A Novel and Potent Thrombolytic Fusion Protein Consisting of Anti-Insoluble Fibrin Antibody and Mutated Urokinase.

Tissue plasminogen activator (tPA) is used clinically because it has a higher binding specificity for insoluble fibrin (IF) than urokinase (UK), but even pro-tPA has catalytic activity against substrates other than IF. UK has the advantage that it is specifically activated on IF; however, it binds IF weakly. Previously, we established a monoclonal antibody (mAb) that recognizes a pit structure formed only in IF. Here, we developed a new mAb against the pit, 1101, that does not affect coagulation or fibrinolysis, and prepared a fusion protein of UK with humanized 1101 Fab to transport UK selectively to IF. In IF-containing lesions, UK is cleaved by plasmin at two sites, Lys158/Ile159 and Lys135/Lys136. Cleavage of the former leads to activation of UK; however, because activated UK is linked by S-S bonds before and after cleavage, it is not released from the fusion. Cleavage at the latter site causes UK to leave the fusion protein; hence, we mutated Lys135/Lys136 to Gly135/Gly136 to prevent release of UK. This engineered UK-antibody fusion, AMU1114, significantly decreased the reduction of plasma plasminogen levels in vivo relative to UK. In a photochemically induced mouse model of thrombus, the vascular patency rate was 0% (0/10) in the control, 50% (5/10) in the tPA treatment group, and 90% (9/10) in the AMU1114 treatment group. Although no death was observed 1 hour after administration of each thrombolytic agent, some mice died within 24 hours in all treatment groups, including control. These data indicate the need for further basic studies of AMU1114.

Tenecteplase vs Alteplase Before Endovascular Therapy in Basilar Artery Occlusion.

OBJECTIVE: To investigate the efficacy of tenecteplase (TNK), a genetically modified variant of alteplase with greater fibrin specificity and longer half-life than alteplase, prior to endovascular thrombectomy (EVT) in patients with basilar artery occlusion (BAO). METHODS: To determine whether TNK is associated with better reperfusion rates than alteplase prior to EVT in BAO, clinical and procedural data of consecutive patients with BAO from the Basilar Artery Treatment and Management (BATMAN) registry and the Tenecteplase vs Alteplase before Endovascular Therapy for Ischemic Stroke (EXTEND-IA TNK) trial were retrospectively analyzed. Reperfusion >50% or absence of retrievable thrombus at the time of the initial angiogram was evaluated. RESULTS: We included 110 patients with BAO treated with IV thrombolysis prior to EVT (mean age 69 [SD 14] years; median NIH Stroke Scale score 16 [interquartile range (IQR) 7-32]). Nineteen patients were thrombolysed with TNK (0.25 mg/kg or 0.40 mg/kg) and 91 with alteplase (0.9 mg/kg). Reperfusion >50% occurred in 26% (n = 5/19) of patients thrombolysed with TNK vs 7% (n = 6/91) thrombolysed with alteplase (risk ratio 4.0, 95% confidence interval 1.3-12; p = 0.02), despite shorter thrombolysis to arterial puncture time in the TNK-treated patients (48 [IQR 40-71] minutes) vs alteplase-treated patients (110 [IQR 51-185] minutes; p = 0.004). No difference in symptomatic intracranial hemorrhage was observed (0/19 [0%] TNK, 1/91 [1%] alteplase; p = 0.9). CONCLUSIONS: TNK may be associated with an increased rate of reperfusion in comparison with alteplase before EVT in BAO. Randomized controlled trials to compare TNK with alteplase in patients with BAO are warranted. CLINICALTRIALSGOV IDENTIFIERS: NCT02388061 and NCT03340493. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that TNK leads to higher reperfusion rates in comparison with alteplase prior to EVT in patients with BAO.

Ultrastructural, Confocal and Viscoelastic Characteristics of Whole Blood and Plasma After Exposure to Cadmium and Chromium Alone and in Combination: An Ex Vivo Study.

