Functional Relevance of Von Willebrand Factor in Mouse Model of Hepatic Ischemia- Reperfusion Injury

Hepatic ischemia-reperfusion (I/R) injury is a liver damage occurring during liver surgeries such as hepatic resection or transplantation, and denotes the major basis for graft dysfunction after transplantation. Although cellular and molecular mechanisms of hepatic I/R injury are complex and remain to be clarified, an excessive inflammatory response is assumed to play a role in this regard. An adhesive protein von Willebrand factor (VWF) plays a pivotal role in platelet thrombus formation and is recently understood as a key protein in a cross-talk between inflammation and thrombosis. In this context, we assumed that VWF may also be involved in the pathophysiology of hepatic I/R injury, in which VWF-dependent platelet activation or inflammatory responses could play a role at liver sinusoidal milieu. To test this hypothesis, we have used a mouse experimental model of hepatic I/R injury. Mice were anesthetized with sodium pentobarbital and a midline laparotomy was then performed on a heating pad. Blood supply for left lateral and median lobes of liver (approximately 70% of the liver mass) was interrupted by cross-clamping the hepatic artery and portal vein with a microvascular atraumatic clip for 90 min. Then a clip was taken off to provoke the reperfusion of hepatic blood flow, which was monitored on the surface of left lateral lobe by Laser Doppler flowmetry (ALF21, Advance Co, Tokyo, Japan). The hepatic blood flow was measured again 24 h after reperfusion and mice were then sacrificed for blood collection and histological analysis of liver tissue. We compared 16 wild-type (WT) and 12 VWF-gene deleted (knock-out; KO) mice (from The Jackson Laboratory, Bar Harbor, ME), all of which were 8-12 weeks of age, healthy and fertile. Excess blood loss was not observed in all mice (WT or KO) during whole surgical process. As compared to WT mice, restoration of hepatic blood flow was significantly greater in VWF-KO mice at 24 h after reperfusion (WT; 61± 17% vs. KO; 87 ± 17%, expressed as the percentage of pre-ischemic value). Consistent with the hepatic blood flow, the time-course analysis of serum alanine aminotransferase (ALT) at several time points after reperfusion revealed the lesser liver damages of KO mice (WT; 6898 ± 3270 and 1313 ± 621 IU/L vs. KO; 3043 ± 1320 and 478 ± 330 IU/L, at 3 h and 24 h after reperfusion, respectively). In addition, histological analysis confirmed that neutrophil infiltration in the liver tissue of KO mice was significantly reduced as compared to WT mice at 24 h after reperfusion. These impaired hepatic blood flow and ALT values as well as intensified neutrophil infiltration in WT mice were significantly improved to an extent comparable to those of KO mice by the bolus injection of recombinant human ADAMTS13 (3 μg/mouse equivalent to 2800 U/kg, n=12) just prior to the I/R operation. Our results altogether indicate that VWF-dependent inflammatory responses with neutrophil recruitment at ischemic sites are involved in pathophysiology of hepatic I/R injury, and functional regulation of VWF by ADAMTS 13 may serve as a promising therapeutic option for hepatic I/R injury.