The Fibrinolytic Potential of the Normal Primate following the Generation of Thrombin In Vivo

1990 ◽  
Vol 63 (03) ◽  
pp. 476-481 ◽  
Author(s):  
Alan R Giles ◽  
Michael E Nesheim ◽  
Steven W Herring ◽  
Hugh Hoogendoorn ◽  
David C Stump ◽  
...  
Keyword(s):  
Time Course ◽  
Factor Xa ◽  
Lipid Vesicles ◽  
Experimental Period ◽  
In Vivo Studies ◽  
Full Time ◽  
Primate Model ◽  
Fibrinolytic Potential

SummaryParameters of the fibrinolytic system were studied in a primate model where the generation of thrombin was promoted in vivo. The procoagulant stimulus used was a combination of human factor Xa in combination with phosphatidylcholine/phos-phatidylserine lipid vesicles (PCPS) as the source of coagulant active phospholipid. The dosage of each component was formulated to provide a gradation of thrombin generating potential assessed prior to in vivo study in an in vitro clotting assay. These ranged from 25.25 - 36.60 pMole/kg (factor Xa) and 18.85 - 56.30 nMole/kg (PCPS). In each case, the ratio of the dose of factor Xa/PCPS was maintained at 0.65 (pMole factor Xa/ nMole PCPS). Individual dosage combinations producing recalcification clotting times in vitro of 15, 20, 25 and 30 s were used in detailed in vivo studies. Previous studies in dogs had confirmed the thrombin generating potential of factor Xa/PCPS infusions and demonstrated an associated activation ot protein C and increased fibrinolytic activity. This has now been extensively characterized in the chimpanzee as follows: 10 min after the infusion of the highest dose (36.6 pMole factor Xa/56.3 nMole PCPS kg bodyweight), the level of circulating t-PA had risen to 900 ng/ml (antigen), 885 IU/ml (functional). Dosage was observed with the lowest dose of 12.25 pMole factor Xa and 18.85 nMole PCPS being associated with relatively minor increases in circulating t-PA activity. There were no changes in u-PA at any dosage during the full time course of the experimental period (90 min). Plasminogen activation was also apparent with alpha-2 antiplasmin levels falling to 30 - 40% of pre-infusion levels at the highest dosages. There was also a significant consumption of fibrinogen and evidence of active fibrinolysis manifested by major increases in the levels of FDP, D-dimer and B-beta 1-42. The data strongly suggested that this was predominantly fibrinolysis rather than fibrinogenolysis and that the fibrinolytic response observed resulted from a major release of t-PA from available stores consequent to thrombin generation and presumably subsequent fibrin generation. These data illustrate the enormous fibrinolytic potential of the intact normal primate and may provide a model for study of the mechanism(s) by which the regulation of t-PA availability can be up- or down-regulated in health and disease.

scholarly journals In vitro and in vivo studies of the novel antithrombotic agent BAY 59-7939-an oral, direct Factor Xa inhibitor

2005 ◽  
Vol 3 (3) ◽  
pp. 514-521 ◽  
Author(s):  
E. PERZBORN ◽  
J. STRASSBURGER ◽  
A. WILMEN ◽  
J. POHLMANN ◽  
S. ROEHRIG ◽  
...  
Keyword(s):  
Factor Xa ◽  
In Vivo Studies ◽  
Factor Xa Inhibitor ◽  
The Novel ◽  
Direct Factor ◽  
Antithrombotic Agent

Trajectory-Based Tissue Engineering for Cartilage Repair: Correlation Between Maturation Rate and Integration Capacity

2013 ◽  
Author(s):  
Matthew B. Fisher ◽  
Nicole Söegaard ◽  
David R. Steinberg ◽  
Robert L. Mauck
Keyword(s):  
Tissue Engineering ◽  
Time Course ◽  
Biomechanical Properties ◽  
Time Dependent ◽  
In Vivo Studies ◽  
Surgical Approaches ◽  
General Hypothesis ◽  
Vitro Assay

