IT’S ALWAYS A CHALLENGE when an anticoagulated patient is admitted to the hospital with a serious bleed or develops one in-house. Based on these results, the structural requirement for a compound to function as an effective heparin antidote and the possible mechanism involved in heparin neutralization were established.Published in the May 2013 issue of Today’s Hospitalist

The above finding was further validated by using a synthetic LMWP analogue-CRRRRRRR-and it was found that its heparin-neutralizing ability was increased by changing from a monomeric to a dimeric structure of this analogue peptide. By aligning the sequences with the potency in heparin neutralization of these LMWP fragments, it was found that retention of potency similar to that of protamine required the presence of at least 2 arginine clusters in the LMWP fragments such as the sequence of VSRRRRRRGGRRRR seen in the most potent LMWP fraction-TDSP5. A typical structural scaffold made by arginine clusters in the middle and nonarginine residues at the N-terminal of the peptide sequence was observed for all these LMWP fragments. By using a peptide mass spectrometry (MS) fingerprint mapping technique, the amino acid sequences of the microheterogeneous LMWP fragments in all these 5 elution fractions were readily identified. Among these 5 fractions, TDSP4 and TDSP5 contained 3 LMWP peptide fragments, and they were found to retain the complete heparin-neutralizing function of protamine. Five distinct fractions were eluted at NaCl concentration ranging from 0.4 to 1.0 M and were denoted as TDSP1 to TDSP5, in increasing order of eluting ionic strength. These fragments were fractionated using a heparin affinity chromatography, and their relative binding strengths toward heparin were elucidated. A number of LWMP fragments, composed of highly cationic peptides with molecular weights ranging from 700 to 1900 d, were prepared by digestion of native protamine with the protease thermolysin. To further examine the utility of this compound as an ultimate nontoxic protamine substitute, detailed studies on the purification and characterization of LMWP including the precise amino acid sequence, structure-function relationship, and possible mechanism were conducted. The feasibility of this hypothesis was proven previously by using a peptide mixture produced from proteolytic digestion of protamine. Low molecular weight protamine (LMWP) appears to be a promising solution for heparin neutralization without the protamine-associated catastrophic toxic effects.

Antidote for heparin