Effects of Compounds from Passiflora edulis Sims f. flavicarpa Juice on Blood Coagulation and on Proteolytic Enzymes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interference of the blood clot on granulation tissue formation after tooth extraction. Histomorphological study in rats.

The interference of a blood clot in the first postoperative hours of dental extraction wounds was studied in rats. Sixty male albino rats were divided into two groups: Group I, immediately after extraction of right maxillary incisor the gingival mucosa was approximated and sutured; Group II, after 6 to 8 minutes postoperatively the blood clot was removed with saline irrigation and absorbent paper cones. The mucosa was then approximated and sutured. Six animals in each group were sacrificed after 12 hours, 1, 4, 7 and 10 days. There was a profound delay in healing in Group II since, although a new blood clot was later formed, it was not organized. The quality and the constitution, maintenance and retraction of the clot are the regulating factors in connective tissue formation during alveolar healing.

Blood cells attachment after root conditioning and PRP application: An in vitro study

Aim: Root conditioning is aimed at smear layer removal and at dental matrix collagen exposure, which may promote periodontal regeneration. This in vitro study assessed smear layer removal, collagen fiber exposure and the influence of PRP (platelet-rich plasma) application on adhesion of blood cells to the root surface using scanning electron microscopy (SEM). Materials and methods: Scaled root samples (n = 160) were set in five groups and conditioned with: group I - control group (saline solution); group II (EDTA 24%); group III (citric acid 25%); group IV (tetracycline hydrochloride 50 mg/ml); group V (sodium citrate 30%). Eighty samples were assessed using the root surface modification index (RSMI). The other eighty samples were set in two groups. The first group (n = 40) received PRP gel application with a soft brush and the second group (n = 40) received PRP application and then a blood drop. The fibrin clot formation was assessed in the first group and the blood cells adhesion was assessed in the second group using the BEAI (blood elements adhesion index). A previously trained, calibrated, and blind examiner evaluated photomicrographs. Statistical analysis was performed using the Kruskal-Wallis's and Dunn's tests. Results: Group III attained the best results for RSMI and BEAI. Moreover, it was the only group showing fibrin clot formation. Conclusion: Citric acid was the most efficient conditioner for smear layer removal, collagen fiber exposure and blood cell adhesion. Moreover, it was the only group showing fibrin clot formation after PRP application. Clinical significance: This study demonstrated that root conditioning followed by PRP application may favor blood cell adhesion on root surface which may optimize periodontal healing.

Fresh autogenous rib cartilage graft to the malar process of rats with and without removal of the perichondrium: a histological study.

A comparison was made of autogenous grafts of rib cartilage with and without removal of the perichondrium, applied to the malar process of rats. Seventy-two male albino rats were divided into two groups according to the kind of graft received by each animal. The experimental periods were 5, 10, 20, 30, 60 and 120 postoperative days. The results showed that, in the control group, the grafts maintained their vitality for the whole experimental period and the perichondrium was biologically integrated into the host bed. Appositional growth was also observed. The treated animals showed intense resorption of the grafts and more intense bone neoformation. The newly formed bone was in intimate contact with the graft in both groups.

Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-Å resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin β-turn of the second inner loop pivots at Val64 and Asp70 by 60°. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 β-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle. © 1993 American Chemical Society.

Factor XIII Val34Leu is a genetic factor involved in the aetiology of venous thrombosis

A mutation in the factor XIII gene (FXIII Val34Leu) gene was recently reported to confer protection against myocardial infarction, but its relationship with venous thrombosis is unknown. In addition, a mutation in the 5'-untranslated region of the FXII gene (46 C→T) was identified which is associated with low plasma levels of the protein. Its prevalence in patients with venous thrombosis is also unknown. We investigated the frequency of the FXIII Val34Leu and FXII 46 C→T mutations in 189 patients with deep venous thrombosis and in 187 age-, gender- and race-matched controls. FXIII Val34Leu was detected in 38.6% of the patients and in 41.2% of the controls. Interestingly, homozygosity for the FXIII mutation was found in 1.6% of the patients and in 9.6% of the controls. yielding an odds ratio (OR) for venous thrombosis of 0.16 (95% CI: 0.05-0.5). The OR for heterozygotes was 1.1 (95% CI: 0.7-1.7). The FXII 46 C→T mutation was detected in 46.0% of the patients and in 48.6% of the controls. The OR for heterozygotes was 0.9 (95% CI: 0.6-1.4) and for homozygotes the OR was 0.8 (95% CI: 0.3-1.9). Our data indicate that the FXII 46 C→T mutation is unlikely to be a major risk factor for venous thrombotic disease. In contrast, the homozygous state for FXIII Val34Leu is a strong protective factor against venous thrombosis, which emerges as a novel generic factor involved in the aetiology of thrombophilia.

