Porcine Von Willebrand Factor Research Articles

Current Barriers to Clinical Liver Xenotransplantation.

Preclinical trials of pig-to-nonhuman primate liver xenotransplantation have recently achieved longer survival times. However, life-threatening thrombocytopenia and coagulation dysregulation continue to limit preclinical liver xenograft survival times to less than one month despite various genetic modifications in pigs and intensive pharmacological support. Transfusion of human coagulation factors and complex immunosuppressive regimens have resulted in substantial improvements in recipient survival. The fundamental biological mechanisms of thrombocytopenia and coagulation dysregulation remain incompletely understood. Current studies demonstrate that porcine von Willebrand Factor binds more tightly to human platelet GPIb receptors due to increased O-linked glycosylation, resulting in increased human platelet activation. Porcine liver sinusoidal endothelial cells and Kupffer cells phagocytose human platelets in an asialoglycoprotein receptor 1-dependent and CD40/CD154-dependent manner, respectively. Porcine Kupffer cells phagocytose human platelets via a species-incompatible SIRPα/CD47 axis. Key drivers of coagulation dysregulation include constitutive activation of the extrinsic clotting cascade due to failure of porcine tissue factor pathway inhibitor to repress recipient tissue factor. Additionally, porcine thrombomodulin fails to activate human protein C when bound by human thrombin, leading to a hypercoagulable state. Combined genetic modification of these key genes may mitigate liver xenotransplantation-induced thrombocytopenia and coagulation dysregulation, leading to greater recipient survival in pig-to-nonhuman primate liver xenotransplantation and, potentially, the first pig-to-human clinical trial.

A 12.3-kb Duplication Within the VWF Gene in Pigs Affected by Von Willebrand Disease Type 3.

Von Willebrand Disease (VWD) type 3 is a serious and sometimes fatal hereditary bleeding disorder. In pigs, the disease has been known for decades, and affected animals are used as models for the human disease. Due to the recessive mode of inheritance of VWD type 3, severe bleeding is typically seen in homozygous individuals. We sequenced the complete porcine VWF (Von Willebrand Factor) complementary DNA (cDNA) and detected a tandem duplication of exons 17 and 18, causing a frameshift and a premature termination codon (p.Val814LeufsTer3) in the affected pig. Subsequent next generation sequencing on genomic DNA proved the existence of a 12.3-kb tandem duplication associated with VWD. This duplication putatively originates from porcine Short Interspersed Nuclear Elements (SINEs) located within VWF introns 16 and 18 with high identity. The premature termination truncates the VWF open reading frame by a large part, resulting in an almost entire loss of the mature peptide. It is therefore supposed to account for the severe VWD type 3. Our results further indicate the presence of strong, nonsense-mediated decay in VWF messenger RNA (mRNA) containing the duplication, which was supported by the almost complete absence of the complete VWF protein in immunohistochemistry analysis of the VWD-affected pig. In the past, differentiation of wild-type and heterozygous pigs in this VWD colony had to rely on clinical examinations and additional laboratory methods. The present study provides the basis to distinguish both genotypes by performing a rapid and simple genetic analysis.

Pig-to-Baboon Liver Xenoperfusion Utilizing GalTKO.hCD46 Pigs and Glycoprotein Ib Blockade.

Although transplantation of genetically-modified porcine livers into baboons has yielded recipient survival for up to 7 days, survival is limited by profound thrombocytopenia, which becomes manifest almost immediately after revascularization, and by subsequent coagulopathy. Porcine von Willebrand's factor (VWF), a glycoprotein that adheres to activated platelets to initiate thrombus formation, has been shown to constitutively activate human platelets via their glycoprotein Ib (GPIb) receptors. Here we report our pig-to-primate liver xenoperfusion model and evaluate whether targeting the GPIb-VWF axis prevents platelet sequestration. Methods: 12 baboons underwent cross-circulation with the following extracorporeal livers: one allogeneic control with a baboon liver, 4 xenogeneic controls with a GalTKO.hCD46 pig liver, 3 GalTKO.hCD46 pig livers in recipients treated with αGPIb antibody during perfusion, and 4 GalTKO.hCD46 pig livers pretreated with DDAVP in recipients treated with αGPIb antibody during perfusion. Results: All perfused livers appeared grossly and macroscopically normal, and produced bile. Xenograft liver perfusion experiments treated with αGPIb antibody may show less platelet sequestration during the initial two hours of perfusion. Portal venous resistance remained constant in all perfusion experiments. Platelet activation studies demonstrated platelet activation in all xenoperfusions, but not in the allogeneic perfusion. Conclusion: These observations suggest that primate platelet sequestration by porcine liver and the associated thrombocytopenia are multifactorial, and perhaps partially mediated by a constitutive interaction between porcine VWF and the primate GPIb receptor. Control of platelet sequestration and consumptive coagulopathy in liver xenotransplantation will likely require a multi-faceted approach in our clinically relevant perfusion model. DISCLOSURES:Ayares, D.: Employee, Revivicor. Pierson III, R.: Grant/Research Support, Revivicor.

