Protease Generation by Chondroitin Sulfate, Semisynthetic OSCS Preparations and Contaminants Isolated From Heparin.

Abstract 3133

Poster Board III-70

The main contaminant in heparins recalled by the FDA in 2008 was reported to be oversulfated chondroitin sulfate (OSCS) (Kishimoto et al 2008). The adverse reactions were reportedly linked to contact activation and the generation of kallikrein. Because of the molecular and biologic heterogeneity of the contaminants isolated from heparin and LMWHs and the potential presence of other carryover contaminants it was hypothesized that heparin contaminants may also activate other proteases such as plasmin, C1 esterase factor Xa (FXa), and thrombin by direct or indirect mechanisms. To investigate the protease activation profile of heparin, porcine chondroitin sulfate (PCS) and OSCS preparations of porcine (POSCS), bovine (BOSCS) shark (SOCS) and squid (SqOSCS) origin along with contaminants isolated from five different recalled batches of heparin were profiled for the generation of kallikrein, plasmin, C1 esterase, FXa, and thrombin. Normal human pooled plasma (NHP) was supplemented with each of the different agents over a concentration range of 100ug/ml to 0.1ug/ml. Specific substrates for kallikrein (Chromozyme PK), plasmin (Chromozyme PL), C1 esterase (S2302), FXa (Chromozyme Xa), and thrombin (Chromozyme Th) were used to measure the generation of specific enzymes. The PCS produced relatively weaker generation of kallikrein, however, the contaminants isolated from the recalled batches of heparin produced a much stronger generation of this enzyme (absorbance 0.45-0.70). The different OSCS preparations produced a lower generation of kallikrein (absorbance .34-.52). While heparin and PCS produced significant activation of C1 esterase, the contaminants produced an even stronger activation of C1 esterase (abs .33-.44). The OSCS preparations from different sources produce relatively weaker activation which was comparable to the starting material. The C1 esterase activation profile was not proportional to the kallikrein activation. The PCS, UFH, and contaminants along with the different OSCS preparations produced varying degrees of plasmin generation. UFH and PCS also produced significant generation of plasmin which was higher than their C1 esterase and prekallikrein generation. With the exception of PCS and UFH, all of the agents produced varying degrees of Xa and thrombin generation. The generation of Xa was higher by all of the OSCS preparations in comparison to the IIa generation. Interestingly each of the OSCS preparations and different contaminants exhibited their own specific protease generation profile. There was no correlation between the kallikrein formation and the generation of other enzymes. These data highly suggest that the molecular composition of OSCS's and the contaminants play a major role in the mediation of the pathophysiologic responses observed with the use of contaminated heparin. The presence of other glycosaminoglycans may influence the protease generation profile of contaminants present in recalled heparins. Moreover, the relative generation of these proteases may also be influenced by the pathologic predisposition of patients where the generation of these proteases may influence the overall pathophysiologic response.