Given the limitations of current surgical approaches to treat articular cartilage injuries, tissue engineering (TE) approaches have been aggressively pursued over the past two decades. Although biochemical and biomechanical properties on the order of the native tissue have been achieved (1–5), several in-vitro and in-vivo studies indicate that increased tissue maturity may limit the ability of engineered constructs to remodel and integrate with surrounding cartilage, although results are highly variable (2, 6–8). Thus, “static” measures of construct maturity (e.g. compressive modulus) upon implantation may not be the best indicators of in-vivo success, which likely requires implanted TE constructs to mature, remodel, and integrate with the host over time to achieve optimal results. We recently introduced the concept of “trajectory-based” tissue engineering (TB-TE), which is based on the general hypothesis that time-dependent increases in construct maturation in-vitro prior to implantation (i.e. positive rates) may provide a better predictor of in-vivo success (9). As a first step in evaluating this concept, in the current study we hypothesized that time-dependent increases in equilibrium modulus (a metric of growth) would be correlated to ability of constructs to integrate to cartilage using an in-vitro assay. To test this hypothesis, the current objective was to determine and model the time course of maturation of TE constructs during in-vitro culture and to assess the ability of these constructs to integrate to cartilage at various points during their maturation.

scholarly journals Comparative Studies on the Anticoagulant Profile of Branded Enoxaparin and a New Biosimilar Version

2020 ◽  
Vol 26 ◽  
pp. 107602962096082
Author(s):  
Dalia Qneibi ◽  
Eduardo Ramacciotti ◽  
Ariane Scarlatelli Macedo ◽  
Roberto Augusto Caffaro ◽  
Leandro Barile Agati ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Thrombin Generation ◽  
Area Under The Curve ◽  
Factor Xa ◽  
In Vivo Studies ◽  
In Vitro Tests ◽  
Thrombin Time

Low molecular weight heparins (LMWH) represent depolymerized heparin prepared by various methods that exhibit differential, biochemical and pharmacological profiles. Enoxaparin is prepared by benzylation followed by alkaline depolymerization of porcine heparin. Upon the expiration of its patent, several biosimilar versions of enoxaparin have become available. Heparinox (Sodic enoxaparine; Cristália Produtos Químicos Farmacêuticos LTDA, Sao Paulo, Brazil) is a new biosimilar form of enoxaparin. We assessed the molecular weight and the biochemical profile of Heparinox and compared its properties to the original branded enoxaparin (Lovenox; Sanofi, Paris, France). Clotting profiles compared included activated clotting time, activated partial thromboplastin time (aPTT), and thrombin time (TT). Anti-protease assays included anti-factor Xa and anti-factor IIa activities. Thrombin generation was measured using a calibrated automated thrombogram and thrombokinetic profile included peak thrombin, lag time and area under the curve. USP potency was determined using commercially available assay kits. Molecular weight profiling was determined using high performance liquid chromatography. We determined that Heparinox and Lovenox were comparable in their molecular weight profile. Th anticoagulant profile of the branded and biosimilar version were also similar in the clot based aPTT and TT. Similarly, the anti-Xa and anti-IIa activities were comparable in the products. No differences were noted in the thrombin generation inhibitory profile of the branded and biosimilar versions of enoxaparin. Our studies suggest that Heparinox is bioequivalent to the original branded enoxaparin based upon in vitro tests however will require further in vivo studies in animal models and humans to determine their clinical bioequivalence.

