O-linked glycosylation and functional incompatibility of porcine von Willebrand factor for human platelet GPIb receptors

Background: Xenograft rejection is associated with vascular inflammation, thrombocytopenia and the accelerated consumption of coagulation factors. Primary biological incompatibilities of the xenograft in the regulation of clotting appear to amplify pathological processes associated with rejection. The functional incompatibility of porcine von Willebrand factor (vWF) expressed within the xenograft vasculature may heighten interactions with the primate platelet receptor GPIb, hence augmenting formation of platelet microthrombi and vascular injury. Here, we address the functional impact of O-linked glycosylation of the vWF A1 domain on primate platelet activation. Methods: Recombinant human or porcine vWF A1-domains were transiently over-expressed in COS-7 cells as FLAG-tagged fusion protein, linked to plasma membranes via GPI anchors. O-linked glycosylation was blocked by the addition of phenyl-alpha-GalNAc2 to cultures. Expressed vWF-A1 domains were characterized utilizing cytofluometric- and Western blot analyses. Results: Cytofluometric analysis confirmed equivalent levels of human and porcine vWF A1-domain expression irrespective of the levels of O-linked glycosylation. Differential glycosylation patterns of vWF-A1 under these conditions were confirmed by Western blot analyses. Native porcine vWF A1-domains had enhanced human platelet activation potential when compared with human recombinant vWF A1. However, the loss of O-linked glycosylation abolished differences in aggregatory responses between human and porcine vWF A1 domains. Conclusions: Various degrees of O-linked glycosylation of vWF-A1-domains modulate levels of functional interaction with platelet receptor GPIb and consequent platelet aggregation responses in vitro. These data may have implications for outcomes of xenotransplantation. We speculate that alterations in glycosylation of vWF and other adhesion proteins associated with the targeting of the alpha1,3-Gal-epitope in mutant swine may have salutatory effects on the primate platelet activation observed in these xenografts

Impact of O-linked glycosylation of the VWF-A1-domain flanking regions on platelet interaction

This study investigated the functional impact of O-linked glycosylation of von Willebrand Factor (VWF) A1 domains on the interaction with platelet receptors. Native or mutant VWF-A1-domains were transiently overexpressed on COS-7 cells as membrane glycosylphosphaticlylinositol (GPI)-anchored FLAG-tagged fusion proteins. Crytofluometric analysis assured comparable levels of A1-domain expression among native and mutant homologues as well as for different culture conditions. Expressing native VWF-A1-domains under O-linked glycosylation blocking conditions increased the platelet aggregatory responses observed for fully glycosylated forms. Utilizing a neuronal network for prediction of O-linked glycosylation of mammalian proteins, threonine (T) and serine (S) residues located in the VWF-A1-loop flanking regions - not: in the loop itself - were determined to be glycosylated n-terminal at amino acids T485, S490, T492 and T493 and c-terminal at T705. Simultaneous selective charge-to-alanine mutation of S490, T492 and T493 led to gain in aggregatory responses. When compared with native forms, equivalent alterations of T485 did not dictate functional differences. Any alanine-substitution for T705 revealed a substantial loss in aggregatory effects - possibly as a result of structural desintegration of the VWF-A1-binding site for glycoprotein (GP) Ib. These data suggest specific O-linked glycosylation of the amino-terminal VWF-A1-loop-flanking region to have a negative regulatory impact on the A1-domain affinity of nonactivated human VWF for human platelct-GPIb

Recipient levels and function of von Willebrand factor prior to liver transplantation and its consumption in the course of grafting correlate with hepatocellular damage and outcome

Von Willebrand factor (vWF) is a major platelet adhesion molecule at sites of vascular injury, such as observed in ischemia/reperfusion injury following orthotopic liver transplantation (OLT). Thirty-three OLT patients were divided into groups with elevated or low markers of hepatocellular damage (high and low-HD). Whole-blood aggregometry was performed to evaluate platelet function. Multimeric analysis was utilized to evaluate functional vWF levels in the course of OLT. Donor and recipient demographics were comparable among groups. Low-HD patients demonstrated better preserved coagulation parameters on POD 1-6 if contrasted to high-HD patients. One year graft survival for the high-HD group was lower than low-HD patients (P = 0.037). Preoperative vWF-dependent platelet aggregation and functional vWF plasma levels correlated directly with alanine transaminase levels early after OLT as did the decrease of functional vWF to reperfusion. In summary, these data suggest that vWF may serve as a significant mediator of platelet recruitment and hepatocellular injury in the graft following reperfusion

ADP-dependent platelet function prior to and in the early course of pediatric liver transplantation and persisting thrombocytopenia are positively correlated with ischemia/reperfusion injury

Little is known about the role of platelets in relation to ischemia/reperfusion injury (IRI) of the liver graft especially in children. Thrombocyte function was prospectively analysed in 21 consecutive pediatric liver transplantation (pLT) patients by platelet aggregometry secondary to adenosine diphosphate (ADP), collagen, and the von Willebrand factor activator ristocetin (VWF:rco). Post-OP serum levels of ALT were used to divide patients into groups with high (highHD, n = 8) and low (lowHD, n = 13) hepatocellular damage. Clinically, highHD-patients showed impaired plasmatic coagulation and elevated serum bilirubin levels early after pLT when compared with lowHD-patients. Further, platelet counts markedly decreased between pre-OP and postreperfusion (postrep.) in the highHD group (P = 0.003) and did not recuperate by POD6. In lowHD individuals thrombocytopenia improved from both pre-OP (P < 0.05) and postrep. (P < 0.001) respectively towards POD6. Experimental thrombocyte testing revealed that before graft reperfusion only ADP-dependent platelet aggregation correlated with reperfusion injury, thrombocytopenia and early graft function. During the first 48 h after graft reperfusion, all inducers tested demonstrated elevated platelet aggregation levels in the highHD group. Our data suggest a possible role of platelets and their aggregative status in liver IRI subsequent to clinical pLT. Reperfusion-independent ADP-triggered platelet function may be a determinant for IRI in the pediatric hepatic graft recipient

Platelet activation and increased tissue factor expression on monocytes in reperfusion injury following orthotopic liver transplantation

Platelets have been implicated in the pathogenesis of liver damage after orthotopic liver transplantation (OLT). Early graft dysfunction is frequently caused by reperfusion injury subsequent to cold ischemia (IRI). Therefore, we investigated activation of the pivotal haemostatic cells, platelets and monocytes, from patients with elevated markers of IRI and from patients with uneventful course (control-group), respectively during the first week after OLT. Flow cytometry analysis of citrate anticoagulated blood samples revealed that platelets from IRI patients became significantly activated within 48 h after OLT in vivo, with increased surface presentation of P-selectin, CD40L, thrombospondin-1 and tissue-factor. Platelet activation in IRI patients on post-transplant day 2 was accompanied by significantly enhanced tissue-factor expression on peripheral blood monocytes, significant elevated levels of C-reactive protein and hepatocellular damage. Towards post-transplant day 4, levels of platelet-derived microparticles rose significantly in IRI patients if contrasted to control patients. Thus, activated cellular haemostasis is involved in the early inflammatory response of hepatocellular damage subsequent to reperfusion of the transplanted liver. Targeting distinct activation patterns of platelets and monocytes in an early phase of hepatic grafting may counteract the extent of IRI via inhibition of micro-thrombus formation and inflammation without exacerbating the existing bleeding risk