Gel Filtration

Tangen, O.; Hj, Berman; Marfey, Peter S.

doi:10.1055/s-0038-1654301

Cited by 254 publications

(21 citation statements)

References 4 publications

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“…The amount of thrombin generated in the presence of cells grown in serum-free medium was equivalent to the amount found in identically passaged cells grown in serum-containing medium. After 20 min of incubation with factor Xa (20 ng) and prothrombin (90 pug), 3.1 units of thrombin per ml was generated in dishes containing cells grown in serumcontaining medium, while 2.9 units of thrombin per ml was generated by cells grown in serum-free medium.…”

Section: Resultsmentioning

confidence: 99%

Prothrombin is activated on vascular endothelial cells by factor Xa and calcium.

Rodgers¹,

Shuman²

1983

Proc. Natl. Acad. Sci. U.S.A.

124

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Vascular endothelial cells derived from adult bovine aorta (ABAE) treated with factor Xa and calcium were found to activate prothrombin. In contrast, nonvascular cells (human foreskin fibroblasts, bovine corneal endothelial cells, or human fetal lung cells) had either no or very little effect on prothrombin activation. In the presence of 6 x 105 ABAE cells, 20 ng of factor Xa converted 90 ,.g of prothrombin into 80 units of thrombin after 45 min at 37C. Exogenous factor V was not required for prothrombin activation, but thrombin generation was enhanced 2-to 4-fold by the addition of factor V (500-2,500 ng/ml). Treatment of ABAE cells with anti-bovine factor V IgG markedly inhibited prothrombin activation by factor Xa and calcium. In cells grown in serum-free medium for 3 months, the amount of factor V activity was equivalent to that found in cells grown with serum, which suggests that these cells probably synthesize factor V. Sparse ABAE cells increased prothrombin activation by factor Xa 6-fold compared to activation in confluent cells. Although previous thrombin treatment of ABAE cells did not enhance prothrombin activation, addition of dansyl arginine-4-ethyl piperidine amide markedly inhibited activation of 125I-labeled prothrombin by factor Xa, indicating that thrombin formation is necessary for optimal prothrombin activation. These data indicate that aortic endothelium may provide a physiologically important surface for activation of prothrombin as well as a mechanism for optimal formation of clots at sites of vascular injury.In an unperturbed state, vascular tissue is generally considered to be inert with regard to activation of blood clotting (1). (2,4,5). In separate experiments, ABAE cells were grown in serum-free medium (6, 7). In these studies, ABAE cells were grown without serum for two passages over a period of 3 months. In experiments in which disrupted cells were assayed, confluent ABAE cells were mechanically homogenized.Coagulation Proteins. All coagulation proteins used in these studies were purified from human plasma. Prothrombin and factor X were purified as described (8) and had specific activities of 34 units/mg and 160 units/mg, respectively. Factor V was provided by Joseph P. Miletich (Washington University, St. Louis, MO) and had a specific activity of 170 units/mg (9). Factor X was activated by RVV immobilized to Sepharose (10); the factor X-activating system consisted of factor X (0.03 ml of 2 mg/ml solution), washed RVV beads (0.03 ml), cephalint (0.015 ml of stock solution), and 100 mM CaCl2 (0.01 ml). This mixture was incubated at 37°C for 1 hr with frequent mixing. After centrifugation to sediment'the beads, the supernatant was assayed for factor Xa as described (11). In a clotting assay (11), factor Xa at 1 ng/ml yielded a clotting time of 38 sec. Factor V activity was measured using the method of Lewis and Ware (12). Protein was assayed by the method of Bradford (13).Prothrombinase Assay. Cultured adherent cells in 35-mm Petri dishes were washed twice with a buffer of ...

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Section: Resultsmentioning

confidence: 99%

Prothrombin is activated on vascular endothelial cells by factor Xa and calcium.

Rodgers¹,

Shuman²

1983

Proc. Natl. Acad. Sci. U.S.A.

124

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“…To remove any contaminating erythrocytes and leukocytes, the plasma was centrifuged again at 200 ϫ g for 15 min. The resulting platelet-rich plasma was gel-filtered into HEPES-Tyrode's buffer (136 mM NaCl, 2 mM KCl, 10 mM Na 2 CO 3 , 0.5 mM NaH 2 PO 4 , 0.4 mM MgCl 2 , 10 mM HEPES, 4.4 mM glucose, pH 7.4) as described previously (44). Fifty ng/ml PGI 2 was added to the enriched platelet suspension.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of Threonine 558 in the Carboxyl-terminal Actin-binding Domain of Moesin by Thrombin Activation of Human Platelets

