A mouse model to study the alterations in haemostatic and inflammatory parameters induced by Lonomia achelous caterpillar haemolymph.

A mouse model was established to reproduce the haemorrhagic syndrome which occurs in humans after accidental contact with the hairs of the caterpillar Lonomia achelous (LA) and measures the haemostatic and inflammatory alterations that occur as a result of this contact. Mice were injected intradermally with different doses (0.4, 0.8 and 1.6 mg/animal) of L. achelous haemolymph (LAH). Haematological (haemoglobin, haematocrit, platelet count, differential leukocyte count), haemostatic (fibrinogen, plasminogen, factor XIII [FXIII], fibrinolytic activity) and inflammatory parameters (tumour necrosis factor alpha [TNF-α], nitric oxide [NO]) were measured at different times up to 48 h. C57BL/6 mice responded to LAH injection, in terms of these parameters, in a manner similar to that seen in humans, whereas the BALB/c mice were unresponsive. In C57BL/6 mice injected with LAH, time course measurements showed: a) a reduction in the haemoglobin, haematocrit, fibrinogen, FXIII and plasminogen levels, b) no effect on the platelet count and c) immediate leukocytosis and an increase in the fibrinolytic activity in plasma. An inflammatory response (TNF-α) was observed within 1 h post-injection, followed by a more persistent increase in serum NO. These findings suggest that C57BL/6 mice represent a useful model of the haemorrhagic syndrome observed in humans who have suffered contact with the caterpillar, permitting a deeper understanding of the role of the inflammatory response in the haematological and haemostatic manifestations of this syndrome.

Plasma fibrinogen level and the risk of major cardiovascular diseases and nonvascular mortality: an individual participant meta-analysis.

CONTEXT: Plasma fibrinogen levels may be associated with the risk of coronary heart disease (CHD) and stroke. OBJECTIVE: To assess the relationships of fibrinogen levels with risk of major vascular and with risk of nonvascular outcomes based on individual participant data. DATA SOURCES: Relevant studies were identified by computer-assisted searches, hand searches of reference lists, and personal communication with relevant investigators. STUDY SELECTION: All identified prospective studies were included with information available on baseline fibrinogen levels and details of subsequent major vascular morbidity and/or cause-specific mortality during at least 1 year of follow-up. Studies were excluded if they recruited participants on the basis of having had a previous history of cardiovascular disease; participants with known preexisting CHD or stroke were excluded. DATA EXTRACTION: Individual records were provided on each of 154,211 participants in 31 prospective studies. During 1.38 million person-years of follow-up, there were 6944 first nonfatal myocardial infarctions or stroke events and 13,210 deaths. Cause-specific mortality was generally available. Analyses involved proportional hazards modeling with adjustment for confounding by known cardiovascular risk factors and for regression dilution bias. DATA SYNTHESIS: Within each age group considered (40-59, 60-69, and > or =70 years), there was an approximately log-linear association with usual fibrinogen level for the risk of any CHD, any stroke, other vascular (eg, non-CHD, nonstroke) mortality, and nonvascular mortality. There was no evidence of a threshold within the range of usual fibrinogen level studied at any age. The age- and sex- adjusted hazard ratio per 1-g/L increase in usual fibrinogen level for CHD was 2.42 (95% confidence interval [CI], 2.24-2.60); stroke, 2.06 (95% CI, 1.83-2.33); other vascular mortality, 2.76 (95% CI, 2.28-3.35); and nonvascular mortality, 2.03 (95% CI, 1.90-2.18). The hazard ratios for CHD and stroke were reduced to about 1.8 after further adjustment for measured values of several established vascular risk factors. In a subset of 7011 participants with available C-reactive protein values, the findings for CHD were essentially unchanged following additional adjustment for C-reactive protein. The associations of fibrinogen level with CHD or stroke did not differ substantially according to sex, smoking, blood pressure, blood lipid levels, or several features of study design. CONCLUSIONS: In this large individual participant meta-analysis, moderately strong associations were found between usual plasma fibrinogen level and the risks of CHD, stroke, other vascular mortality, and nonvascular mortality in a wide range of circumstances in healthy middle-aged adults. Assessment of any causal relevance of elevated fibrinogen levels to disease requires additional research.

A novel mutation (deletion of Aalpha-Asn 80) in an abnormal fibrinogen: fibrinogen Caracas VI. Consequences of disruption of the coiled coil for the polymerization of fibrin: peculiar clot structure and diminished stiffness of the clot.

