Abstract 804: Noninvasive Integrated SPECT/CT Molecular Imaging of Activated Factor XIII Activity in Thrombosis

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Farouc A Jaffer ◽  
Jose L Figueiredo ◽  
Gregory Wojtkiewicz ◽  
Hanwen Zhang ◽  
Purvish Patel ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Human Plasma ◽  
Half Life ◽  
Factor Xiii ◽  
Control Agent ◽  
Isotropic Resolution ◽  
Novel Approach ◽  
Imaging Strategy

Background : Activated factor XIII (FXIIIa) is a blood transglutaminase that mediates fibrinolytic resistance and is a hallmark of acute thrombi. Noninvasive molecular imaging of FXIIIa may offer a novel approach to identify acute thrombi and to gauge fibrinolytic resistance in vivo. Here we developed and validated a FXIIIa thrombosis imaging strategy using noninvasive integrated SPECT/CT. Methods: A FXIIIa-targeted peptide agent (F13) was synthesized using NQEQVSPLTLLK chelated to DOTA and then labeled with 111 InCl 3 . A control agent (C13, 111 In-NAEQVSPLTLLK) was analogously synthesized. In vitro validation of the F13 agent was performed in human plasma clots. Next, the in vivo blood-half life of F13 was determined in mice (n=4). In vivo thrombosis studies (n=15 mice) were then performed using 10% ferric chloride jugular venous thrombi aged 1 hour or 16 hours. Mice were intravenously injected with 200 μCi of F13 or C13. After 4 hours, mice underwent integrated CT angiography (72 μm isotropic resolution) and SPECT imaging (32 minute acquisition). In situ thrombi were then resected for radioactivity and weight measurements. Results : Human plasma clots incubated with F13 showed 280–740% greater counts per minute (CPM) than controls (p<0.01). F13 binding was dose-dependent and >90% inhibited by pretreatment with iodoacetamide, an alkylating agent. The blood half-life of F13 was calculated to be 16 minutes. In one hour thrombi, in vivo SPECT/CT imaging revealed strong focal F13 SPECT signal in the co-registered ipsilateral venous thrombi but not the contralateral normal jugular vein. One hour thrombi in the F13 group had 15-fold greater radioactivity than the C13 group (4.6±3.6% vs. 0.3±0.2% injected dose per gram tissue, IDGT, p<0.01). Compared to 1 hour thrombi, 16 hour old thrombi had 4-fold less F13 radioactivity (1.1%±0.1% IDGT, p<0.05). Conclusions : Blood transglutaminase FXIIIa can be noninvasively detected using a FXIIIa-sensitive and specific imaging agent for integrated SPECT/CT. The current in vivo results further validate that activated factor XIII is a hallmark of acute thrombi and declines in activity over time. This clinically translatable imaging strategy could permit visualization of FXIIIa in patients with thrombotic syndromes.

Zymogen-Like FXa Is An Effective Pro-Hemostatic To Reverse The Anticoagulant Effects Of Direct FXa Inhibitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2383-2383
Author(s):  
Nabil K Thalji ◽  
Sunita Patel-Hett ◽  
Reema Jasuja ◽  
Joachim Fruebis ◽  
Debra Pittman ◽  
...  
Keyword(s):  
Blood Loss ◽  
Human Plasma ◽  
Active Site ◽  
Half Life ◽  
Thrombin Generation ◽  
Fold Increase ◽  
Normal Human Plasma ◽  
Normal Human

