Augmentation of ultrasound-induced clot disruption by nongas-filled microparticles

The augmentation of ultrasound-induced clot disruption by echocardiographic contrast microbubbles may be a direct mechanical erosive effect of the clot by the microparticles. To further assess this hypothesis, we evaluated the rate and extent of clot disruption by using external ultrasound and nongas-filled microparticles (HAEMACCEL and HAES). Human blood clots were used in this in vitro study. The percent clot reduction using the combination of ultrasound and microparticles was dose dependent and significantly higher than that with microparticles alone.     

Fundoplication wrap disruption: an experimental study in rats.

Antireflux surgery usually gives long lasting control of gastroesophageal reflux disease, but late failures can occur from fundoplication wrap disruption. Disruption presumably occurs when physiological mechanical stresses cause the sutures to pull out of the fundoplication wrap. We hypothesized that complete fundoplications (fundus sutured to fundus) would withstand disruptive forces better than partial fundoplications (fundus sutured to esophagus). Forty-eight rats underwent fundoplication (24 partial and 24 complete). Fundoplication wraps were disrupted by distending the stomach (bursting pressure technique) and by distracting the wrap in a tensiometer (breaking strength technique). Bursting pressures were similar in the partial (103.7 +/- 13.5 mmHg) and complete (100.5 +/- 13.1 mmHg) fundoplication wraps (P = 0.93, not significant). In both groups, all disruptions occurred by sutures tearing through the stomach wall. Breaking strength was also equivalent for the two types of wrap. Partial wraps disrupted at 6.69 +/- 1.49 N and complete wraps disrupted at 6.52 +/- 1.28 N (P = 0.77, not significant). Sutures tore out of the stomach side of the partial wrap in five rats and out of the esophagus in the other seven rats with partial wraps. Disruption occurred by sutures tearing through the stomach in all rats with complete fundoplications. This experimental study in the rat did not show any difference in the ability of partial and complete fundoplication wraps to withstand disruptive forces.     

Ivermectin and coagulation: an in vitro study

The prothrombin time, partial thromboplastin time with kaolin, and thrombin clotting time of plasma derived from healthy human volunteers were unaltered after in vitro addition of therapeutic concentrations (20-90 ng ml-1) of ivermectin. No difference in these coagulation tests, relative to untreated controls, was observed after 12 hours' incubation with the drug.     

Dusart syndrome: a new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure

Fibrinogen Dusart is a congenital dysfibrinogenemia (A-alpha 554 Arginine-->Cysteine) associated with severe thrombotic disorder, high incidence of thrombotic embolism, and abnormal fibrin polymerization. This thrombotic disorder was attributed to an abnormal clot thrombolysis with reduced plasminogen binding to fibrin and defective plasminogen activation by tissue plasminogen activator. The purpose of this work was to assess whether clot architecture could be involved in the thromboresistance of the fibrin Dusart and the high incidence of embolism. An important change in Dusart fibrin clot structure was identified with dramatic decrease of gel porosity (Ks), fiber diameters (d), and fiber mass-length ratios (mu) derived from permeation analysis. In addition, rigidity of the Dusart clot was found to be greatly increased compared with normal fibrin. We provide evidence that both thrombolysis resistance and abnormal rigidity of the fibrin Dusart are related to this abnormal architecture, which impairs the access of fibrinolytic enzymes to the fibrin and which is responsible for a brittle clot that breaks easily, resulting in a high incidence of embolism. Indeed, when restoring a normal clot structure by adding dextran 40 (30 mg/mL) before coagulation, clot thrombolysis and clot rigidity recovered normal values. This effect was found to be dose- dependent. We conclude that clot architecture is crucial for the propensity of blood clot to be degraded and that abnormal clot structure can be highly thrombogenic in vivo. The alpha-C domains of fibrinogen are determinant in fibrin clot structure.     

