Influence of root-surface conditioning with acid and chelating agents on clot stabilization.

OBJECTIVE: To compare the adhesion and maturation of blood components on chemically conditioned root surfaces. METHOD AND MATERIALS: Clinical root samples of human teeth were obtained (n = 150) and manually scaled. Five groups of 30 samples were treated as follows: (1) saline solution irrigation (control); (2) 24% EDTA gel; (3) 25% citric acid solution; (4) tetracycline solution (50 mg/mL); and (5) 30% sodium citrate solution. After these treatments, 15 samples of each group received a blood drop and were analyzed by SEM. The remaining 15 had their surface morphology evaluated for collagen fibrils exposure by SEM. Photomicrographs were analyzed according to the score of adhesion of blood components. Kruskal-Wallis and Dunn multiple comparison tests were employed. RESULTS: The control group was characterized by the absence of blood elements on the surface. The best result was observed in the citric acid group, which had a dense fibrin network with blood elements adhered. The EDTA group showed a moderate fibrin network formation. In contrast, a scarce fibrin network and a few cells were present in the tetracycline samples, and an absence of blood elements was found on sodium citrate specimens. The citric acid group was statistically different from the control group (P < .01). No differences were found among the control, EDTA, tetracycline, and sodium citrate groups (P > .05). CONCLUSION: Under these experimental conditions, citric acid is indicated to stabilize clots on the root surface, which act as a scaffold for connective tissue cell development.

An in-vitro model for comparing the efficiency of wound-rinsing solutions.

OBJECTIVE: To develop an in-vitro model to evaluate the efficiency of wound-rinsing solutions in removing adherent, hydrophobic, denatured proteins. We hypothesised that saline solutions would be less effective than surfactant-containing solutions in removing denatured proteins. METHOD: Prepared slides containing dried blood plasma or fibrin were incubated for up to one hour in histological troughs filled with one of four test solutions: physiological saline solution, Ringer's solution, a surfactant-containing solution and an antiseptic. The concentration of dissolved proteins was measured using a modified Biuret test. Results were analysed by plotting protein concentration against the incubation time. RESULTS: During the incubation period, the protein concentration increased in all of the test solutions, with the lowest concentration reported in the two saline-based solutions. These stayed clear, while the surfactant-containing solution become opaque, indicating that the surfactant had encased hydrophic substances, such as denatured proteins. CONCLUSION: Ringer's solution and saline are inappropriate solvents for adhering wound coatings. A sterile, surfactant-containing wound rinsing solution contains the essential properties for thorough and gentle cleansing of chronic wounds. DECLARATION OF INTEREST: None.

Ultrastructure of platelets and fibrin networks of asthmatic mice exposed to selenium and Withania somnifera.

Platelets and fibrin play an important role in allergic processes, including allergic asthma. The asthmatic BALB/c mouse model was used to induce asthma, and asthmatic mice were treated with the anti-inflammatory plant Withania somnifera, separately and in combination with the antioxidant selenium. Selenium is an important supplement in asthma, because asthmatics may have a selenium deficiency. Hydrocortisone was used as positive control. Results indicate control mice possess major thick fibers, minor thin fibers, and tight round activated platelets with typical pseudopodia formation. Minor fibers of asthmatic mice have a netlike appearance covering the major fibers whereas the platelets form loosely connected, granular aggregates. Hydrocortisone made the fibrin more fragile and platelet morphology changes from a tight activated platelet to a more granular activated platelet, not closely fused to each other. The plant extracts, separately and in combination with selenium did not affect the fragility of the fibrin and reversed the formation of the dense minor netlike layer over the major fibers, and the platelets formed a dense aggregate. Asthmatic mice treated with selenium showed a dense minor fibrin layer; however, the platelets formed a dense aggregate. We conclude that the anti-inflammatory products affect the stability of fibrin networks but not platelet stability (seen with hydrocortisone). Selenium does not affect the stability of the fibrin networks, but affects platelet stability. These results suggests that asthmatic patients should indeed use an antioxidant supplement, e.g. selenium, because it stabilizes activated platelets, together with anti-inflammatory products.

A multiparameter test of clot formation and fibrinolysis for in-vitro drug screening.

