Comparative Studies on the HIT Antibody Mediated Platelet Aggregation / Serotonin Release by Synthetic Pentasaccharide and Two Chemoenzymatically Synthesized Heptasaccharides
Authors: Walenga Jeanine M. , Aranda Chris , Linhardt Robert , Liu Jian , Lewis Mary , Hoppensteadt Debra , Fareed Jawed 
Published: October 2019

Abstract
Abstract 2231
Synthetic oligosaccharides such as the Pentasaccharide (Arixtra) and its derivatives are antithrombotic agents which are clinically used in the management of thrombotic indications. These agents are claimed to be devoid of triggering the generation of HIT antibodies and therefore do not produce HIT syndrome. Several additional synthetic oligosaccharides are also developed for the management of thrombotic indications. More recently, two novel ultra low molecular weight heparins (ULMWHs) were synthesized chemoenzymatically. These ULMWHs are both heptasaccharides with AT pentasaccharide-binding sites within their structures which is comparable to the pentasaccharide. The IC50 of the anti-Xa effects of the agents are comparable to pentasaccharide, ranging from 0.7 to 1.0 ug/ml in comparison to pentasaccharide which is 0.8 ug/ml. All agents produced comparable anticoagulant effects in the Heptest clotting time. The purpose of this study is to compare the effects of the pentasaccharide and the two heptasaccharides namely ULMWH1 and ULMW2 in the HIT mediated platelet aggregation and serotonin release assays. In addition platelet factor 4 release in whole blood was also studied. The HIT mediated platelet aggregation studies were carried out utilizing a HIT antibody positive pool plasma preparation. PRP collected from 10 individual donors (250ul) was mixed with 200ul of HIT pool plasma and equilibrated at 37° C for 3 minutes. 50ul of 1, 10, and 100 ug/ml of each of these agents was added to trigger the platelet aggregation responses. Enoxaparin was used as a positive control in the same concentration ranges. The serotonin release assay was carried out using the standard method in the same concentration range monitoring the release of 14C serotonin with each of these agents. The PF4 release was also measured using an ELISA method for serotonin measurement in whole blood samples incubated with each of these agents at concentrations of 0, 10 and 100ug/ml. The pentasaccharide and the two heptasaccharides did not produce any aggregation of platelets in the HIT aggregation assay at all concentrations whereas Enoxaparin at concentrations of > 1ug/ml produces positive aggregation responses. In the 14C assay none of the agents produced any release of serotonin however Enoxaparin produced 14C release at all concentration studied. Similarly, the pentasachhardide and heptasaccharides did not produce any platelet factor 4 from the whole blood incubation studies, however Enoxaparin produced a measurable release of platelet factor 4. Interestingly, unlike Enoxaparin, the anti-Xa and heptest effects of these agents were not neutralized by platelet factor 4 or protamine sulfate. These results demonstrate that the pentasaccharide and chemoenzymatically synthesized ULMWH1 and ULMWH2 do not meditate HIT antibody induced aggregation and serotonin release. Therefore, these heptasaccharides may exhibit comparable safety profile to the pentasaccharide in heparin compromised patients.

Disclosures:
No relevant conflicts of interest to declare.