Genetics of fibrin clot structure: a twin study

Coronary artery thrombosis following plaque rupture is an important feature of myocardial infarction, and studies have highlighted the role of coagulation in this condition. Although genetic and environmental influences on the variance in coagulation protein concentrations have been reported, there are no data on the heritability of structure/function of the final phenotype of the coagulation cascade, the fibrin clot. To assess genetic and environmental contributions to fibrin structure, permeation and turbidity studies were performed in 137 twin pairs (66 monozygotic, 71 dizygotic). The environmental influence (e2) on pore size (Ks) (e2 = 0.61 [95% confidence interval (CI), 0.45-0.80]) and fiber size (e2 = 0.54 [95% CI, 0.39-0.73]) was greater than the heritability (h2 = 0.39 [95% CI, 0.20-0.55] and 0.46 [95% CI, 0.27-0.62], respectively). After correction for fibrinogen levels, the environmental effect persisted for Ks (e2 = 0.61), but genetic influence assumed a greater importance in determining fiber size (h2 = 0.73). Multivariate analysis revealed an overlap in the influence of genetic and environmental factors on fibrinogen levels, Ks, and fiber size. Factor XIII B subunit showed environmental and genetic correlation with fibrinogen and fiber size and a genetic correlation with Ks. The results indicate that genetic and environmental influences are important in determining fibrin clot structure/function.     

A novel mutation (deletion of Aalpha-Asn 80) in an abnormal fibrinogen: fibrinogen Caracas VI. Consequences of disruption of the coiled coil for the polymerization of fibrin: peculiar clot structure and diminished stiffness of the clot.

An abnormal fibrinogen was identified in a 10-year-old male with a mild bleeding tendency; several years later, the patient developed a thrombotic event. Fibrin polymerization of plasma from the propositus and his mother, as measured by turbidity, was impaired. Plasmin digestion of fibrinogen and thrombin bound to the clot were both normal. The structure of clots from both plasma and purified fibrinogen was characterized by permeability, scanning electron microscopy and rheological measurements. Permeability of patients' clots was abnormal, although some measurements were not reliable because the clots were not mechanically stable. Consistent with these results, the stiffness of patients' clots was decreased approximately two-fold. Electron microscopy revealed that the patients' clots were very heterogeneous in structure. DNA sequencing of the propositus and his mother revealed a new unique point mutation that gives rise to a fibrinogen molecule with a missing amino acid residue at Aalpha-Asn 80. This new mutation, which would disrupt the alpha-helical coiled-coil structure, emphasizes the importance of this part of the molecule for fibrin polymerization and clot structure. This abnormal fibrinogen has been named fibrinogen Caracas VI.     

Ultrasonic determination of clot deposition rates in a milk-based, in-vitro procedure for thrombogenicity assessment

BACKGROUND AND AIM OF THE STUDY: Thrombosis and thromboembolism remain the main problems associated with mechanical heart valves. We have devised a milk-based clotting technique to simulate in-vitro clinical incidence of thrombosis. Early results with the technique revealed good correlation between milk clot deposition and clinical thrombosis, but were limited in their ability to predict the course of clot deposition, as deposition could only be measured upon dismantling the apparatus. METHODS: Clot deposition was observed ultrasonically for both steady and pulsatile flows of a milk preparation through a cylindrical test chamber containing axisymmetric test bodies similar in shape to those used in earlier studies of thrombosis. Echo ultrasound images were recorded at regular time intervals, depicting the interface between clot deposits and flowing milk. From these images, the thickness of the clot deposit could be determined as a function of time. RESULTS: Milk clot deposition on the test bodies, while observed, could not be measured accurately due to faint reflections arising from significant differences between the angle of incidence and the angle of reflection of the ultrasound beam. However, measurement was possible at the wall of the test chamber where deposition rates revealed steady growth of clot, following an initial 'lag', with growth continuing until a maximum thickness is reached. In some experiments shedding of parts of the deposit was observed. In pulsatile flow, wall clot deposition rates and final clot thickness attained were significantly lower than in steady flow. CONCLUSION: Ultrasonic measurement of clot deposition rates is possible in our thrombogenicity assessment apparatus on surfaces perpendicular to the line of incidence of the ultrasound beam. With suitably designed viewing windows in an artificial heart, such measurements should enable the time course of clot deposition on artificial valves to be determined, with a view to identifying initial deposition sites and the dynamics of clot growth. Observations of the growth of clot on the test chamber wall in this study suggest that both the rate of deposition and nature of deposit formed are strongly influenced by fluid mechanical properties such as shear and mass transfer rates. In particular, our results appear to suggest a different structure of deposit, whose rate of deposition is relatively slow, under conditions of high shear.     

A new thrombectomy catheter device (AngioJet) for the disruption of thrombi: An in vitro study.

In this study we examined a new thrombectomy catheter device. Different kinds of in vitro generated thrombi and cadaver thrombi were disrupted in test tubes. The mean disruption rate (and disruption time for 1 g of thrombus) was 225 +/- 65 mg/sec (5 +/- 2 sec) for whole-blood, 117 +/- 60 mg/sec (12 +/- 9 sec) for fibrin, 41 +/- 18 mg/sec (30 +/- 18 sec) for mixed, 70 +/- 42 mg/sec (17 +/- 5 sec) for unorganized, 45 +/- 8 mg/sec (22 +/- 4 sec) for partly, and 5 +/- 1 mg/sec (216 +/- 29 sec) for completely organized cadaver thrombi (P < 0.05). More than 99% of fragmented particles of whole-blood thrombi were 0-12 microm in diameter. The particle size of fibrin, mixed, and cadaver thrombi was similar, with 25%-40% of particles between 0-12 microm, 55%-71% >12-24 microm, and 2%-7% >24 microm. The device may be effectively used in the therapy of massive pulmonary embolism or acute peripheral and coronary artery syndromes when medical thrombolysis is contraindicated and organization of thrombus is absent. Further studies need to be performed to investigate the potential effects of particle microembolization. Cathet. Cardiovasc. Intervent. 47:381-389, 1999.     

The mechanism of in vitro clot lysis induced by vascular plasminogen activator

The contribution of vascular plasminogen activator (v-PA) to the lysis of whole blood and plasma clots was investigated. v-PA released into the circulation after infusion of deamino-D-arginine vasopressin (DDAVP) was shown to bind quantitatively to plasma clots. Its apparent molecular weight, determined by the SDS-PAGE fibrin-agarose underlay method, was approximately 68,000 daltons, and its activity was quenched by antibodies against human tissue plasminogen activator (t-PA). Clots prepared from post-DDAVP plasma or post-DDAVP whole blood, rich in v- PA, did not lyse when incubated in imidazole buffer or normal plasma, as determined by the release of 125I from radiolabeled clots. However, clots made of v-PA-poor plasma or whole blood, incubated in v-PA-rich plasma, underwent substantial lysis. The concentration of PA in clots incubated in v-PA-rich plasma progressively increased in relation to the initial concentration of v-PA in the surrounding plasma. The results suggest that, at low concentrations of circulating v-PA, a hemostatic plug will lyse at a very low rate. However, when the v-PA concentration in the clot environment is increased, v-PA will accumulate progressively onto fibrin and induce thrombolysis.     

