Heparin degradation

In developing a reactor such as the one just described, it is important to understand important design parameters, such as the radial distribution of the enzyme within the catalyst particles, the kinetics of heparin degradation catalyzed by immobilized heparinase, the flow properties in the reactor, and the effect of in vivo factors such as blood proteins which bind to the substrate. These parameters and how they can be evaluated are now discussed. 

The heparin degradation rate at any radial position inside the catalyst particle is proportional to the bound heparinase concentration at that position. If the immobilized enzyme concentration is not uniform, the conventional analysis of simultaneous diffusion and reaction within a porous catalytic particle must be modified. The reaction rate within the catalyst particle will have an explicit radial dependence introduced via the enzyme concentration, as well as a dependence on the substrate concentration. 

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)]. 

The reaction rates are based on the total gel volume. The kinetics of heparin degradation [Reaction (14)] follow Michaelis-Menten kinetics (52) ... 

Heparinase Activity. Several assays were used to follow heparinase activity. These assays followed (1) the disappearance of heparin, (2) the appearance of heparin degradation products, or (3) the loss of the physiological function of heparin in anticoagulation. The basis of these assays and explanations as to when they are routinely used are listed below. 

In a second experiment, the effect of both heparin concentration and flow rate on heparin degradation was examined. The same size column as was used in the previous experiment was employed. As shown in Figure 6, at low flow rates this small column was fully capable of degrading very large quantities of heparin (more than 100-fold in excess of clinically used amounts) in a single pass. 

Dietrich, C. P. Studies on the Induction of Heparin Degrading Enzymes in Flavobacterium Heparinum Biochemistry 8, 3342-3347 (1969). 

Dietrich, C. P. Novel Heparin Degradation Products. Isolation and Characterization of Novel Disaccharides and Oligosaccharides Produced From Heparin by Bacterial Degradiation , Biochem. J. 108,647-654(1968). 

A heparin-degrading enzyme isolated from a transplantable murine mastocytoma hydrolysed some of the bonds in macromolecular mastocytomal [ S]heparin, yielding products similar in size to commercial heparins. ... 

The structure required for a polysaccharide to induce the synthesis of a heparin-degrading system by F. heparinum has been studied. It was concluded that the polysaccharide must contain a uronic acid residue (with a free carboxy-group) attached to either a 2-acetamido-2-deoxy- or 2-deoxy-2-sulphamido-hexose residue by an a-(l - 4)-linkage. Neither O- nor A sulphated residues are essential for inducing the synthesis of this enzyme system. The reason why some heparan sulphates are more effective than heparin in inducing enzymic activity was discussed. 

A number of laboratory tests are available to measure the phases of hemostasis described above. The tests include platelet count, bleeding time, activated partial thromboplastin time (aPTT or PTT), prothrombin time (PT), thrombin time (TT), concentration of fibrinogen, fibrin clot stabifity, and measurement of fibrin degradation products. The platelet count quantitates the number of platelets, and the bleeding time is an overall test of platelet function. aPTT is a measure of the intrinsic pathway and PT of the extrinsic pathway. PT is used to measure the effectiveness of oral anticoagulants such as warfarin, and aPTT is used to monitor heparin therapy. The reader is referred to a textbook of hematology for a discussion of these tests. 

Alkali-catalyzed cleavage of glycosidic bonds of glycosaminoglycur-onates occurs by way of -elimination.223 By proper choice of the experimental conditions, it seems possible to achieve a controlled degradation of heparin by -elimination, producing fragments apparently preserving... 

Scheme 4. —Smith Degradation of Heparin (Arbitrary Sequence). (R = remnant from a D-glucuronic acid residue.)...

Extracts of Flavobacterium heparinum that had been induced to grow on heparin-like polysaccharides contain a number of enzymes that may ultimately degrade heparin and heparan sulfate to monosaccharides.137,145 240 The enzymes that cause the primary cleavage of heparinlike chains are heparinase (EC 4.2.2.7) and heparanase (EC 4.2.2.8, formerly called heparitinase241,242). 

There are various inhibitors within the coagulation system that counterregulate activation of the coagulation cascade. Among them, antithrombin III (AT-III) and protein C (PC) are the most important (SI). AT-III binds in the presence of heparin the activated factors F-IXa, F-Xa, and F-IIa (thrombin). PC is activated by a complex formed between thrombin and thrombomodulin, a surface protein of endothelial cells. Once activated, PC in the presence of protein S (PS) specifically degrades activated factors F-Va and F-VIIIa. PC decreases in the course of sepsis in relation to the severity of the condition (L15). Experimental studies have... 

Connaghan D. G Francis C. E., Ryan D. H., Marder V. J. Prevalence and clinical implications of heparin-associated false positive tests for serum fibrin(ogen) degradation products. Am J Clin Pathol 1986 86,304-10. 

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