Chang LC, Lee HF, Yang ZQ and Yang VC Low Molecular Weight Protamine (LMWP) as Nontoxic Heparin/Low Molecular Weight Heparin Antidote (I): Preparation and Characterization AAPS PharmSci 2001; 3 (3) article 17 (https://www.pharmsci.org/scientificjournals/pharmsci/journal/01_17.html).

Figures and Tables

Figure 1.Chromatograms from a heparin affinity chromatography of (A) low molecular weight protamine; (B) protamine; and (C) antithrombin III.

Table 1. Properties of Protamine and Low Molecular Weight Protamine (LMWP)

^aElution concentration from a heparin affinity column

^bDetermined by using the reverse-phase high performance liquid chromatography method

^cDetermined by using the matrix assisted laser desorption - time of flight mass spectrometric analysis

Figure 2. Neutralization of heparin by protamine and low molecular weight protamine as measured by the anti-Xa chromogenic assay. The dashed line represents the control (ie, 100% neutralization).

Figure 3. (A) Reverse-phase high performance liquid chromatography chromatogram of protamine. Flow rate: 1.0 mL/min; temperature: 30oC; column: Alltech C4 (0.46 x 25 cm, 5 µm inner diameter). Gradient was prepared by mixing 0.1% trifluoroacetic acid (TFA) with acetonitrile (10%-20% over 90 minutes). Absorbance was monitored at 215 nm. (B) Amino acid sequence and molecular weight of the fractions in protamine.

Table 2. Sequences Identified by the Mass-Mapping Approach for Peptides in Low Molecular Weight Protamine (LMWP) Fractions

^aMass was calculated as the average M+1 of the free base

^bDetermined by using the matrix assisted laser desorption - time of flight mass spectrometric analysis

*See Figure 3 for information

#See Figure 1 for information

Figure 4. (A) Chromatograms of the CRRRRRRR (CR₇ ) monomer and dimer from a heparin affinity column. (B) Heparin neutralization by protamine, CR₇ monomer, and CR₇ dimer as measured by the anti-Xa assay. The dashed line represents the control (ie, 100% neutralization).

Table 3. Efficacy in Heparin Neutralization by Protamine, Low Molecular Weight Protamine Fractions, and CRRRRRRR (CR7) Peptides

*See Table 2 for information

?Both protamine and TDSP3 are heterogeneous compounds. Efficacy was determined by using an approximate molecular weight of 4200 and 1200 d for protamine and TDSP3, respectively.