully intact. Furthermore, rHA-Infestin-4 was highly protective in a murine model of ischemic stroke. CONCLUSIONS: These results identify rHA-Infestin-4 as a promising agent to achieve powerful protection from ischemic cardiovascular and cerebrovascular events without affecting hemostasis.

Fistula between cystic artery pseudoaneurysm and cystic bile duct cause of acute anemia one year after laparoscopic cholecystectomy.

We present a case of hemorrhage from a cystic artery pseudoaneurysm one year after laparoscopic cholecystectomy. A 78-year-old male with a history of recurrent melena, hematemesis, and right upper abdominal pain was admitted to our emergency department. His blood pressure was 60/30 mm Hg with a pulse rate of 100 beats per minute. Hemoglobin was 7.6 g/dL and white blood cell count 19500/mm(3). Computed tomography scan of the abdomen and selective digital subtraction arteriography showed a pseudoaneurysm in the region of the former bed of the gallbladder. During gastroscopy, a pulsatile bleeding out of the papilla of Vater was found. Surgery by the open approach confirmed the presence of a cystic artery pseudoaneurysm and showed an additional fistula between the pseudoaneurysm and the cystic bile duct. Resection of the pseudoaneurysm and revision of the common bile duct with implantation of a T-tube was performed. The patient recovered well and was discharged from our hospital three weeks after surgery.

Recurrent severe gastrointestinal bleeding and malabsorption due to extensive habitual megacolon.

Dilatation of the colon and the rectum, which is not attributable to aganglionosis, is a rare finding and can be the result of intractable chronic constipation. We report a rare case of a 29-year-old male patient with impressive megacolon, in whom Hirschsprung's or Chagas disease was ruled out. In the present case, dilatation of the colon was most likely due to a behavioral disorder with habitual failure of defecation. Chronic stool retention led to a bizarre bulging of the large bowel with displacement of the other abdominal organs and severe occult blood loss. Because of two episodes of life-threatening gastrointestinal bleeding despite conventional treatment of constipation, a surgical approach for bowel restoration was necessary. Temporary loop ileostomy had to be performed for de-pressurization of the large bowel and the subsequent possibility for effective antegrade colonic lavage to remove impacted stools. Shortly after the operation, the patient was healthy and could easily manage the handling of the ileostomy. However, the course of the megacolon in this young adult cannot be predicted and the follow-up will have to reveal if regression of this extreme colonic distension with reestablishment of regular rectal perception will occur.

Physicochemical characterisation of rVIII-SingleChain, a novel recombinant single-chain factor VIII.

rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational modifications of FVIII products are N-glycosylation of asparagine residues and tyrosine sulphations. Here, the physicochemical properties, thrombin cleavage products and post-translational modifications of rVIII-SingleChain were investigated and compared against commercially available recombinant FVIII (rFVIII) products with a predominant two-chain structure (B-domain deleted rFVIII and full-length rFVIII). rVIII-SingleChain was expressed in Chinese hamster ovary (CHO) cells and purified by chromatographic methods. Physicochemical properties of rVIII-SingleChain or thrombin-derived cleavage products were assessed using size-exclusion chromatography, reversed-phase chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Analysis of the respective carbohydrate structures was performed after release of N-glycans by PNGase F followed by fluorescence labelling and high-performance liquid chromatography. Proteolysis by trypsin generated the corresponding peptides, which were analysed for sulphated tyrosines by liquid chromatography-electrospray ionisation time of flight-mass spectrometry. rVIII-SingleChain was shown to be of high purity and homogeneity, and presented a well-defined single-chain molecule with predominant ß-sheet conformation. The coagulation-relevant thrombin-activation products of rVIII-SingleChain were comparable with those obtained by activation of commercially available rFVIII products. rVIII-SingleChain post-translational modifications were similar to other CHO cell-derived rFVIII products for N-glycopattern and tyrosine sulphation. In conclusion, rVIII-SingleChain is of high homogeneity and purity, and provides an expected cleavage pattern on activation, setting the basis for optimal efficacy in the patient.

Reboxetine acutely stimulates cortisol, ACTH, growth hormone and prolactin secretion in healthy male subjects.

In this single-blind study the effects of acute oral administration of the selective noradrenaline reuptake inhibitor reboxetine on the cortisol (COR), ACTH, growth hormone (GH) and prolactin (PRL) secretion were examined in 12 healthy male volunteers. In a randomized order, the subjects received placebo or reboxetine (4 mg) at 0800 h on two different days. After insertion of an intravenous catheter, blood samples were drawn 1 hour prior to the administration of placebo or reboxetine, at time of administration, and during the time of 5 hours thereafter at periods of 30 minutes. Serum concentrations of COR, GH, and PRL as well as plasma levels of ACTH were determined in each blood sample by means of double antibody RIA, fluoroimmunoassay and chemiluminescence immunometric assay methods. The area under the curve (AUC) value was used as parameter for the COR, ACTH, GH, and PRL response. Using t-tests for paired samples, statistical analysis revealed significant stimulatory effects of reboxetine on COR, ACTH, GH, and PRL secretion, compared to placebo (mean AUC values+/-S.E.M. (a) reboxetine: COR 127893.20+/-8125.75 nmol/l x min; ACTH 2385.68+/-387.19 pmol/l x min; GH 56026.59+/-15594.87 pmol/l x min; PRL 113961.60+/-10280.44 pmol/l x min; (b) placebo: COR 83672.19+/-5225.20 nmol/l x min; ACTH 1449.83+/-190.67 pmol/l x min; GH 9308.16+/-3402.75 pmol/l x min; PRL 64663.28+/-7283.62 pmol/l x min). Mean arterial blood pressure and heart rate were significantly increased by reboxetine, too. Our results suggest that besides COR, ACTH and GH secretion, the release of PRL is also under the control of the noradrenergic systems in man.