BACKGROUND/AIMS: Heavy metal pollution is increasing in the environment, contaminating water, food and air supplies. This can be linked to many anthropogenic activities. Heavy metals are absorbed through the skin, inhalation and/or orally. Irrespective of the manner of heavy metal entry in the body, the blood circulatory system is potentially the first to be affected following exposure and adverse effects on blood coagulation can lead to associated thrombotic disease. Although the plasma levels and the effects of cadmium (Cd) and chromium (Cr) on erythrocytes and lymphocytes have been described, the environmental exposure to heavy metals are not limited to a single metal and often involves metal mixtures, with each metal having different rates of absorption, different cellular, tissue, and organ targets. Therefore the aim of this study is to investigate the effects of the heavy metals Cd and Cr alone and whether Cr synergistically increases the effect of Cd on physiological important processes such as blood coagulation. METHODS: Human blood was exposed to the heavy metals ex vivo, and thereafter morphological analysis was performed with scanning electron- and confocal laser scanning microscopy (CLSM) in conjunction with thromboelastography®. RESULTS: The erythrocytes, platelets and fibrin networks presented with ultrastructural changes, including varied erythrocytes morphologies, activated platelets and significantly thicker fibrin fibres in the metal-exposed groups. CLSM analysis revealed the presence of phosphatidylserine on the outer surface of the membranes of the spherocytic erythrocytes exposed to Cd and Cr alone and in combination. The viscoelastic analysis revealed only a trend that indicates that clots that will form after heavy metal exposure, will likely be fragile and unstable especially for Cd and Cr in combination. CONCLUSION: This study identified the blood as an important target system of Cd and Cr toxicity.

Neprilysin Inhibits Coagulation through Proteolytic Inactivation of Fibrinogen.

Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aß), the main pathological component of Alzheimer's disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bß-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions.

Role of pH Changes on Transforming Growth Factor-ß1 Release and on the Fibrin Architecture of Platelet-rich Fibrin When Layered with Biodentine, Glass Ionomer Cement, and Intermediate Restorative Material.

INTRODUCTION: The purpose of this study was to evaluate the influence of pH that is due to setting reaction of Biodentine, glass ionomer cement (GIC), and intermediate restorative material (IRM) on transforming growth factor-ß1 (TGF-ß1) release and on the fibrin architecture of platelet-rich fibrin (PRF). METHODS: PRF was obtained from 8 volunteers and layered over the freshly prepared GIC, IRM, and Biodentine mixtures. TGF-ß1 release was estimated by using enzyme-linked immunosorbent assay (ELISA), and fibrin structure of PRF was analyzed by using scanning electron microscope at 1 and 5 hours. RESULTS: Biodentine, GIC, and IRM increased the TGF-ß1 release in comparison with that of control group (PRF alone) at both 1 and 5 hours. Biodentine released significantly more TGF-ß1 than GIC and IRM at 1 hour. At 5 hours both GIC and Biodentine released significantly more TGF-ß1 than IRM. The fibrin architecture of the Biodentine group was similar to that of control group at both 1 and 5 hours. In GIC and IRM groups the fibrillar structure of fibrin was collapsed, ill-defined, and cloudy with very thick fibers and irregularly reduced porosities. CONCLUSIONS: Biodentine induces larger amount of TGF-ß1 release and also maintains the integrity of fibrin structure when compared with GIC and IRM when layered over PRF.

Plasma fibrin clot properties in postmenopausal women: effects of hormone therapy.

OBJECTIVE: Postmenopausal women are at risk of thromboembolic events. It is unclear whether menopause alters fibrin clot properties. The aim of our study was to assess the effects of menopause and hormone therapy on clot characteristics. METHODS: Ex vivo plasma clot permeability, turbidity, and susceptibility to lysis were determined in 70 premenopausal and 70 postmenopausal women (a case-control study). From the postmenopausal group, 30 women were randomly assigned (1:1) to a 24-week oral or transdermal treatment with 17ß-estradiol, combined with norethisterone acetate (2 mg + 1 mg/d or 0.05 mg + 5 mg/d, respectively). RESULTS: Compared with premenopausal women (aged 29.2 ±â€Š2.60 y), postmenopausal women (aged 49.7 ±â€Š3.4 y; P = 0.009) were characterized by higher fibrinogen levels (by 36.8%), lower C-reactive protein levels (by 36.9%), and lower clot permeability (by 10.5%); also after adjustment for fibrinogen (all P < 0.05), with no difference in turbidimetric or fibrinolytic variables. Estrogen plus progestogen therapy led to higher maximal absorbency of fibrin gels by 6.0% (P < 0.05), whereas all other fibrin variables remained unchanged. Compared with transdermal hormone therapy, 24-week oral therapy was associated with higher absorbency of plasma clots by 16% (P < 0.05), without any other changes in fibrin characteristics. CONCLUSIONS: Menopause age is associated with the formation of denser fibrin clots. Estrogen plus progestogen therapy has a minor effect on plasma fibrin properties, but leads to the formation of thicker and more branched fibrin fibers, particularly during oral administration.

