Plasminogen activator inhibitor-1 is locked in active conformation and polymerizes upon binding ligands neutralizing its activity.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serpin super-family, forms a covalent complex with its target proteinases, such as tissue and urokinase plasminogen activators. Thus, PAI-1 controls the physiological and pathological proteolysis. An abnormal expression of PAI-1 has been observed in different diseases, which can be treated by returning the proteolysis back to normal physiological levels. It has been reported that some PAI-1 inhibitors neutralize its activity by accelerating the conversion of PAI-1 into a latent form. We have found small organic chemicals that also neutralize PAI-1 activity, but by a different mechanism. Using the NBD fluorescent probe [N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] incorporated into the reactive center loop (RCL) of PAI-1, we measured the kinetics of conversion from an active to a latent form. Unexpectedly, we found that some inhibitors of PAI-1 arrest this serpin in its active form instead of increasing the speed of conversion. Using docking calculations, we located two possible binding sites for these chemicals. The sites are in proximity of the P1/P1' amino acids of the RCL of PAI-1. Binding in this area can inactivate PAI-1 and additionally create a steric obstacle on the RCL making insertion of this loop between the A3 and A5 strands more difficult; hence abolishing a necessary step in the conversion of this protein into the latent form. Additionally, PAI-1 inhibitors link the RCL of one PAI-1 molecule with the strand 3C and strand 4C or helix A and strand 1B regions of the other PAI-1 molecule aiding polymerization or stabilizing the junction of the two. The polymerization of PAI-1 reduces PAI-1 activity by encapsulating the critical RCL fragment inside the formed PAI-1/PAI-1 polymers.

Differential effects of clivarin and heparin in patients undergoing hip and knee surgery for the generation of anti-heparin-platelet factor 4 antibodies.

The pathophysiology of heparin-induced thrombocytopenia (HIT) syndrome is mediated via a heterogeneous group of heparin(s)-platelet factor 4 (H-PF4) complexes bound to their antibodies. These anti-H-PF4 (AHPF4) antibodies that are capable of binding to the FcgammaRIIA receptor [cluster of differentiation (CD) 32] on platelets, resulting in platelet activation, widely vary in their specific activities as platelet activation (functionality). Predisposing factors related to specific pathologic conditions may also contribute to the generation of these antibodies and their relative functionality during HIT syndrome. To understand this phenomenon, a sub-study was carried out in patients undergoing elective total hip and knee replacement surgery (ECHOS Study) and who were treated with unfractionated heparin (UFH) and a low-molecular-weight heparin (LMWH; Clivarin). Approximately 600 patients per arm [UFH=7,500 anti-Xa U twice a day (b.i.d.) subcutaneous (s.c.) and clivarin=4200 U once daily (o.d.) s.c.], age >40 years, received prophylactic treatment for a minimum of 11-14 days. Plasma samples were collected at pre-dose, days 2-4, days 11-14 and at follow-up 6-8 weeks after discharge and were analyzed for AHPF4 antibody titers. Functionality of the enzyme-linked immunosorbant assay (ELISA)-positive AHPF4 antibodies to cause platelet activation was tested by 14C-serotonin release assay (SRA). Both UFH and clivarin treatments in orthopedic surgical patients resulted in a progressive generation of AHPF4 antibodies. The relative prevalence/functionality of AHPF4 antibodies in clivarin arm was markedly lower (two- to threefold, p<0.001) as compared to UFH at each time point. Most of the samples in clivarin group were found to be SRA negative, suggesting the presence of AHPF4 antibodies that did not activate platelets (nonfunctional). Within the UFH arm, the relative prevalence/functionality of AHPF4 antibodies was much higher (p<0.002) in knee group compared to the corresponding hip group. This study, for the first time, reports on the elevated levels of AHPF4 antibodies generated by heparin associated with the pathogenesis of knee surgery. Clinical significance of the differential generation of HIT-associated antibodies remains unexplored at this time.