A large spectrum of methods has been used in both routine and scientific studies of the hemostatic system. The particular interest of the investigators has been focused on methods simultaneously evaluating clotting and fibrinolysis processes. The aim of the present study was to develop an optical method for overall evaluation of clot formation and lysis (CL-test) that could be used in drug screening. The CL-test was performed in citrate plasma diluted with Tris-buffered saline. Thrombin was applied for plasma clotting (0.5 IU/ml), while tissue plasminogen activator (60 ng/ml) was used for fibrinolysis activation. Clot formation and lysis were monitored in thermostatic conditions (37 degrees C) as a continuous record of transmittance change. By means of own computer program, kinetic parameters of the processes studied and plasma overall clot formation and fibrinolysis potential, expressed as the area under the clotgeneration and fibrinolysis curves, were calculated. The CL-test was developed and checked by evaluation of the effect, on clot formation and lysis, of various concentrations of acetylsalicylic acid (a drug that affects hemostasis), aprotinin (fibrinolysis activator) and venoruton (fibrinolysis inhibitor). The obtained results confirmed that the test we propose for monitoring clot formation, stabilization and lysis is sensitive and enables precise estimation of the processes studied. In our opinion, it can be a useful tool in drug screening investigations.

Comparative ultrastructural analyses of platelets and fibrin networks using the murine model of asthma.

The murine Balb/c asthma model has been used successfully for a number of in vivo immunological applications and for testing novel therapeutics, and it is a reliable, clinically relevant facsimile of the human disease. Here we investigate whether this model can be used to study other components of the human body, e.g. ultrastructure. In particular, we investigate the effect of the phytomedicine Euphorbia hirta (used to treat asthma), on the ultrastructure of fibrin as well as platelets, cellular structures that both play an important role in the coagulation process. Hydrocortisone is used as positive control. Ultrastructure of the fibrin networks and platelets of control mice were compared to mice that were asthmatic, treated with two concentrations of hydrocortisone and one concentration of the plant material. Results indicate control mice possess major, thick fibers and minor thin fibers as well as tight round platelet aggregates with typical pseudopodia formation. Minor fibers of asthmatic mice have a netlike appearance covering the major fibers, while the platelets seem to form loosely connected, granular aggregates. Both concentrations of hydrocortisone make the fibrin more fragile and that platelet morphology changes form a tight platelet aggregate to a more granular aggregate not closely fused to each other. We conclude that E. hirta does not impact on the fragility of the fibrin and that it prevents the minor fibers to form the dense netlike layer over the major fibers, as is seen in untreated asthmatic mice. This ultrastructural morphology might give us better insight into asthma and the possible new treatment regimes.

Foodstate vitamin C complex may beneficially affect haemostasis and fibrin network structure in hyperlipidaemic patients.

This randomized, placebo-controlled, double-blind, crossover study on 25 free-living hyperlipidaemic volunteers aspired to prove the hypothesis that supplementation for 8 weeks with a FoodState Vitamin C complex (500 mg vitamin C, 160 mg bioflavonoids, 600 mg magnesium and 900 mg vitamin B complex) may improve haemostatic factors and fibrin network structures. Of the haemostatic factors measured, only median plasmin-antiplasmin complex (PAP) and thrombin-antithrombin complex (TAT) concentrations were both significantly decreased with FoodState Vitamin C complex compared with placebo [PAP, -4.05% (-23.39, -0.23) versus 1.81% (-8.95, 8.09); TAT, -5.81% (-18.47, 0.39) versus 0.12% (-8.03, 13.5)]. As for fibrin network structures, only compaction was significantly increased from baseline to end [49.95% (47.55, 53.70) to 51.85% (48.55, 56.65)] by FoodState Vitamin C complex supplementation. No significant changes were found in plasma fibrinogen, plasminogen activator inhibitor 1 activity, tissue plasminogen activator antigen, D-dimer, serum lipids or lipoprotein (a) concentrations. In conclusion, the decreases in TAT and PAP are possibly an indication that the FoodState Vitamin C complex decreased the initiation of haemostasis, which in turn led to a compensatory reduction in fibrinolysis. FoodState Vitamin C complex may therefore be protective of cardiovascular disease by causing a new reduced steady state of haemostatic balance and less rigid clots (increased compaction).

Mechanisms of poly-N-acetyl glucosamine polymer-mediated hemostasis: platelet interactions.

BACKGROUND: Investigations were performed to determine whether poly-N-acetyl glucosamine (p-GlcNAc) induces hemostasis by the activation of platelets. METHODS: Platelets were isolated from human blood, fixed in the presence poly-N-acetyl glucosamine fibers, and visualized with scanning electron microscopy. Platelet activation surface markers were measured by fluorescence multiphoton microscopy. Platelet aggregation in the presence of p-GlcNAc fibers and integrin receptor blockers was measured. RESULTS: Scanning electron microscopy indicated that contact of platelets with poly-N-acetyl glucosamine fibers resulted in platelet activation. Fluorescent microscopy showed that contact of platelets with the marine polymer increased intracellular levels of free calcium and resulted in surface exposure of platelet phosphatidylserine, P selectin, and the alphaIIbbeta3 integrin. Antibody inhibitors of the platelet alphaIIbbeta3 integrin inhibited p-GlcNAc to stimulate fibrin polymerization. CONCLUSION: Poly-N-acetyl glucosamine fiber material promotes hemostasis by the activation of platelets.

