81 research outputs found
Isolation and characterization of an antifactor V antibody causing activated protein C resistance from a patient with severe thrombotic manifestations
No full textBlood. 2002 Jun 1;99(11):3985-92.
Isolation and characterization of an antifactor V antibody causing activated
protein C resistance from a patient with severe thrombotic manifestations.
Kalafatis M, Simioni P, Tormene D, Beck DO, Luni S, Girolami A.
Department of Chemistry, Cleveland State University, The Cleveland Clinic
Foundation, Cleveland, OH 44115, USA. [email protected]
A 44-year-old woman with a history of severe thrombotic manifestations presented
with a markedly reduced activated protein C-sensitivity ratio (APC-SR). DNA
sequencing of and around the regions encoding the APC cleavage sites in the
factor Va molecule excluded the presence of the factor VLeiden mutation and of
other known genetic mutations. No antiphospholipid antibodies were present in the
patient's plasma and both prothrombin time and activated partial thromboplastin
time were normal. The total immunoglobulin fraction was isolated from the
patient's plasma and found to induce severe APC resistance when added to normal
plasma and to factor V-deficient plasma supplemented with increasing
concentrations of factor V. Immunoblotting and immunoprecipitation experiments
with the total immunoglobulin fraction purified from the patient's plasma
demonstrated that the antibody recognizes factor V, is polyclonal, and has
conformational epitopes on the entire factor V molecule (heavy and light chains,
and B region). Thus, the immunoglobulin fraction interferes with the
anticoagulant pathway involving factor V. The inhibitor was isolated by
sequential affinity chromatography on protein G-Sepharose and factor V-Sepharose.
The isolated immunoglobulin fraction inhibited factor Va inactivation by APC
because of impaired cleavage at Arg306 and Arg506 of the heavy chain of the
cofactor. The isolated immunoglobulin fraction was also found to inhibit the
cofactor effect of factor V for the inactivation of factor VIII by the
APC/protein S complex. Our data provide for the first time the demonstration of
an antifactor V antibody not related to the presence of antiphospholipid
antibodies, which is responsible for thrombotic rather than hemorrhagic symptoms.
PMID: 12010798 [PubMed - indexed for MEDLINE
Case study of physiotherapy treatment of a patient with Bell's palsy
No full textThesis title: Physiotherapy treatment of a patient with Bell's palsy Author: Dominic Kalafatis Work placement: Ustředni Vojenská Nemocnice in Prague Summary In this bachelor thesis, which was written and composed by myself, it is divided in two parts, the general part and the special part. The general part mainly is the theoretical part in which it is included the whole anatomy of the face, the facial and neck muscles. The cranial nerves and specifically the facial nerve, the seventh cranial nerve. Kinesiology of the facial muscles for any facial expression. All these components will be described in this part. Secondly, the special part which is the most important part of the whole bachelor thesis, is the part of the case of my patient with Bell's palsy/ Facial paresis. There will be the whole anamnesis, the initial kinesiologic examinations, therapy sessions, the final kinesiologic examinations in which there are also the improvements of my patient and finally the evaluation of the therapies. The last part of my bachelor thesis it is composed from my bibliography which contains the literature which I used to write the general part of my bachelor thesis. The list of figures and tables from the whole thesis. The abbreviations and finally the last thing is the ethics committee. Key words: Facial..
