Humanized von Willebrand factor reduces platelet sequestration in ex vivo and in vivo xenotransplant models

Abstract

The transplantation of organs across species offers the potential to solve the shortage of human organs. While activation of human platelets by human von Willebrand factor (vWF) requires vWF activation by shear stress, contact between human platelets and porcine vWF (pvWF) leads to spontaneous platelet adhesion and activation. This non‐physiologic interaction may contribute to the thrombocytopenia and coagulation pathway dysregulation often associated with xenotransplantation of pig organs in nonhuman primates. Pigs genetically modified to decrease antibody and complement‐dependent rejection (GTKO.hCD46) were engineered to express humanized pvWF (h*pvWF) by replacing a pvWF gene region that encodes the glycoprotein Ib‐binding site with human cDNA orthologs. This modification corrected for non‐physiologic human platelet aggregation on exposure to pig plasma, while preserving in vitro platelet activation by collagen. Organs from pigs with h*pvWF demonstrated reduced platelet sequestration during lung (p ≤ .01) and liver (p ≤ .038 within 4 h) perfusion ex vivo with human blood and after pig‐to‐baboon lung transplantation (p ≤ .007). Residual platelet sequestration and activation were not prevented by the blockade of canonical platelet adhesion pathways. The h*pvWF modification prevents physiologically inappropriate activation of human or baboon platelets by porcine vWF, addressing one cause of the thrombocytopenia and platelet activation observed with xenotransplantation.

Knock‐out of N‐glycolylneuraminic acid attenuates antibody‐mediated rejection in xenogenically perfused porcine lungs

“…Non‐human primate and human platelets bind pig VWF through their GPIb receptors in the absence of shear stress, resulting in over‐activation and adhesion to pig xenograft endothelium 39 . To mitigate clotting cascade activation, various methods have been validated to block this “non‐physiologic” interaction 40 : pre‐depletion of pvWF from pig endothelium before organ procurement; 41 blood treatment with a humanized aGPIb Fab fragment, 6B4, which selectively blocks the binding site of platelet GP1B by VWF; and genetically modifying pvWF to remove or “humanize” the epitopes that interact non‐physiologically with human GP1b 28 . Accordingly, in this study we treated four pairs of lungs with 1‐deamino‐8‐d‐arginine vasopressin (DDAVP), an analog of vasopressin, to deplete porcine endothelial VWF‐stores according to a clinically well‐established practice, 42 and treated the human blood perfusate for one lung in each pair with GP1b 43 .…”

“…Neu5GcKO lungs are perfused with human blood. The upstream trigger for thromboxane release and the injury exhibited by GalTKO.hCD46.Neu5GcKO lungs within 8 h of ex vivo perfusion may be attributable to incomplete control of complement activation, 46,47 vWF‐ 28 or selectin‐mediated 48 platelet adhesive interactions, 49,50 innate immune cell activation via absence of HLA‐E 51 or human CD47, 52 or incompatibility between human pro‐ and anti‐coagulant molecules and pig thromboregulatory molecules 1,52,53 . Evaluating each of these hypotheses separately may be limited by logistical constraints regarding production of informative pig phenotypes.…”

“…39 To mitigate clotting cascade activation, various methods have been validated to block this "non-physiologic" interaction 40 : pre-depletion of pvWF from pig endothelium before organ procurement; 41 blood treatment with a humanized aGPIb Fab fragment, 6B4, which selectively blocks the binding site of platelet GP1B by VWF; and genetically modifying pvWF to remove or "humanize" the epitopes that interact non-physiologically with human GP1b. 28 Accordingly, in this study we treated four pairs of lungs with 1deamino-8-d-arginine vasopressin (DDAVP), an analog of vasopressin, to deplete porcine endothelial VWF-stores according to a clinically well-established practice, 42 and treated the human blood perfusate for one lung in each pair with GP1b. 43 Although we failed to detect any statistically significant difference associated with these interventions in these two pairs of experiments, platelet activation and sequestration tended to be attenuated, as previously reported in association with different pig genetics.…”

“…Four experiments using GalTKO.hCD46.Neu5GcKO lungs (Table 1) were conducted after additionally treating the pig lung donor with Desmopressin (DDAVP®) given over 2 days prior to lung procurement as previously described, to stimulate vWF depletion in pulmonary vascular endothelium 28 . DDAVP treatment alone ( n = 2, one lung in each pair) was compared to additional treatment with 6B4, a monoclonal antibody Fab that blocks binding of human GP1B to porcine vWF ( n = 2), added to the blood perfusate of the contralateral lung 28 . These four experiments are excluded from the statistical analysis, but associated data are reported as supplemental information.…”

“…28 DDAVP treatment alone (n = 2, one lung in each pair) was compared to additional treatment with 6B4, a monoclonal antibody Fab that blocks binding of human GP1B to porcine vWF (n = 2), added to the blood perfusate of the contralateral lung. 28 These four experiments are excluded from the statistical analysis, but associated data are reported as supplemental information.…”