Presence Of Fibrin Research Articles

Vocal fold nodule vs. vocal fold polyp: answer from surgical pathologist and voice pathologist point of view

SummaryVocal fold polyps and nodules are caused by inflammation caused by stress or irritation. Our study involved looking retrospectively at 30 patients. A clinical diagnosis was established by an otolaryngologist and voice pathologist through videostroboscopic analysis. Histological sections after removal of the vocal fold lesion were available for microscopic examination. All of these cases have also been previously evaluated and photographed. A clinical diagnosis of either a polyp or a laryngeal nodule have been made and documented. Two pathologists made the histological evaluation separately initially unaware of the clinical diagnosis. Their impression was recorded. After the clinical diagnosis become available and comparison between the clinical and pathological diagnosis were made, a retrospective review of the tissue was attempted to reconcile the differences. In conclusion, after careful histological evaluation, no definitive histological distinction can be made between laryngeal nodules and polyps. Our data showed no distinction between the two entities when compared for the presence of edema, fibrin, inflammation, and amyloid-like material. A statistically significant difference was found in the size of the specimen and the presence of telangiectasias. Based on our data, a biopsy larger than 0.3 cm could be a polyp and a biopsy less than 0.3 cm could be a nodule.

β 2-glycoprotein I, anti-β 2-glycoprotein I, and fibrinolysis

β 2-glycoprotein-I (β 2GPI) is a phospholipid-binding plasma protein that consists of five homologous domains. Domain V is distinguished from others by bearing a positively charged lysine cluster and hydrophobic extra C-terminal loop. β 2GPI has been known as a natural anticoagulant regulator. β 2GPI exerts anticoagulant activity by inhibition of phospholipid-dependent coagulation reactions such as prothrombinase, tenase, and factor XII activation. It also binds factor XI and inhibits its activation. On the other hand, β 2GPI inhibits anticoagulant activity of activated protein C. According to the data from knockout mice, β 2GPI may contribute to thrombin generation in vivo. Phospholipid-bound β 2GPI is one of the major target antigens for antiphospholipid antibodies present in patients with antiphospholipid syndrome (APS). Binding of pathogenic anti-β 2GPI antibodies increases the affinity of β 2GPI to the cell surface and disrupts the coagulation/fibrinolysis balance on the cell surface. These pathogenic antibodies activate endothelial cells via signal transduction events in the presence of β 2GPI. Impaired fibrinolysis has been reported in patients with APS. Using a newly developed chromogenic assay, we demonstrated lower activity of intrinsic fibrinolysis in euglobulin fractions from APS patients. Addition of monoclonal anti-β 2GPI antibodies with β 2GPI also decreased fibrinolytic activity in this assay system. β 2GPI is proteolytically cleaved by plasmin in domain V (nicked β 2GPI) and becomes unable to bind to phospholipids, reducing antigenicity against antiphospholipid antibodies. This cleavage occurs in patients with increased fibrinolysis turnover. Nicked β 2GPI binds to plasminogen and suppresses plasmin generation in the presence of fibrin, plasminogen, and tissue plasminogen activator (tPA). Thus, nicked β 2GPI plays a role in the extrinsic fibrinolysis via a negative feedback pathway loop.

Platelet membrane fragments enhance the procoagulant effect of recombinant factor VIIa in studies with circulating human blood under conditions of experimental thrombocytopenia

The mechanism of action of recombinant factor VIIa (rFVIIa), which is being considered as an alternative treatment for the control of bleeding episodes in patients with thrombocytopenia, has not been fully characterized. This study was undertaken to explore the effects of rFVIIa and platelet microvesicles on hemostasis in an experimental model of thrombocytopenia. Damaged arterial segments were exposed to thrombocytopenic blood (shear rate 600 s −1) either with or without the addition of rFVIIa and/or platelet microvesicles. The presence of fibrin and platelets on the subendothelium were morphometrically quantified and immunolocalization techniques and electron microscopy were used for a more detailed analysis. Both rFVIIa and platelet microvesicles consistently improved fibrin formation on the damaged vascular subendothelium, and microvesicles were shown to be localized at different levels of the fibrin lattice. Further, under conditions of moderate thrombocytopenia, addition of platelet microvesicles potentiated the procoagulant action of rFVIIa. This effect may be due to the phospholipid surface provided by the platelet microvesicles. These studies support the concept that, under conditions of thrombocytopenia, both rFVIIa and platelet microvesicles enhance fibrin formation at sites of vascular damage.