Abstract 2117: TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma
No full textAbstract
Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), the optimized form of the endogenous death ligand TRAIL, shows therapeutic potential for cancer due to its ability to induce apoptosis in cancer cells independent of p53, while exhibiting minimal toxicity to normal cells. Despite this, a majority of breast cancers display resistance to rhTRAIL treatment due to up-regulation of pro-apoptotic proteins, down-regulation of anti-apoptotic proteins, and/or up-regulation of death receptors (DR) 4 and 5. To overcome rhTRAIL resistance, natural compounds have been investigated as sensitizing agents. This study considered the application of the naturally occurring flavonol Quercetin (Q). Q has been shown to have the ability to up-regulate DR5 and down-regulate anti-apoptotic proteins in cancer cells, and thereby, making Q a favorable choice to be employed as a sensitizing agent. The intention of this study was to ascertain the capacity of Q to sensitize rhTRAIL-resistant triple negative breast cancer BT-20 cells and hormone-dependent breast cancer MCF-7 cells to rhTRAIL-induced apoptosis and elucidate the underlying mechanism for Q’s sensitization. Q demonstrated the ability to intensify rhTRAIL’s pro-apoptotic effects in the breast cancer BT-20 and MCF-7 cell lines as detected through Annexin V/PI assays followed by FACS analysis. In comparison to single agent treatments, the cotreatment of Q and rhTRAIL enhanced the induction of the extrinsic pathway of apoptosis as marked by PARP cleavage (a hallmark of apoptosis), activation of caspase 8, and activation of the executioner caspases 3 and 7. The mechanism for Q’s augmentation in breast cancer was determined to be through the down-regulation of c-FLIPL (caspase 8 inhibitor) in a dose-dependent manner. Furthermore, Q promoted the ubiquitination of c-FLIPL facilitating the proteasome-mediated degradation of c-FLIPL in breast cancer. An additional mechanism for Q’s sensitization was displayed in breast cancer BT-20 cells. Q up-regulated DR5 membrane and protein expression in breast cancer BT-20 cells in a dose-dependent manner. RT-PCR analysis revealed that Q’s influence on DR5 expression occurred at the transcriptional level in those breast cancer cells. Thus, these data suggest that the cotreatment of Q and rhTRAIL possesses the therapeutic potential to be an effective anti-breast carcinoma regimen.
Citation Format: Jasmine M. Manouchehri, Michael Kalafatis. TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2117. doi:10.1158/1538-7445.AM2017-2117</jats:p
Phenotype and Genotype Expression in Pseudohomozygous Factor V <sup>LEIDEN</sup>
No full text
Abstract
—The presence of a DNA mutation is frequently used to define a disease or a risk state. Because DNA typing has become easy and convenient in contrast to protein characterization, it is generally assumed that a mutation if present (or not) at the DNA level will be also present (or not) in the corresponding protein. However, discrepancies between phenotype and genotype can occur. A point mutation in the coagulation factor V gene (G
1691
→A, resulting in an Arg
506
→Gln amino acid substitution in the factor V molecule [factor V
LEIDEN
], leading to activated protein C resistance) is the most common genetic risk factor for familial thrombophilia. A pseudohomozygous factor V
LEIDEN
phenotype would occur if a heterozygous individual for factor V
LEIDEN
also did not express the “normal” (non-Leiden) factor V allele. However, to date, no data have been available to confirm the presence of only the factor V
LEIDEN
form in the plasma of these individuals. Platelet mRNA from 2 presumed pseudohomozygous patients and their family members was isolated, the amplified partial cDNAs were sequenced or restricted, and the allelic bands were quantified. Both patients were found to be heterozygous for the G
1691
→A substitution at both the DNA and mRNA levels. The presence of either the normal or mutated form of factor V in the patients’ plasmas was investigated using a monoclonal antibody to factor V that recognizes an epitope located between residues 307 and 506 of the factor Va heavy chain. No normal factor V could be detected in the plasmas of the 2 propositi. The present data demonstrate absence of a correlation between genotype at position 1691 (at the DNA and mRNA levels) and the corresponding phenotype data found in the plasmas of patients with pseudohomozygous factor V
LEIDEN
. Overall, these data suggest the existence of heterogeneous genetic “lesions,” which interfere with factor V expression, processing, secretion, and/or stability. Because the presence of the factor V
LEIDEN
molecule in plasma is directly related to pathology, identification and quantification of the circulating forms of factor V in plasma may be required for the diagnosis of individuals with activated protein C resistance.
