Aged garlic extract (AGE) has been shown previously to have moderate cholesterol-lowering and blood pressure-reducing effects. We have now investigated whether platelet function, a potential risk factor for cardiovascular disease, can be inhibited by AGE administration. In a randomized, double-blind study of normal healthy individuals (n = 34), both men and women, the effect of AGE was evaluated in doses between 2.4 and 7.2 g/d vs. equal amounts of placebo. Platelet aggregation and adhesion were measured at 2-wk intervals throughout the study. Threshold concentrations for epinephrine and collagen increased moderately during AGE administration compared with the placebo and baseline periods. Only at the highest supplementation level did AGE show a slight increase in the threshold level of ADP-induced aggregation. Platelet adhesion to collagen, fibrinogen and von Willebrand factor was investigated by perfusing whole blood through a laminar flow chamber under controlled flow conditions. Adherence of platelets was inhibited by AGE in a dose-dependent manner when collagen was the adhesive surface perfused at low shear rates ( approximately 30 s(-1)). At high shear rates (1200 s(-1)), AGE also inhibited platelet adhesion to collagen but only at higher intake levels. Adhesion to von Willebrand factor was reduced only at 7.2 g/d AGE, but adherence to fibrinogen was potently inhibited at all levels of supplementation. Thus, AGE exerts selective inhibition on platelet aggregation and adhesion, platelet functions that may be important for the development of cardiovascular events such as myocardial infarction and ischemic stroke. We briefly review the effect of garlic preparations in general on cardiovascular risk factors and point out differences between AGE and other garlic preparations that we feel are important to explain the efficacy of AGE.

To cite this article: Adams TE, Li W, Huntington JA. Molecular basis of thrombomodulin activation of slow thrombin. J Thromb Haemost 2009; 7:1688-95.Summary. Background: Coagulation is a highly regulated process where the ability to prevent blood loss after injury is balanced against the maintenance of blood fluidity. Thrombin is at the center of this balancing act. It is the critical enzyme for producing and stabilizing a clot, but when complexed with thrombomodulin (TM) it is converted to a powerful anticoagulant. Another cofactor that may play a role in determining thrombin function is the monovalent cation Na + . Its apparent affinity suggests that half of the thrombin generated is in a Na + -free ÔslowÕ state and half is in a Na + -coordinated ÔfastÕ state. While slow thrombin is a poor procoagulant enzyme, when complexed to TM it is an effective anticoagulant. Methods: To better understand this molecular transformation we solved a 2.4 Å structure of thrombin complexed with EGF domains 4-6 of TM in the absence of Na + and other cofactors or inhibitors. Results: We find that TM binds as previously observed, and that the thrombin component resembles structures of the fast form. The Na + binding loop is observed in a conformation identical to the Na + -bound form, with conserved water molecules compensating for the missing ion. Using the fluorescent probe p-aminobenzamidine we show that activation of slow thrombin by TM principally involves the opening of the primary specificity pocket. Conclusions: These data show that TM binding alters the conformation of thrombin in a similar manner as Na + coordination, resulting in an ordering of the Na + binding loop and an opening of the adjacent S1 pocket. We conclude that other, more subtle subsite changes are unlikely to influence thrombin specificity toward macromolecular substrates.

Protein C inhibitor (PCI) is a widely distributed, multifunctional member of the serpin family of protease inhibitors, and has been implicated in several physiological processes and disease states. Its inhibitory activity and specificity are regulated by binding to cofactors such as heparin, thrombomodulin and phospholipids, and it also appears to have non-inhibitory functions related to hormone and lipid binding. Just how the highly conserved serpin architecture can support the multiple diverse functions of PCI is a riddle best addressed by protein crystallography. Over the last few years we have solved the structure of PCI in its native, cleaved and protein-complexed states. They reveal a conserved serpin fold and general mechanism of protease inhibition, but with some unique features relating to inhibitory specificity/promiscuity, cofactor binding and hydrophobic ligand transport.