Abstract Oral anticoagulants are the mainstay of treatment for prothrombotic disorders. The emerging oral factor Xa (FXa) inhibitors, which include rivaroxaban and apixaban, have been shown to be highly effective anticoagulants in several clinical scenarios, including venous thromboembolism and non-valvular atrial fibrillation. Compared to warfarin, direct FXa inhibitors have less variable pharmacokinetics, may not require routine monitoring of coagulation parameters, and have comparable to a somewhat lower bleeding risk. Despite these advantages, no approved strategy has been developed to reverse the anticoagulant effects of these drugs in the event of life-threatening bleeding or emergent need for surgery. This represents an urgent unmet clinical need. Our group has recently developed a panel of FXa mutants that are more zymogen-like than wild-type (wt)-FXa. These “zymogen-like” FXa variants have lower activity in in vitro assays compared to wt-FXa due to impaired active site maturation. Furthermore, the variants have longer plasma half-lives (>30 minutes) in vitro compared to wt-FXa (1-2 minutes) due to diminished reactivity with antithrombin III (ATIII) and tissue factor pathway inhibitor (TFPI). Remarkably however, binding to FVa rescues the activity of these zymogen-like FXa variants and as a result they are highly effective procoagulants in vivo in the setting of hemophilia (Nat. Biotech; 2011, 29:1028-33). We hypothesized that these variants could also be effective procoagulants to overcome the effects of direct FXa inhibitors. Furthermore, since direct FXa inhibitors bind the FXa active site, we expect them to compete with ATIII and TFPI for FXa binding and prolong their half-lives. We tested both of these hypotheses in in vitro coagulation studies and in vivo hemostasis models. Rivaroxaban dose-dependently inhibited thrombin generation in thrombin generation assays (TGA) when added to normal human plasma. Specifically, 500 nM rivaroxaban, the expected therapeutic steady-state plasma concentration, decreased peak thrombin generation to ∼10% of normal, and addition of 3 nM of the FXa zymogen-like variant FXaI16L restored peak thrombin generation to 105% of normal. Higher concentrations of rivaroxaban (2.5 µM) completely abrogated thrombin generation in this assay, but 10 nM FXaI16L restored thrombin generation to 72% of normal under these conditions. We compared these data to results obtained with other proposed reversal strategies. Gla-domainless, catalytically inactive FXa (GD-FXaS195A), which has been shown to reverse the effects of rivaroxaban by scavenging the inhibitor, restored thrombin generation in the presence of 500 nM rivaroxaban, but required high concentrations (1 µM; >300-fold greater than FXaI16L) to be effective. In addition, activated prothrombin complex concentrates (FEIBA), which have been shown to have some ex vivo efficacy, were ineffective under our assay conditions. In tail-clip hemostasis studies in mice, rivaroxaban dose-dependently increased blood loss, with 50 mg/kg rivaroxaban resulting in 217% of normal blood loss. Addition of FXaI16L (200 mg/kg) reduced rivaroxaban-induced blood loss to 141% of normal. To examine the effect of rivaroxaban on the half-life of FXa, we pre-incubated FXaI16L or wt-FXa with or without rivaroxaban in normal human plasma and then performed TGA experiments after various incubation times. When wt-FXa or FXaI16L were pre-incubated in plasma in the absence of rivaroxaban, their half-lives were 4.6 minutes and 1.37 hours, respectively. Remarkably, when wt-FXa or FXaI16L were incubated in plasma in the presence of 500 nM rivaroxaban, their respective half-lives were prolonged to 9.4 hours (123-fold increase) and 18.1 hours (13.2-fold increase). These results suggest that a zymogen-like FXa variant, FXaI16L, can reverse the effects of rivaroxaban in vitro and in vivo. Furthermore, FXaI16L is a bypassing agent that only requires catalytic amounts of protein, in contrast to scavengers or “true” antidotes like GD-FXaS195A that require stoichiometric concentrations. This indicates that much lower quantities of FXaI16L may be effective in vivo. We also showed that rivaroxaban dramatically prolongs the half-life of FXa in plasma, possibly by competing with ATIII and TFPI for FXa binding. This work provides a starting point for the development of a long half-life reversal strategy for the emerging FXa inhibitors. Disclosures: Patel-Hett: Pfizer: Employment. Jasuja:Pfizer: Employment. Fruebis:Pfizer: Employment. Pittman:Pfizer: Employment. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; Alnylam: Consultancy.

scholarly journals Diagnosis of the Prethrombotic State. Introduction

1977 ◽  
Author(s):  
Jan J. Sixma
Keyword(s):  
Half Life ◽  
Blood Platelets ◽  
Clotting Factors ◽  
Platelet Factor ◽  
Short Half Life ◽  
Novel Approach ◽  
Increased Risk ◽  
Coagulant Activity

