Health care resource use, diagnostic delay and disease burden in transthyretin amyloid cardiomyopathy in Sweden.

AIMS: To estimate healthcare resource use and direct healthcare costs of Transthyretin Amyloid Cardiomyopathy (ATTR-CM) in Sweden over 12 months across severity stages as defined by the New York Heart Association (NYHA). Secondary to investigate the current diagnostic trajectory for patients with ATTR-CM in Sweden. METHODS: A stratified inclusion of patients with a confirmed diagnosis of ATTR-CM in different NYHA classes. Data was extracted from medical records in two cardiology clinics in Sweden. Healthcare resource use data were retrospectively collected for 12 months. RESULTS: 38 patients were included, of whom 7 were in NYHA class II, 20 in class III and 4 in class IV. The total cost of health care per patient increased from SEK 69,000 (€6800) in NYHA stage II, SEK 219,000 (€21,500) in NYHA stage III, to SEK 638,000 (€62,900) in stage IV, mainly due to an increase in inpatient stays. Mean time (standard deviation, SD) from any cardiac related diagnosis prior to ATTR-CM diagnosis was 3.5 (3.1) years. CONCLUSIONS: Advanced ATTR-CM stages are associated with significant healthcare costs, as patients more often require resource-intensive inpatient care. The current diagnostic trajectory of ATTR-CM in this study was characterized by a diagnostic delay of several years.

This study shows that both healthcare resource use and healthcare costs increased considerably with a higher degree of ATTR-CM severity.The diagnostic trajectory of ATTR-CM in this study was characterized by a diagnostic delay of several years.Greater disease awareness and a lower threshold for screening risk groups for TTR-amyloidosis is prompted to establish an earlier diagnosis.

A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate.

BACKGROUND: Platelet polyphosphate is an inorganic procoagulant polymer of orthophosphate units that is stored in dense granules and is released upon platelet activation. Here, we describe an assay to measure polyphosphate on the surface of procoagulant human platelets. METHODS AND RESULTS: Recombinant Escherichia coli-expressed exopolyphosphatase deletion mutant PPX_Δ12 labeled with fluorescent Alexa488 dye was used as a polyphosphate probe in flow cytometry. PPX_Δ12-Alexa488-signal dose-dependently increased with long-chain polyphosphate binding to platelets. In contrast, short-chain polyphosphate that is found in the supernatant of activated platelets, did not bind to the platelet surface. Both exopolyphosphatase treatment and polyphosphate pre-incubation abolished PPX_Δ12-Alexa488 binding to polyphosphate on platelets. Stimulation of platelets with thrombin receptor agonist Trap6, and P2Y12 receptor activator ADP increased polyphosphate accumulation on platelet surfaces and PPX_Δ12-Alexa488 signal in a dose-dependent manner. CONCLUSION: This study indicates that long-chain polyphosphate binds to platelet plasma membranes and presents a promising diagnostic assay to measure this interaction on human platelets in platelet-rich plasma. Future investigations will aim to determine if polyphosphate can be used as a novel biomarker of thrombosis. © 2016 International Clinical Cytometry Society.

Polyphosphate nanoparticles on the platelet surface trigger contact system activation.

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII.

Neutralizing blood-borne polyphosphate in vivo provides safe thromboprotection.

Polyphosphate is an inorganic procoagulant polymer. Here we develop specific inhibitors of polyphosphate and show that this strategy confers thromboprotection in a factor XII-dependent manner. Recombinant Escherichia coli exopolyphosphatase (PPX) specifically degrades polyphosphate, while a PPX variant lacking domains 1 and 2 (PPX_Δ12) binds to the polymer without degrading it. Both PPX and PPX_Δ12 interfere with polyphosphate- but not tissue factor- or nucleic acid-driven thrombin formation. Targeting polyphosphate abolishes procoagulant platelet activity in a factor XII-dependent manner, reduces fibrin accumulation and impedes thrombus formation in blood under flow. PPX and PPX_Δ12 infusions in wild-type mice interfere with arterial thrombosis and protect animals from activated platelet-induced venous thromboembolism without increasing bleeding from injury sites. In contrast, targeting polyphosphate does not provide additional protection from thrombosis in factor XII-deficient animals. Our data provide a proof-of-concept approach for combating thrombotic diseases without increased bleeding risk, indicating that polyphosphate drives thrombosis via factor XII.

The polyphosphate/factor XII pathway in cancer-associated thrombosis: novel perspectives for safe anticoagulation in patients with malignancies.

