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1.  Low molecular weight protamine (LMWP) as nontoxic heparin/low molecular weight heparin antidote (II): In vitro evaluation of efficacy and toxicity 
AAPS PharmSci  2001;3(3):15-23.
Patients undergoing anticoagulation with heparin or low molecular weight heparin (LMWH) require a superior antidote that possesses more selective biological actions and a better safety profile than protamine. We had previously developed 2 low molecular weight protamine (LMWP) fractions (TDSP4 and TDSP5) from thermolysin-digested protamine as potential nontoxie, heparin-neutralizing agents. In this, the second article in this series, studies focused on in vitro evaluation of heparin/LMWH-neutralizing efficacy and putative toxicity. These LMWP fractions, particularly TDSP5, were effective and fully capable of neutralizing a broad spectrum of heparin-induced anticoagulant activities (ie, aPTT, anti-Xa, and anti-IIa activities). Additionally, these LMWP fractions could neutralize the activities of commercial LMWH. As assessed by the anti-Xa assay, TDSP5 was as effective as, although less potent than, protamine in reversing the activity of Mono-Embolex (molecular weight 5000–7000) and 2 other different sizes (molecular weight of 3000 and 5000 d) of LMWH preparations. Furthermore, compared with protamine, TDSP5 exhibited a much-reduced toxicity and thus an improved safety profile, as reflected by its reduced ability to activate the complement system and cross-react with the antiprotamine antibodies, which are 2 primary indices of protamine toxicity.
PMCID: PMC2751013  PMID: 11741269
Heparin/LMWH neutralization; protamine toxicity; aPTT clotting assay; anti-Xa assay; complement Activation; immunogenicity; cross-reactivity
2.  Low molecular weight protamine (LMWP) as nontoxic heparin/low molecular weight heparin antidote (I): Preparation and characterization 
AAPS PharmSci  2001;3(3):7-14.
Low molecular weight protamine (LMWP) appears to be a promising solution for heparin neutralization without the protamine-associated catastrophic toxic effects. The feasibility of this hypothesis was proven previously by using a peptide mixture produced from proteolytic digestion of protamine. 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. 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. These fragments were fractionated using a heparin affinity chromatography, and their relative binding strengths toward heparin were elucidated. 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. 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. 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. 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 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. 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. 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.
PMCID: PMC2751012  PMID: 11741268
Heparin LMWH neutralization; Protamine toxicity; LMWP peptide sequences; MS fingerprint mapping; Mechanism of heparin neutralization

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