Regulation of glucose transporters in human peritoneal mesothelial cells.

BACKGROUND: Risk factors for peritoneal fibrosis and mesothelial cell (MsC) injury in CAPD are infections and bioincompatibility of the dialysate, including high glucose concentrations. To study a potential link between dialysate and glucose toxicity in MsC, we investigated the expression of facilitative glucose transporters (GLUT), which could contribute to glucose toxicity. METHODS: After induction of cell differentiation, MsC were incubated in regular medium or medium with 60 mM D-glucose, 30 mM glucose plus 30 mM mannitol, 60 mM mannitol, PD effluent, or with a cytokine mix. Expression of GLUT1, GLUT3, SGLT and GAPDH/L32 was studied by RNase protection assay. MsC were incubated under identical conditions with 14C-fluoro-deoxy-glucose for 30 minutes and glucose uptake was measured. To estimate Vmax and Km, 14C-fluoro-deoxy-glucose uptake rates were determined over a range of 0.6 to 10 mM unlabeled glucose. RESULTS: The cytokine mix significantly stimulated GLUT1 expression (3-fold) and GLUT3 (1.7-fold). There was a 1.4-fold increase in GLUT1 (p<0.05) and a 1.7-fold increase in GLUT3 (p<0.05) after incubation in high glucose but not in mannitol or PD-effluent controls. Glucose uptake studies confirmed this increase after incubation in 30 mM (p<0.05) and 60 mM glucose solutions. Kinetic studies showed the Km was approximately 3.7 mM for this transport. CONCLUSIONS: GLUT mRNA expression and glucose uptake are induced by high ambient glucose concentrations and cytokines. Unlike many other cells, MsC are not able to protect themselves from increased glucose concentrations by downregulation of GLUTs. The intracellular glucose concentration may therefore increase during CAPD, affecting growth factor expression and glycosylation, and contributing to glucose toxicity.

A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk.

Currently used anticoagulants prevent thrombosis but increase bleeding. We show an anticoagulation therapy without bleeding risk based on a plasma protease factor XII function-neutralizing antibody. We screened for antibodies against activated factor XII (FXIIa) using phage display and demonstrated that recombinant fully human antibody 3F7 binds into the FXIIa enzymatic pocket. 3F7 interfered with FXIIa-mediated coagulation, abolished thrombus formation under flow, and blocked experimental thrombosis in mice and rabbits. We adapted an extracorporeal membrane oxygenation (ECMO) cardiopulmonary bypass system used for infant therapy to analyze clinical applicability of 3F7 in rabbits. 3F7 provided thromboprotection as efficiently as heparin, and both drugs prevented fibrin deposition and thrombosis within the extracorporeal circuit. Unlike heparin, 3F7 treatment did not impair the hemostatic capacity and did not increase bleeding from wounds. These data establish that targeting of FXIIa is a safe mode of thromboprotection in bypass systems, and provide a clinically relevant anticoagulation strategy that is not complicated by excess bleeding.

Experience with ultrasound scissors and blades (UltraCision) in open and laparoscopic liver resection.

OBJECTIVE: The authors used new ultrasonically activated scissors and blades in open and laparoscopic liver resections to investigate their capabilities. SUMMARY BACKGROUND DATA: Despite standardized techniques for liver resection, the surgical death rate ranges from 4% to 20%. Dissection of liver parenchyma may cause considerable blood loss. Further complications include liver failure, hematoma, infections, and bile leakage. The surgical technique is an important factor in preventing intraoperative and postoperative complications. Various techniques have been developed for safe and careful dissection of the liver parenchyma. In addition to blunt dissection using the "finger fracture" technique, various ultrasonic dissectors, water jet dissectors, laser systems, and specially prepared suction devices have been used, but none of these techniques can achieve complete hemostasis during dissection. METHODS: The instrument was used in open and laparoscopic liver resections. It works by means of a longitudinally vibrating blade or scissors in tissue dissection, coagulation, and preparation. Denaturation of protein and coagulation of vessels up to 2 to 3 mm is possible as a result of the vibration. In this prospective study of a consecutively sampled case series of 41 patients, the author sought to gain experience in handling this instrument and in its capabilities, and they also measured the extent of intraoperative and postoperative blood loss. RESULTS: The UltraCision was used for 64 open liver resections in 39 patients and for 2 laparoscopic liver resections in 2 patients. Blood loss in laparoscopic resections was less than 50 mL; in open resections it averaged 820 mL. Eleven patients (28%) needed blood transfusions. There were no biliary leakages or abscesses. One patient died after postoperative bleeding leading to fatal liver failure after 4 weeks. Handling of the instrument and cutting and coagulation quality were satisfactory. CONCLUSIONS: The advantages over other resection techniques are limited heat and smoke generation and the lack of current flow through the patient. The handling and coagulation and cutting quality of the UltraCision appeared satisfactory and safe. The new instrument can be recommended for laparoscopic and open resections of the liver.