Cancer is an established risk factor for venous thromboembolism (VTE) and VTE is the second leading cause of death in patients with cancer. The incidence of cancer-related thrombosis is rising and is associated with worse outcomes. Despite our growing understanding on tumor-driven procoagulant mechanisms including cancer-released procoagulant proteases, expression of tissue factor on cancer cells and derived microvesicles, as well as alterations in the extracellular matrix of the cancer cell milieu, anticoagulation therapy in cancer patients has remained challenging. This review comments on a newly discovered cancer-associated procoagulant pathway. Experimental VTE models in mice and studies on patient cancer material revealed that prostate cancer cells and associated exosomes display the inorganic polymer polyphosphate on their plasma membrane. Polyphosphate activates blood coagulation factor XII and initiates thrombus formation via the intrinsic pathway of coagulation. Pharmacologic inhibition of factor XII activity protects mice from VTE and reduces thrombin coagulant activity in plasma of prostate cancer patients. Factor XII inhibitors provide thrombo-protection without impairing hemostatic mechanisms and thus, unlike currently used anticoagulants, do not increase bleeding risk. Interference with the polyphosphate/factor XII pathway may provide the novel opportunity for safe anticoagulation therapy in patients with malignancies.

The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis.

Cancer is a leading cause of thrombosis. We identify a new procoagulant mechanism that contributes to thromboembolism in prostate cancer and allows for safe anticoagulation therapy development. Prostate cancer-mediated procoagulant activity was reduced in plasma in the absence of factor XII or its substrate of the intrinsic coagulation pathway factor XI. Prostate cancer cells and secreted prostasomes expose long chain polyphosphate on their surface that colocalized with active factor XII and initiated coagulation in a factor XII-dependent manner. Polyphosphate content correlated with the procoagulant activity of prostasomes. Inherited deficiency in factor XI or XII or high-molecular-weight kininogen, but not plasma kallikrein, protected mice from prostasome-induced lethal pulmonary embolism. Targeting polyphosphate or factor XII conferred resistance to prostate cancer-driven thrombosis in mice, without increasing bleeding. Inhibition of factor XII with recombinant 3F7 antibody reduced the increased prostasome-mediated procoagulant activity in patient plasma. The data illustrate a critical role for polyphosphate/factor XII-triggered coagulation in prostate cancer-associated thrombosis with implications for anticoagulation without therapy-associated bleeding in malignancies.

Plasma contact system activation drives anaphylaxis in severe mast cell-mediated allergic reactions.

BACKGROUND: Anaphylaxis is an acute, potentially lethal, multisystem syndrome resulting from the sudden release of mast cell-derived mediators into the circulation. OBJECTIVES AND METHODS: We report here that a plasma protease cascade, the factor XII-driven contact system, critically contributes to the pathogenesis of anaphylaxis in both murine models and human subjects. RESULTS: Deficiency in or pharmacologic inhibition of factor XII, plasma kallikrein, high-molecular-weight kininogen, or the bradykinin B2 receptor, but not the B1 receptor, largely attenuated allergen/IgE-mediated mast cell hyperresponsiveness in mice. Reconstitutions of factor XII null mice with human factor XII restored susceptibility for allergen/IgE-mediated hypotension. Activated mast cells systemically released heparin, which provided a negatively charged surface for factor XII autoactivation. Activated factor XII generates plasma kallikrein, which proteolyzes kininogen, leading to the liberation of bradykinin. We evaluated the contact system in patients with anaphylaxis. In all 10 plasma samples immunoblotting revealed activation of factor XII, plasma kallikrein, and kininogen during the acute phase of anaphylaxis but not at basal conditions or in healthy control subjects. The severity of anaphylaxis was associated with mast cell degranulation, increased plasma heparin levels, the intensity of contact system activation, and bradykinin formation. CONCLUSIONS: In summary, the data collectively show a role of the contact system in patients with anaphylaxis and support the hypothesis that targeting bradykinin generation and signaling provides a novel and alternative treatment strategy for anaphylactic attacks.

Challenges in oral administration of metronidazole dissolved in drinking water to rhesus monkeys (Macaca mulatta).

Intestinal pathogens such as Entamoeba spp. and Giardia spp. protozoans are not uncommon among rhesus macaques (Macaca mulatta) in research facilities. These infections affect the health of the macaques, potentially causing severe diarrhea, and also pose a risk of zoonotic transmission to human caretakers. Infections must therefore be treated, but no standard treatment for intestinal protozoans in macaques has been developed. Metronidazole is commonly used to treat infections with Giardia spp. and Entamoeba spp. in veterinary medicine, but evidence-based information on effectiveness and dosages for nonhuman primates is lacking, and administration of drugs to nonhuman primates is challenging. The authors designed a study to determine whether oral administration of metronidazole dissolved in drinking water would be successful in rhesus macaques. They monitored daily fluid intake of macaques given water with or without metronidazole and with or without flavored syrup. Metronidazole addition, with or without flavored syrup, resulted in a decrease in fluid intake. Although it was possible to administer metronidazole in drinking water to some macaques, the authors conclude that this strategy is not a practical clinical method because of variation in the amount of water and metronidazole ingested by the macaques.