Structural perturbation of proteins in low denaturant concentrations.

The presence of very low concentrations of the widely used chemical denaturants, guanidinium chloride and urea, induce changes in the tertiary structure of proteins. We have presented results on such changes in four structurally unrelated proteins to show that such structural perturbations are common irrespective of their origin. Data representative of such structural changes are shown for the monomeric globular proteins such as horseradish peroxidase (HRP) from a plant, human serum albumin (HSA) and prothrombin from ovine blood serum, and for the membrane-associated, worm-like elongated protein, spectrin, from ovine erythrocytes. Structural alterations in these proteins were reflected in quenching studies of tryptophan fluorescence using the widely used quencher acrylamide. Stern-Volmer quenching constants measured in presence of the denaturants, even at concentrations below 100 mM, were higher than those measured in absence of the denaturants. Both steady-state and time-resolved fluorescence emission properties of tryptophan and of the extrinsic probe PRODAN were used for monitoring conformational changes in the proteins in presence of different low concentrations of the denaturants. These results are consistent with earlier studies from our laboratory indicating structural perturbations in proteins at the tertiary level, keeping their native-like secondary structure and their biological activity more or less intact.

Relationship between prothrombin fragment 1+2 and helicobacter pylori associated chronic gastritis

Helicohacter pylori [H. pylori] have been suggested to be an independent risk factor for cardiovascular and cerebral stroke; through the activation of clotting system following chronic inflammation. Among proinflammatory cytokines, tumour necrosis factor-alpha [TNF-alpha] and interleukin-8 [TL-8] which have been previously demonstrated to activate the clotting system in human. This study aimed to investigate: [i] if there is a difference between the circulating levels of prothromhin fragment 1+2 [F1+2]; a sensitive marker of thrombin generation in patients with chronic gastritis according to N. pylori status, [ii] the relationship between circulating and mucosal levels of TNF-alpha, IL-8 and Fl+2 and [iii] If H. pylori eradication modifies the level of these cytokins and clotting system activation. Forty-two patients with chronic gastritis were diagnosed with upper endoscopy, and enrolled in this study, divided into 2 groups on the basis of histopathological examination of gastric antral biopsies and culture. Group I: included 28 H. pylori positive patients, and group H: 14 H. pyIori negative patients chronic gastnitis. A triple eradication therapy for H. pylori [omeprazole, amoxacillin and tinidazole] was given for the 28 H. pylori positive patients [Group I] and H. pylori eradication was evaluated after 2 months. Gastric mucosal levels of TNF-alpha and IL-8 as well as plasma levels of TNF-alpha, IL-8 and F1+2 were measured in group II and before and 2 months after H. pylori eradication in group I. Our results showed that: patients with H pylori positive gastritis had a significant increase in the mean value of plasma F1+2 mucosal and plasma levels of TNF-alpha and IL-8 compared with group II [P<0.001]. There was a significant positive correlation between plasma level of F1+2 and plasma levels of TNF-alpha and IL-8 [r=0.89, p<0.00, r=0.88, p<0.000] respectively and between the plasma levels of F1+2 and gastric mucosal levels of TNF-alpha and IL-S [r=0.89, P<0.000; r=0.86 P<0.001] respectively. There was a significant reduction in the mean value of plasma levels of TNF-alpha. IL-8 and F1+2 as well as fibrinogen 2 months later after H. pylori eradication. While in-group II [H pylori negative chronic gastrius] there was no significant correlation between the studied parameters. In conclusion, the present study showed a close relationship between plasma levels of prothrombin F1+2, and both plasma and gastric mucosal values of TNF-alpha and IL-8 in cases with H. pylori associated chronic gastritis. These findings suggest that H. pylori may represent a trigger factor for clotting system activation through persistent inflammatory stimulation, cytokines production and consequently H. pylori eradication may reduce the risk of hyper-coagulability state or thrombotic events which may be associated in these patients

Study of some coagulation and fibrinolytic parameters in schistosomal cases

Schistosomiasis is a major health problem in various parts of the world and Egypt. The disease is well known for the bleeding tendency that may result from diminished synthesis of coagulation factors, fibrinolytic system activation and consumption coagulopathy. This work attempted to evaluate the role of the ascitic fluid milieu in the hypercoagulability that may be observed in schistosomal hepatic fibrosis. Three parameters were assayed; prothrombin fragment 1+2[PF 1 + 2], thrombin-antithrombin complex [TAT] and fibrin monomers [FM]. These parameters were assayed both in the plasma and ascitic fluid simultaneously. The study was carried out on 18 schistosomal patients with hepatic fibrosis and tense ascites. The values of PF 1+2, TAT were significantly correlated both in plasma and ascitic fluid, while FM although was higher than the expected values yet was not significantly associated to either of the two other parameters. We concluded from this study that the ascitic fluid can be an important source of pro-coagulant material to the plasma in ascitic patients with schistosomal hepatic fibrosis

Detection of PF3 availability in whole blood from volunteers and beta-thalassemia/HbE patients: a promising method for prediction of thrombotic tendency.

The platelet factor 3 (PF 3) plays a very important role in activation of coagulation factors and is regarded to be available during activation of platelets. However, membrane fraction of erythrocytes is also shown to have PF 3-like activity, suggesting that the abnormal erythrocytes may accelerate the activation of platelet by forming thrombin on their abnormal membrane or by way of other factors of the abnormal erythrocytes, and may increase the availability of PF 3 in whole blood (WB). To examine this hypothesis, we developed a method for determination of PF 3 activity, because the method now available for the PF3 determination could not detect changes in PF 3 activity with time. The principles of our method were as follows: 1) The reaction system was adjusted so that the amount of thrombin generated in a fixed reaction time correlates with the amount of PF 3. 2) To avoid inhibition of thrombin activity by antithrombin III, a synthetic thrombin inhibitor, MD 805, was added to the system and the activity of thrombin generated was measured by synthetic thrombin substrate S-2238 using A405 as an indicator of the availability of PF3. The results obtained by the method were the following: WB taken from volunteers showed A405 of 0.12 +/- 0.02 at 30 minutes after blood collection and then the A405 increased to 0.27 +/- 0.03 at 90 minutes. However, one volunteer showed the value of 0.59 at 90 minutes, though the value at 30 minutes was 0.16. The platelet number in his WB did not change during the study.(ABSTRACT TRUNCATED AT 250 WORDS)