Heparinized beads

Determination of Anti-Clotting Activities of Covalently Bound Heparin Beads... 

One of the samples which contained 5 mg. of covalently bonded heparinized beads had only 41 minutes clotting time which is very much shorter than the other sample, but still longer than the ionically bonded heparin complex beads. This shortened clotting time was attributed to an air bubble which was trapped by a mechanical error while the technician was adding the blood into the tube dropwise. The technician predicted a priori that this particular sample would have a low value for clotting time. 

Figure 2. Inactivation of thrombin (T) by antithrombin III (AT) on heparinized beads in a chromatography column (27).

We have also developed a mechanical wound assay whereby a heparin bead is placed into the wound site. Unlike the laser assay, this system allows for drug manipulation as the bead can be pre-treated with pharmacological agents that may modulate hemocyte migratory responses. Similar to laser-induced wounds, untreated bead implantation results in rapid accumulation of hemocytes (Fig. le), while pre-treatment of beads with inhibitors of known signaling pathways important for cell migration,... 

Fig. 1. Wound healing and inflammation in Drosophila embryos, (a) A laser ablation wound to a moesinGFP expressing Drosophila embryo will, by a purse-string mechanism, (b) seal shut over a period of a few hours, (c) This same laser wound will also recmit hemo-cytes that, by driving GFP specifically in these cells, can be imaged by widefield microscopy. (d) Alternatively, the wound site can be imaged by Differential Interference Contrast microscopy to give a general idea of wound size (dotted line) and hemocyte presence ( ). (e) The insertion of a heparin bead into the wound site will also lead to the recruitment of hemocytes that will surround and encapsulate the bead, (f) A hemocyte undergoing developmental dispersal viewed by confocal microscopy.

Heparin beads (Sigma) or Affigel blue beads (Bio-Rad Laboratories). 

GAG binding sites are generally identified by alanine scanning mutagenesis studies of the chemokine of interest. However, biochemical methods can be used to predict the regions that are involved, which can be subsequently confirmed by restricted mutagenesis. An example of such a method involves the use of protein bound to heparin beads, which is subsequently covalently stabilized by chemical... 

This results in almost the same specific surface area as found in capillary packed with macroporous silica beads. Next, the heparin was covalently immobilized on the surface of an etched capillary through a spacer using silane chemistry. 

Fig. 1. Chemokines have different relative affinities for heparin in an inunobilized-heparin competitive binding assay. Radiolabeled chemokines were incubated with either Heparin-Sepharose beads (O), Sepharose beads (A), or without beads ( ) in the presence of increasing concentrations of heparin. Competition assays with MCP-1 were performed as described in the Methods section. Each point represents the mean S.E.M. of triplicate determinations, and the experiment shown is a representative of 2-A experiments for each chemokine. The maximal binding was 3000-5000 cpm, which corresponded to 15-20% of the total added radioactivity.

Trimethylaminopolystyrene-heparin ionically bonded complex beads synthesized from 1. 

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. 

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],... 

Fio. 9. Effect of agitation speed on rate of heparin degradation catalyzed by immobilized heparinase at T = 37°C, pH 7.4, E = 230 units/mL, and Cb = 0.1 mg/mL. Ratio of volume of fluid phase to volume of beads, 200 1. Each point is mean of 10 independent experiments [from Bernstein et al. (50)]. 

For each bead set and heparin concentration, the experimentally determined effectiveness factor was compared to the predicted effectiveness factor. The Thiele modulus, , was calculated from... 

To test the model predictions, the single-pass clearance of heparin was determined as a function of time in three different sheep. For each experiment, the volume of beads, the initial enzyme loading, the animal s antithrombin level, the hematocrit, the inlet heparin concentration, and the half-life of the enzyme were used to generate model predictions for the clearance of heparin as a function of time. 

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. 

In Vitro Clotting Tests. The plasma recalcification time of citrated human plasma was determined in the presence of heparin-PVA beads and control PVA beads without heparin. Various amounts (10-200 mg) of gel were incubated with 0.5 mL of plasma at room temperature for 5 min. After the addition of 0.5 mL of0.025M CaCl2, the time to clot was noted by tilting the test tube gently each minute, until the beads clumped together or were found to stick to the test tube wall. 

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). 

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