Factor V Leiden and factor II G20210A mutations in patients with recurrent abortion

Recurrent abortion (RA) represents an intriguing problem in obstetric practice in which genetic and acquired factors may play a role. In the present investigation we sought to assess the possibility that inherited thrombophilia might determine the risk of RA. We therefore investigated the prevalence of two genetic abnormalities frequently associated with venous thrombosis [factor V Leiden (FVL) and factor II G20210A] in 56 patients with primary or secondary abortion and in 384 healthy control women. Polymerase chain reaction amplification followed by digestion with the restriction enzymes MnlI and HindIII was used to define the FVL and FII G20210A genotypes respectively. FVL was found in 4/56 patients (7.1%) and in 6/384 controls (1.6%), yielding an odds ratio (OR) for RA related to FVL of 4.9 [95% confidence interval (CI): 1.3-17.8]. FII G20210A was detected in 2/56 (3.6%) patients and in 4/384 (1%) controls (OR for RA: 3.5, CI: 0.6-19.7). In conclusion, FVL and FII G20210A mutations in patients with RA were more prevalent in comparison with controls. These data support a role for both mutations as determinants of the risk of RA and strengthen the notion that thrombophilia plays a role in this clinical entity.

Structure of tick anticoagulant peptide at 1.6 Å resolution complexed with bovine pancreatic trypsin inhibitor

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at t.6 Å resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 Å, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 Å. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (CyS5(T)- Cys 15(T)) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 Å to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 Å with the guanidinium group forming a cation-π-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 Å in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N- terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.

The thrombocyte aggregation process in the turtle Phrynopys hilarii (Chelonia). An ultrastructural study

This study investigates the thrombocyte aggregation process in the South American fresh water turtle (Phrynopys hilarii) using electron microscopy. Blood was taken from surgically exposed lateral neck vessels often turtles Phrynopys hilarii during the spring and summer seasons, when the mean temperature is 37°C. Blood samples were fixed with Karnovsky solution for processing by transmission electron microscopy. The turtle thrombocytes were spindle-shaped with lobulated nuclei. Prominent vesicles and canaliculi were found throughout the cytoplasm. The cytoplasm organelles showed an agranular endoplasmatic reticulum, Golgi complex near the centrioles and scattered free ribosomes. These cells are similar to bird thrombocytes but distinct from fish and frog thrombocytes. Blood clotting time was 5 min ± 30 sec measured by the Lee and White method. Structural alterations resulting from the aggregation process occurred after activation. Thrombocytes developed numerous filopodial projections, an increased number of vacuoles and changed from spindle to spherical shape. P. hilarii thrombocytes have different morphologic characteristics compared to other non-mammalian vertebrate cells. These cells can participate in the aggregation process, as observed in birds.

Role of natural killer cells in antitumor resistance

Natural killer cells constitute a population of lymphocytes able to non-specifically destroy virus-infected and some kinds of tumor cells. Since this lytic activity was shown by non-immunized animals the phenomenon is denominated natural killer (NK) activity and contrasts with specific cytotoxicity performed by cytolytic T lymphocytes (CTLs) because it does not depends on MHC-restricted peptides recognition. In fact, the main feature of most functional receptors of NK cells (NKRs) is their ability to be inhibited by different kinds of class I MHC antigens. In the middle of the 1950's, Burnet & Thomas forged the concept of tumor immunosurveillance and NK cells can be considered one of the main figures in this phenomenon both for effector and regulatory functions. In the present review the early studies on the biology of NK cells were revisited and both their antitumor activity and dependence on the activation by cytokines are discussed.

Evaluation of adverse effects of long-term oral administration of carprofen, etodolac, flunixin meglumine, ketoprofen, and meloxicam in dogs

Objective - To evaluate adverse effects of long-term oral administration of carprofen, etodolac, flunixin meglumine, ketoprofen, and meloxicam in dogs. Animals - 36 adult dogs. Procedures - Values for CBC, urinalysis, serum biochemical urinalyses, and occult blood in feces were investigated before and 7, 30, 60, and 90 days after daily oral administration (n = 6 dogs/group) of lactose (1 mg/kg, control treatment), etodolac (15 mg/kg), meloxicam (0.1 mg/kg), carprofen (4 mg/kg), and ketoprofen (2 mg/kg for 4 days, followed by 1 mg/kg daily thereafter) or flunixin (1 mg/kg for 3 days, with 4-day intervals). Gastroscopy was performed before and after the end of treatment. Results - For serum γ-glutamyltransferase activity, values were significantly increased at day 30 in dogs treated with lactose, etodolac, and meloxicam within groups. Bleeding time was significantly increased in dogs treated with carprofen at 30 and 90 days, compared with baseline. At 7 days, bleeding time was significantly longer in dogs treated with meloxicam, ketoprofen, and flunixin, compared with control dogs. Clotting time increased significantly in all groups except those treated with etodolac. At day 90, clotting time was significantly shorter in flunixin-treated dogs, compared with lactose-treated dogs. Gastric lesions were detected in all dogs treated with etodolac, ketoprofen, and flunixin, and 1 of 6 treated with carprofen. Conclusions and Clinical Relevance - Carprofen induced the lowest frequency of gastrointestinal adverse effects, followed by meloxicam. Monitoring for adverse effects should be considered when nonsteroidal anti-inflammatory drugs are used to treat dogs with chronic pain.

