Identification and analyses of inhibitors targeting apolipoprotein(a) kringle domains KIV-7, KIV-10, and KV provide insight into kringle domain function.

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 µm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6-2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).

Lp(a) is not associated with diabetes but affects fibrinolysis and clot structure ex vivo.

Lipoprotein (a) [Lp(a)] is a low density lipoprotein (LDL) with one apolipoprotein (a) molecule bound to the apolipoprotein B-100 of LDL. Lp(a) is an independent risk factor for cardiovascular disease (CVD). However, the relationship of Lp(a) to diabetes and metabolic syndrome, both known for increased CVD risk, is controversial. In a population based study on type two diabetes mellitus (T2DM) development in women, Lp(a) plasma levels showed the well known skewed distribution without any relation to diabetes or impaired glucose tolerance. A modified clot lysis assay on a subset of 274 subjects showed significantly increased clot lysis times in T2DM subjects, despite inhibition of PAI-1 and TAFI. Lp(a) plasma levels significantly increased the maximal peak height of the clot lysis curve, indicating a change in clot structure. In this study Lp(a) is not related to the development of T2DM but may affect clot structure ex vivo without a prolongation of the clot lysis time.

The new oral immunomodulating drug DiNAC induces brachial artery vasodilatation at rest and during hyperemia in hypercholesterolemic subjects, likely by a nitric oxide-dependent mechanism.

OBJECTIVES: To investigate if the immunomodulator drug DINAC (1) affects arterial dimensions in asymptomatic patients with hypercholesterolemia, (2) has effects on leucocyte markers of inflammation and (3) has in vitro effects on nitric oxide synthase (NOS) in human umbilical vein endothelial cells (HUVEC). METHODS AND RESULTS: One hundred and fifty-three patients with asymptomatic hypercholesterolemia were randomized to either 100 or 500 mg of DINAC or placebo in a double-blind, parallel-group fashion for 24 weeks. Treatment at the highest dose induced a significant increase in resting brachial artery diameter measured by ultrasound and also induced a significant increase in vessel diameter during hyperemia. However, flow-mediated vasodilation (FMD) and the vasodilatory response to nitroglycerin, lipid levels or leukocyte count were unaltered. Expression of several cell surface markers of inflammation, like CD11b and CD25, were reduced by treatment. In vitro, DINAC counteracted TNF-alpha induced reductions in NO levels and in NOS protein and mRNA levels. CONCLUSION: The immunomodulator drug DINAC increased brachial artery diameter at rest and during hyperemia in asymptomatic subjects with hypercholesterolemia without affecting blood lipid levels. Based on parallel in vitro studies this effect is likely due to an enhancement of NOS activity.

Antithrombotic effects of ximelagatran plus acetylsalicylic acid (ASA) and clopidogrel plus ASA in a human ex vivo arterial thrombosis model.

It was the objective of this study to compare the antithrombotic effects and bleeding profiles of the oral direct thrombin inhibitor ximelagatran, an anticoagulant, and the antiplatelet agent clopidogrel on top of steady-state acetylsalicylic acid (ASA) in a human arterial thrombosis model. Healthy male volunteers (n=62) received ASA (160 mg once daily), plus either clopidogrel for 6 days (loading dose 300 mg, then 75 mg once daily), or a single dose of ximelagatran (36 or 72 mg) on Day 6. Changes in total thrombus area (TTA) under low shear rate (LSR; 212 s(-1)) and high shear rate (HSR; 1690 s(-1)) conditions were measured, using the ex vivo Badimon perfusion chamber model pre-dose and 2 and 5 hours after dosing on Day 6, and capillary bleeding times (CBT) were determined. Ximelagatran plus ASA significantly reduced TTA under LSR and HSR, compared with ASA alone. Ximelagatran plus ASA reduced TTA more than clopidogrel plus ASA under LSR after 2 hours (36 mg, P=0.0011; 72 mg, P<0.0001) and 5 hours (72 mg, P=0.0057), and under HSR after 2 and 5 hours (72 mg, P<0.05). Compared with ASA alone, CBT was markedly prolonged by clopidogrel plus ASA (ratio 6.4; P<0.0001) but only slightly by ximelagatran plus ASA (72 mg ximelagatran, ratio 1.4; P=0.0010). Both drug combinations were well tolerated. Oral ximelagatran plus ASA has a greater antithrombotic effect in this human ex vivo thrombosis model and a less pronounced prolongation of bleeding time than clopidogrel plus ASA.

No pharmacokinetic or pharmacodynamic interaction between atorvastatin and the oral direct thrombin inhibitor ximelagatran.

In this randomized, 2-way crossover study, the potential for interaction was investigated between atorvastatin and ximelagatran, an oral direct thrombin inhibitor. Healthy female and male volunteers (n = 16) received atorvastatin 40 mg as a single oral dose and, in a separate study period, ximelagatran 36 mg twice daily for 5 days plus a 40-mg oral dose of atorvastatin on the morning of day 4. In the 15 subjects completing the study, no pharmacokinetic interaction was detected between atorvastatin and ximelagatran for all parameters investigated, including melagatran (the active form of ximelagatran) area under the plasma concentration versus time curve (AUC) and maximum plasma concentration, atorvastatin acid AUC, and AUC of active 3-hydroxy-3-methyl-glutaryl-coenzyme-A (HMG-CoA) reductase inhibitors. Atorvastatin did not alter the melagatran-induced prolongation of the activated partial thromboplastin time, and both drugs were well tolerated when administered in combination. In conclusion, no pharmacokinetic or pharmacodynamic interaction between atorvastatin and ximelagatran was observed in this study.