Coagulation disorders in patients with cirrhosis and severe sepsis.

In patients with cirrhosis, severe sepsis may stimulate the extrinsic coagulation pathway resulting in thrombin generation and fibrin formation. To compare 23 patients with severe sepsis to 13 infected patients without severe sepsis and 18 patients without infection. Zymogen forms of clotting factors involved in the extrinsic pathway (i.e., factors VII + X, V, prothrombin), and the presence of soluble fibrin were assessed. Zymogen forms of clotting factors were significantly lower, while Child-Pugh score and the proportion of patients with soluble fibrin were higher in the severe-sepsis group than in the other groups. Decreased zymogen levels were independently correlated with an elevated Child-Pugh score and the presence of severe sepsis. In the severe-sepsis group, after adjustment for the severity of cirrhosis, decreased zymogen levels were associated with significant increases in the relative risk ratios of in-hospital death. Cirrhotic patients with severe sepsis have decreased blood levels of zymogen forms of factors VII+X, V, and prothrombin, which may be due not only to the severity of cirrhosis but also, at least in part, to the consumption of these zymogens by the extrinsic coagulation pathway.

Hemostatic effect of activated recombinant factor VII (rFVIIa) in liver disease: studies in an in vitro model

Background/Aims: There is clinical evidence for the efficacy of activated recombinant factor VII (rFVIIa) in patients with cirrhosis. The exact mechanism of action of rFVIIa in this clinical condition is unknown. We have explored effects of rFVIIa on hemostasis in cirrhotic patients using an in vitro perfusion technique. Methods: Blood samples were drawn from control donors or from 11 patients previously diagnosed with cirrhosis (seven Child-Pugh B and four Child-Pugh C) and anticoagulated with low molecular weight heparin. rFVIIa was added to blood samples at therapeutic concentrations (0.5 or 1 μg/ml of plasma) and blood was recirculated through annular chambers containing damaged vascular segments. Presence of platelets and fibrin on the subendothelium were morphometrically quantified. Results: Cirrhotic patients showed a diminished platelet interaction with the subendothelium compared to healthy donors (17.3% (9.28–28.88%) vs. 26.16% (19.96–54.5%), P<0.05). After addition of rFVIIa to cirrhotic samples, no differences in platelet covered surface were observed. However, fibrin formation was significantly improved after the addition of rFVIIa (from 51.81% (3.02–86.68%) to 86.94% (30.03–93.18%) and 89.05% (45.65–93.84%), respectively, P<0.05). Conclusions: Our data confirm a defective interaction of platelets with the subendothelium in cirrhotic patients. rFVIIa improved local fibrin formation at damaged sites and this mechanism could explain the beneficial action of rFVIIa in cirrhotic patients.

Avaliação do dímero D (D-Di) na doença hipertensiva específica da gravidez (DHEG)

