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Thrombin heparinized beads

Figure 2. Inactivation of thrombin (T) by antithrombin III (AT) on heparinized beads in a chromatography column (27). Figure 2. Inactivation of thrombin (T) by antithrombin III (AT) on heparinized beads in a chromatography column (27).
Anticoagulant properties are due to the formation of a complex between heparin and antithrombin (ATIII) heparin increases ATIII activity, inhibiting thrombin, which is responsible for the formation of the clot [8]. Although this complex is already characterized by a weak affinity, the exact mechanism of association between heparin and antithrombin is not exactly known. A multistep protocol of immobilization of heparin on silica beads permitted high-performance chromatographic phases to be obtained. Thus, it has been possible to evidence a slightly stronger affinity of heparin for antithrombin than for thrombin. [Pg.301]

Heparinlike sulfated dextran derivatives, like carboxymethyldextran benzylamide sulfonates (CMDBS), have been immobilized on silica beads. By high-performance liquid affinity chromatography (HPLAC), they allow a good recovery of thrombin, with a yield of 80%. The affinity constant was estimated K lO M ) and was found superior to the value obtained between thrombin and heparin. On the contrary, the affinity of dextran derivatives for ATIII is estimated at a lower value than that of heparin [17],... [Pg.302]

Displacement by plasma of radiolabeled thrombin and radio-labeled thrombin-antithrombin III inactive complex from a heparinized surface was measured and found to be significant for example, removing 63% of the thrombin and 90% of the complex that could not be removed by phosphate-buffered saline alone. Heparin-poly(vinyl alcohol) (PVA) gel beads with a very low heparin release rate, prepared by acetal coupling of the heparin to the PVA, adsorbed thrombin and potentiated the inactivation of thrombin by antithrombin 111, as measured by both thrombin time and chromogenic substrate assays. [Pg.150]

The thrombin time was determined similarly by incubation of 2 IU of crude bovine thrombin (10 IU/mL, Miles Laboratories) with the beads for 5 min at room temperature in an albumin-coated glass tube, followed by 0.2 mL of citrated human plasma. The time to clot was noted by tilting the test tube gently every few seconds. PBS, after incubation with heparin-PVA beads for 5-60 min, was analyzed for the presence of heparin using both toluidine blue and the thrombin time test (PBS in place of gel beads). [Pg.152]

In Vitro Activity of Bound Heparin. The prolongation of the thrombin time caused by the addition of heparin-PVA beads to plasma is contrasted with the negligible effect of PVA beads without heparin in Figure la. A similar prolongation was observed for the plasma recalcification time (Figure lb). In both cases, clumping or adhesion of the beads caused presumably by fibrin was the end point of the assay. [Pg.155]

Figure 2 shows that thrombin binds to both heparin-PVA beads and PVA beads without heparin. After a load of crude bovine thrombin (62 IU) followed by PBS and chromogen, color yields of 89% and 81% were obtained for the heparin-PVA and PVA gel, respectively, indicating the presence of active thrombin on the columns. Passing 15 mL of 20% (w/w) bovine albumin through the same columns followed by chromogen lowered the color yield for... [Pg.155]

Figure la. Thrombin time of plasma in the presence of PVA (o) and heparin-PVA ( ) beads. Thrombin incubated with beads prior to plasma addition (2 IU of crude bovine thrombin, 0.2 mL of citrated human plasma, and 7 mg of heparin g gel) (13). [Pg.155]

Thrombin Binding Affinity. The affinity of thrombin for PVA or heparin-PVA was analyzed by loading crude bovine thrombin (Parke-Davis, 62 U, 18 nmoles) onto a PVA or heparin-PVA "bead" column, washing with 100 mL PBS and/or 15 mL 20% (w/v) bovine... [Pg.569]

Preliminary thrombin adsorption isotherms were prepared by incubating 100 mg of heparin-PVA (control PVA) beads in centrifuge tubes (15 mL) with 0.6 mL of thrombin solution (3-1200 U/mL). Unlabelled purified human thrombin was spiked with I-labelled purified bovine thrombin. After adsorption for 15 minutes, the beads were washed twice with 5 mL of PBS and the supernatants counted. The amount of thrombin/gram of gel was determined. [Pg.569]

Loading Sequence. Two similar columns of heparin-PVA beads were prepared ( ). The first column was loaded with thrombin followed by antithrombin III the sequence was reversed for the second column. The thrombin load was either crude bovine (62 U, 18 nmoles), pure bovine (23 U, 0.35 nmoles), or pure human (1072 U, 9.4 nmoles). The antithrombin 111 load was either crude human (3 mg 48 nmoles, 15 mL of defibrinated plasma) or purified human (1.5 mg, 24 nmoles, 0.29 mg/mL). After loading each protein, 100 mL of PBS was passed through the column and the residual thrombin activity measured by the chromogenic substrate method. [Pg.569]

A series of experiments were made with heparin-PVA bead columns. Thrombin (100 U crude bovine, II50 U purified human or 100 U purified bovine) was loaded onto the columns and the excess removed with PBS. Antithrombin III ( 2000-2500 U purified human) was then loaded, and the columns eluted with PBS, (w/v) bovine serum albumin, or plasma fractions. In some cases heat defibrinated or arvinized plasma was used instead of purified human antithrombin III as a crude source of antithrombin III, as well as a displacing agent. The inactivation of thrombin by anti thrombin III was shown in a separate experiment using the chromogenic substrate assay. [Pg.570]

Thrombin Inactivation of Antithrombin III on Heparin-PVA Bead Columns. Bovine and human thrombins loaded onto heparin-PVA columns, followed by chromogenic substrate, resulted in colour yields of... [Pg.571]

Figure 3 Displacement of purified bovine I-thrombin from heparin-PVA "beads by arvinized plasma. Each column was loaded with 100 U of thrombin, eluted with 100 mL of PBS and then further eluted with arvinized plasma or 5% (w/v) bovine albumin at a flowrate of 60 mL/hr. (Reproduced with permission from Ref. 28. Copyright 1981, Gordon and Breach, Science Publishers Inc.)... Figure 3 Displacement of purified bovine I-thrombin from heparin-PVA "beads by arvinized plasma. Each column was loaded with 100 U of thrombin, eluted with 100 mL of PBS and then further eluted with arvinized plasma or 5% (w/v) bovine albumin at a flowrate of 60 mL/hr. (Reproduced with permission from Ref. 28. Copyright 1981, Gordon and Breach, Science Publishers Inc.)...
Purified Bovine I-thrombin from Heparin-PVA "Beads" in Centrifuge Tubes... [Pg.574]

Analyses of the effluent produced by passing arvinized plasma through a heparin-PVA "bead" column previously loaded with 100 U of purified bovine I-thrombin showed that 46.8 8.9% (SD, n = 5) of the recovered radioactivity (labelled thrombin) passed through... [Pg.574]


See other pages where Thrombin heparinized beads is mentioned: [Pg.54]    [Pg.31]    [Pg.153]    [Pg.153]    [Pg.159]    [Pg.160]    [Pg.569]    [Pg.570]    [Pg.571]    [Pg.572]    [Pg.574]    [Pg.577]   
See also in sourсe #XX -- [ Pg.154 ]




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