Pharmacodynamics analysis
The pharmacodynamic effects of inclacumab were assessed by determination of PLA formation, measurement of P-selectin inhibition, and the measurement of free and total soluble P-selectin plasma concentrations. Measurement of PLA formation was performed in whole blood using a validated flow cytometry method. After blood collection at the clinical research center, blood samples were activated with thrombin receptor-activating peptide (TRAP) to induce P-selectin expression on the platelet surface and subsequent PLA formation. Adding TRAP ex vivo results in activation of platelets and consequent increased P-selectin cell surface expression, resulting in more-robust PLA formation [The inhibition of the interaction between P-selectin and PSGL-1 by inclacumab was measured in clinical samples using a validated surface plasmon resonance (SPR) assay at BioAgilytix Europe GmbH (Hamburg, Germany). The effect of inclacumab was measured as percent inhibition of spiked P-selectin fused to immunoglobulin (Psel-FC) binding to glycosulfopeptide (GSnP-6), a peptide analog that structurally resembles the N-terminal domain of PSGL-1. Initially, biotinylated GSnP-6 was injected on the streptavidin chip to immobilize the peptide for analysis. Serum samples collected from participants were then injected over the GSnP-6 sensor chip in the presence of Psel-FC. The pre-dose sample for each patient was used as the donor-specific negative control (without Psel-FC) and positive controls (in the presence of Psel-FC). The percentage of P-selectin inhibition for each sample was then calculated based on the measured response units of the sample and the respective negative and positive control values for each patient. P-selectin inhibition measured using the SPR assay has a theoretical range between 0 (no inhibition) and 100% (complete inhibition). To enable interpretation, P-selectin inhibition values above 100% and below 0% were set to the boundary result value of 100% and 0%, respectively.Soluble P-selectin is a truncated form of P-selectin that may be produced by alternative splicing, cleaved from the cell surface, or secreted from activated platelets [