A doença hipertensiva específica da gravidez (DHEG), na sua forma pura, caracteriza-se pelo aparecimento, em grávida normotensa, após a vigésima semana de gestação, da tríade sintomática: hipertensão, proteinúria e edema. A DHEG está associada a vasoconstrição generalizada, anormalidades da coagulação e deposição de fibrina na microcirculação da placenta. A fibrina compromete a perfusão adequada da placenta, contribuindo para a prematuridade, o baixo peso fetal e a mortalidade neonatal. Os dímeros D (D-Di) constituem os menores fragmentos dos produtos de degradação da fibrina (pdf) e são produzidos após a lise, pela plasmina, da ligação cruzada da fibrina. Níveis plasmáticos aumentados de D-Di indicam tanto uma exacerbação da formação de fibrina como um aumento da ação da plasmina. O presente trabalho teve por objetivo avaliar e comparar os níveis plasmáticos de D-Di, obtido por Elisa (Stago) em gestantes-controle (n = 26), gestantes com DHEG nas formas leve e moderada (n = 23) e gestantes com DHEG na forma grave (n = 20), visando a investigar a possível utilização desse marcador laboratorial para diagnóstico e/ou prognóstico da doença. As médias e os desvios padrões obtidos para os três grupos avaliados foram 1.146,6 ± 311,2; 1.060,3 ± 259,2 e 1.497,8 ± 435,3ng/ml, respectivamente. A análise estatística das médias dos resultados obtidos revelou um aumento significativo de D-Di plasmático no grupo com DHEG grave, comparando-se aos grupos com DHEG leve/moderada (p < 0,001) e grupo-controle (p < 0,01). Considerando-se os resultados obtidos, pode-se sugerir que há uma exacerbação da coagulação na forma grave da DHEG e que o sistema fibrinolítico está atuante buscando remover a excessiva quantidade de fibrina formada. O D-Di constitui uma potencial ferramenta para se avaliar a gravidade da doença.

Roles of Low Specificity and Cofactor Interaction Sites on Thrombin during Factor XIII Activation: COMPETITION FOR COFACTOR SITES ON THROMBIN DETERMINES ITS FATE

Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin.

Structure-Function Analysis of the Streptokinase Amino Terminus (Residues 1–59)

Streptokinase (SK) binds to plasminogen (Pg) to form a complex that converts substrate Pg to plasmin. Residues 1-59 of SK regulate its capacity to induce an active site in bound Pg by a nonproteolytic mechanism and to activate substrate Pg in a fibrin-independent manner. We analyzed 24 SK mutants to better define the functional properties of SK-(1-59). Mutations within the alphabeta1 strand (residues 17-26) of SK completely prevented nonproteolytic active site induction in bound Pg and rendered SK incapable of protecting plasmin from inhibition by alpha2-antiplasmin. However, when fibrin-bound, the activities of alphabeta1 strand mutants were similar to that of wild-type (WT) SK and resistant to alpha2-antiplasmin. Mutation of Ile1 of SK also prevented nonproteolytic active site induction in bound Pg. However, unlike alphabeta1 strand mutants, the functional defect of Ile1 mutants was not relieved by fibrin, and complexes of Ile1 mutants and plasmin were resistant to alpha2-antiplasmin. Plasmin enhanced the activities of alphabeta1 strand and Ile1 mutants, suggesting that SK-plasmin complexes activated mutant SK.Pg complexes by hydrolyzing the Pg Arg561-Val562 bond. Mutational analysis of Glu39 of SK suggested that a salt bridge between Glu39 and Arg719 of Pg is important, but not essential, for nonproteolytic active site induction in Pg. Deleting residues 1-59 rendered SK dependent on plasmin and fibrin to generate plasminogen activator (PA) activity. However, the PA activity of SK-(60-414) in the presence of fibrin was markedly reduced compared with WT SK. Despite its reduced PA activity, the fibrinolytic potency of SK-(60-414) was greater than that of WT SK at higher (but not lower) SK concentrations due to its capacity to deplete plasma Pg. These studies define mechanisms by which the SK alpha domain regulates rapid active site induction in bound Pg, contributes to the resistance of the SK-plasmin complex to alpha2-antiplasmin, and controls fibrin-independent Pg activation.

Increased local procoagulant action: a mechanism contributing to the favorable hemostatic effect of recombinant FVIIa in PLT disorders.