Heparin Therapy In Dispholidus Typus Envenomation: An Experimental Study

1981 ◽  
Author(s):  
B A Bradlow ◽  
P M Atkinson ◽  
M Rebello ◽  
M C Gaillard
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Therapy ◽  
In Vivo Studies ◽  
Intrinsic Pathway ◽  
Heparin Resistance ◽  
Very High ◽  
Direct Activator

The coagulant action of Dispholidus typus venom was relatively resistant to inhibition by heparin in vitro. Heparin concentrations that inhibited coagulation due to either intrinsic pathway or Russell’s viper venom activation had little effect on coagulation due to D. Typus venom. At very high heparin to venom ratios, similar to ratios attainable in vivo this resistance could be overcome. The resistance could not be attributed to an abnormal thrombin produced by the venom since the thrombin produced from purified prothrombin by venom action reacted similarly to the thrombin produced by Factor Xa activation with purified antithrombin III. Thrombin produced from whole plasma by venom action also reacted similarly to physiological thrombin with antithrombin III in a crossed immunoelectrophoresis system. Incubation of venom with heparin and with antithrombin III did not alter the activities of these inhibitors. The heparin resistance may therefore be due to the fact that the venom is a direct activator of prothrombin. In vivo studies in rabbits indicated that heparin administered simultaneously with venom delayed the onset and reduced the severity of disseminated intravascular coagulation. Heparin administered later was much less effective. Early heparin therapy may be of value in human victims when specific antivenom is not available.

scholarly journals The coagulant-active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex (Factor IX) concentrates in rabbits

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 401-407 ◽  
Author(s):  
AR Giles ◽  
ME Nesheim ◽  
H Hoogendoorn ◽  
PB Tracy ◽  
KG Mann
Keyword(s):  
Factor Xa ◽  
Lipid Vesicles ◽  
Factor Ix ◽  
Major Determinant ◽  
Phospholipid Content ◽  
Clotting Factors ◽  
Prothrombin Complex Concentrates ◽  
Vitro Assay

In vitro evaluation of prothrombin complex concentrates in a thrombin generation assay, using DAPA and purified components of the prothrombinase complex, demonstrated significant levels of coagulant- active “phospholipid replacing” activity. Quantification of this activity showed a significant correlation (r = 0.8747, p less than 0.01) with thrombogenicity measured in vivo in a stasis model in rabbits. Extracted lipid material retained full phospholipid replacing activity in the vitro assay. Thin-layer chromatographic characterization confirmed the presence of phospholipids with known coagulant activity in vitro. In vivo, the extracted material was nonthrombogenic but augmented the thrombogenicity of purified factor Xa. Substitution of a synthetic coagulant-active phospholipid (phosphatidylcholine-phosphatidylserine lipid vesicles) for the extracted phospholipid produced a similar augmentation of a factor-Xa- induced thrombogenicity in vivo. It is concluded that the coagulant- active phospholipid content of prothrombin complex concentrates is a major determinant of thrombogenicity but requires the presence of activated clotting factors for its expression in vivo.

Coagulation cascade proteases and tissue fibrosis

2002 ◽  
Vol 30 (2) ◽  
pp. 194-200 ◽  
Author(s):  
R. C. Chambers ◽  
G. J. Laurent
Keyword(s):  
Growth Factor ◽  
Factor Xa ◽  
Tissue Growth ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Proteolytic Activation ◽  
Surface Receptors ◽  
Relative Contribution

Fibrotic disorders of the liver, kidney and lung are associated with excessive deposition of extracellular matrix proteins and ongoing coagulation-cascade activity. In addition to their critical roles in blood coagulation, thrombin and the immediate upstream coagulation proteases, Factors Xa and VIIa, influence numerous cellular responses that may play critical roles in subsequent inflammatory and tissue repair processes in vascular and extra-vascular compartments. The cellular effects of these proteases are mediated via proteolytic activation of a novel family of cell-surface receptors, the protease-activated receptors (PAR-1, −2, −3 and −4). Although thrombin is capable of activating PAR-1, −3 and −4, there is accumulating in vitro evidence that the profibrotic effects of thrombin are predominantly mediated via PAR-1. Factor Xa is capable of activating PAR-1 and PAR-2, but its mitogenic effects for fibroblasts are similarly mediated via PAR-1. These proteases do not exert their profibrotic effects directly, but act via the induction of potent fibrogenic mediators, such as platelet-derived growth factor and connective tissue growth factor. In vivo studies using proteolytic inhibitors, PAR-1 antagonists and PAR-1-deficient mice have provided evidence that coagulation proteases play a key role in tissue inflammation and in a number of vascular pathologies associated with hyperproliferation of smooth muscle cells. More recently, coagulation proteases have also been shown to play a role in the pathogenesis of fibrosis but the relative contribution of their cellular versus their procoagulant effects awaits urgent evaluation in vivo. These studies will be informative in determining the potential application of PAR-1 antagonists as antifibrotic agents.