An abnormal fibrinogen was identified in a 10-year-old male with a mild bleeding tendency; several years later, the patient developed a thrombotic event. Fibrin polymerization of plasma from the propositus and his mother, as measured by turbidity, was impaired. Plasmin digestion of fibrinogen and thrombin bound to the clot were both normal. The structure of clots from both plasma and purified fibrinogen was characterized by permeability, scanning electron microscopy and rheological measurements. Permeability of patients' clots was abnormal, although some measurements were not reliable because the clots were not mechanically stable. Consistent with these results, the stiffness of patients' clots was decreased approximately two-fold. Electron microscopy revealed that the patients' clots were very heterogeneous in structure. DNA sequencing of the propositus and his mother revealed a new unique point mutation that gives rise to a fibrinogen molecule with a missing amino acid residue at Aalpha-Asn 80. This new mutation, which would disrupt the alpha-helical coiled-coil structure, emphasizes the importance of this part of the molecule for fibrin polymerization and clot structure. This abnormal fibrinogen has been named fibrinogen Caracas VI.

The effects of additional carbohydrate in the coiled-coil region of fibrinogen on polymerization and clot structure and properties: characterization of the homozygous and heterozygous forms of fibrinogen Lima (Aalpha Arg141-->Ser with extra glycosylation).

Fibrinogen Lima is an abnormal fibrinogen with an Aalpha Arg141-->Ser substitution resulting in an extra N-glycosylation at Aalpha Asn139, which seems to be responsible for the impairment of fibrin polymerization. We have studied the polymerization and properties of clots made from both plasma and purified fibrinogen of both the homozygous and heterozygous forms. The clot permeation studies with both plasma and purified protein revealed a normal flux through the network for the heterozygous form but very decreased permeation in the homozygous form. Consistent with turbidity results, the clot network of the homozygous form, seen by scanning electron microscopy, was tight and composed of thin fibers, with many branch points, while the appearance of clots from the heterozygous form was similar to that of control clots, but in both cases the fibers were more curved than those of control clots. The rheological properties of clots from the homozygous form were also altered, with rigidity being increased in plasma clots, but decreased in the purified system, a consequence of the balance between numbers of branch points and fiber curvature. From these results it seems that the extra carbohydrate moiety, located in the alpha coiled-coil region close to the betaC domains, impairs the protofibril lateral association process, giving rise to thinner, more curved fibers, with the structural anomalies being most pronounced in the clots from the homozygous plasma. These studies support a model for fibrin polymerization in which the betaC-betaC interactions are involved in lateral aggregation.

Thrombolytic effect of lonomin V in a rabbit jugular vein thrombosis model.

Thrombolytic efficacy of lonomin V (LV), a protein isolated from Lonomia achelous caterpillars haemolymph, administered either as a single intravenous bolus or as a continuous infusion, was evaluated in a rabbit jugular vein thrombosis model, and compared with those of single-chain tissue-type plasminogen activator (sct-PA) and two-chain urokinase-type plasminogen activator (tcu-PA). As a bolus LV, at doses of 100 000 IU/kg body weight (bw) produced an activator-induced thrombolysis (AIL) of 50.94% +/- 12.4 compared with 14.4% +/- 10.8 for tcu-PA at the same dose. As a continuous infusion at doses of 200 000 IU/kg bw LV produced an AIL of 45.8%, whereas sct-PA and tcu-PA produced an AIL of 69.9 and 33.7%, respectively. Fibrinogen, plasminogen and alpha-2-antiplasmin levels decreased significantly with the higher doses of LV, sct-PA, and tcu-PA. Factor XIII levels were significantly reduced in a dose-dependent manner only with LV. In conclusion, LV produces a dose-dependent thrombolysis in combination with a decrease in factor XIII activity.

Mode of perturbation of Asahi fibrin assembly by the extra oligosaccharides.

Steric hindrance by the backbone of extra oligosaccharides at gamma-Asn 308 may cause the repulsive force to widen the junction at the D:D interface, and thus, interfere with the longitudinal elongation and lateral association of protofibrils.

Lonomia genus caterpillar envenomation: clinical and biological aspects.