</jats:p
Identification des domaines fonctionnels du facteur Willebrand humain a l'aide d'anticorps monoclonaux
No full textCNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc
Coagulation Factor V: A Plethora of Anticoagulant Molecules
No full textPURPOSE OF REVIEW: Thrombin is necessary for survival and is produced after activation of prothrombin by prothrombinase at the site of a vascular injury. While the enzyme component of prothrombinase alone, factor Xa, bound to a membrane surface can activate prothrombin, incorporation of the cofactor molecule, factor Va, into prothrombinase results in a five orders of magnitude increase in the catalytic efficiency of factor Xa that provides the physiologic pathway for thrombin generation. While the kinetic constants and the identity of peptide bonds cleaved in prothrombin to generate alpha-thrombin have been long established, the peptidyl portions of the factor Va molecule responsible for its interactions with factor Xa, prothrombin, and the lipid surface are still the subject of intense investigation. In this review, we summarize the current state of knowledge with respect to the interactions of the factor Va molecule with the various components of prothrombinase. RECENT FINDINGS: Binding sites for factor Xa have been identified on both the heavy and light chains of factor Va. Two amino acid regions that interact with factor Xa have been delineated on the heavy chain of the cofactor. It has also been demonstrated that the carboxyl-terminal portion of the heavy chain of factor Va contains hirudin-like motifs and appears to be responsible for the interaction of factor Va with prothrombin. This region of the molecule is important for procofactor activation by thrombin as well as cofactor function. Finally, the membrane-binding site of factor Va is contributed by several elements of the light chain and involves both electrostatic and hydrophobic interactions. SUMMARY: The absence or dysfunction of factor Va leads to hemorrhagic diseases while prolonged existence of the active cofactor species is associated with thrombosis. Thus, modulation of the incorporation of factor Va into prothrombinase in vivo by using synthetic peptides that have the potential to impair factor Va binding to any of the components of prothrombinase, will allow for control of the rate of thrombin generation at the site of vascular damage. As a consequence, a systematic definition of the regions of factor Va governing its incorporation within prothrombinase will provide the scaffold for the synthesis of potent anticoagulant molecules that could modulate thrombin formation and suppress excessive clotting in thrombotic individuals
Amino Acids Glu323, Tyr324, Glu330, and Val331 of Factor VA Heavy Chain Are Essential for Expression of Cofactor Activity
No full textWe have recently demonstrated that amino acid region 323-331 of factor Va heavy chain (9 amino acids, AP4\u27) contains a binding site for factor Xa (Kalafatis, M., and Beck, D. O. (2002) Biochemistry 41, 12715-12728). To ascertain which amino acids within this region are important for the effector and receptor properties of the cofactor with respect to factor Xa, we have synthesized three overlapping peptides (5 amino acids each) spanning the amino acid region 323-331 and tested them for their effect on prothrombinase complex assembly and function. Peptide containing amino acids 323EYFIA327 alone was found to increase the catalytic efficiency of factor Xa but had no effect on the fluorescent anisotropy of active site-labeled factor Xa (human factor Xa labeled in the active site with Oregon Green 488; [OG488]-EGR-hXa). In contrast, peptide containing the sequence 327AAEEV331 was found to interact with [OG488]-EGR-hXa with half-maximal saturation reached at approximately 150 microm, but it was unable to produce a cofactor effect on factor Xa. Peptide 325FIAAE329 inhibited prothrombinase activity and was able to partially decrease the fluorescent anisotropy of [OG488]-EGR-hXa but could not increase the catalytic efficiency of factor Xa with respect to prothrombin. A control peptide with the sequence FFFIA did not increase the catalytic efficiency of factor Xa, whereas a peptide with the sequence AAEMI was impaired in its capability to interact with [OG488]-EGR-hXa. Two mutant recombinant factor Va molecules (Glu323 --\u3e Phe/Tyr324 --\u3e Phe, factor VaFF; Glu330 --\u3e Met/Val331 --\u3e Ile, factor VaMI) showed impaired cofactor activity when used at limiting cofactor concentration, whereas the quadruple mutant (Glu323 --\u3e Phe/Tyr324 --\u3e Phe and Glu330 --\u3e Met/Val331 --\u3e Ile, factor VaFF/MI) had no cofactor activity under similar experimental conditions. Our data demonstrate that amino acid residues Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are critical for expression of factor Va cofactor activity
Computational Approaches to Investigate the Effects of Sensory Stimuli in Brain
No full textA fundamental goal of neuroscience is to understand how the brain responds to sensory stimuli. We can employ various computational approaches to solve research problems pertaining to this goal. This thesis, specifically, aims to investigate two such problems using computational approaches.