Recombinant FVIIa (rFVIIa) has been shown to improve hemostasis in patients with thrombocytopenia and to prevent or control bleeding episodes in patients with inherited deficiencies of major PLT glycoproteins, but the mechanism of action is not well understood. Effects of rFVIIa on hemostasis were explored with an in vitro perfusion technique. Blood samples, from healthy donors or from patients with congenital defects of PLT glycoprotein IIb-IIIa (GPIIb-IIIa), were anticoagulated with low-molecular-weight heparin. Experimental thrombocytopenia (<6000 PLTs/microL) was induced by a filtration procedure. rFVIIa was added to blood samples at therapeutic concentrations. A severe GPIIb-IIIa impairment was also induced by exposure of normal blood samples to a specific antibody. Perfusion studies were performed through annular chambers containing damaged vascular segments. The presence of fibrin and PLTs on the perfused subendothelium was morphometrically quantified. Under conditions of experimental thrombocytopenia, addition of rFVIIa enhanced fibrin formation in a dose-dependent manner (p < 0.05). Improvements in local fibrin generation and partial restoration of PLT interactions were also observed after incubation of blood from patients with Glanzmann's thrombasthenia with rFVIIa at 5 microg per mL (180 microg/kg). Similar improvements were observed in blood samples incubated with antibodies to GPIIb-IIIa. rFVIIa in whole normal blood also enhanced fibrin formation but PLT deposition was unaffected. Evaluation of prothrombin fragments 1 and 2 in the perfusates confirmed that rFVIIa increased thrombin generation in all cases. Our data indicate that rFVIIa promotes a procoagulant activity at sites of vascular damage. This mechanism could explain the beneficial hemostatic effect of rFVIIa in patients with thrombocytopenia or with Glanzmann's thrombasthenia.

Inhibition of Plasminogen Activation by Lipoprotein(a): CRITICAL DOMAINS IN APOLIPOPROTEIN(a) AND MECHANISM OF INHIBITION ON FIBRIN AND DEGRADED FIBRIN SURFACES

Similarity between the apolipoprotein(a) (apo(a)) moiety of lipoprotein(a) (Lp(a)) and plasminogen suggests a potentially important link between atherosclerosis and thrombosis. Lp(a) may interfere with tissue plasminogen activator (tPA)-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoagulable state in vivo. A fluorescence-based system was employed to study the effect of apo(a) on plasminogen activation in the presence of native fibrin and degraded fibrin cofactors and in the absence of positive feedback reactions catalyzed by plasmin. Human Lp(a) and a physiologically relevant, 17-kringle recombinant apo(a) species exhibited strong inhibition with both cofactors. A variant lacking the protease domain also exhibited strong inhibition, indicating that the apo(a)-plasminogen binding interaction mediated by the apo(a) protease domain does not ultimately inhibit plasminogen activation. A variant in which the strong lysine-binding site in kringle IV type 10 had been abolished exhibited substantially reduced inhibition whereas another lacking the kringle V domain showed no inhibition. Amino-terminal truncation mutants of apo(a) also revealed that additional sequences within kringle IV types 1-4 are required for maximal inhibition. To investigate the inhibition mechanism, the concentrations of plasminogen, cofactor, and a 12-kringle recombinant apo(a) species were systematically varied. Kinetics for both cofactors conformed to a single, equilibrium template model in which apo(a) can interact with all three fibrinolytic components and predicts the formation of ternary (cofactor, tPA, and plasminogen) and quaternary (cofactor, tPA, plasminogen, and apo(a)) catalytic complexes. The latter complex exhibits a reduced turnover number, thereby accounting for inhibition of plasminogen activation in the presence of apo(a)/Lp(a).

Association of fibrin and interleukin 8 in human oral squamous cell carcinoma.

We have recently demonstrated that fibrin induces a specific, dose- and time-dependent upregulation of the angiogenic factor interleukin 8 (IL-8) from human oral squamous cell carcinoma (OSCC) cells in vitro. In this study we begin to test the hypothesis that fibrin induces IL-8 expression from tumor cells in vivo by studying their in vivo association in OSCC. The presence of fibrin(ogen) was initially evaluated in 20 archival human OSCCs by means of immunohistochemistry with a polyclonal antibody. The presence of fibrin and IL-8 was then studied in 19 sections from 8 different patients' head and neck tumors (including 6 OSCCs) by means of immunohistochemistry with a monoclonal antibody against fibrin. These 8 tumors had been treated with inhibitors of new fibrin formation and degradation immediately after surgical removal. Fibrin staining was found in 100% of the tumor sections tested. IL-8 staining was found in the cytoplasm of tumor cells in 100% of the studied tumors, including areas adjacent to fibrin. These data demonstrate an in vivo association between fibrin and IL-8 in OSCC. These studies support our hypothesis that fibrin induces expression of protumorigenic factors such as IL-8 from tumor cells in vivo.