Nakamura

Amieva

Furthmayr

1995

Journal of Biological Chemistry

189

183

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The phosphorylation and localization of the membrane-linking protein moesin was analyzed during early activation of platelets with thrombin. Activated platelets elaborate filopodia and spread to assume flat pancake-like shapes, and moesin is localized in filopodia and cell body. In resting platelets, approximately 25% of moesin molecules are phosphorylated as shown by metabolic labeling with 32 P i and by isoelectric focusing. Within seconds after exposure to thrombin, phosphorylation increases, reaching a maximum of 35% labeled molecules by 1 min, followed by a decrease to a new basal level within 5 min. This modification affects a single residue, Thr 558, which is located within or close to a binding site for F-actin. Rapid shifts (0 -100%) in the number of phosphorylated molecules are observed in the presence of inhibitors of serine/threonine kinases and phosphatases. Inhibitors affecting tyrosine phosphorylation also modulate phosphorylation at this site suggesting that the enzymes involved in the modification of Thr 558 are regulated by tyrosine phosphorylation. Platelets respond to both extremes of modification by forming extremely long filopodia and the absence of spreading on glass. Completely phosphorylated moesin is concentrated together with F-actin in the center of the cell. The rapid modification of moesin at or near its actin-binding domain suggests a model for regulated membrane-cytoskeleton interaction during cell activation.Platelets are anucleate, membrane-bound particles that participate in the process of hemostasis and thrombosis. They can be activated by a wide variety of stimuli and predictably respond to such stimuli by changing shape, by releasing the content of storage granules, by adhesion, and by aggregation (1, 2). These phenomena require drastic changes in the organization of the cytoskeleton and thus make platelets a powerful model system for the study of relationships between activating stimuli and cytoskeletal organization. The signal transduction events that mediate platelet cytoskeletal rearrangements have proven to be largely mediated through phosphorylation and dephosphorylation of cytoskeleton-associated and membrane proteins (3, 4). Such modifications result in changes in proteinprotein interactions and/or subcellular distribution and allow for the formation of new protein complexes between membrane components and the cytoskeleton. Following activation by physiological stimuli, for example, phosphorylation of myosin light chain, actin-binding protein, P-selectin, glycoprotein IIIa, glycoprotein Ib, talin, vinculin, cofilin, pp60 c-src , and focal adhesion kinase (pp125 FAK) is induced and regulates some of the molecular changes involved in platelet aggregation and release (5-22).Moesin is a member of the protein 4.1 family (23, 24) and shares considerable structural homology with ezrin and radixin (25-30). These proteins are often co-expressed in a great variety of cell types, where they are localized at the cytoplasmic face of the membrane in filopodia and other microextens...

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“…Platelets were filtered through Sepharose 2B by using a modification of the method of Tangen et al (7). Three milliliters of PRP was applied to the top of a 1.7 x 17.5 cm column previously equilibrated with calcium-free Tyrode's buffer, pH 7.4, containing albumin (3.5 mg/ml).…”

Section: Methodsmentioning

confidence: 99%

Subcellular localization and secretion of factor V from human platelets.

Chesney¹,

Pifer²,

Colman³

1981

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT' Factor V, a plasma protein cofactor necessary for optimal conversion of prothrombin to thrombin, is also present in considerable concentration in blood platelets (9.9 units per 109 platelets). Subcellular fractionation by. two methods has localized factor V in the a granules of-unstimulated platelets. ADP and epinephrine cause release of4.6% and 6.4%, respectively, ofthe total factor V, a process completely inhibited by cyclooxygenase alkylation by aspirin. In contrast, collagen causes release of 25% of platelet factor V, a process only partially suppressed by aspirin. Secretion of factor V depends on the availability of metabolic energy, because antimycin A,. an inhibitor of aerobic metabolism, and 2-deoxyglucose, an inhibitor of anaerobic glycolysis, together almost totally inhibited the secretion of factor V induced by collagen. The data establish that factor V is not normally available on unstimulated platelets but can be secreted from a granules upon stimulation with physiological agents such as ADP, epinephrine, and collagen. Because factor V is known to serve as a receptor for factor Xa, the exposure of factor V on platelets consequent to release would accelerate the process of blood coagulation.One year after the discovery of factor V in plasma, Ware et al.(1) recognized that platelets also contain an accelerator of prothrombin conversion. Though this coagulant activity was more stable to heat inactivation and storage than was plasma factor V, it was generally assumed to be the plasma factor adsorbed to the platelet surface (2, 3). In 1975, Breederveld et aL (4), using gel filtration to isolate platelets from plasma, found minimal activity of factor V associated with platelets but were able to increase the V activity by freeze-thawing. 0sterud et aL (5) confirmed this observation and showed that platelets contain an activated form offactor V as well as a platelet activator ofplasma factor V. Kane et aL (6) have presented convincing evidence that factor Xa binds to the platelet membrane at a binding site that is identical to or requires the presence of factor V.Despite these important observations, several questions remain in evaluating the influence of platelet factor V on the hemostatic and possible thrombotic processes. What is the sub, cellular localization offactor V? Are prostaglandin synthesis and metabolic energy required for its release? Do collagen, ADP, and epinephrine release factor V as thrombin does? This study attempts to answer these questions. MATERIALS AND METHODSBovine serum albumin (fraction V), ADP, epinephrine, and bovine fibrinogen (fraction 1, type IV, 93% clottable) were supplied by Sigma. Human fibrinogen (grade L, 95%

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Gel Filtration

Cited by 254 publications

References 4 publications

Prothrombin is activated on vascular endothelial cells by factor Xa and calcium.

Prothrombin is activated on vascular endothelial cells by factor Xa and calcium.

Phosphorylation of Threonine 558 in the Carboxyl-terminal Actin-binding Domain of Moesin by Thrombin Activation of Human Platelets

Subcellular localization and secretion of factor V from human platelets.

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