Antithrombotic and antiplatelet activities of pelargonidin in vivo and in vitro.

Pelargonidin is a well-known red pigment found in plants, and has been reported as having important biological activities that are potentially beneficial for human health. However, the possible roles of pelargonidin as an anticoagulant and the underlying mechanism have not yet been elucidated. We tested the effect of pelargonidin and its glucoside-conjugated form, pelargonidin-3-glucoside, on the clotting times, such as activated partial thromboplastin time (aPTT) and prothrombin time (PT), and the activities and productions of thrombin and activated factor X (FXa). Furthermore, the effects of pelargonidin on the fibrin polymerization, platelet aggregation, and the ratio of plasminogen activator inhibitor-1 (PAI-1) to tissue plasminogen activator were determined. Pelargonidin, but not pelargonidin-3-glucoside, prolonged the aPTT and PT, and inhibited the activity and production of thrombin and FXa in human umbilical vein endothelial cells. Furthermore, pelargonidin inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation and elicited anticoagulant effects in mice. In addition, pelargonidin significantly reduced PAI-1 to t-PA ratio. Collectively, these results indicate that the anthocyanin pelargonidin possesses antithrombotic activity, and can be beneficial in preventing thrombus formation, thus improving blood circulation.

Effects of riboflavin and amotosalen photoactivation systems for pathogen inactivation of fresh-frozen plasma on fibrin clot structure.

BACKGROUND: Fresh-frozen plasma (FFP) transfusion carries a risk of viral transmission from donor to recipient. Riboflavin (Mirasol) and amotosalen (Intercept) are two pathogen inactivation (PI) methods that may enhance the safety of FFP for transfusion. Our study investigated the effects of Mirasol and Intercept treatment on fibrin formation and clot structure. STUDY DESIGN AND METHODS: FFP underwent either Mirasol or Intercept treatment, and aliquots were taken before addition of the compound, before illumination (after addition of compound only), and after treatment (addition of compound plus illumination). All samples underwent turbidimetric analysis, lysis analysis, assessment of clot permeation, and analysis by laser scanning confocal microscopy. RESULTS: After treatment, there was a decrease in optical density of the fibrin network for Mirasol and Intercept, lag time to fibrin formation was prolonged for Mirasol and lysis time for Intercept, clot permeability was significantly decreased, and clot density was increased for both. CONCLUSIONS: Our study shows that plasma treated with Mirasol and Intercept produces denser clots consisting of thinner fibers and warrants further studies to evaluate the clinical significance of these structural changes in fibrin clot formation.

Alteplase Reduces Downstream Microvascular Thrombosis and Improves the Benefit of Large Artery Recanalization in Stroke.

BACKGROUND AND PURPOSE: Downstream microvascular thrombosis (DMT) is known to be a contributing factor to incomplete reperfusion in acute ischemic stroke. The aim of this study was to determine the timing of DMT with intravital imaging and to test the hypothesis that intravenous alteplase infusion could reduce DMT in a transient middle cerebral artery occlusion (MCAO) rat stroke model. METHODS: Rats were subjected to 60-minute transient MCAO. Alteplase (10 mg/kg) was administered 30 minutes after the beginning of MCAO. Real-time intravital fluorescence microscopy through a dura-sparing craniotomy was used to visualize circulating blood cells and fibrinogen. Cerebral microvessel patency was quantitatively evaluated by fluorescein isothiocyanate-dextran perfusion. RESULTS: Immediately after MCAO, platelet and leukocyte accumulation were observed mostly in the venous compartment. Within 30 minutes after MCAO, microthrombi and parietal fibrin deposits were detected in postcapillary microvessels. Alteplase treatment significantly (P=0.006) reduced infarct volume and increased the percentage of perfused vessels during MCAO (P=0.02) compared with saline. Plasma levels of fibrinogen from alteplase-treated rats showed a rapid and profound hypofibrinogenemia. In vitro platelet aggregation demonstrated that alteplase reduced platelet aggregation (P=0.0001) and facilitated platelet disaggregation (P=0.001). These effects were reversible in the presence of exogenous fibrinogen. CONCLUSIONS: Our data demonstrate that DMT is an early phenomenon initiated before recanalization. We further show that alteplase-dependent maintenance of downstream perfusion during MCAO improves acute ischemic stroke outcome through a fibrinogen-dependent platelet aggregation reduction. Our results indicate that early targeting of DMT represents a therapeutic strategy to improve the benefit of large artery recanalization in acute ischemic stroke.