In vitro comparison of complementary interactions between synthetic linear/branched oligo/poly- l-lysines and tissue plasminogen activator by means of high-performance monolithic-disk affinity chromatography

The recently discovered serine protease called tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots. t-PA works by converting plasminogen into its active form, plasmin, dissolving the major component of blood clots, fibrin. The activation of plasminogen by t-PA is enhanced by the presence of fibrin, and this is probably due to the fact that both plasminogen and t-PA possess high affinity binding sites for fibrin. Besides fibrin, fibrin monomers and some fibrin(ogen) degradation products, certain synthetic polymers (for instance, poly- l-lysines) can provide the same stimulation of plasminogen activation. The recently developed high-performance monolithic-disk chromatography, HPMDC, could become the most convenient way to study biological pairs of interest. The inherent speed of HPMDC isolation facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences, such as solvents or temperature, is reduced. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting. The step-by-step modeling of hypothetical affinity pairs between t-PA and different types of oligo/polymer forms of linear and branched lysine derivatives obtained both by initiated polycondensation and solid-phase peptide synthesis using HPMDC seemed to be possible and a quite useful tool. The results of quantitative evaluation of such affinity interactions were compared with those established for natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The role of steric structure of lysine ligands was observed and analyzed. The results allowing to make the practical choice of affinity systems will be used for development of fast and efficient analytical and preparative methods for the downstream processes of recombinant production of this valuable enzyme.

Is severe deficiency of ADAMTS-13 specific for thrombotic thrombocytopenic purpura? Yes

Is severe deficiency of ADAMTS-13 specific for thrombotic thrombocytopenic purpura? Yes

Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.

Inflammatory breast cancer shows angiogenesis with high endothelial proliferation rate and strong E-cadherin expression.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Improved understanding of the mechanisms responsible for the differences between IBC and non-IBC might provide novel therapeutic targets. We studied 35 consecutive patients with IBC, biopsied prior to the initiation of chemotherapy. Angiogenesis was evaluated by Chalkley counting and by assessment of endothelial cell proliferation (ECP) and vessel maturity. The presence of fibrin, expression of the hypoxia marker carbonic anhydrase IX (CA IX) and epithelialcadherin (E-cadherin) expression were immunohistochemically detected. The same parameters were obtained in a group of 104 non-IBC patients. Vascular density, assessed by Chalkley counting (P<0.0001), and ECP (P=0.01) were significantly higher in IBC than in non-IBC. Abundant stromal fibrin deposition was observed in 26% of IBC and in only 8% of non-IBC (P=0.02). Expression of CA IX was significantly less frequent in IBC than in non-IBC with early metastasis (P=0.047). There was a significant positive correlation between the expression of CA IX and ECP in IBC (r=0.4, P=0.03), implying that the angiogenesis is partly hypoxia driven. However, the higher ECP in IBC and the less frequent expression of CA IX in IBC vs non-IBC points at a role for other factors than hypoxia in stimulating angiogenesis. Strong, homogeneous E-cadherin expression was found at cell–cell contacts in all but two IBC cases, both in lymphovascular tumour emboli and in infiltrating tumour cells, challenging our current understanding of the metastatic process. Both the intense angiogenesis and the strong E-cadherin expression may contribute to the highly metastatic phenotype of IBC.