Preliminary in vitro studies on the Marsypianthes chamaedrys (boia-caá) extracts at fibrinoclotting induced by snake venoms.

The extract of Marsypianthes chamaedrys, a plant used against snakebites, in the present study was shown to inhibit fibrinoclotting induced by several Brazilian snake venoms or thrombin. These data indicate that this extract affected thrombin-like enzymes. In this first report we determine some features of the components present in the extract regarding the antifibrinoclotting action. Our results show that active components responsible for those effects are thermo-resistant and are concentrated in the methanolic fraction.

Prourokinase mutant that induces highly effective clot lysis without interfering with hemostasis.

Prourokinase (proUK) is a zymogenic plasminogen activator that at pharmacological doses is prone to nonspecific activation to urokinase. This has handicapped therapeutic exploitation of its fibrin-specific physiological properties. To attenuate this susceptibility without compromising specific activation of proUK on a fibrin clot, a Lys300-->His mutation (M5) was developed. M5 had a lower intrinsic activity and, therefore, remained stable in plasma at a 4-fold higher concentration than did proUK. M5 had a higher 2-chain activity and induced more rapid plasminogen activation and fibrin-specific clot lysis in vitro. Sixteen dogs embolized with radiolabeled clots were infused with saline, proUK, tissue plasminogen activator, or M5. The lower intrinsic activity allowed a higher infusion rate with M5, which induced the most rapid and efficient clot lysis (50% clot lysis by approximately 600 microg/kg M5 versus approximately 1200 microg/kg proUK). In association with this, M5 caused neither a significant increase in the primary bleeding time nor secondary bleeding (total blood loss). By contrast, these measurements increased 4-fold and 5-fold, respectively, with proUK and >5-fold and 8-fold, respectively, with tissue plasminogen activator. Clot lysis by M5 and hemostasis were further evaluated in 6 rhesus monkeys. M5 again induced rapid clot lysis without a significant increase in the primary bleeding time, and secondary bleeding did not occur. In conclusion, a site-directed mutation designed to improve the stability of proUK in blood at therapeutic concentrations induced superior clot lysis in vitro and in vivo without causing significant interference with hemostasis.

Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana.

A protease, phytolacain G, has been found to appear on CM-Sepharose ion-exchange chromatography of greenish small-size fruits of pokeweed, Phytolacca americana L, from ca. 2 weeks after flowering, and increases during fruit enlargement. Reddish ripe fruit of the pokeweed contained both phytolacain G and R. The molecular mass of phytolacain G was estimated to be 25.5 kDa by SDS-PAGE. Its amino acid sequence was reconstructed by automated sequence analysis of the peptides obtained after cleavage with Achromobacter protease I, chymotrypsin, and cyanogen bromide. The enzyme is composed of 216 amino acid residues, of which it shares 152 identical amino acid residues (70%) with phytolacain R, 126 (58%) with melain G, 108 (50%) with papain, 106 (49%) with actinidain, and 96 (44%) with stem bromelain. The amino acid residues forming the substrate binding S(2) pocket of papain, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Trp, Met, His, Ala, and Ser in phytolacain G, respectively. As a consequence of these substitutions, the S(2) pocket is expected to be less hydrophobic in phytolacain G than in papain.

Chinese herbs nephropathy-associated slimming regimen induces tumours in the forestomach but no interstitial nephropathy in rats.

Chinese herbs nephropathy (CHN), a rapidly progressive interstitial fibrosis of the kidney, has been described in approximately 100 young Belgian women who had followed a slimming regimen containing some Chinese herbs. In 4 patients multifocal transitional cell carcinomas (TCC) were observed. Aristolochic acid (AA), suspected as the causal factor of CHN, is a well known carcinogen but its ability to induce fibrosis has never been demonstrated. The objective of this study was to evaluate the latter using doses of AA, durations of intoxication and delays of sacrifice known to yield tumours in rats. We also tested the hypothesis that a possible fibrogenic role of AA was enhanced by the other components of the slimming regimen. Male and female rats were treated orally with 10 mg isolated AA/kg per day for 5 days/week, or with approximately 0.15 mg AA/ kg per day 5 days/week contained in the herbal powder together with the other components prescribed in the slimming pills for 3 months. The animals were killed respectively 3 and 11 months later. At sacrifice, animals in both groups had developed the expected tumours but not fibrosis of the renal interstitium. Whether the fibrotic response observed in man is due to species and/or strain related differences in the response to AA or to other factors, remains to be determined. Interestingly, despite the addition of fenfluramine and diethylpropion, two drugs incriminated in the development of valvular heart disease, no cardiac abnormalities were observed.

