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1.  Low molecular weight protamine as nontoxic heparin/low molecular weight heparin antidote (III): Preliminary in vivo evaluation of efficacy and toxicity using a canine model 
AAPS PharmSci  2001;3(3):24-31.
Heparin employed in cardiovascular surgeries often leads to a high incidence of bleeding complications. Protamine employed in heparin reversal, however, can cause severe adverse reactions. In an attempt to address this clinical problem, we developed low molecular weight protamine (LMWP) as a potentially effective and less toxic heparin antagonist. A homogeneous 1880-d peptide fragment, termed LMWP-TDSP5 and containing the amino acid sequence of VSRRRRRRGGRRRR was derived directly from protamine by enzymatic digestion of protamine with thermolysin. In vitro studies demonstrated that TDSP5 was capable of neutralizing various anticoagulant functions of both heparin and commercial low molecular weight heparin preparations. In addition, TDSP5 exhibited significantly reduced crossreactivity toward mouse sera containing antiprotamine antibodies. TDSP5 showed a decrease in its potential in activating the complement system. All of these findings suggested the possibility of markedly reduced protamine toxicity for TDSP5.
In this article, we conducted preliminary in vivo studies to further demonstrate the feasibility and utility of using LMWP as a nontoxic clinical protamine substitute. Dogs were chosen as test animals because they were known to magnify the typical human response to protamine. By using a full spectra of biological and clinical assays for heparin, including the anti-IIa and anti-Xa chromogenic assays and the activated partial, thromboplastin time and TCT clotting assays, TDSP5 showed that it could completely neutralize all these different anticoagulant functions of heparin in dogs. Although administration of protamine in dogs produced a significant reduction in mean arterial blood pressure (−14.9 mm Hg) and elevation in pulmonary artery systolic pressure (+5.0 mm Hg), the use of TDSP5 in dogs did not elicit any statistically significant change in any of the variables measured. Furthermore, the use of LMWP also significantly reduced the protamine-induced transient thrombocytopenic and granulocytopenic responses. The white blood cell counts and platelet counts decreased to 82.1% and 60.0% of baseline, respectively, in dogs given intravenous protamine compared to 97.8% and 88.6% of baseline in dogs receiving TDSP5. These preliminary findings indicated that LMWP could potentially provide an effective and safe means to control both heparin- and protamine-induced complications.
doi:10.1208/ps030319
PMCID: PMC2751014  PMID: 11741270
Heparin Neutralization; Protamine Toxicity; aPTT/TCT Heparin Clotting Assays; Anti-IIa Anti-Xa Chromogenic Assays; Hemodynamic/Hematologic Responses
2.  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.
doi:10.1208/ps030318
PMCID: PMC2751013  PMID: 11741269
Heparin/LMWH neutralization; protamine toxicity; aPTT clotting assay; anti-Xa assay; complement Activation; immunogenicity; cross-reactivity

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