Persons who have been in contact with Lonomia achelous or Lonomia obliqua caterpillars present external and internal bleeding and opening of recently healed wounds. Hematological tests show normal platelet count, prolonged prothrombin time, activated partial thromboplastin time and thrombin time, totally corrected by normal plasma. Decreased fibrinogen (Fg), factor (F) V, FXIII, plasminogen and alpha(2)-antiplasmin with increased FVIII: C, von Willebrand factor, Fg degradation products and D dimers. Tissue plasminogen activator, plasminogen activator inhibitor and protein C varied. In L. achelous biological fluids, compounds with anticoagulant or procoagulant properties have been identified. In L. obliqua bristle extracts, mainly procoagulant activities have been identified. Subcutaneous injections of L. achelous crude extracts and a semipurified fraction reduce Fg, plasminogen and FXIII in rabbits. Intravenous injections of a very purified fraction of L. achelous in rabbits produce lysis of preformed thrombi, a decrease of Fg, plasminogen, alpha(2)-antiplasmin, FXIII and inhibition of postthrombolytic thrombus growth. Subcutaneous injections of L. obliqua bristle extracts prolong prothrombin time and activated partial thromboplastin time and reduce FXIII. Intravenous injections of crude bristle extract and a purified fraction of L. obliqua induce disseminated intravascular coagulation.

Lonomia genus caterpillar toxins: biochemical aspects.

In 1967 we reported for the first time five cases of an acquired bleeding disorder in humans which developed after contact with saturnidae caterpillars. Since that time, other cases have been reported in Brazil, French Guyana, Peru, Paraguay and Argentina. The caterpillars have been identified as Lonomia achelous (LA) in Venezuela and northern Brazil and as Lonomia obliqua (LO) in southern Brazil. All patients present pain and a burning sensation at the site of contact. Within a few hours hematomas and hematuria are seen in combination with intracerebral and intraperitoneal hemorrhage (in some cases also renal failure). Hematological tests show: mild anemia with leucocytosis; prolonged PT, PTT and ThT; decreased fibrinogen, factor V, factor XIII, plasminogen and alpha2-antiplasmin levels; increased factor VIII:c, von Willebrand factor, and FDPs/D-dimers levels with normal ATIII and platelets. Factor VII, factor II and PC levels varied. Several activities similar to or directed against blood clotting factors have been identified in LA: fibrinolytic enzymes, which degrade fibrinogen producing abnormal FDPs; prothrombin activators: one direct and one factor Xa-like; a thermostable factor V activator; a thermolabile factor V inhibitor; a factor XIII proteolytic/urokinase-like activity; and a kallikrein-like activitiy. In LO three activities have been described: a prothrombin activator called 'Lonomia obliqua prothrombin activator protease' (LOPAP); a factor X activator; and a phospholipase A(2)-like activity called Lonomiatoxin. No fibrinolytic activity has been described in LO. Subcutaneous injection of crude hemolymph and some chromatographic fractions of LA induce a decrease in fibrinogen, plasminogen and factor XIII. Intravenous injection of factor XIII proteolytic/urokinase-like activity induce a dose-dependent thrombolysis with a decrease in plasmatic factor XIII without hemorrhagic manifestations. Intradermal injection of LO bristle extracts in rats and rabbits produce incoagulability whereas intravenous injection of LOPAP induced DIC in mice.

Fibrinogen Caracas V, an abnormal fibrinogen with an Aalpha 532 Ser-->Cys substitution associated with thrombosis.

A new dysfibrinogenemia associated with thrombophilia has been identified in a Venezuelan kindred. Thrombin and Reptilase times were prolonged and the accelerating capacity of the patient's fibrin on the t-PA-induced plasminogen activation was decreased. In addition the affinity of fibrinogen for plasminogen was diminished. Permeability and electron microscopy studies revealed that the abnormal clot was made up of thin and densely packed fibres giving rise to a reduced fibrin gel porosity. This was confirmed by turbidity studies showing a decreased fibre mass/length ratio. Affected members were heterozygous for an Aalpha 532 Ser-->Cys mutation as demonstrated by genetic analyses. This abnormal fibrinogen has been designated as Fibrinogen Caracas V. The family study showed a convincing association between the mutation and thrombotic manifestations. The thrombotic tendency may be ascribed to lack of accelerating capacity of fibrin to induce fibrinolysis caused by an abnormal clot structure with thin fibres and reduced porosity.

Thrombotic dysfibrinogenemia. Fibrinogen "Caracas V" relation between very tight fibrin network and defective clot degradability.

