Solvent accessibility of the thrombin-thrombomodulin interface.

The kinetics of solvent accessibility at the protein-protein interface between thrombin and a fragment of thrombomodulin, TMEGF45, have been monitored by amide hydrogen/deuterium (H/2H) exchange detected by MALDI-TOF mass spectrometry. The interaction is rapid and reversible, requiring development of theory and experimental methods to distinguish H/2H exchange due to solvent accessibility at the interface from H/2H exchange due to complex dissociation. Association and dissociation rate constants were measured by surface plasmon resonance and amide H/2H exchange rates were measured at different pH values and concentrations of TMEGF45. When essentially 100% of the thrombin was bound to TMEGF45, two segments of thrombin became completely solvent-inaccessible, as evidenced by the pH insensitivity of the amide H/2H exchange rates. These segments form part of anion-binding exosite I and contain the residues for which alanine substitution abolishes TM binding. Several other regions of thrombin showed slowing of amide exchange upon TMEGF45 binding, but the exchange remained pH-dependent, suggesting that these regions of thrombin were rendered only partially solvent-inaccessible by TMEGF45 binding. These partially inaccessible regions of thrombin form both surface and buried contacts into the active site of thrombin and contain residues implicated in allosteric changes in thrombin upon TM binding.

Electrostatic dependence of the thrombin-thrombomodulin interaction.

The rate constants for the binding interaction between thrombin and a fully active fragment of its anticoagulant cofactor, thrombomodulin, have been determined by surface plasmon resonance. At physiological ionic strength, the k(a) was 6.7x10(6) M(-1) s(-1 )and the dissociation rate constant was 0.033 s(-1). These extremely fast association and dissociation rates resulted in an overall binding equilibrium constant of 4.9 nM, which is similar to previously reported values. Changing the ionic strength from 100 mM to 250 mM NaCl caused a tenfold decrease in the association rate while the dissociation rate did not change significantly. A similar effect was observed with tetramethylammonium chloride. A Debye-Hückel plot of the data had a slope of -6 and an intercept at 0 ionic strength of 10(9) M(-1) s(-1). The same slope and intercept were obtained for data that was collected in the presence of glycerol to slow the association rates. These results show that the thrombin-TM456 interaction is extremely rapid and nearly completely electrostatically steered. An association model is presented in which TM456 approaches thrombin along the direction of the thrombin molecular dipole.

Mechanism of thrombin clearance by human astrocytoma cells.

Astroglial cells secrete a variety of factors that contribute to the regulation of neurite initiation and continued outgrowth, among them proteases and protease inhibitors. An alteration in the balance between these proteins has been implicated in Alzheimer's disease, resulting in an accumulation of thrombin:protease nexin 1 (PN1) complexes in the brains of these patients. This report aims at providing a biochemical explanation for this phenomenon. We show that human astrocytoma cells bind and internalize thrombin and thrombin:PN1 complexes efficiently by a PN1-dependent mechanism. Binding was potently inhibited by soluble heparin and did not occur with the mutant PN1 (K7E) deficient in heparin binding. Receptor-associated protein, an antagonist of the low-density lipoprotein receptor-related protein (LRP), inhibited internalization of thrombin by the astrocytoma cells, but did not affect cell-surface binding. The results are consistent with a mechanism by which astrocytoma cells clear thrombin in a sequential manner: thrombin is first complexed with PN1, then bound to cell-surface heparins, and finally internalized by LRP. This mechanism provides a link between the neuronal growth regulators thrombin and PN1 and proteins genetically associated with Alzheimer's disease, such as LRP.