Correction to: New clotting disorders that cast new light on blood coagulation and may play a role in clinical practice.

The original version of the article unfortunately contained few errors.

Effects of Embryonic Development Stage and Cryoprotective Agents on Galleria Mellonella Embryo Cryopreservation.

BACKGROUND: The increased research use of the honeycomb pest Galleria mellonella has created the need for cryopreservation. The diverse characteristics of G. mellonella embryos at different stages may affect embryo survival upon cryopreservation due to differential dehydration, cold resistance, cryoprotectant toxicity and permeability. OBJECTIVE: The study aimed to determine the ability of G. mellonella to survive cryogenic condition in the early and late embryonic developmental stages (24h and 75h post-oviposition, PO). MATERIALS AND METHODS: A modified protocol was used to select the proper embryonic stage by comparing the early and late embryonic stages under two procedures of dechorionation-permeabilization (D/P) dilutions. The embryos at the early stage were used for testing the toxicity and viscosity effects of various cryoprotective agents. Various cryoprotectant treatments for improving the hatch rate were studied. RESULTS: The survival rates of embryos at the early stages (24h PO) were 51.5% and 69.5% respectively after cryoprotectant loading in 12% ethylene glycol (EG) for 30 min and dehydration in vitrification solution for for 10min. These survival rates of embryos at the late stages (75h PO), however, decreased to 22.9% and 34.0%, respectively. D/P treatment with Tween 20 and Tween 80 (2:6) slightly improved the survival of embryos at the 24h PO stage after immersion in liquid nitrogen (LN) from 0.1% to 0.6%. The increased exposure time in dehydration treatment from 14 to 30min in two vitrification solutions, EG and methanol (MeOH), slightly increased the survival rate of cryopreserved embryos from 0% and 0.3% to 1.1% and 1.4%, respectively, while the vitrification solution containing dimethyl sulfoxide (Me2SO or DMSO) slightly decreased the hatch rate from 1% to 0.6%. CONCLUSION: The survival rate of G. mellonella embryos after cryopreservation was affected by the oviposition period, eggshell permeability to cryoprotectant, cryoprotectant type, as well as their concentration and exposure times.

Heparin, coumarin, protein C, antithrombin, fibrinolysis and other clotting related resistances: old and new concepts in blood coagulation.

The concept of resistance in blood coagulation has become important. In the past it was limited to the resistance shown by some patients to heparin, coumarin or aspirin. Subsequently, it was demonstrated that a mutation in a single clotting factor, FV, showed resistance to activated protein C. Since activated protein C is supposed to downregulate aFV and aFVIII, their persistence in the circulation gives origin to a hypercoagulable state. Recently antithrombin resistance has been defined. Several prothrombin abnormalities (dysprothrombinemias) have been shown to be resistant to the action of antithrombin. This is associated with the occurrence of a trombophilic state. Prothrombin may therefore be associated like FV with both a bleeding condition (prothrombin deficiency) and a thrombophilic state (some dysprothrombinemias). Finally, thrombomodulin resistance has been defined in liver cirrhosis. These patients often show an increased ratio between FVIII levels and protein C. This imbalance may be partly responsible for the frequent presence of portal vein thrombosis seen in these patients. All these studies have greatly increased the complexity of the clotting mechanisms and interactions. They have cast light on clinical events which had remained unknown or ill-defined.

New clotting disorders that cast new light on blood coagulation and may play a role in clinical practice.

Recently several variants of clotting factors have shown a peculiar behavior so that they appear as new defects. The factors involved are FII, FV and FIX. Prothrombin deficiency is usually associated with bleeding. Recently a few prothrombin abnormalities involving Arg396 mutations, have been demonstrated to show antithrombin resistance with the consequent appearance of a thrombophilic state and venous thromboses in young age. The same is true for an abnormal FIX (FIX Padua). The thrombotic manifestations in the latter condition are also venous. The abnormal FIX (FIX Padua) is characterized by a great increase in FIX activity whereas FIX antigen is only slightly increased. The condition is due to an Arg338Lys mutation. The increased intrinsic clotting activity of this abnormal FIX is being investigated as a useful therapeutic approach in homophile B patients. Another new clotting disorder is represented by two abnormal FV (FV East Texas and FV Amsterdam). These are characterized by a deletion of part of the B domain of FV resulting in a "short" FV. The condition is characterized by a mild bleeding tendency due to high levels of Tissue Factor pathway inhibitor. The "short" factor V is in fact resistant to the action of Tissue Factor pathway inhibitor which is sharply increased in these patients. These new clotting entities have again demonstrated that the study of patients who show a tendency to venous thrombosis or a mild bleeding condition that cannot be explained on the basis of our current concepts of blood coagulation, may represent "new" coagulation disorders. All persons interested in thrombotic or hemorrhagic disorders should be informed about these new clinical and laboratory conditions.

Drug-Induced Thrombophilic or Prothrombotic States: An Underestimated Clinical Problem That Involves Both Legal and Illegal Compounds.

