Dual effect of histone H4 on prothrombin activation

Abstract

Essentials Prothrombin converts slowly to thrombin upon interaction with histone H4. Histone H4 may also affect the reactivity of prothrombin toward factor Xa. Histone H4 enhances or inhibits activation by factor Xa depending on cofactor Va. The results reveal an unanticipated dual effect of histone H4 on prothrombin activation by factor Xa.

Summary: Background Recent studies have documented the ability of prothrombin to convert to the mature protease thrombin upon interaction with histone H4. The effect is abrogated by mutation of the catalytic Ser and requires the Gla domain. Objectives To explore the effect of histone H4 on the reactivity of prothrombin to its physiological activator factor (F) Xa, free or assembled in the prothrombinase complex. Methods The effect of histone H4 on prothrombin activation by FXa and prothrombinase is studied with kinetic assays. The potential epitope of prothrombin recognizing histone H4 is explored with electrostatic calculations using recent crystal structures. Results and Conclusions Binding of histone H4 has a dual effect on prothrombin activation by FXa that is of mechanistic significance: it enhances the reaction > 10-fold in the absence of cofactor Va, but produces complete inhibition in the presence of cofactor. Histone H4 binding to prothrombin produces very slow autoactivation independent of the coagulation cascade and promotes slow thrombin generation by FXa in the absence of phospholipids. In addition, histone H4 has a rapid and drastic inhibitory effect on prothrombin activation by prothrombinase that is likely to dominate pathophysiology.

Keywords: blood coagulation factors; enzyme precursors; prothrombin; thrombin; thrombosis.

Figure 1. Effect of histone H4 on prothrombin activation by factor Xa

Effect of histone H4 on the initial velocities of thrombin generation in the absence (a) or presence (d) of phospholipids, and in the presence of cofactor Va and phospholipids (g). The fold change measures the ratio between the initial velocity in the presence (v_i) versus and absence (v₀) of histone H4. The concentration of histone H4 producing half of the maximal effect is (a) 0.9±0.2 μM, (d) 1.0±0.2 μM and (g) 3.5±0.5 μM (g). The effect of histone H4 was also monitored by following the disappearance of prothrombin over time using SDS-PAGE under reducing conditions. In all assays, DAPA was added to inhibit thrombin and meizothrombin activity. Factor Xa cleaves prothrombin at R155, R271 and R320. Cleavage at R155 generates prethrombin-1 (Pre-1) and fragment-1 (F1) composed of the Gla domain and kringle-1. Cleavage at R271 produces prethrombin-2 (Pre-2) and fragment 1.2 (F1.2) composed of the Gla domain and the two kringles, and additional cleavage of prethrombin-2 at R320 generates the A and B chains. Direct cleavage of R320 generates the B chain and fragment 1.2.A (F1.2.A), where fragment 1.2 is attached to the A chain. In the presence of 3 μM histone H4, prothrombin activation by factor Xa is enhanced with (e, f) or without (b, c) phospholipids, but is inhibited in the presence of cofactor Va and phospholipids (h, i). The inhibition completely abrogates thrombin generation at high concentrations.

Figure 2. Regions of prothrombin for potential interaction with histone H4

The crystal structure of prothrombin devoid of residues 146–167 in the linker connecting the two kringles [3] reveals the general assembly of the modular components of the zymogen (GD=Gla domain, K1=kringle-1, K2=kringle-2, Ac=A chain, Bc=B chain). The left panel shows acidic (red) and hydrophobic (gold) residues with solvent exposure >70% in the Gla domain and adjacent kringle-1 that constitute prime candidates for mutagenesis screens aimed at identifying the epitope of prothrombin recognizing histone H4. Two of these residues (Y44 and D118) are associated with naturally occurring mutations causing severe bleeding [–22]. The panel at right shows the electrostatic potential surface (red=negative, blue=positive), contoured at ±2kT/e under conditions of 140 mM NaCl, pH 7.4. The surface identifies the Gla domain and neighbor kringle-1 as regions of charge complementarity to the positively charged histone H4.