A rapid method for isolation of fibrinogen from human plasma by precipitation with polyethylene glycol 6,000

Vila, Virtudes; Regañón, Edelmiro; Llopis, F.; Aznar, Justo

doi:10.1016/0049-3848(85)90248-8

Cited by 23 publications

(11 citation statements)

References 14 publications

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“…Trout fibrinogen was isolated using a modified method from Vila et al (1985). Trout fibrinogen was isolated using a modified method from Vila et al (1985).…”

Section: Methodsmentioning

confidence: 99%

The thromboxane mimetic, U‐46619, induces the aggregation of fish thrombocytes

Hill

Rowley

1996

Br J Haematol

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Fish blood contains nucleated cells termed thrombocytes which are thought to be functionally analogous to mammalian platelets. The present study was undertaken to assess the aggregatory response of peripheral blood thrombocytes from the rainbow trout, Oncorhynchus mykiss. Incubation of Percoll density gradient purified thrombocytes with the thromboxane A2 mimetic, U-46619 (0.03-10 microM), alone or in the presence of human or trout fibrinogen, elicited a dose-dependent aggregatory response. A greater amount of aggregation was observed in the presence of fish, rather than human, fibrinogen (e.g. c 55% compared with 15% maximal aggregation respectively). This response was inhibited by pre-incubation of thrombocytes with the specific thromboxane A2 receptor antagonist, GR32191 (0.01-10 microM) with an IC50 value of 5.7 microM. Ultrastructural and aggregometry studies of thrombocyte aggregation at various time periods (1-12 min) after incubation with U-46619 (0.5 microM) revealed clear differences between the amount and dynamics of thrombocyte clumping in the presence of human, compared with trout, fibrinogen (final concentration 400 micrograms ml-1). Thrombocytes rapidly underwent shape change and aggregation after only 1 min incubation without the initial involvement of any other cell types. The maximum degree of aggregation was achieved after 4-8 min with larger aggregates formed in the presence of trout compared than human fibrinogen. Ultrastructurally, the thrombocytes in these aggregates displayed a number of changes compared with non-stimulated cells, including increased pseudopodial activity, a more pronounced canalicular system and condensation of the nuclear heterochromatin. After 12 min incubation the clumps of thrombocytes showed progressive disaggregatory behaviour, with some cells reverting back to their normal in vivo appearance. Neutrophilic granulocytes, present as contaminants in the thrombocyte suspensions, were occasionally found attached to the thrombocyte aggregates, perhaps suggesting a specific interaction similar to that occurring in mammals. Finally, and also of some significance, was the finding that trout thrombocytes exhibit phagocytic activity in vitro towards cell debris and contaminating bacteria, indicating that this cell type may possess both haemostatic and immunological capacities.

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“…Trout fibrinogen was isolated using a modified method from Vila et al (1985). Trout fibrinogen was isolated using a modified method from Vila et al (1985).…”

Section: Methodsmentioning

confidence: 99%

The thromboxane mimetic, U‐46619, induces the aggregation of fish thrombocytes

Hill

Rowley

1996

Br J Haematol

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“…The fibrinogen precipitate was dissolved in 18 mM phosphate buffer, pH 7 . 8, 150 mM NaCl (Vila et al, 1985). Contaminating fibronectin was removed from the fibrinogen solution by chromatography on a gelatin-Sepharose 4B (Pharmacia) column (Ruoslahti et al, 1982).…”

Section: Preparation Of Plateletsmentioning

confidence: 99%

The effect of thrombin on the dynamic exchange between intraplatelet and extraplatelet fibrinogen