Platelet and fibrin deposition on coronary stents in minipigs: effect of hirudin versus heparin.

OBJECTIVES: The present study was designed to test the hypothesis that the direct thrombin hirudin is more efficient than heparin in reducing thrombus formation after coronary stenting. BACKGROUND: Despite aggressive anticoagulation, subacute thrombosis of coronary stents is a major complication associated with these new devices. METHODS: In 19 minipigs indium-111-labeled thrombocytes and iodine-125-labeled fibrinogen were injected 14 to 19 h before coronary implantation of tantalum balloon-expandable stents. In group 1 (n = 6, seven stents), a bolus of heparin (100 U/kg body weight) was given before stenting. Group 2 (n = 6, 11 stents) received both dextran (500 ml) and heparin (a 100-U/kg bolus followed by a continuous infusion of 50 U/kg per h). In group 3 (n = 7, 13 stents), hirudin (recombinant desulphatohirudin HV 1 [CGP 39393] [1 mg/kg]) was given before stent implantation, followed by an infusion of 1 mg/kg per h. All animals were pretreated with aspirin (250 mg intravenously). RESULTS: Activated partial thromboplastin time was prolonged to > 1.8 times control values in groups 2 and 3. Histologic examination after perfusion fixation 12 h after stenting showed a variable extent of thrombus on all stents. Medial tear was observed in three stents in group 1, six stents in group 2 and six stents in group 3. The number of platelets on all stents averaged 116.2 (range 22 to 522) x 10(6) in group 1, 64.3 (range 11 to 169) x 10(6) in group 2 and 19.7 (range 9 to 38) x 10(6) in group 3 (p < 0.05 vs. group 1 and vs. group 2). The increase in platelet deposition, associated with medial tear in all groups, was lowest in the hirudin group. Similarly, fibrin deposition was lowest on stents in hirudin-treated animals. CONCLUSIONS: Recombinant hirudin significantly reduces platelet and fibrin deposition on coronary stents compared with the reduction achieved with combined heparin, dextran and aspirin.

[The anticoagulant action of a factor from the nondialyzed fraction of the ammoniacal extract of the lungwort, Pulmonaria mollissima]. / Protivosvertyvaiushchee deistvie antikoagulianta nedializuemoi fraktsii ammiachnogo ékstrakta iz travy medunitsy miagchaishei.

It has been established that a non-dialyzed fraction of ammoniacal extract from P. molissima contains an anticoagulant-glycopeptide. In in vivo experiments, it produces stable hypocoagulemia, primarily blocking fibrin self-assemblage, and prevents the animal's death from thromboplastinemia. It has been found that the aforesaid action does not exert any adverse effect on the blood contents, arterial pressure, respiration or urinary renal function.

Effects of low and high dose oral contraceptives on blood coagulation and thrombogenesis induced by vascular subendothelium exposed to flowing human blood.

We investigated the effect of oral contraceptives with low and high estrogen concentration on blood coagulation and thrombogenesis, induced by vascular subendothelium of rabbit aorta exposed to flowing human blood. Twenty healthy women intending to take oral contraceptives were studied [1] before drug ingestion (control), and subsequently during the intake of oral contraceptives with [2] low estrogen content (20 micrograms ethinyl estradiol and 150 micrograms desogestrel per day) and [3] high estrogen content (50 micrograms ethinyl estradiol and 125 micrograms desogestrel per day). All experiments were performed between day 17 and 21 of the menstrual cycle and drug effects were studied during the third tablet cycle. Deposition of fibrin, platelets and platelet thrombi on vascular subendothelium was tested at a defined blood flow and wall shear rate (10 ml/min, 650 s-1) and was quantified by morphometrical techniques. Treatment with the low and high dose contraceptive increased the plasma levels of ethinyl estradiol (728 +/- 139 and 1438 +/- 212 vs. 0 fmol/l [low and high dose vs. control], means +/- SEM, P less than 0.001) and fibrinogen (2.3 +/- 0.1 and 2.6 +/- 0.1 vs. 2.0 +/- 0.1 g/l, P less than 0.05); and decreased antithrombin III activity (95 +/- 3 and 92 +/- 3 vs. 101 +/- 3 %, P less than 0.05). Fibrin deposition on vascular subendothelium was enhanced by the high dose contraceptive only (47 +/- 4 vs. 35 +/- 4 % coverage of the subendothelial surface with fibrin, high dose vs. control, P less than 0.05). The subendothelial deposition of platelets and platelet thrombi was not changed by contraceptive treatment. These results indicate that treatment with high dose contraceptives leads to an increase of fibrin-subendothelial interactions, whereas low dose contraceptives do not significantly alter the blood-subendothelium interactions. observed in this ex vivo model of thrombogenesis.