Table 1.

Activity and characterization of antiheparin virus-like particles.

Particle	Maximum charge relative to WT[a]	Clotting time [s][b]	Heparin inhibition [%][c]	Retention time, [NaCl] at elution[d]	ZP [mV][e]
No heparin	n/a	54 ± 2	100	n/a	n/a
Heparin only	n/a	118 ± 4	0	n/a	n/a
K16M	−180	117 ± 1	2 ± 2	–	−0.8 ± 0.6
WT	0	73 ± 2	70 ± 3	–	0.6 ± 0.5
N10R	180	86 ± 3	50 ± 5	60.3 min, 0.31M	0.5 ± 0.4
T18R	180	57 ± 1	95 ± 2	42.3 min, 0.21M	0.7 ± 0.5
D14R	360	54 ± 1	100 ± 2	63.3 min, 0.33M	2.9 ± 1.2
WT-(R8G2)95	40[f]	55 ± 1	92 ± 2	–	−0.6 ± 0.3
K16M-(R5)50	−110[g]	67 ± 1	80 ± 2	–	−11.5 ± 2.0

^[a]The acylation or replacement of an amine-containing residue is counted as −1; the addition of a positively-charged residue counts as +1.

^[b]Determined by the aPTT assay as described in the text. Error limits are the standard deviation from a minimum of three independent experiments.

^[c]Inhibition of heparin’s effect on blood plasma clotting (100 % = clotting time in the absence of heparin; 0 % = clotting time in the presence of heparin but no candidate inhibitor).

^[d]Retention time on cation exchange chromatography (HiTrap SP HP column, 10 mM KP_i, pH 7, eluted with NaCl gradient by increasing by 12.5 mM min⁻¹). A dash indicates that the particle eluted in the void volume without added NaCl, and therefore does not interact with the cation exchange resin.

^[e]Zeta potential determined in 25 mM KP_i, pH 7.

^[f]Includes the removal of 4 positive charges per subunit by acylation of sidechain and N-terminal amino groups (−720) plus the addition of a maximum of 760 positive charges by attachment of 95 G₂R₈ chains.

^[g]Includes the removal of one positive charge per subunit by K16M substitution (−180), another by N-acylation (−180), and the addition of 250 charges by attachment of 50 R₅ chains.