2013 scientific sessions Sol Sherry distinguished lecture in thrombosis: polyphosphate: a novel modulator of hemostasis and thrombosis.

Polyphosphate is a highly anionic, linear polymer of inorganic phosphates that is found throughout biology, including in many infectious microorganisms. Recently, polyphosphate was discovered to be stored in a subset of the secretory granules of human platelets and mast cells, and to be secreted on activation of these cells. Work from our laboratory and others has now shown that polyphosphate is a novel, potent modulator of the blood clotting and complement systems that likely plays roles in hemostasis, thrombosis, inflammation, and host responses to pathogens. Therapeutics targeting polyphosphate may have the potential to limit thrombosis with fewer hemorrhagic complications than conventional anticoagulant drugs that target essential proteases of the blood clotting cascade.

Clinical usefulness of measuring prothrombin time and soluble fibrin levels in Japanese patients with atrial fibrillation receiving rivaroxaban.

BACKGROUND: Rivaroxaban is currently used to prevent stroke in patients with atrial fibrillation. Measuring coagulation function may help clinicians to understand the effects of this drug and the associated risk of bleeding. METHODS AND RESULTS: Rivaroxaban was given to 136 patients with non-valvular atrial fibrillation. Mean age was 74.5±9.0 years (men: 63.2%) and mean CHADS2 score (±SD) was 1.8±1.2. Prothrombin times (PTs) and plasma soluble fibrin (SF) levels were examined in 84 out of 136 patients at baseline and at least 2 weeks thereafter. In 48 patients we were able to collect blood at exact times, namely just before and 3h after rivaroxaban administration, corresponding to the trough and peak concentrations. Mean peak PT in 48 patients was 17.1±3.6s and median peak SF level was 1.46µg/mL. Multiple regression analysis showed that female sex, high brain natriuretic peptide, and high dose were independent factors prolonging the peak PT. Patients with peak PTs ≥20s experienced significantly more bleeding events. Among 29 of 46 patients newly treated with rivaroxaban without any previous anticoagulant, we examined coagulation function at the exact trough and peak times. In 29 patients, peak PT was significantly more prolonged than the baseline or trough PT (p<0.001 for both), whereas trough PT was comparable to the baseline PT. In contrast, both trough and peak SF levels in these newly treated patients were significantly reduced than at baseline (p=0.003 and p<0.001, respectively). CONCLUSIONS: In Japanese patients with non-valvular atrial fibrillation receiving rivaroxaban, a prolonged peak PT (≥20s) could indicate increased risk of bleeding, and both trough and peak SF levels were reduced relative to baseline. PT and SF are both valuable measures of coagulation status in patients receiving rivaroxaban, regardless of prior anticoagulant history.

Ozone dosing alters the biological potential and therapeutic outcomes of plasma rich in growth factors.

BACKGROUND AND OBJECTIVE: Until now, ozone has been used in a rather empirical way. This in-vitro study investigates, for the first time, whether different ozone treatments of plasma rich in growth factors (PRGF) alter the biological properties and outcomes of this autologous platelet-rich plasma. MATERIAL AND METHODS: Human plasma rich in growth factors was treated with ozone using one of the following protocols: a continuous-flow method; or a syringe method in which constant volumes of ozone and PRGF were mixed. In both cases, ozone was added before, during and after the addition of calcium chloride. Three ozone concentrations, of the therapeutic range 20, 40 and 80 µg/mL, were tested. Fibrin clot properties, growth factor content and the proliferative effect on primary osteoblasts and gingival fibroblasts were evaluated. RESULTS: Ozone treatment of PRGF using the continuous flow protocol impaired formation of the fibrin scaffold, drastically reduced the levels of growth factors and significantly decreased the proliferative potential of PRGF on primary osteoblasts and gingival fibroblasts. In contrast, treatment of PRGF with ozone using the syringe method, before, during and after the coagulation process, did not alter the biological outcomes of the autologous therapy. CONCLUSION: These findings suggest that ozone dose and the way that ozone combines with PRGF may alter the biological potential and therapeutic outcomes of PRGF.