Immunosuppressive effects of factor IX products: an in vitro study

The effects of a recombinant factor IX product (BeneFix), and of five plasma-derived factor IX products, AlphaNine, Immunine, Konyne, Mononine and Replinine on in vitro peripheral blood mononuclear cell (PBMC) immune function were compared in a blinded study. We assessed the effects of these products on Con-A-induced lymphocyte proliferation and interleukin-2 and interleukin-10 secretion, expression of lymphocyte activation markers, and nitric oxide secretion by stimulated mouse peritoneal macrophages. At 1 mL-1 for 48 h, Konyne reduced Con-A-induced mitogenesis by 50% (P < 0.05); AlphaNine, Mononine and BeneFix had no effect. At 10 IU mL-1, Con-A-induced mi- togenesis was at control levels with Mononine and BeneFix, but was reduced to <15% (P < 0.05) with each of the other products. IL-2 and IL-10 secretion by Con-A-stimulated lymphocytes was also markedly depressed by all the products tested except Mononine and BeneFix. Dialysis of these products did not substantially affect these results. Flow cytometric analysis of lymphocyte activation markers following Con-A stimulation showed that Konyne also decreased IL-2 receptor alpha and beta chain (CD25 and CD122) induction on PBMC. Konyne also inhibited nitric oxide secretion to levels <18% of controls. These results indicate that certain factor IX products, including some of purported higher purity, substantially depress in vitro immune function. The importance of these findings to in vivo immune function in haemophilia B patients remains to be established.     

Clot lysis by gastric juice: an in vitro study.

Gastric juice from patients with peptic ulcer disease and from patients with no upper gastrointestinal abnormality was studied in order to assess its effect on a formed fibrin clot. In both groups of patients gastric juice caused a marked increase in fibrinolysis as evidenced by a shortening of the euglobulin clot lysis time. This plasmin mediated fibrinolytic activity was found to be heat labile and only present in an acid environment. Addition of tranexamic acid or sucralfate to gastric juice almost completely reversed this effect, whereas pepstatin was only partially effective. It is probable that acid dependent proteases other than pepsin are responsible for the marked fibrinolysis. The ulcer healing agent sucralfate might be useful in those patients at risk of bleeding or rebleeding from active peptic ulcer disease.     

Insulin resistance in uremia: an in vivo and in vitro study

Insulin-mediated glucose metabolism was examined in vivo and in vitro in a chronically uremic (4-week) rat model established by a 90% nephrectomy. Using the euglycemic insulin clamp technique, uremic rats demonstrated a 28% reduction (P less than .01) in total body glucose disposal compared with pair-fed controls. Suppression of hepatic glucose production by insulin was not impaired. The ability of insulin to promote glycogen synthesis by the soleus muscle in vitro was normal in uremic rats. In contrast, the ability of insulin to enhance both glycolysis and glucose oxidation by the soleus muscle was significantly reduced (P less than .01) in uremic rats. These results provide evidence that at least two intracellular metabolic defects, ie, in the glycolytic and glucose oxidative pathways, contribute to the insulin resistance of chronic uremia.     

Impact of architectural disruption on adrenocortical steroidogenesis in vitro

The adrenal cortex is an architecturally complex tissue, with cellular zonation thought to determine steroidogenesis. The impact that disruption of this tissue's architecture has on steroidogenesis in vitro, particularly adrenal androgen (AA) production, is unclear. We hypothesized that the extent of architectural disruption during tissue preparation would impact the study results. To test this hypothesis, we compared adrenocortical steroidogenesis in freshly prepared tissue slices, minces, and cell suspensions. Normal human adrenals (n = 5, three males and two females, age range 17-43 yr) were obtained at the time of organ donation. The three adrenal tissue preparations were incubated in serum-free medium with 10 microM pregnenolone substrate +/- 1 microM ACTH. The production of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, and cortisol in the media were measured by radioimmunoassay. Initial time course intubations using adrenals from a single donor generally demonstrated that minces and suspensions had a greater steroid production compared with slices. In another series of 6-hr incubations using adrenals from four donors, production of dehydroepiandrosterone sulfate was found to be quite sensitive to architectural disruption, i.e. slices less than minces less than suspensions (0.88 vs. 2.1 vs. 3.0 microg/gm tissue, respectively, P < 0.0001). Alternatively, cortisol and androstenedione production was higher in minces compared with slices or suspensions (25.6 vs. 37.7 vs. 18.7 ng/gm tissue, P < 0.0028, and 254 vs. 709 vs. 456 ng/gm tissue, P < 0.0042, respectively). Production of dehydroepiandrosterone was apparently not significantly affected by the type of tissue preparation (28.2 vs. 22.2 vs. 31.2 ng/gm tissue, P < 0.297, respectively). It is unlikely that generalized tissue disruption alone accounted for the observed differences, as the trends among tissue preparations were not consistent among steroids. We conclude that the type of tissue preparation of fresh adrenal tissue impacts significantly on steroidogenesis in vitro.     