Timecourse of fibrin deposition and removal after arterial injury

Atherosclerosis is a major disease of our aging society. It models of atherosclerosis and in-stent neointimal formation is commonly treated by angioplasty, in which a balloon is inflated in the artery to dilate the vessel and crush the primary lesion, or by stenting, in which a metallic brace is placed in the vessel after angioplasty. These treatments may result in immediate occlusive complications (1–8% of patients) or restenosis, a subsequent reblocking of the artery within 6 months of surgery (up to 60% of cases) [1–3]. Restenosis is due to a number of processes: intimal hyperplasia, vessel remodelling and/or thrombus deposition. Animal models of stenosis have shown that both vascular remodelling and neointimal hyperplasia determine the final luminal area [4,5]. Additionally, luminal narrowing may result from inclusion and absorption of agents of the thrombotic state (platelets, fibrin and erythrocytes) [6–8]. Thrombus and the presence of fibrin may play an active role in many other instances of restenosis. While normal aorta contains little fibrin or fibrinogen, early atherosclerotic plaques contain fibrin(ogen) present as long threads surrounding macrophages and other cells in the artery wall [9]. Although thrombi are not associated with uncomplicated plaques, insoluble fibrin and fibrinogen are concentrated in the shoulder region and necrotic core of the plaque [10,11]. Fibrin(ogen) has chemotactic and mitogenic effects and may act as a scaffold for smooth muscle cell migration and cell recruitment, adding to the size of the plaque [12,13]. Angioplasty results in localised areas of arterial wall damage, including endothelial denudation and, in extreme cases, medial stretching and splitting [1,2]. When the endothelium is removed, there is a loss of thrombosis inhibition normally present, platelets are deposited and adhesive molecules exposed [6,14]. There are several animal

Tratamiento tópico sobre úlceras varicosas de evolución tórpida con ampollas monodosis estériles de ciprofloxacino. La efectividad frente a suero fisiológico

ObjectiveTo determine the efficacy of topical application of sterile ciprofloxacin solution and physiologic serum as local treatment of venous ulcers compared with that of physiologic serum alone. DesignTherapeutic crossover trial. Material And MethodsA crossover study was performed. Patients with varicose lesions of more than 3 weeks’ duration (n = 63) were included. The mean age was 76.33 ± 16.79 years. There were 21 men (33%; 69.9 ± 19.9 years) and 42 women (67%; 80.2 ± 12.3 years). The sample was divided in two groups: group A (n = 63) received moisture semiocclusive therapy with single-dose ciprofloxacin ampoules and physiologic serum (NaCl 0.9%) applied by syringe 20 cc and disposable needle plus cotton gauzes (3-4 per application); group B (n = 63) received physiologic serum alone, used for a maximum of 2 weeks. ResultsIn the crossover study, the response was positive in 29 patients in group B and in 41 in group A (65 vs 46%; p = 0.000). The percentage decrease in wound area was 31.2 vs 9.3%; 1.46 vs 0.47 cm2, p = 0.00 (group A vs group B). The results of cultures for the crossover study were: 37.5% Pseudomonas, 17.6% sterile, 17.1% group D Streptococcus, and 14.1% saprophyte flora. ConclusionsCombined treatment of long-standing varicose lesions with topical ciprofloxacin and physiologic serum seems to achieve a greater initial response than the use of physiologic serum alone. The presence of fibrin at the beginning of treatment with serum does not seem to modify subsequent response to fibrin. Addition of topical ciprofloxacin to lesions without fibrin improves response compared with physiologic serum alone.

Aspirin alters the cardioprotective effects of the factor XIII Val34Leu polymorphism.