scholarly journals Temporal analysis of mRNA expression profiles in Orientia infected C3HeB/FeJ mouse

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Chien-Chung Chao ◽  
Ruoting Yang ◽  
Zhiwen Zhang ◽  
Tatyana Belinskaya ◽  
Chye-Teik Chan ◽  
...  
Keyword(s):  
Post Infection ◽  
Mammalian Cells ◽  
Time Course ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Global Changes

Abstract Background Scrub typhus causes up to 35% mortality if left untreated. One billion people living in the endemic regions are at risk. In spite of its heavy disease burden in some of the most populated areas in the world, there is no vaccine available. Although the disease can be effectively treated by proper antibiotics, timely and accurate diagnosis remains a challenge. Orientia tsutsugamushi infects a variety of mammalian cells in vitro and replicates in the cytoplasm of the infected cells. Microarray analysis has been used extensively to study host-pathogen interactions in in vitro models to understand pathogenesis. However there is a lack of in vivo studies. Results In this study, C3HeB/FeJ (C3H) mice were infected by O. tsutsugamushi via the intraperitoneal route and monitored gene expression at 10 different time points post infection. We observed two distinct types of expression profiles in the genes that we analyzed. There are two valleys (4–18 h and 2–4 days) with low number of differentially expressed genes (DEG) with three peaks with high number of DEG at 2 h, 1-day and 7-day post infection. Further analysis revealed that pathways like complement and coagulation cascade, and blood clotting cascade pathways showed significant global changes throughout entire time course. Real time quantitative Polymerase Chain Reaction (RT-qPCR) confirmed the change of expression for genes involved in complement and coagulation cascade. These results suggested dynamic regulation of the complement and coagulation cascades throughout most of the time post infection while some other specific pathways, such as fatty acid metabolism and tryptophan metabolism, are turned on or off at certain times post infection. Conclusions The findings highlight the complex interconnection among all different biological pathways. It is conceivable that specific pathways such as cell growth control and cell development in the host are affected by Orientia in the initial phase of infection for Orientia to grow intracellularly. Once Orientia is replicating successfully inside the host as infection progresses, the infection could activate pathways involved in cellular immune responses to defend for host cell survival and try to eliminate the pathogen.

Photoresponses of human rods in vivo derived from paired-flash electroretinograms

1997 ◽  
Vol 14 (1) ◽  
pp. 73-82 ◽  
Author(s):  
David R. Pepperberg ◽  
David G. Birch ◽  
Donald C. Hood
Keyword(s):  
Time Course ◽  
Light Adaptation ◽  
Impulse Response Function ◽  
Test Flash ◽  
Rod Photoreceptor ◽  
Full Time ◽  
Flash Intensity ◽  
Rod Response