Estudo morfológico comparativo das cascas e folhas de Stryphnodendron adstringens, S. polyphyllum e S. obovatum - Leguminosae

The genus Stryphnodendron (S.) belongs to the family Leguminosae, subfamily Mimosoideae, which includes mostly trees of tropical and subtropical South America. Extracts of the stem bark are used traditionally by the local population to treat leucorrhoea and diarrhoea, as anti-inflammatory and antiseptic agents (antimicrobial) and to promote blood clotting and wound healing, and in a few cases of gastric ulcers. A review of the literature presented a previous morpho-anatomical study only for S. adstringens (Mart.) Coville. The aim of the present work is to compare morpho-anatomically the stem bark and leaves of three species of Stryphnodendron, known popularly as barbatimão: S. adstringens, S. polyphyllum and S. obovatum, in order to help botanical identification and contribute to quality control. Macro- and microscopical evaluation of the stem barks revealed no significant differences among the species. Morphological analyses of the leaves revealed differences in size, coloration, and pubescence. The leaves of S. adstringens are the largest, glabrous, and concolor. The leaves of S. polyphyllum are smaller, pubescent, and discolor; whereas the leaves of S. obovatum are the same size as those of S. polyphyllum, however, they are glabrous, and discolor.

Cytokine gene variants and venous thrombotic risk in the BRATROS (BRAZILIAN THROMBOSIS STUDY)

Introduction: Venous thrombosis (VT) and inflammation are two closely related entities. In the present investigation we assessed whether there is a relation between genetic modifiers of the inflammatory response and the risk of VT. Materials and methods: 420 consecutive and unrelated patients with an objective diagnosis of deep VT and 420 matched controls were investigated. The frequencies of the following gene polymorphisms were determined in all subjects: TNF-α- 308 G/A, LT-α+ 252 A/G, IL-6-174 G/C, IL1-ra 86 bp VNTR, IL-10-1082 A/G and CD-31 125 C/G. Results: Overall odds ratio (OR) for VT related to TNF-α- 308 G/A, LT-α+ 252 A/G, IL-6-174 G/C, A1 allele (4 bp repeat) of the IL1-ra 86 bp VNTR, IL-10-1082 A/G and CD-31 125 C/G were respectively: 1.0 (CI95: 0.8-1.5), 1.3 (CI95: 1.0-1.7), 1.1 (CI95: 0.9-1.5), 1.6 (CI95: 1-2.5), 1.2 (CI95: 0.8-1.7) and 0.8 (CI95: 0.6-1.1). A possible interaction between polymorphisms was observed only for the co-inheritance of the mutant alleles of the LT-α+ 252 A/G and IL-10-1082 G/A polymorphisms (OR = 2; CI95: 1.1-3.8). The risk of VT conferred by factor V Leiden and FII G20210A was not substantially altered by co-inheritance with any of the cytokine gene polymorphisms. Conclusions: Cytokine gene polymorphisms here investigated did not significantly influence venous thrombotic risk. © 2006 Elsevier Ltd. All rights reserved.

Highlights from the I international symposium of thrombosis and anticoagulation in internal medicine, October 23-25, 2008, Sao Paulo, Brazil

The importance of thrombosis and anticoagulation in clinical practice is rooted firmly in several fundamental constructs that can be applied both broadly and globally. Awareness and the appropriate use of anticoagulant therapy remain the keys to prevention and treatment. However, to assure maximal efficacy and safety, the clinician must, according to the available evidence, choose the right drug, at the right dose, for the right patient, under the right indication, and for the right duration of time. The first International Symposium of Thrombosis and Anticoagulation in Internal Medicine was a scientific program developed by clinicians for clinicians. The primary objective of the meeting was to educate, motivate and inspire internists, cardiologists and hematologists by convening national and international visionaries, thought-leaders and dedicated clinician-scientists in Sao Paulo, Brazil. This article is a focused summary of the symposium proceedings. © Springer Science+Business Media, LLC 2009.