Expression of bacterial beta-glucuronidase in human bile: an in vitro study

BACKGROUND: Bacterial beta-glucuronidase causes deconjugation of bilirubin diglucuronide resulting in the precipitation of calcium bilirubinate, which contributes to biliary sludge and stone formation. This process is attributed to enzyme activity produced by the aerobic enterobacteriaceae such as Escherichia coli and Klebsiella sp. The presence of Clostridium sp. was detected in 48 of 56 intrahepatic stones by using polymerase chain reaction techniques and cultured Clostridium perfringens from 14 of 18 unblocked biliary stents. Such bacteria are reported to produce beta-glucuronidase activity. The aim of this study was to determine the proportion of biliary bacteria isolated from pigment stones and stents that produce beta-glucuronidase and to compare the enzyme activity expressed by the different bacteria in human bile. METHODS: A total of 202 bacteria were isolated from blocked and unblocked biliary stents and pigment ductal stones recovered from patients. Of these, 61 bacteria expressed beta-glucuronidase activity in brain heart infusion broth. These 61 bacteria were subsequently grown in human bile under aerobic or anaerobic conditions to the early stationary phase and assayed for beta-glucuronidase activity by using rho-nitrophenyl beta-D glucuronide as substrate. Results were normalized and reported as units of enzyme activity per milligram protein of the bacteria. RESULTS: C. perfringens produced beta-glucuronidase enzyme activity that was 34-fold higher than that for E coli, Staphylococcus, Corynebacterium sp., Bacillus sp., Enterococcus sp., Acinetobacter sp., Streptococcus sp., and Klebsiella sp. CONCLUSION: C. perfringens with its higher enzyme activity is more important in the deconjugation of bilirubin diglucuronide than E coli and Klebsiella sp.     

The elasticity of an individual fibrin fiber in a clot

A blood clot needs to have the right degree of stiffness and plasticity to stem the flow of blood and yet be digestable by lytic enzymes so as not to form a thrombus, causing heart attacks, strokes, or pulmonary emboli, but the origin of these mechanical properties is unknown. Clots are made up of a three-dimensional network of fibrin fibers stabilized through ligation with a transglutaminase, factor XIIIa. We developed methods to measure the elastic moduli of individual fibrin fibers in fibrin clots with or without ligation, using optical tweezers for trapping beads attached to the fibers that functioned as handles to flex or stretch a fiber. Here, we report direct measurements of the microscopic mechanical properties of such a polymer. Fibers were much stiffer for stretching than for flexion, as expected from their diameter and length. Elastic moduli for individual fibers in plasma clots were 1.7 +/- 1.3 and 14.5 +/- 3.5 MPa for unligated and ligated fibers, respectively. Similar values were obtained by other independent methods, including analysis of measurements of fluctuations in bead force as a result of Brownian motion. These results provide a basis for understanding the origin of clot elasticity.     

ITF对PAF引起的肠上皮细胞骨架F-actin破坏的抑制作用

目的:探讨血小板活化因子(PAF)对肠上皮细胞骨架F-actin的影响以及肠三叶因子(ITF)对此影响的抑制作用.方法:体外培养人结肠腺癌细胞株Caco-2,分为4组.对照组:不加刺激物及干预因素;实验组:加入PA F,终浓度分别为0、50、100和200nmo1/L,作用24 h;PAF 100 nmol/L,分别作...