The purpose of this symposium is to highlight new developments in methods that may detect patients with increased risk for arterial or venous thrombosis. Some of the techniques presented rely on the presence of changed clotting factors, fibrinogen in particular, or of released peptides. A novel approach is the use of antibodies directed specifically against complexes of inhibitors with activated factors.Blood platelets play an important role particularly in arterial thrombosis.Radio-immuno-assays have been worked out for two secretion products from platelets: platelet factor 4 and beta-thromboglobulin. Theoretically the sensitivity of these tests will be limited by the short half life of the substances. A possible useful approach is therefore the study of the properties of released platelets since it has been demonstrated that these platelets may have a normal survival. The platelet coagulant activity as predictor of thrombosis may fit in here. Other properties of released platelets such as the exposure of actin or the decreased uptake of serotonin are currently under investigation. Spontaneous aggregation in vivo or in vitro as well as a short half life of labeled platelets has been found in various thromboembolic diseases.The predictive value of various tests should be evaluated in prospective studies. An example of such an approach is given in the paper that concludes the symposium.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

Presence and Possible Origin of ɛ(γ-Glutamyl)Lysine Isodipeptide in Human Plasma

1992 ◽  
Vol 67 (01) ◽  
pp. 060-062 ◽  
Author(s):  
J Harsfalvi ◽  
E Tarcsa ◽  
M Udvardy ◽  
G Zajka ◽  
T Szarvas ◽  
...  
Keyword(s):  
Human Plasma ◽  
Fibrinolytic Therapy ◽  
Normal Human Plasma ◽  
Normal Human ◽  
Hplc Technique ◽  
Significant Change

Summaryɛ(γ-glutamyl)lysine isodipeptide has been detected in normal human plasma by a sensitive HPLC technique in a concentration of 1.9-3.6 μmol/1. Incubation of in vitro clotted plasma at 37° C for 12 h resulted in an increased amount of isodipeptide, and there was no further significant change when streptokinase was also present. Increased in vivo isodipeptide concentrations were also observed in hypercoagulable states and during fibrinolytic therapy.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

scholarly journals HSP90-dependent PUS7 overexpression facilitates the metastasis of colorectal cancer cells by regulating LASP1 abundance

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Dan Song ◽  
Ming Guo ◽  
Shuai Xu ◽  
Xiaotian Song ◽  
Bin Bai ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Intellectual Development ◽  
Molecular Mechanisms ◽  
Hsp90 Inhibitor ◽  
Pseudouridine Synthase ◽  
Novel Approach ◽  
Cell Metastasis ◽  
Underlying Mechanisms

Abstract Background Pseudouridine synthase (PUS) 7 is a member of the PUS family that catalyses pseudouridine formation. It has been shown to be involved in intellectual development and haematological malignancies. Nevertheless, the role and the underlying molecular mechanisms of PUS7 in solid tumours, such as colorectal cancer (CRC), remain unexplored. This study elucidated, for the first time, the role of PUS7 in CRC cell metastasis and the underlying mechanisms. Methods We conducted immunohistochemistry, qPCR, and western blotting to quantify the expression of PUS7 in CRC tissues as well as cell lines. Besides, diverse in vivo and in vitro functional tests were employed to establish the function of PUS7 in CRC. RNA-seq and proteome profiling analysis were also applied to identify the targets of PUS7. PUS7-interacting proteins were further uncovered using immunoprecipitation and mass spectrometry. Results Overexpression of PUS7 was observed in CRC tissues and was linked to advanced clinical stages and shorter overall survival. PUS7 silencing effectively repressed CRC cell metastasis, while its upregulation promoted metastasis, independently of the PUS7 catalytic activity. LASP1 was identified as a downstream effector of PUS7. Forced LASP1 expression abolished the metastasis suppression triggered by PUS7 silencing. Furthermore, HSP90 was identified as a client protein of PUS7, associated with the increased PUS7 abundance in CRC. NMS-E973, a specific HSP90 inhibitor, also showed higher anti-metastatic activity when combined with PUS7 repression. Importantly, in line with these results, in human CRC tissues, the expression of PUS7 was positively linked to the expression of HSP90 and LASP1, and patients co-expressing HSP90/PUS7/LASP1 showed a worse prognosis. Conclusions The HSP90-dependent PUS7 upregulation promotes CRC cell metastasis via the regulation of LASP1. Thus, targeting the HSP90/PUS7/LASP1 axis may be a novel approach for the treatment of CRC.