The effect of sibutramine, a serotonin-norepinephrine reuptake inhibitor, on platelets and fibrin networks of male Sprague-Dawley rats: a descriptive study.

Sibutramine is used in the treatment of obesity due to its ability to influence feelings of hunger and satiety by inhibiting the re-uptake of serotonin and noradrenalin in the central nervous system (CNS). Sibutramine use has been associated with numerous adverse events in particular cardiovascular complications possibly due to the formation of thrombi. This ultrastructural descriptive study investigated the effect of sibutramine on blood coagulation, specifically the effect on morphology of platelets and fibrin networks using scanning electron microscopy. Male Sprague-Dawley rats treated with either a recommended therapeutic dose [low dosage 1.32 mg/kg] or a toxicological higher dose [high dosage 13.2 mg/kg] of sibutramine for 28 days were used and compared to control animals. Blood samples were collected and plasma smears were prepared for platelet evaluation. Following the addition of thrombin to the plasma samples, the morphology of the fibrin clots was evaluated. Platelet evaluation by scanning electron microscopy revealed morphology typical of a prothrombotic state with a characteristic excessive platelet activation in both low-dose (LD) and high-dose (HD) rats. The fibrin clots of sibutramine-treated rats, LD and HD revealed fused thick fibers with thin fibers forming a net-like structure over the thick fibers which differ considerably from the organized structure of the control animals. It can be concluded that sibutramine alters the ultrastructure of platelets and fibrin networks creating a prothrombotic state.

Histone deacetylase inhibitor treatment attenuates coagulation imbalance in a lethal murine model of sepsis.

BACKGROUND: Sepsis has a profound impact on the inflammatory and hemostatic systems. In addition to systemic inflammation, it can produce disseminated intravascular coagulation, microvascular thrombosis, consumptive coagulopathy, and multiple organ failure. We have shown that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACI), improves survival in a lethal model of cecal ligation and puncture (CLP) in mice, but its effect on coagulation remains unknown. The goal of this study was to quantify the impact of SAHA treatment on coagulopathy in sepsis. METHODS: C57BL/6J mice were subjected to CLP, and 1 hour later given intraperitoneally either SAHA dissolved in dimethyl sulfoxide (DMSO) or DMSO only. Sham-operated animals were handled in similar manner without CLP. Blood samples were collected by cardiac puncture and evaluated using the TEG 5000 Thrombelastograph Hemostasis Analyzer System. RESULTS: Compared with the sham group, all animals in DMSO vehicle group died within 72 hours, and developed coagulopathy that manifested as prolonged initial fibrin formation and fibrin cross-linkage time, and decreased clot formation speed, platelet function, and clot rigidity. SAHA treatment significantly improved survival and was associated with improvement in fibrin cross-linkage and clot formation, as well as platelet function and clot rigidity, without a significant impact on the clot initiation parameters. CONCLUSION: SAHA treatment enhances survival and attenuates sepsis-associated coagulopathy by improving fibrin cross-linkage, rate of clot formation, platelet function, and clot strength. HDACI may represent a novel therapeutic strategy for correcting sepsis-associated coagulopathy.

Fibrin reaction after uveitic cataract surgery: treatment and prevention.

PURPOSE: To describe the management with intracameral recombinant tissue plasminogen activator (rtPA) of severe fibrin effusion after phacoemulsification, and its prevention with sustained-release dexamethasone intravitreal implant. METHODS: A 59-year-old man with chronic HLA-B27-associated uveitis underwent phacoemulsification followed by a sustained-release dexamethasone intravitreal implant. RESULTS: A postoperative severe fibrin reaction was completely resolved after intracameral injection of rtPA. At the time of the phacoemulsification of the fellow eye, a dexamethasone implant was injected 5 days prior to surgery, with no fibrin formation. CONCLUSIONS: Intracameral rtPA may be successfully used in the management of severe fibrin reaction. Dexamethasone intravitreal implant to control postoperative inflammation may take several days until it achieves high concentrations in the vitreous.