et al. 1997

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Summary.We examined the distribution of platelet fibrinogen and the exchange between intra-and extra-platelet fibrinogen in unstimulated and thrombin-stimulated platelets. In unstimulated platelets 60% of platelet fibrinogen was found in the soluble platelet fraction and 40% in the insoluble one. In platelets activated with thrombin, changes took place in the distribution of intraplatelet fibrinogen but not in the total fibrinogen content. At у 0 . 5 U/ml of thrombin the fibrin(ogen) content of the insoluble and soluble fractions was approximately 80% and 20%, respectively. When we evaluated how extraplatelet fibrinogen affects the content and distribution of intraplatelet fibrinogen, we found that when unlabelled fibrinogen was added to unstimulated and thrombin-stimulated platelets the content and distribution of intraplatelet fibrinogen remained unaltered. However, when 125 I-fibrinogen was added, it was incorporated into unstimulated and thrombin-stimulated platelets. In unstimulated platelets, 70% of the incorporated 125 I-fibrinogen was in the soluble fraction and 30% in the insoluble. In thrombin-stimulated platelets the distribution of the incorporated 125 I-fibrinogen was 62% and 38% in soluble and insoluble fractions respectively. MoAb to GPIIbIIIa produced 80% and 60% inhibition of 125 I-fibrinogen incorporation by unstimulated and thrombin-stimulated platelets. Our data showed dynamic exchange between intraplatelet and extraplatelet fibrinogen both in unstimulated and thrombin-stimulated platelets mediated mainly by GPIIb-IIIa.

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“…The precipitate was dissolved in 10 mM phos phate buffer, 150 mM NaCl, pH 7.4 (original plasma volume), and precipitated with 2 M acetate buffer, pH 4.4 (2/1, vol/vol). The precipitate obtained was dissolved in 36 mM phosphate buffer, pH 7.8 (origi nal plasma volume), and again precipitated with 30% saturated ammonium sulfate [20], The fibrino gen precipitate was dissolved in 18 mM phosphate buffer, 150 mA/ NaCl, pH 7.8 (1/4 original plasma volume). Contaminating fibronectin was removed from the fibrinogen solution by chromatography on a gelatin-Sepharose 4B (Pharmacia) column [21].…”

Section: Fibrinogen Preparationmentioning

confidence: 99%

Unstimulated and Thrombin-Stimulated Platelets Binding to Immobilized Fibrinogen and Fibrin on Polystyrene Supports

Martínez‐Sales

Vila²,

Regañón³

et al. 1995

Pathophysiol Haemos Thromb

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The binding of unstimulated and thrombin-stimulated platelets was studied with immobilized fibrinogen and fibrin on polystyrene. The amount of fibrinogen bound to the polystyrene support was 2 μg/tube, which represents 2.35 μg/cm². Immobilized fibrin was obtained by adding thrombin (5 nM) to immobilized fibrinogen. The number of unstimulated ¹¹¹ln-platelets bound to immobilized fibrinogen and fibrin was similar (3.2 ± 0.3 × 10⁶ and 3.1 ± 0.4 × 10⁶ platelets/μg fibrin-(ogen), respectively). The platelet binding steadily increased. In the first 2 min, the binding rate was 0.23 × 10⁶ platelets/μg fibrinogen/min. The binding rate then increased rapidly and saturation was reached at 10 min. The extent of the adhesion of resting platelets to immobilized fibrinogen is about one half that of the same platelets stimulated with thrombin. In thrombin-stimulated ¹¹¹In-platelets, the binding to immobilized fibrinogen and fibrin is time dependent, and saturation is reached at 5 min. The early rate of thrombin-stimulated platelet binding to fibrinogen is about twice that of binding to fibrin (1.25 and 0.74 × 10⁶ platelets/μg fibrin(ogen)/min, respectively). In saturation conditions, 1 μg fibrinogen binds 5.7 ± 0.6 × 10⁶ thrombin-stimulated platelets and 1 μg fibrin binds 4.6 ± 0.5 × 10⁶ thrombin-stimulated platelets. Our results indicate that the rate of platelet aggregation is faster than fibrin formation, and the rate of fibrinogen-platelet binding is faster than that of fibrin-platelet binding. Therefore, after thrombin stimulation, the binding of platelets to fibrin must be secondary to the binding of platelets to fibrinogen.

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A rapid method for isolation of fibrinogen from human plasma by precipitation with polyethylene glycol 6,000

Cited by 23 publications

References 14 publications

The thromboxane mimetic, U‐46619, induces the aggregation of fish thrombocytes

The thromboxane mimetic, U‐46619, induces the aggregation of fish thrombocytes

The effect of thrombin on the dynamic exchange between intraplatelet and extraplatelet fibrinogen

Unstimulated and Thrombin-Stimulated Platelets Binding to Immobilized Fibrinogen and Fibrin on Polystyrene Supports

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