Intraperitoneal Injection and Recovery of VWF and FVIII and Differences From Intravenous and Subcutaneous Injection.
Authors: Shi Qizhen , Kuether Erin L. , Zhang Guowei , Schroeder Jocelyn A. , Fahs Scot A. , Montgomery Robert R. 
Published: October 2019

Abstract
Abstract 3178
Poster Board III-117
von Willebrand factor (VWF) is a multivalent adhesive protein that serves as a carrier protein for factor VIII (FVIII) and an adhesive link between platelets and the injured blood vessel wall. Both VWF and FVIII are large adhesive proteins and intravenous infusion studies in humans suggest that both proteins remain intravascular in contrast to other coagulation proteins (e.g. FIX). When FIX is injected intraperitoneally (IP) it does achieve access to the vasculature. We were interested in investigating whether infusion of VWF and FVIII by either IP or subcutaneous (SQ) injection would result in efficient absorption of these large proteins into the vascular circulation. To address these questions, FVIIInull, VWFnull, or FVIII/VWF double knockout mice were infused with human plasma-derived or recombinant VWF and/or FVIII at a dose of 50 U / kg by IP, SQ, or intravenous (IV) injection. Plasma samples were collected at various time points after infusion. The levels of FVIII were determined by chromogenic assay and VWF was measured by ELISA assay. VWF multimer structure was analyzed using non-reducing SDS-agarose electrophoresis. Both VWF and FVIII were absorbed into the blood circulation after IP injection with a peak between 2 to 4 hours with levels similar to those observed in mice infused intravenously. In contrast, neither VWF nor FVIII was detected in the plasma following SQ injection. Although IV injection achieved peak plasma levels quickly, both human VWF and FVIII rapidly decreased during the first 2 hours following IV injection. However, if we compared VWF and FVIII in the plasma of infused mice after the 2 hour time point, the half-life (t1/2) of both proteins following IV injection was not significantly different from those obtained by IP administration. Following both IV and IP infusion of VWF, the multimeric structure of circulating VWF was similar to that observed in the infusate, indicating the entire spectrum of VWF multimers was absorbed into the vasculature using either the IV or IP route. The t1/2 of human plasma-derived VWF and FVIII in mouse plasma was 1.56 ± 0.72 hrs (n = 33) and 3.49 ± 1.25 hrs (n = 21), respectively, which was significantly shorter than those obtained from recombinant human VWF and FVIII infusions (2.17 ± 0.33 hrs, and 5.03 ± 2.55 hrs, n = 18, respectively). As expected, endogenous murine FVIII was restored in VWFnull mice when VWF was infused, whether by IP or IV injection. Interestingly, we found the t1/2 of infused mouse plasma-derived VWF was significantly longer (7.59 ± 2.07 hrs, n = 16) than those obtained from human VWF infusion. These results demonstrate that both VWF and FVIII can be efficiently absorbed into the blood circulation following IP but not SQ injection. Whether similar results would be obtained in humans or larger animals must await further study.

Disclosures
Montgomery: GTI Diagnostics: Consultancy; Baxter: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees.