Plasma cross-linked fibrin degradation product (XLFbDP) assays in an in vivo model of fibrinolysis.

Assumptions regarding the elaboration of plasma cross-linked fibrin degradation products (XLFbDPs) in vivo were tested using an experimental model in which particulate human fibrin was infused into rabbits and the products of lysis monitored with an immunoassay utilizing DD-3B6/22, a monoclonal antibody to human cross-linked derivatives. XLFbDPs were generated following the infusion of a suspension of cross-linked fibrin, attaining a peak between 40 and 60 min, then falling at a rate approximating a plasma half-life of 2 h. The major in vivo products of lysis of cross-linked fibrin, identified by SDS-PAGE of immunoextracted plasma, were D-dimer and high-molecular-weight moieties. Peak levels of XLFbDPs achieved correlated with the amount of fibrin administered. Since XLFbDP levels were no higher when fibrin infusion was followed by infusions of streptokinase and human plasminogen, it is concluded that endogenous mechanisms of lysis were already maximally stimulated. Infusions of non-cross-linked (NXL) fibrin or of fibrinogen led to much smaller, but measurable, rises in XLFbDP. In the latter group, XLFbDP levels rose further following fibrinolytic therapy. Treatment with epsilon aminocaproic acid (EACA) caused partial (greater than 50%) inhibition of lysis while pre-treatment with nitrogen mustard, inducing leucopenia, virtually abolished the appearance of XLFbDPs in the circulation. This implies that fibrinolytic responses are substantially dependent upon cellular functions sensitive to nitrogen mustard

Purification and characterization of a prothrombin activator from the venom of the Australian brown snake, Pseudonaja textilis textilis

A simple procedure, involving chromatography on concanavalin A-Sepharose and gel filtration, has been developed for the purification of a prothrombin activator from the venom of the Australian brown snake Pseudonaja textilis textilis. The prothrombin activator, which is a major venom component, is a high molecular weight protein (Mr greater than or equal to 200,000) which yields a number of subunits when examined by SDS-PAGE. It is related antigenically to the venom prothrombin activator of the taipan Oxyuranus scutellatus. P. textilis prothrombin activator is able to coagulate citrated plasma, warfarin plasma, and Factor V- and Factor X-deficient plasmas; to convert purified human prothrombin to thrombin; and to hydrolyse the peptide p-nitroanilide substrate S-2222. Calcium ions and phospholipids had little if any effect on the rates of coagulation of citrated plasma or S-2222 hydrolysis catalysed by this enzyme

Identification of D dimer-E complex in disseminated intravascular coagulation

Serum fibrin degradation products in a patient with severe disseminated intravascular coagulation (caused by fulminant pneumococcal sepsis), were characterized using immunoprecipitation, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and crossed immunoelectrophoresis. These revealed a spectrum of fragments identified as high molecular weight (HMW) complexes, a component with mobility on SDS PAGE similar to that of fibrinogen X ("X"), D dimer and E. By their electrophoretic characteristics and reactions with antisera to fragments E and D it was found that most of the D dimer and E were noncovalently complexed as D dimer-E, and that there was relatively little free D dimer and free E. This pattern of FDP (HMW complexes, "X" and D dimer-E) has also been identified during the lysis of crosslinked fibrin by plasmin. The HMW complexes and "X" are believed to be crosslinked X oligomers and crosslinked Y-Y or Y-D respectively

Fibrinolysis as a feature of disseminated intravascular coagulation (DIC) after Pseudonaja textilis textilis envenomation

Blood was obtained from four patients envenomated by the Australian common brown snake, Pseudonaja textilis textilis. This elapid snake has one of the most toxic venoms in the world, containing extremely potent neurotoxic and coagulant components. The latter is a potent complete prothrombinase, converting prothrombin to α-thrombin, and comprises more than 30 % of the total venom protein. The four envenomated patients developed a typical consumption coagulopathy. Serial serum and plasma samples from patients were studied by immunoaffinity adsorption, 2-alanine precipitation of fibrinogen and fibrinogen-related products, and 2-dimensional immunoelectrophoresis, and assay for crosslinked fibrin degradation products as D dimer, using the monoclonal antibody, DD-3B6/22. These procedures showed the virtually complete disappearance of fibrinogen, accompanied by the appearance of large quantities of fibrinogen and fibrin degradation products consisting of both crosslinked and noncrossed species. With recovery, a homogeneous high molecular weight fibrinogen was observed. The data suggest that the prothrombin activator of this venom causes the generation of thrombin which subsequently converts fibrinogen to fibrin and stimulates partial crosslinking of both α and γ-chains. The resultant disseminated intravascular coagulation is accompanied by very active secondary fibrinolysis which apparently limits the extent of any microvascular thrombosis but which may contribute to a bleeding tendency

Measurement of crosslinked fibrin degradation products. An immunoassay using monoclonal antibodies

We have prepared a monoclonal antibody which recognises an antigenic determinant on D-dimer, a specific fragment resulting from the degradation of crosslinked fibrin. This antibody has been used in the development of an enzyme-linked immunoassay for D dimer and related degradation products containing crosslinked gamma-gamma chains, to provide a simple assay of circulating crosslinked fibrin degradation products suitable for clinical use. Since these crosslinked fibrin degradation products are characteristic of fibrinolysis, as distinct from fibrinogenolysis, their measurement should aid in the diagnosis, evaluation and monitoring of thrombotic and thrombolytic states. In preliminary studies, low concentrations of crosslinked fibrin derivatives were detected in normal sera. High levels were found in 30/30 patients with disseminated intravascular coagulation and in the majority of patients having deep venous thrombosis or pulmonary embolism

Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity

The snake venom group C prothrombin activators contain a number of components that enhance the rate of prothrombin activation. The cloning and expression of full-length cDNA for one of these components, an activated factor X (factor Xa)-like protease from Pseudonaja textilis as well as the generation of functional chimeric constructs with procoagulant activity were described. The complete cDNA codes for a propeptide, light chain, activation peptide (AP) and heavy chain related in sequence to mammalian factor X. Efficient expression of the protease was achieved with constructs where the AP was deleted and the cleavage sites between the heavy and light chains modified, or where the AP was replaced with a peptide involved in insulin receptor processing. In human kidney cells (H293F) transfected with these constructs, up to 80% of the pro-form was processed to heavy and light chains. Binding of the protease to barium citrate and use of specific antibodies demonstrated that gamma-carboxylation of glutamic acid residues had occurred on the light chain in both cases, as observed in human factor Xa and the native P. textilis protease. The recombinant protease caused efficient coagulation of whole citrated blood and citrated plasma that was enhanced by the presence of Ca2+. This study identified the complete cDNA sequence of a factor Xa-like protease from P. textilis and demonstrated for the first time the expression of a recombinant form of P. textilis protease capable of blood coagulation

Global, regional, and national disability-adjusted life years (DALYs) for 306 diseases and injuries and healthy life expectancy (HALE) for 188 countries, 1990-2013: Quantifying the epidemiological transition

Background The Global Burden of Disease Study 2013 (GBD 2013) aims to bring together all available epidemiological data using a coherent measurement framework, standardised estimation methods, and transparent data sources to enable comparisons of health loss over time and across causes, age-sex groups, and countries. The GBD can be used to generate summary measures such as disability-adjusted life-years (DALYs) and healthy life expectancy (HALE) that make possible comparative assessments of broad epidemiological patterns across countries and time. These summary measures can also be used to quantify the component of variation in epidemiology that is related to sociodemographic development. Methods We used the published GBD 2013 data for age-specific mortality, years of life lost due to premature mortality (YLLs), and years lived with disability (YLDs) to calculate DALYs and HALE for 1990, 1995, 2000, 2005, 2010, and 2013 for 188 countries. We calculated HALE using the Sullivan method; 95 uncertainty intervals (UIs) represent uncertainty in age-specific death rates and YLDs per person for each country, age, sex, and year. We estimated DALYs for 306 causes for each country as the sum of YLLs and YLDs; 95 UIs represent uncertainty in YLL and YLD rates. We quantified patterns of the epidemiological transition with a composite indicator of sociodemographic status, which we constructed from income per person, average years of schooling after age 15 years, and the total fertility rate and mean age of the population. We applied hierarchical regression to DALY rates by cause across countries to decompose variance related to the sociodemographic status variable, country, and time. Findings Worldwide, from 1990 to 2013, life expectancy at birth rose by 6·2 years (95 UI 5·6-6·6), from 65·3 years (65·0-65·6) in 1990 to 71·5 years (71·0-71·9) in 2013, HALE at birth rose by 5·4 years (4·9-5·8), from 56·9 years (54·5-59·1) to 62·3 years (59·7-64·8), total DALYs fell by 3·6 (0·3-7·4), and age-standardised DALY rates per 100 000 people fell by 26·7 (24·6-29·1). For communicable, maternal, neonatal, and nutritional disorders, global DALY numbers, crude rates, and age-standardised rates have all declined between 1990 and 2013, whereas for non-communicable diseases, global DALYs have been increasing, DALY rates have remained nearly constant, and age-standardised DALY rates declined during the same period. From 2005 to 2013, the number of DALYs increased for most specific non-communicable diseases, including cardiovascular diseases and neoplasms, in addition to dengue, food-borne trematodes, and leishmaniasis; DALYs decreased for nearly all other causes. By 2013, the five leading causes of DALYs were ischaemic heart disease, lower respiratory infections, cerebrovascular disease, low back and neck pain, and road injuries. Sociodemographic status explained more than 50 of the variance between countries and over time for diarrhoea, lower respiratory infections, and other common infectious diseases; maternal disorders; neonatal disorders; nutritional deficiencies; other communicable, maternal, neonatal, and nutritional diseases; musculoskeletal disorders; and other non-communicable diseases. However, sociodemographic status explained less than 10 of the variance in DALY rates for cardiovascular diseases; chronic respiratory diseases; cirrhosis; diabetes, urogenital, blood, and endocrine diseases; unintentional injuries; and self-harm and interpersonal violence. Predictably, increased sociodemographic status was associated with a shift in burden from YLLs to YLDs, driven by declines in YLLs and increases in YLDs from musculoskeletal disorders, neurological disorders, and mental and substance use disorders. In most country-specific estimates, the increase in life expectancy was greater than that in HALE. Leading causes of DALYs are highly variable across countries. Interpretation Global health is improving. Population growth and ageing have driven up numbers of DALYs, but crude rates have remained relatively constant, showing that progress in health does not mean fewer demands on health systems. The notion of an epidemiological transition - in which increasing sociodemographic status brings structured change in disease burden - is useful, but there is tremendous variation in burden of disease that is not associated with sociodemographic status. This further underscores the need for country-specific assessments of DALYs and HALE to appropriately inform health policy decisions and attendant actions. © 2015 Elsevier Ltd