Vascular thrombosis, both arterial and venous, is a condition associated with significant morbidity and mortality. There are multiple risk factors for thrombosis, both congenital and acquired, and in the majority of cases, these risk factors are not modifiable. Over the past 2 decades, multiple drugs (both illegal and legal) have been associated with increased risk of thrombosis. However, due to limited scientific literature regarding the prothrombotic tendencies of these drugs, there is a concomitant limited understanding of the pathophysiology of drug-induced thrombosis. As drugs are one of the few modifiable risk factors for thrombosis, further study and dissemination of knowledge regarding drug-associated and drug-induced thrombosis are essential and have the potential to lead to decreased future incidence of thrombosis. The mechanisms at the basis of the thrombophilic activity of these drugs are variable and sometimes still ill recognized. Increased levels of clotting factors, reduction in coagulation natural inhibitors, decreased fibrinolysis, activated clotting factors, increased blood viscosity, endothelial damage, and increased platelet number and activation are the most frequent causes. Arterial steal or coronary arteries no flow has also been implicated. In some cases due to the intake of several drugs, more than one mechanism is present in a given patient. The purpose of the present review is to analyze all the drugs demonstrated to be potentially thrombotic. It is hoped that a prudent use or nonuse of these drugs might result in a reduction of thrombosis-associated diseases.

Prethrombotic, prothrombotic, thrombophilic states, hypercoagulable state, thrombophilia etc.: semantics should be respected even in medical papers.

The study of prothrombotic or thrombophilic states have drawn considerable attention during the past two decades. This was the result of the increasing number of thrombotic events, both arterial and venous reported all over the world but especially in the developed countries. This wealth of studies and papers have not always respected the semantical significance of the various terms used, namely prethrombotic state, hypercoagulable state, thrombophilic or prothrombotic state, thrombophilia, susceptibility to thrombosis and procoagulant state. This review is an attempt to adhere to a correct Semantic format in order to avoid confusion and misinterpretations. This is of fundamental importance in order to avoid the wrong attribution of a thrombosis to a hypercoagulable or a prethrombotic state.

Thrombotic Events in Asymptomatic FXII Deficiency versus Symptomatic FXI Deficiency: Surprising Observations.

OBJECTIVE: To evaluate the impact of an asymptomatic congenital clotting defect (FXII deficiency) versus that of a similar but symptomatic defect (FXI deficiency) on protection from thrombosis. PATIENTS AND METHODS: All patients with FXII or FXI deficiency and thrombosis were gathered from a time-unlimited PubMed search that was carried out twice and from personal records. Combined defects were excluded. The defect had to be proven by the demonstration of a suited hereditary pattern and by a specific clotting assay. Only patients with a factor activity level of <30% of normal were selected. RESULTS: Twenty-eight patients with an FXII deficiency presented with arterial thrombosis, mainly myocardial infarction, and 29 showed venous thrombosis; for FXI deficiency, these figures were 43 and 10, respectively. The ratio of arterial and venous thrombosis was 0.96 and 4.3, respectively, for FXII and FXI deficiency. CONCLUSIONS: Factor FXII deficiency supplies no protection from arterial or venous thrombosis. FXI deficiency shows no protection from arterial thrombosis but appears to guarantee protection from venous thrombosis. A symptomatic, namely bleeding, condition (FXI deficiency) provides protection from venous thrombosis whereas an asymptomatic one (FXII deficiency) does not.

The effect of Tween 80 on eggshell permeabilization in Galleria mellonella (L.) (Lepidoptera, Pyralidae).

The development of a species-specific protocol for dechorionation and permeabilization of insect eggs is a necessary prerequisite to cryopreserve the embryos. Here we tested different procedures based on heptane or the surfactant Tween 80 as an alternative to alkane, evaluating their efficacy and toxicity on the early (24 h post-oviposition) and late (75 h post-oviposition) stage embryos. Heptane efficiently permeabilized the eggs of G. mellonella but the hatching rate ranged from 0.1 to 4.2 percent in the early stage and from 4.3 to 11.2 percent in the late stage. The embryos treated with 1.25 percent NaOCl + 0.08 percent Tween 80 for 2 min showed the same shrinkage and reswelling percentages as eggs exposed to heptane for 10 sec, with a significantly higher hatching percentage in the early (68.2 +/- 1.5 percent) and late stages (22.4 +/- 3.7 percent). Thus, 0.08 percent Tween 80 allows sufficient permeabilization of G. mellonella embryos without the high toxicity of alkane.

Lipids and NADPH-dependent superoxide production in plasma membrane vesicles from roots of wheat grown under copper deficiency or excess.

The effects of in vivo copper on the lipid composition of root plasma membrane and the activities of membrane-bound enzymes, such as NADPH-dependent oxidases and lipoxygenase, were studied. Plants were grown in hydroponic culture for 11 d without Cu supply or in the presence of 50 microM Cu. Control plants were supplied with 0.3 microM Cu. Growth of roots was severely affected in the 50 microM Cu-grown plants, whereas roots grown in Cu-deficient solution did not show any difference in comparison with the control. The 50 microM Cu concentration caused an increase in the leakage of K(+) ions as well. Excess metal supply resulted in a decrease in the total lipid content of plasma membrane, a higher phospholipid amount and a reduction of steryl lipids (free sterols, steryl glycosides and acylated steryl glycosides). Cu depletion in the growth solution had only a slight effect on the plasma membrane lipid composition. In comparison with the control, only the excess of Cu caused a decrease in the lipid to protein ratio as well as a change in the phospholipid composition, with a lower phosphatidylcholine to phosphatidylethanolamine ratio. The degree of unsaturation of root plasma membranes decreased following the 0 Cu treatment and even more after the 50 microM Cu supply. Plasma membranes of wheat grown under metal deficiency and excess showed increased NADPH-dependent superoxide-producing oxidase activities, whereas membrane-bound lipoxygenase was not increased or activated due to Cu treatments. The consequences of changes in plasma membrane lipid composition and activated oxygen production as a result of Cu treatments are discussed.