Fibrinogen Caracas V is a thrombotic dysfibrinogenemia with an Aalpha 532 Ser-->Cys mutation characterized by a tight fibrin network formed of thin fibers responsible for a less porous clot than a normal one. In the present work, fibrinogen Caracas V is further characterized in order to understand the relationship between the structural defect and thrombophilia. This thrombotic disorder has been attributed to a tight fibrin network responsible for a decreased permeation of flow through the clot, leading to defective thrombus lysis due to a diminished availability of fibrinolytic enzymes to the inner fibrin surface. Correction of clot structure anomaly, by addition of dextran 40 to fibrinogen before clotting, induces an improvement in fibrin degradation that was attributed to an increase in porosity. The pulmonary embolism observed in this family has been related to an hyper rigidity of the clot, an anomaly that is also corrected by dextran. Furthermore, this abnormal fibrinogen binds more albumin than does normal fibrinogen, a phenomenon attributed to the mutation of serine in Aalpha-532 by cysteine. Therefore, this fibrinogen shows a striking similarity to the fibrinogen Dusart, allowing us to confirm that the alphaC-terminal part of fibrinogen plays an important role in fibrin structure, and to conclude that the anomaly of fibrin network observed in fibrinogen Caracas V is responsible for a deficient thrombus lysis.

Experimental thrombosis I: relation with fibrinogen and other haemostatic parameters.

Epidemiological studies have shown that the haemostatic parameters Fibrinogen (Fg), Factor VII (F VII), Factor VIII (F VIII), von Willebrand factor (vWF), Tissue Plasminogen Activator (t-PA), Plasminogen Activator Inhibitors (PAI) are risk factors/markers of ischemic cardiovascular disease. Ferritin (sFER) and Leukocytosis have also been implicated. In the present study we have followed the levels of fibrinogen, von Willebrand factor and thrombomodulin in relation to lipids, iron and the appearance of atherosclerotic lesions in New Zealand rabbits fed with a cholesterol enriched diet for a two-month period compared with a group of control rabbits. Hematocrit and white blood cell count (WBC) were measured in parallel. In hyperchlesterolemic rabbits the levels of fibrinogen and von Willebrand factor increased progressively, showing a positive correlation with the increasing cholesterol levels. There was an increase in soluble thrombomodulin beginning at the eighth week of study. In addition, these animals showed gross intimal atherosclerotic lesions in the whole extension of their aortas. Immunohistochemical studies showed the presence of fibrin(ogen) related antigen throughout the arterial wall and in the central portions of the atheromas. In the control group there was no formation of atherosclerotic plaques and all haemostatic, haematological and biochemical parameters were within the normal range. WBC and sFER levels were unaffected in both groups. Our results show that increased levels of fibrinogen and von Willebrand factor, known coronary risk factors, are strongly associated with the formation of atherosclerotic plaques in rabbits. The plaques contain a considerable amount of fibrinogen related antigen.

The action of Lonomia achelous caterpillars venom on human factor V.

The bleeding syndrome produced by contact with the Lonomia achelous caterpillars is characterized by a decrease of fibrinogen, factor XIII, plasminogen, and factor V with normal platelets. In this study, we report the effect of crude hemolymph and some semipurified chromatographic fractions on human factor V. Incubation of factor V with crude hemolymph resulted in an increase in procoagulant activity, followed by a subsequent decline in factor V activity. Identical results were obtained with fraction I, whereas with fraction II there was only a decrease in activity reaching its minimum at 30 minutes. fraction III did not modify the activity of factor V. All concentrations of fraction I tested produced an initial rise and subsequent fall in activity. However, at lower relative concentrations of fraction I, more sustained increases in activity were observed. The activator and inactivator activities present in fraction I show differences in temperature and pH stability, susceptibility to different inhibitors, and in SDS/PAGE pattern. The factor V activator is a thermostable protein, with maximum activity at acid pH and is inhibited by o-phenantroline, EDTA, and EGTA, while the factor V inactivator is thermolabile, presents maximum activity at basic pH, precipitates at pH 5.0, and is completely inhibited by iodoacetic acid and TLCK. It is partially blocked by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and p-chloromercuribenzoic acid. These results suggest that the activator should be a metallo-proteinase, while the inactivator is a serine or cysteine proteinase with a serine, histidine, or cysteine residue in the active site.

The action of Lonomia achelous caterpillar venom on some blood coagulation and fibrinolysis parameters of the rabbit.

The bodily secretions of the Lonomia achelous caterpillar cause a severe and often fatal acquired bleeding diathesis in humans. The rabbit was selected as model animal in an attempt to understand the mode of action of the venom. The animals were injected subcutaneously with either hemolymph or chromatographically purified fractions. Injections of hemolymph produced a drop in fibrinogen and factor XIII levels and an increase in fibrinogen degradation products (FDP). In addition one batch of hemolymph decreased plasminogen levels. The chromatographically semipurified fraction II decreased both fibrinogen and plasminogen. The effect was dose dependent but, unlike in humans, there was a fairly rapid return to baseline values. In conclusion, the response to Lonomia achelous venom in the rabbit is similar to the response seen in humans, but with a more rapid recovery.