AbstractIn the human eye, domination of the electroretinogram (ERG) by the b−wave and other postreceptor components ordinarily obscures all but the first few milliseconds of the rod photoreceptor response to a stimulating flash. However, recovery of the rod response after a bright test flash can be analyzed using a paired-flash paradigm in which the test flash, presented at time zero, is followed at time t by a bright probe flash that rapidly saturates the rods (Birch et al., 1995). In ERG experiments on normal subjects, the hypothesis that a similar method can be used to obtain the full time course of the rod response to test flashes of subsaturating intensity was tested. Rod-only responses to probe flashes presented at varying times t after the test flash were used to derive a family of amplitudes A(t) that represented the putative rod response to the test flash. These rod-only responses to the probe flash were obtained by computational subtraction of the cone-mediated component of each probe flash response. With relatively weak test flashes (11–15 scot-td-s), the time course of the rod response to the test flash derived in this manner was consistent with a four-stage impulse response function of time-to-peak ≃170 ms. A(170), the amplitude of the derived response at 170 ms, increased with test flash intensity (Itest) to a maximum value Amo and exhibited a dependence on Itest given approximately by the relation, A(170)/Amo = 1 - exp(-kItest), where k = 0.092 (scot-td-s)−1. In steady background light, the falling (i.e. recovery) phase of the derived response began earlier, and the sensitivity parameter k was reduced several-fold from its dark-adapted value. As the sensitivity, kinetics, and light-adaptation properties of the derived response correspond closely with those of photocurrent flash responses previously obtained from isolated rods in vitro, it was concluded that the response derived here from the human ERG approximates the course of the massed in vivo rod response to a test flash.

Studies in Man and Experimental Animals of a Low Molecular Weight Heparin Fraction

1981 ◽  
Vol 45 (03) ◽  
pp. 214-218 ◽  
Author(s):  
D P Thomas ◽  
R E Merton ◽  
W E Lewis ◽  
T W Barrowcliffe
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Specific Activity ◽  
Ex Vivo ◽  
High Specific Activity ◽  
Factor Xa ◽  
In Vivo Studies ◽  
Low Molecular Weight

SummaryIn vitro and in vivo studies were carried out on a commercially prepared low molecular weight heparin fraction. By APTT assay the fraction had a specific activity of half that of unfractionated mucosal heparin, yet retained full potency by anti-Xa assay (both clotting and chromogenic substrate). When administered intravenously to human volunteers, the anti-Xa/APTT ratio remained the same as it was in vitro. However, after subcutaneous injection, the ratio increased and anti-Xa activity could not be fully neutralized ex vivo by PF4. The fraction was as effective as unfractionated heparin in preventing experimental serum-induced thrombosis, suggesting that a heparin fraction with high specific activity by anti-Factor Xa assay compared to APTT activity may be an effective drug for the prophylaxis of venous thrombosis.

scholarly journals Utilizing the Combination of Binding Kinetics and Micro-Pharmacokinetics Link in Vitro α-Glucosidase Inhibition to in Vivo Target Occupancy

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 493
Author(s):  
Wang ◽  
Ji ◽  
Li ◽  
Wang ◽  
Gong ◽  
...  
Keyword(s):  
Simulation Model ◽  
Rate Constants ◽  
Inhibitory Activity ◽  
Time Course ◽  
Dynamic Change ◽  
Binding Kinetics ◽  
In Vivo Studies ◽  
Epicatechin Gallate

Many compounds with good inhibitory activity (i.e., high affinity) within in vitro experiments failed in vivo studies due to a lack of efficacy from limited target occupancy (TO) in the drug discovery process. Recently, it was found that rate constants of the formation and dissociation of the binary drug-target complex, rather than affinity, often govern in vivo efficacy. Therefore, the binding kinetics (BK) properties of compound-target interaction are emerging as a pivotal parameter. However, it is obvious that BK rate constants of the compound against target would not be directly linked to the in vivo TO unless the compound concentration in the target vicinity at any time point (TPK) can be evaluated. Here, we developed a novel simulation model to quantitate the dynamic change of target engagement over time in rat with a combined use of BK and TPK features of Epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) on the basis of α-glucosidase (AGH). Analysis of the results displayed that the percent of maximum AGH occupancies by the ECG were varied significantly from 48.9 to 95.3% and by the EGCG slightly from 96 to 99.8%; that the time course of above 70% engagement by ECG spanned a range from 0 to 0.64 h and by EGCG a range of 1.5 to 8.9 h in four different intestinal segments of the rat. It was clearly analyzed how each parameter in the simulation model effected on the in vivo the AGH engagement by ECG and EGCG. Our results provide a novel approach for assessing the potential inhibitory activity of the compounds against AGH.

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