scholarly journals Plasma cells shape the mesenchymal identity of ovarian cancers through transfer of exosome-derived microRNAs

2021 ◽  
Vol 7 (9) ◽  
pp. eabb0737
Author(s):  
Zhengnan Yang ◽  
Wei Wang ◽  
Linjie Zhao ◽  
Xin Wang ◽  
Ryan C. Gimple ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Ovarian Cancer Cells ◽  
Mesenchymal Phenotype ◽  
Ovarian Cancers ◽  
Novel Approach

Ovarian cancer represents a highly lethal disease that poses a substantial burden for females, with four main molecular subtypes carrying distinct clinical outcomes. Here, we demonstrated that plasma cells, a subset of antibody-producing B cells, were enriched in the mesenchymal subtype of high-grade serous ovarian cancers (HGSCs). Plasma cell abundance correlated with the density of mesenchymal cells in clinical specimens of HGSCs. Coculture of nonmesenchymal ovarian cancer cells and plasma cells induced a mesenchymal phenotype of tumor cells in vitro and in vivo. Phenotypic switch was mediated by the transfer of plasma cell–derived exosomes containing miR-330-3p into nonmesenchymal ovarian cancer cells. Exosome-derived miR-330-3p increased expression of junctional adhesion molecule B in a noncanonical fashion. Depletion of plasma cells by bortezomib reversed the mesenchymal characteristics of ovarian cancer and inhibited in vivo tumor growth. Collectively, our work suggests targeting plasma cells may be a novel approach for ovarian cancer therapy.

scholarly journals THER-05. GENETICALLY STABLE POLIOVIRUS VECTOR CARRYING H3.3K27M ANTIGEN FOR TREATMENT OF DIFFUSE MIDLINE GLIOMA BY INTRAMUSCULAR INJECTION

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii472-iii472
Author(s):  
Mubeen Mosaheb ◽  
Daniel Landi ◽  
Elena Dobrikova ◽  
Michael Brown ◽  
Yuanfan Yang ◽  
...  
Keyword(s):  
T Cell ◽  
Antigen Presentation ◽  
Intramuscular Injection ◽  
Cd8 T Cell ◽  
Cytokine Profiles ◽  
Cell Immunity ◽  
Novel Approach ◽  
Diffuse Midline Glioma

Abstract BACKGROUND H3 K27M-mutant diffuse midline glioma (DMG) is invariably lethal. Viruses naturally engage innate immunity, induce antigen presentation, and mediate CD8 T cell priming against foreign antigens. Polioviruses, in particular, are uniquely tropic for dendritic cells (DC) and potently activate DC, inducing Th1-dominant cytokine profiles, CD8 T cell immunity, and enhanced epitope presentation. Thus, poliovirus is ideally suited for vectored delivery of signature tumor neoantigens, e.g. the H3 K27M feature of DMG. However, poliovirus vector design is inherently limited by genetic instability and the underlying neuropathogenicity of poliovirus. METHODS We created a genetically stable, polio:rhinovirus chimera vector devoid of neuropathogenicity and modified for stable expression of the HLA-A2 restricted H3.3 K27M antigen (RIPO (H3.3)). RESULTS RIPO(H3.3) infects, activates, and induces H3.3K27M antigen presentation in DCs in vitro. Given intramuscularly in vivo, RIPO(H3.3) recruits and activates DCs with Th1-dominant cytokine profiles, efficiently primes H3.3K27M-specific CD8 T cells, induces antigen-specific CD8 T cell migration to the tumor site, delays tumor growth, and enhances survival in murine tumor models. CONCLUSION This novel approach leverages the unique ability of polioviruses to activate DCs while simultaneously introducing the H3.3 K27M antigen. In this way, DCs are activated optimally in situ, while being simultaneously infected to express/present tumor antigen. RIPO(H3.3), given by intramuscular injection, will be evaluated in a clinical trial for children with H3 K27M-mutant diffuse midline glioma.

Sign in / Sign up

Export Citation Format

Share Document