The first problem deals with the modulation of plantar cutaneous feedback with Transcutaneous Electrical Nerve Stimulation (TENS or E-stim). Plantar cutaneous feedback or tactile feedback from the foot sole is weakened in case of peripheral neuropathy and results in fall-related accidents. Previous studies show E-stim applied on the distal-tibial nerve elicits tactile feedback (Eletrotactile feedback) from heels. We put forward three hypotheses���a) Simple Augmentation Theory, b) Stochastic Resonance Theory, c) Nerve Conduction Block Theory���and used psychophysics to explain how electrotactile feedback modulates plantar tactile feedback. Two experiments involving thirteen healthy participants conclusively proved the validity of the simple augmentation theory that states E-stim augments plantar cutaneous feedback by adding more action potentials. This result suggests that E-stim can be used as a potential treatment for peripheral neuropathy.
The second problem sheds light on the response of auditory and prefrontal cortex to complex acoustic stimuli in echolocating bats. We used two neuroimaging techniques���intrinsic signal imaging and calcium Imaging���to find solutions to this problem. Echolocating bats can decode the shape, size, and texture of an object from echoes, which makes them excellent models to understand more about the sound-processing part of the brain. Since there are no such existing studies on neuroimaging in bats, we conducted a proof-of-concept study for both intrinsic signal imaging and calcium imaging. With the help of intrinsic signal imaging, we showed the existence of tonotopic map in auditory cortex. Furthermore, we transfected the brains of free-tailed bats (Tadarida brasiliensis) with an adeno-associated viral vector (AAV) carrying the gene of GCaMP7s (Calcium Indicator) for calcium imaging. After 6 weeks, we saw a rapid and extensive activity in the medial prefrontal cortex of expressing bats in response to downward frequency modulated sweeps mimicking echolocating calls
Spellbinding Effects of the Acidic COOH-Terminus of Factor Va Heavy Chain on Prothrombinase Activity and Function
No full textHuman factor Va (hfVa) is the important regulatory subunit of prothrombinase. Recent modeling data have suggested a critical role for amino acid Arg of hfVa for human prothrombin (hPro) activation by prothrombinase. Furthermore, it has also been demonstrated that hfVa has a different effect than that of bovine fVa on prethrombin-1 activation by prothrombinase. The difference between the two cofactor molecules was also found within the Asn-Arg dipeptide in the human factor V (hfV) molecule, which is replaced by the Asp-Glu sequence in bfV. As a consequence, we produced a recombinant hfV (rhfV) molecule with the substitution NR→DE. rhfV together with the wild-type molecule (rhfV) were expressed in COS7 cells, purified, and tested for their capability to function within prothrombinase. Kinetic studies showed that the of rhfVa for human fXa as well as the and of prothrombinase made with rhfVa for hPro activation were similar to the values obtained following hPro activation by prothrombinase made with rhfVa. Remarkably, sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of hPro activation time courses demonstrated that the rate of cleavage of hPro by prothrombinase reconstituted with rhfVa was significantly delayed with substantial accumulation of meizothrombin, and delayed thrombin generation, when compared to activation of hPro by prothrombinase made with rhfVa. These unanticipated results provide significant insights on the role of the carboxyl-terminal end of the heavy chain of hfVa for hPro cleavage and activation by prothrombinase and show that residues NR regulate at least in part the enzyme-substrate/product interaction during fibrin clot formation
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