Utility and variability of three non-invasive liver fibrosis imaging modalities to evaluate efficacy of GR-MD-02 in subjects with NASH and bridging fibrosis during a phase-2 randomized clinical trial.

BACKGROUND: Given the worldwide prevalence of NAFLD and NASH, there is a need to develop treatments to slow or reverse disease progression. GR-MD-02 (galactoarabino-rhamnogalaturonate) has been shown to reduce hepatic fibrosis in animal studies, and lower serum biomarkers of NASH fibrogenesis in humans. The primary aim of this study was to determine the difference between four-months of treatment with GR-MD-02 or placebo in liver inflammation and fibrosis as measured by iron-corrected T1 (cT1) mapping, a non-invasive magnetic resonance imaging (MRI) biomarker that correlates with the extent of hepatic fibro-inflammatory disease. The secondary aims were to determine change in liver stiffness as measured by magnetic resonance elastography (MRE) and shear-wave ultrasonic elastography (LSM), and to explore test-retest repeatability of the three biomarkers. MATERIALS AND METHODS: Thirty subjects (13 females, 46-71 years) with NASH and advanced fibrosis were recruited. Subjects were randomized to receive 8 mg.kg-1 GR-MD-02 (via IV infusion) or placebo, administered biweekly over a 16-week period. Therapeutic efficacy was examined using cT1, MRE, and LSM. Statistical analyses on group differences in the biomarkers were performed using robust ANCOVA models adjusting for baseline measurement and additional covariates. RESULTS: There was no significant difference in cT1 (p = 0.16) between GR-MD-02 and placebo groups following a 16-week intervention. There was also no significant difference in liver stiffness, measured by MRE (p = 0.80) or LSM (p = 0.63), between groups. Examination of repeatability of the cT1, MRE and LSM revealed coefficient of variations of 3.1%, 11% and 40% respectively. CONCLUSIONS: 8 mg.kg-1 of GR-MD-02 had no significant effect on non-invasive biomarkers of liver inflammation or fibrosis over a 4-month period. Histological confirmation was not available in this study. The high reproducibility of the primary outcome measure suggests that cT1 could be utilized for monitoring longitudinal change in patients with NASH.

Mechanism of low-molecular-weight heparin reversal by platelet factor 4.

Low-molecular-weight heparins (LMWH) exert their anticoagulant effect by accelerating anti-thrombin (AT) inactivation of factor Xa (fXa) and thrombin. To address the hypothesis of a calcium-dependent template mechanism in LMWH activity, we compared the ability of the heparin neutralising agent Platelet Factor 4 (PF4) to inhibit various therapeutic LMWH in a kinetic assay. Neutralization coefficients by PF4 and apparent affinities of PF4 for various LMWH increased in a molecular weight-dependent manner. Protamine sulphate neutralized heparin via a non-specific mechanism. EDTA abolished the calcium-dependent acceleration of the fXa-AT reaction, indicating that the bridging mechanism contributed significantly to LMWH activity. Within a low range of LMWH concentration (<0.2 U/ml), excess AT over PF4 (4:1) had no effect on PF4 activity, indicating that PF4 and AT binding to heparin were independent of each other. Instead, increasing enzyme concentration reversed the negative effect of heparin-bound AT on PF4-dependent neutralization. Inhibition of thrombin by heparin was also neutralized by PF4, albeit to a higher extent than the fXa-AT reaction. Altogether, these results suggested that an interaction of PF4 with protease mediated the association of PF4 to the heparin chain. We propose that PF4 participates in the anti-fXa dependence of LMWH due to its major effect on the anti-thrombin activity of LMWH and that inhibition of fXa via the template mechanism plays an essential role in LMWH activity and pharmacokinetics, because PF4 specifically targets this mechanism.

Dual effect of Platelet Factor 4 on the activities of Factor Xa.

Platelet Factor 4 (PF4) prevents inhibition of blood coagulation proteases by heparin via formation of a putative enzyme-PF4 complex. To investigate the contribution of the latter, the activity of factor Xa (fXa) was determined in chromogenic assays measuring hydrolysis of a peptide substrate S2765 or cleavage of the macromolecular substrate prothrombin in the activating complex, prothrombinase. Upon preincubation with fXa and heparin, PF4 at about 250 nM decreased the k(cat) of S2765 hydrolysis about fivefold and that of prothrombin activation about 25-fold. In the presence of saturating fVa, inhibition of fXa by PF4 was abolished, while in the presence of limiting fVa, PF4 altered the interaction of fXa with fVa. Interestingly, high concentrations of PF4 restored fXa activity toward S2765 and prothrombin, indicating a dual effect of PF4 on fXa activities. These findings suggest that PF4 in the presence of heparin is an allosteric effector of the prothrombinase complex.

Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa by preventing interaction(s) of enzyme with polysaccharide.

Platelet factor 4 (PF4) is a heparin-binding protein which exhibits anti-heparin activities through the inhibition of antithrombin (AT)-dependent reactions with the serine proteases thrombin and factor Xa. PF4 also neutralizes heparan sulfate (HS), a glycosaminoglycan (GAG) present on the surface of endothelial cells, thereby possibly modulating an anticoagulant response. Previous models of PF4 mechanism did not distinguish whether PF4 causes steric hindrance of AT binding to fXa or of AT binding to the surface of the GAG chain. To shed light on the mechanism of PF4, studies of HS/heparin-catalyzed fXa inactivation by AT were undertaken. The results were consistent with PF4 directly interfering with AT binding to fXa rather than AT binding to the GAG chain, since PF4 did not prevent the heparin-dependent increase in AT intrinsic fluorescence. In fact, PF4 mechanism was competitive with respect to AT and non-competitive with respect to fXa, suggesting inhibition of important regulatory/catalytic interactions of fXa with the polysaccharide. Altogether, the results suggested a model by which PF4 bound to proximal (but distinct) sites to AT, resulting in steric interference of fXa binding to both polysaccharide and AT. It is proposed that PF4 inhibited the sequence of events recapitulated in the template mechanism describing heparin-dependent inhibition of fXa.