scholarly journals A modified clot-based assay to measure negatively charged procoagulant phospholipids

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Cathrine Ramberg ◽  
S. Jamaly ◽  
N. Latysheva ◽  
L. Wilsgård ◽  
T. Sovershaev ◽  
...  
Keyword(s):  
Human Plasma ◽  
Tissue Factor ◽  
Extracellular Vesicles ◽  
Whole Blood ◽  
Coagulation Factors ◽  
Commercial Assay ◽  
Coefficients Of Variation ◽  
Postprandial Lipoprotein

AbstractGrowing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL). Current commercial PPL-dependent clotting assays use chemically phospholipid depleted plasma to measure PPL activity. The purpose of our study was to modify the PPL assay by substituting the chemically phospholipid depleted plasma with PPL depleted plasma obtained by ultracentrifugation This in order to get readily access to a sensitive and reliable assay to measure PPL activity in human plasma and cell supernatants. The performance of the assay was tested, including the influence of individual coagulation factors and postprandial lipoproteins and compared to a commercial PPL assay (STA-Procoag-PPL). The two PPL assays displayed similar sensitivity to exogenously added standardized phospholipids. The PPL activity measured by the modified assay strongly correlates with the results from the commercial assay. The intraday- and between-days coefficients of variation ranged from 2–4% depending on the PPL activity in the sample. The modified PPL assay was insensitive to postprandial lipoprotein levels in plasma, as well as to tissue factor (TF) positive EVs from stimulated whole blood. Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF+ EVs.

An Improved Radioenzymatic Assay for Histamine in Human Plasma, Whole Blood, Urine, and Gastric Juice

1979 ◽  
Vol 16 (1-6) ◽  
pp. 259-264 ◽  
Author(s):  
C. Bruce ◽  
W. H. Taylor ◽  
A. Westwood
Keyword(s):  
Gastric Juice ◽  
Human Plasma ◽  
Whole Blood ◽  
Plasma Histamine ◽  
Normal Human Plasma ◽  
Radioenzymatic Assay ◽  
Coefficients Of Variation ◽  
Histamine Methyltransferase ◽  
Normal Human ◽  
The Mean

A radioenzymatic method suitable for the assay of histamine in human blood, urine, plasma, and gastric juice is described. It differs from earlier methods by use of a histamine methyltransferase preparation from pig brain, of high activity tritiated S-adenosylmethionine, and of a heat precipitation step to reduce the previously noted interference from plasma constituents. The method is simpler than those requiring solvent extraction and concentration of histamine, gives recoveries in the range 80–120%, and so eliminates the need for internal standardisation. The method is sensitive and precise with coefficients of variation for blood, urine, and plasma of 5%, 6%, and 13% respectively. The mean ± standard deviation for normal human plasma histamine is 5 ± 4 nmol/l, for whole blood 559 ± 193 nmol/l, and for urine 229 ± 128 nmol/24h.

scholarly journals Thrombin Generation and Cancer: Contributors and Consequences

Cancers ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 100 ◽  
Author(s):  
Caroline Reddel ◽  
Chuen Tan ◽  
Vivien Chen
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Extracellular Vesicles ◽  
Thrombin Generation ◽  
Tumour Growth ◽  
Coagulation Factors ◽  
Cell Types ◽  
Cancer Treatments ◽  
Cancer Associated Thrombosis

The high occurrence of cancer-associated thrombosis is associated with elevated thrombin generation. Tumour cells increase the potential for thrombin generation both directly, through the expression and release of procoagulant factors, and indirectly, through signals that activate other cell types (including platelets, leukocytes and erythrocytes). Furthermore, cancer treatments can worsen these effects. Coagulation factors, including tissue factor, and inhibitors of coagulation are altered and extracellular vesicles (EVs), which can promote and support thrombin generation, are released by tumour and other cells. Some phosphatidylserine-expressing platelet subsets and platelet-derived EVs provide the surface required for the assembly of coagulation factors essential for thrombin generation in vivo. This review will explore the causes of increased thrombin production in cancer, and the availability and utility of tests and biomarkers. Increased thrombin production not only increases blood coagulation, but also promotes tumour growth and metastasis and as a consequence, thrombin and its contributors present opportunities for treatment of cancer-associated thrombosis and cancer itself.

Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Human Factor ◽  
Solid Phase ◽  
Three Dimensional ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Dimensional Structure ◽  
Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

Antithrombin-mediated Inhibition of Factor Vlla-Tissue Factor Complex by the Synthetic Pentasaccharide Representing the Heparin Binding Site to Antithrombin

1996 ◽  
Vol 76 (01) ◽  
pp. 005-008 ◽  
Author(s):  
Jean Claude Lormeau ◽  
Jean Pascal Herault ◽  
Jean Marc Herbert
Keyword(s):  
Binding Site ◽  
Tissue Factor ◽  
Residual Activity ◽  
Coagulation Factors ◽  
Heparin Binding ◽  
Experimental Conditions ◽  
First Order ◽  
Heparin Binding Site ◽  
Coagulant Activity

SummaryWe examined the effect of the synthetic pentasaccharide representing the minimal binding site of heparin to antithrombin on the antithrombin-mediated inactivation of factor Vila bound to tissue factor. This effect was compared to the effect of unfractionated heparin. Using purified recombinant human coagulation factors and either a clotting or an amidolytic assay for the determination of the residual activity of factor Vila, we showed that the pentasaccharide was an efficient antithrombin-dependent inhibitor of the coagulant activity of tissue factor-factor Vila complex. In our experimental conditions, assuming a mean MW of 14,000 for heparin, the molar pseudo-first order rate constants for ATIII-mediated FVIIa inhibition by ATIII-binding heparin and by the synthetic pentasaccharide were found to be similar with respective values of 104,000 ± 10,500 min-1 and 112,000 ± 12,000 min-1 (mean ± s.e.m., n = 3)

Plasma Thromboplastin Component (Christmas Factor, Factor IX) Levels in Stored Human Blood and Plasma

1960 ◽  
Vol 04 (03) ◽  
pp. 376-388 ◽  
Author(s):  
J Dieter Geratz ◽  
John B. Graham
Keyword(s):  
Human Plasma ◽  
Human Blood ◽  
Whole Blood ◽  
Room Temperature ◽  
Close Agreement ◽  
Factor Ix ◽  
Deficient Patient ◽  
Glass Tubes ◽  
Disodium Hydrogen Citrate ◽  
Bank Blood

Summary1. PTC activity was assayed in 26 units of human plasma prepared from whole blood stored for 3 weeks at 4° C. The plasma had been frozen and stored at — 20° C for additional periods ranging from a few days to 4 months. High PTC activity was still present in the plasma at the end of this period, the activity averaging 95% of normal.2. The PTC activity of 19 samples of “reclaimed“ plasma stored for an additional 6 months at — 20° C decreased by an average of 23%. This decrease was statistically significant.3. Liquid plasma kept at room temperature for 5½—7½ months contained no PTC activity.4. Lyophilized plasma stored at room temperature for 6—8 years contained an average of 30% PTC activity. Lyophilized plasma stored at — 20° C for 4 years contained 68% PTC activity.5. ACD and disodium hydrogen citrate anticoagulant solutions served equally well in preserving PTC activity in whole blood stored in glass tubes over a period of 3 weeks at 4° C.6. “Reclaimed“ plasma from outdated bank blood provided effective hemostasis in two operations for the removal of 20 teeth from a severely PTC-deficient patient.7. The high PTC activity of “reclaimed“ plasma was confirmed by the close agreement between the PTC level expected in a PTC deficient patient after transfusion of such plasma and that observed.

Extracellular Vesicles from Human Plasma Show a Distinctive Proteome and miRNome Profile in Patients with Severe Cutaneous Adverse Reactions

2021 ◽  
Author(s):  
Orlando Salinas-Jaramillo ◽  
Alejandra Monroy-Arreola ◽  
Sebastian Herrera-Noreña ◽  
Ana L. Guzmán-Ortiz ◽  
Abrahan Hernández-Hernández ◽  
...  
Keyword(s):  
Human Plasma ◽  
Extracellular Vesicles ◽  
Adverse Reactions ◽  
Severe Cutaneous Adverse Reactions ◽  
Cutaneous Adverse Reactions

Free flow electrophoresis allows quick preparation of extracellular vesicles from cell culture supernatants and human plasma

Cytotherapy ◽  
2021 ◽  
Vol 23 (5) ◽  
pp. S118
Author(s):  
S. Staubach ◽  
T. Tertel ◽  
V. Börger ◽  
C. Grätz ◽  
M. Pfaffl ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Plasma ◽  
Extracellular Vesicles ◽  
Free Flow ◽  
Culture Supernatants

Direct Isolation of Circulating Extracellular Vesicles from Blood for Vascular Risk Profiling in Type 2 Diabetes Mellitus

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Hui Min Tay ◽  
Sheng Yuan Leong ◽  
Xiaohan Xu ◽  
Fang Kong ◽  
Megha Upadya ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
Extracellular Vesicles ◽  
Whole Blood ◽  
Vascular Risk ◽  
Risk Profiling

Extracellular vesicles (EVs) are key mediators of communication among cells, and clinical utilities of EVs-based biomarkers remain limited due to difficulties in isolating EVs from whole blood reliably. We report...

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