Thrombodynamics of microvascular repairs: effects of antithrombotic therapy on platelets and fibrin.

PURPOSE: To evaluate the hypothesis that platelets and fibrin differentially accrue at microvascular anastomoses in arteries versus veins and under different pharmacologic conditions. METHODS: We evaluated mouse arterial and venous anastomoses with intravital fluorescence imaging, using fluorophore-labeled platelets and anti-fibrin antibodies to measure the extent of thrombus component development in the intraluminal anastomotic site. We evaluated systemic heparin or eptifibatide (platelet aggregation inhibitor) to determine their relative influences on thrombus composition. RESULTS: Platelets accumulated rapidly in both arterial and venous repairs, and then fell in number after 10 to 30 minutes of reflow. Fibrin had a relatively steady development over 60 minutes in veins, with a more variable increase in arteries. Heparin reduced platelet accumulation in arteries and fibrin development in veins. Eptifibatide reduced platelets in both arteries and veins and had an apparent effect on lowering the amount of fibrin in veins. CONCLUSIONS: These findings show that platelets have a rapid, transient response, whereas fibrin has a slower, more sustained accrual in both arterial and venous anastomoses. Furthermore, inhibition of either coagulation or platelet aggregation can influence presumably non-targeted components of thrombosis in vascular repairs of both arteries and veins. CLINICAL RELEVANCE: Preventing replantation failure using antithrombotic therapies requires a better understanding of the effect of each pharmacologic compound on the various aspects of thrombogenesis.

Plasminogen controls inflammation and pathogenesis of influenza virus infections via fibrinolysis.

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza.

Ultrastructural characteristics of fibrin clots from canine and feline platelet concentrates activated with calcium gluconate or calcium gluconate plus batroxobin.

BACKGROUND: The aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates (PC) that had been activated with calcium gluconate (CG) or CG plus batroxobin (CGB). Platelets from fibrin clots were classified according their morphological changes. The area of the intercellular space (µm2), the area of the fibrin fibers (µm2), and the width of the fibrin fibers (µm) were determined for the dog clots. The platelet area (µm2), the area of fibrin fibers (µm2), the ratio of the minor and major axes of platelets, the ratio of the major and minor axes of platelets, and the number of α-granules found within platelets were measured for the cat clots. RESULTS: Cat platelets displayed full activation. Dog platelets displayed lysis with loss of normal architecture. In both species, a statistically significant difference was found (P < 0.01) between the fibrin fiber measurements in the PC clots activated with CG and CGB. CONCLUSIONS: The findings suggest that activation with CG caused platelet alpha granules to release their contents. In cats, fibrin production was greater when the PC was activated with CG. In dogs, activation with CG produced thick fibrin fibers.

Influence of nicotine and cotinine impregnation on the first step of periodontal regeneration: clot stabilization.

This study analyzes the clot stabilization on root surfaces of teeth impregnated with cotinine and nicotine and the influence of the scaling in the adhesion of blood components, observing the influence of new exposition to nicotine and/or cotinine after scaling. Fifteen human teeth extracted due to periodontal disease of non-smokers patients were selected and manually scaled. Four dentin blocks were obtained from each tooth (n = 60). Samples received blood application or reimpregnation with nicotine and/or cotinine, depending on the groups. Group 1: PBS immersion + root scaling + blood; group 2: nicotine + root scaling + blood; group 3: nicotine + root scaling + nicotine reapplication + blood; group 4: cotinine + root scaling + blood; group 5: cotinine + root scaling + cotinine reapplication+ blood; group 6: nicotine and cotinine + root scaling + nicotine and cotinine + blood. Samples were kept in 2 ml of each substance for 24 hours. Each group received a blood drop and was analyzed by SEM. The higher amount of blood components was present in teeth exposed to cotinine and the groups submitted to scaling and blood application in comparison with groups that received reapplication of toxic substances after scaling. The greater toxic effect on root dentin surface was after the exposure to nicotine and cotinine. Results suggest that periodontal healing may be delayed in smokers due to the direct inhibition of clot stabilization on the root surface when nicotine and cotinine are present concomitantly.