Invertebrate compounds acting on the hemostatic mechanism.

Physiological secretions from some invertebrates have toxic effects on mammalian blood coagulation and fibrinolytic systems. Some of these effects occur because the substances contained in the secretions resemble the components of the hemostatic system. Some of the substances have been characterized, and have been found to have similar molecular weights or sequences, which may indicate a common ancestry. The components can be divided into five groups: antithrombic agents (group I); inhibitors and activators of the prothrombinase complex (group II); substances that affect platelet function (group III); substances that affect the fibrinolytic mechanism (group IV); and a group of miscellaneous agents whose activities are difficult to group together (group V). In group I special mention of the antithrombin agents in Hirudo medicinalis should be made. In group II, the agents affecting the prothrombinase complex are antistasin from Haementeria officinalis, ghilanten from Haementeria Ghiliani and the tick anticoagulant protein from Ornithodoros moubata, a factor V activator/inhibitor from Lonomia achelous and factor II and factor X activators from L. achelous and Lonomia obliqua. Examples of factors which affect platelet function (group III) are glossina from the black fly Glossina morsitans, calin from H. medicinalis, decorsin (a desintegrin) from Macrobdella decorsa, and FAGA from Stichopus japonicus selenka. The first three of these are inhibitors of platelet aggregation, and the last is an inducer. The plasminogen activators (group IV) from the L. achelous caterpillar and Eutriatoma maculata trigger the fibrinolytic system, whereas hementin from H. officinalis and hementerin from Haementeria depressa are directly fibrinolytic. The last group of substances (group V) include those with factor-XIIa-like activity from D. farinae, kallikrein-like activity and a factor XIII degrading enzyme from L. achelous, destabilase from H. medicinalis and prolixin S (nitroforin 2, or anti-factor-IXa) from Rhodnius prolixus. Some of these components have been well characterized, cloned and prepared in recombinant form, and seem to be very promising from the therapeutic point of view.

Effect on platelet FXIII and partial characterization of Lonomin V, a proteolytic enzyme from Lonomia achelous caterpillars.

Contact with Lonomia achelous caterpillars venom induces a severe bleeding syndrome in humans. A constant finding in all reported cases is a marked decrease of blood coagulation factor XIII (FXIII), which has been attributed to the presence of a proteolytic enzyme, isolated and named Lonomin V, in the hemolymph and hair secretion. In this study, the effect of Lonomin V on transglutaminase activity from human plasma, rabbit plasma, and platelet FXIII was analyzed. The decrease of activity was more pronounced in platelet (A2) when compared with rabbit plasma (AB) and human plasma FXIII (A2B2). This finding might be explained by the differences in FXIII molecular structure. In addition, platelet FXIII molecule was degraded by Lonomin to several fragments of low molecular mass. Lonomin V was stable over a wide range of pH (6-8.5) and temperatures of -70 degrees C, -20 degrees C and between 4 to 24 degrees C, with a progressive decrease at 37 degrees C and total inactivation at 60 degrees C after 2 hours incubation. Diisopropyl fluoro-phosphate, phenylmethylsulfonyl fluoride, tosyl-l-lysine chloromethyl ketone, and iodoacetamide abolished the effect of Lonomin V on FXIII; in contrast dithiothreitol and EDTA-Na enhance the activity. We concluded that Lonomin V is a serine proteinase with a free Cys essential for the enzymatic activity. Due to its proteolytic activity on FXIII, with concomitant impairment of fibrin cross-linking, Lonomin V might be useful in association with thrombolytic drugs for preventing rethrombosis.

[Vitamin K: biochemistry, function, and deficiency. Review]. / La vitamina K: bioquímica, función y deficiencias. Revisión.

Vitamin K is a cofactor for the synthesis of blood coagulation Factors II, VII, IX and X, and inhibitors such as Protein C and S and bone matrix protein. Its active form is a coenzyme in the glutamic acid carboxylation. Vitamin K-dependent factors form enzymatic complexes with calcium and membrane phospholipids. The insufficiency of gamma glutamic carboxylation impairs the hemostatic function. Hereditary deficiencies, antibiotics and oral anticoagulants, decrease the capacity of complex formation giving way to hemorrhage or thrombosis, or bone mass disturbances which are easily treated with administration of Vitamin K. The main causes of Vitamin K deficiency are lack of hepatic storage in newborns, liver insufficiency, malabsorption, dietetic deficiency, therapy with the antibiotics and coumarin administration. For the study of Vitamin K there are methods to measure the Vit K dependent proteins and as well methods to measure specifically the quinonas.