Development of Non-Anticoagulant Derivatives of Biotechnology Derived Sulfaminoheparosan Derivatives to Investigate the Non-Anticoagulant Effects of these Agents.

It is now well known that heparin and related glycosaminoglycans produce some of their non-anticoagulant effects such as the anti-inflammatory, antiproliferative and regulatory actions independent of endogenous serpine (AT and HC-II). Sulfaminoheparosans (SAH) represent a class of biotechnology derived bacterial capsular polysaccharide (K5) derivatives which are epimerized and sulfated to mimic heparin’s biologic effects and exhibit affinity to both AT and HC-II. These SAH can be produced to exhibit molecular profile mimicking the low molecular weight heparins (LMWHs). The disaccharide unit structure of epimerized SAH is GlcA-GlcNSO₃6SO₃(3,6)H) in comparison to heparin which is IdoA2SO₃-GlcNSO₃6SO₃. The anticoagulant and antiprotease profile of a 6 kDa SAH has been found to be similar to Tinzaparin (Maddineni et al. Clin Appl Thromb Hemost 10(1):27–37,2004). As this agent also exhibits various other biologic effects such as the release of TFPI, modulation of adhesion molecules and antiproliferative effects, it was hypothesized that the non-anticoagulant forms of this agent may exhibit some of these effects. In order to produce the non-anticoagulant derivatives in 6 kDa SAH, desulfated derivatives were prepared by removing sulfate groups on the position 2 alone (2 desulfated) and positions 2 and 6 (2,6 desulfated derivatives). Additional modifications in the 2,6 desulfated derivatives included the presence of either free amino group or N-acetyl in position 2. These modifications did not result in the molecular weight profile of the desulfated derivatives. However, in comparison to the parent 6 kDa SAH both the 2-desulfated and 2,6 desulfated derivatives were weaker in the anticoagulant (PT, PTT, Heptest) and antiprotease (anti-Xa, anti-IIa) and protease generation assays. 2,6 desulfation produced a strong decrease in the anticoagulant effects. Furthermore, the desulfation was proportional to a decrease in the affinity to AT and HC-II. None of these agents produced any activation of ADP induced platelet aggregation, however in whole blood flowcytometric studies ADP induced aggregation was augmented by 6 kDa SAH whereas the mono and disulfated derivatives produced variable effects. Heparinase I and II did not produce any digestion of any of these derivatives. Incubation of the 6 kDa SAH and the 2 desulfated and 2,6 disulfated derivatives produced varying degrees of inhibition of the Lewis-lung carcinoma cell cultures which was not dependent on degree of sulfation. These studies suggest that the non-serpine mediated effect of these agents may be independent of the sulfation pattern and the relative anticoagulant effects of these sulfaminoheparosan.