Role of Exosites 1 and 2 in Thrombin Reaction with Plasminogen Activator Inhibitor-1 in the Absence and Presence of Cofactors

Abstract

The cofactors heparin, vitronectin (VN), and thrombomodulin (TM) modulate the reactivity of alpha-thrombin with plasminogen activator inhibitor (PAI-1). While heparin and VN accelerate the reaction by approximately 2 orders of magnitude, TM protects alpha-thrombin from rapid inactivation by PAI-1 in the presence of VN. To understand how these cofactors function, we studied the kinetics of PAI-1 inactivation of alpha-thrombin, the exosite 1 variant gamma-thrombin, the exosite 2 mutant R93,97,101A thrombin, and recombinant meizothrombin in both the absence and presence of these cofactors. Heparin and VN accelerated the second-order association rate constant [k(2) = (7.9 +/- 0.5) x 10(2) M(-)(1) s(-)(1)] of alpha-thrombin with PAI-1 approximately 200- and approximately 240-fold, respectively. The k(2) value for gamma-thrombin [(7.9 +/- 0.7) x 10(1) M(-)(1) s(-)(1)] was impaired 10-fold, but was enhanced by heparin and VN approximately 280- and approximately 75-fold, respectively. Similar to inactivation of gamma-thrombin, PAI-1 inactivation of alpha-thrombin in complex with the epidermal growth factor-like domains 4-6 of TM (TM4-6) was impaired approximately 10-fold. The exosite 2 mutant R93,97,101A thrombin, which was previously shown not to bind heparin, and meizothrombin, in which exosite 2 is masked, reacted with PAI-1 at similar rates in both the absence and presence of heparin [k(2) = (1.3-1.5) x 10(3) M(-)(1) s(-)(1) for R93,97,101A thrombin and k(2) = (3.6-5.1) x 10(2) M(-)(1) s(-)(1) for meizothrombin]. Unlike heparin, however, VN enhanced the k(2) of R93,97,101A thrombin and meizothrombin inactivation approximately 80- and approximately 30-fold, respectively. Continuous kinetic analysis as well as competition kinetic studies in the presence of S195A thrombin suggested that the accelerating effect of VN or heparin occurs primarily by lowering the dissociation constant (K(d)) for formation of a noncovalent, Michaelis-type complex. Analysis of these results suggest that (1) heparin binds to exosite 2 of alpha-thrombin to accelerate the reaction by a template mechanism, (2) VN accelerates PAI-1 inactivation of alpha-thrombin by lowering the K(d) for initial complex formation by an unknown mechanism that does not require binding to either exosite 1 or exosite 2 of alpha-thrombin, (3) alpha-thrombin may have a binding site for PAI-1 within or near exosite 1, and (4) TM occupancy of exosite 1 partially accounts for the protection of thrombin from rapid inactivation by PAI-1 in the presence of vitronectin.

Vitronectin Functions as a Cofactor for Rapid Inhibition of Activated Protein C by Plasminogen Activator Inhibitor-1

“…The kЈ values were calculated as described above and plotted versus PAI-1 concentrations. The maximal inhibition rate constant (k) and K d values were determined by fitting the saturable dependence of kЈ values on PAI-1 concentrations to a hyperbolic equation as described previously (20).…”

“…Determination of Inhibition Stoichiometry-The inhibition stoichiometry for PAI-1 inactivation of APC in the presence of vitronectin and APC E192Q in both the absence and presence of vitronectin were determined by titration of 20 nM APC with increasing concentrations of PAI-1 corresponding to PAI-1/APC molar ratios of 0 -2, in the absence or presence of 600 nM vitronectin in the same TBS buffer system as described previously (20). The residual amidolytic activities of the wild type and mutant enzymes were monitored for up to 12 h at room temperature by the hydrolysis of SpPCa as described above.…”

“…1), previous studies of the vitronectin concentration dependence of thrombin inactivation by PAI-1 has indicated that the rate of reaction slightly decreases at higher concentrations of vitronectin, possibly suggesting that vitronectin may function by a template mechanism (31). However, other kinetic studies, employing several mutant thrombin derivatives, have not confirmed such a cofactor mechanism for vitronectin function (20). Thrombin also contains a Glu at position 192.…”

“…Interestingly, the reactivity of thrombin E192Q with PAI-1 was also markedly improved independent of vitronectin, suggesting that vitronectin may accelerate PAI-1 inhibition of both proteinases by a similar mechanism (data not shown). In the case of thrombin, thrombomodulin occupancy of exosite 1 protects the proteinase from rapid inhibition by PAI-1 in the presence of vitronectin (20). To determine whether complex formation of APC with its cofactor protein S on membrane surfaces protects it from inhibition by the vitronectin-PAI-1 complex, kinetic studies were also carried out in the presence of human protein S (220 nM) on either PC/PS or PC/PS/PE vesicles in the range of 0 -50 g/ml.…”

Conservation of Critical Functional Domains in Murine Plasminogen Activator Inhibitor-1

“…The point mutation at the P1 site (R346A) effectively diminished its inhibitory activity, as shown for human PAI-1. In the presence of glycosaminoglycans such as heparin, PAI-1 becomes an effective inhibitor toward thrombin by a bridging mechanism (17), with an even higher affinity than antithrombin, suggesting that PAI-1 not only regulates fibrinolysis through the plasminogen activation system, but also has potential to regulate the coagulation system. The heparin-binding site of PAI-1 has been identified and is located within several residues of helix D, including Arg 76 (18).…”