The mechanism underlying decreased risk for myocardial infarction in carriers of the Leu34 polymorphism of the factor (F) XIII A-subunit is unclear. Given that acetylation of fibrinogen by aspirin can alter its clotting properties and the presence of fibrin stimulates thrombin-mediated activation of FXIII, we have tested the hypothesis that treatment with aspirin differentially modulates the influence of the FXIII Val34Leu polymorphism on its activation in vivo. The rates of the disappearance of FXIIIA chain and the appearance of its activated form (FXIIIAa) in sequential 30-second blood samples collected at the site of microvascular injury were compared in 14 healthy carriers of the Leu34 allele and 23 Val34 homozygotes both before and after a 7-day aspirin ingestion (75 mg/d), with the use of quantitative Western blotting. The presence of the Leu34 allele was associated with a significant increase in the maximum rate of FXIII activation by thrombin. Although the Leu34-positive and -negative subjects were similar with respect to aspirin-related impairment of thrombin generation, aspirin led to a more pronounced inhibition of the activation of FXIII in the Leu34 carriers as compared with the Val34 homozygotes. Inhibition of FXIII activation by aspirin is enhanced in the Leu34 carriers in vivo, suggesting that these subjects might benefit more than the Leu34-negative subjects from the reduction in risk for myocardial infarction with low-dose aspirin.

Prophylactic heparinization is ineffective in a primate model of hemolytic uremic syndrome.

The presence of abundant fibrin within the glomerular capillaries of patients with Shiga toxin (Stx)-mediated (post-diarrheal) hemolytic uremic syndrome (HUS) has suggested that heparin anticoagulation might be useful in the treatment of this disease. Although controlled studies have failed to show benefit, this lack of therapeutic efficacy could be due to administration of heparin after the thrombotic microangiopathy (TMA) had developed. If benefit could be demonstrated by giving heparin prior to the onset of the HUS, one could consider prophylactic heparinization of those at high risk of progressing from Stx colitis to HUS. We have developed a baboon model of Stx-mediated HUS, and have used this model to study the effect of prophylactic heparinization on the clinical expression and glomerular TMA of Stx-mediated HUS. A single intravenous infusion of Stx-1 at a dose that uniformly produces clinical and nephropathologic features of HUS was given with and without the prior administration of regular and low molecular weight heparin. Therapeutic levels of anticoagulation were achieved, but this did not reduce the clinical severity or lessen the TMA. Heparin anticoagulation may be ineffective because the rate of coagulation that occurs on the surfaces of dysfunctional endothelial surfaces exceeds the capacity of heparin/antithrombin-III inhibitor complexes to bind and block the factor Xa and thrombin that are generated on these surfaces. Our observations fail to support the use of heparin in preventing Stx-mediated HUS.

Inhibition of fibrin-bound thrombin by a covalent antithrombin-heparin complex.

Numerous studies have shown that fibrin-bound thrombin (IIa) is protected from inhibition by antithrombin (AT) + heparin (H) due to the formation of a ternary fibrin.IIa.H complex. We investigated factors affecting the inhibition of fibrin.IIa by a covalent complex of AT and H (ATH). The rate of IIa reaction with ATH was decreased 2-3-fold by fibrin monomer as compared to 57-fold for AT + heparin with high AT affinity. Furthermore, although the reaction of AT + H with a IIa mutant with decreased H binding (RA-IIa) was inhibited 2-3-fold in the presence of fibrin, reaction rates of ATH + RA-IIa were not reduced by fibrin. The relative difference in the effect of fibrin on the ATH reaction with RA-IIa compared to that for reactions of AT + H with RA-IIa is consistent with the fact that, in the absence of fibrin, the rate of the ATH reaction with RA-IIa relative to IIa was much less reduced (8-fold) compared to the corresponding reactions of AT + H (decreased 306 fold). Similarly, the addition of excess H in the absence of fibrin gave only a small decrease in rate of ATH + IIa reaction. However, in the presence of fibrin, the addition of 40-fold excess H decreased the rate of ATH inhibition of IIa by 1 order of magnitude. Experiments with ATH containing low molecular weight heparin chains with low AT affinity showed that IIa inhibition requires ATH with long chains that activate the AT moiety. Finally, electrophoresis of fibrin +/- ((125)I-)IIa +/- ((125)I-)ATH on native and denaturing gels showed that ATH forms ATH-IIa complexes that remain bound to fibrin through the ATH component. Thus, ATH is a potent inhibitor of fibrin-bound IIa, likely due to the formation of fibrin.ATH-IIa as opposed to fibrin.IIa.H ternary complexes.