Pig‐to‐baboon liver xenoperfusion utilizing GalTKO.hCD46 pigs and glycoprotein Ib blockade

Although transplantation of genetically modified porcine livers into baboons has yielded recipient survival for up to 7days, survival is limited by profound thrombocytopenia, which becomes manifest almost immediately after revascularization, and by subsequent coagulopathy. Porcine von Willebrand's factor (VWF), a glycoprotein that adheres to activated platelets to initiate thrombus formation, has been shown to constitutively activate human platelets via their glycoprotein Ib (GPIb) receptors. Here, we report our pig-to-primate liver xenoperfusion model and evaluate whether targeting the GPIb-VWF axis prevents platelet sequestration. Twelve baboons underwent cross-circulation with the following extracorporeal livers: one allogeneic control with a baboon liver, 4 xenogeneic controls with a GalTKO.hCD46 pig liver, 3 GalTKO.hCD46 pig livers in recipients treated with αGPIb antibody during perfusion, and 4 GalTKO.hCD46 pig livers pre-treated with D-arginine vasopressin (DDAVP) in recipients treated with αGPIb antibody during perfusion. All perfused livers appeared grossly and macroscopically normal and produced bile. Xenograft liver perfusion experiments treated with αGPIb antibody may show less platelet sequestration during the initial 2h of perfusion. Portal venous resistance remained constant in all perfusion experiments. Platelet activation studies demonstrated platelet activation in all xenoperfusions, but not in the allogeneic perfusion. These observations suggest that primate platelet sequestration by porcine liver and the associated thrombocytopenia are multifactorial and perhaps partially mediated by a constitutive interaction between porcine VWF and the primate GPIb receptor. Control of platelet sequestration and consumptive coagulopathy in liver xenotransplantation will likely require a multifaceted approach in our clinically relevant perfusion model.

Porcine factor VIII

Provided are porcine factor VIII nucleotide sequences and hybrid sequences encoding human/porcine chimeric factor VIII-type procoagulant proteins. DNAs encoding such chimeric factor VIII-type procoagulant proteins, pharmaceutical compositions containing such chimeric factor VIII-type procoagulant proteins, and methods of treating hemophilia using such chimeric factor VIII-type procoagulant proteins are also provided.

Prolonged Function of Macrophage, von Willebrand Factor-Deficient Porcine Pulmonary Xenografts

Porcine von Willebrand factor (vWF) activates human and primate platelets. Having determined the importance of pulmonary intravascular macrophages (PIMs) in pulmonary xenotransplantation, we evaluated whether, in the absence of PIMs, vWF might play a role in pulmonary xenograft dysfunction. Utilizing a left single-lung transplant model, baboons depleted of anti-alphaGal antibodies received lungs from either vWF-deficient (n = 2); MCP-expressing (n = 5); MCP PIM-depleted (n = 5); or vWF-deficient PIM-depleted swine (n = 3). Two out of three of the PIM-depleted, pvWF deficient grafts survived longer than any previously reported pulmonary xenografts, including PIM-depleted xenografts expressing human complement regulatory proteins. Depletion of PIM's from vWF-deficient lungs, like depletion of PIM's from hMCP lungs, resulted in abrogation of the coagulopathy associated with pulmonary xenotransplantation. Thus, in terms of pulmonary graft survival, control of adverse reactions involving pvWF appears to be equally or even more important than is complement regulation using hMCP expression. However, based on the rapid failure of PIM-sufficient, pvWF-deficient pulmonary xenografts, pVWF-deficient pulmonary xenografts appear to be particularly sensitive to macrophage-mediated damage. These data provide initial evidence that vWF plays a role in the 'delayed' (24 h) dysfunction observed in pulmonary xenotransplantation using PIM depleted hMCP organs.

Shielding of the A1 Domain by the D′D3 Domains of von Willebrand Factor Modulates Its Interaction with Platelet Glycoprotein Ib-IX-V

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 region in the N-terminal D'D3 domains. In this study we demonstrated that the D'D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D'D3 region in VWF augmented binding to GPIbalpha, suggesting an inhibitory role for this region, (ii) the isolated D'D3 region inhibited the GPIbalpha interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D'D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbalpha. Our results support a model of domain translocation in VWF that allows interaction with GPIbalpha. The suggested shielding interaction of the A1 domain by the D'D3 region then becomes disrupted by VWF immobilization.

O‐Linked glycosylation and functional incompatibility of porcine von Willebrand factor for human platelet GPIb receptors

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. 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. 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. 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.

Cross-Species Chemoattraction and Xenograft Failure: Do Neutrophils Play a Role?

Polymorphonuclear granulocytes (PMN, neutrophils) are well known to contribute to the pathogenesis of many congenital disorders (e.g., CR3 deficiency, chronic granulomatous disease, myeloperoxidase deficiency) and acquired disease states (e.g., inflammatory bowel disease, glomerulonephritis, immune vasculitis, rheumatoid arthritis, neutrophil dermatoses, sepsis). They play a key role in mediating ischemia-reperfusion injury following solid organ allotransplantation,andmorerecentevidencesuggeststhat,following early migration into the allograft, neutrophils are capable of amplifying T-cell mediated allograft rejection (1). In xenotransplantation, the major hurdle of hyperacute rejection (HAR) has led research efforts to focus on the role of natural xenoantibodies and complement activation, while the neutrophilwasconsideredtoplayasecondaryroleandwastherefore given little attention. Meanwhile, it seems that experimental prevention of HAR becomes feasible thanks to the genetic modification of donor animals (DAF/CD55 transgenicpigs,-Galknockoutpigsandmice)and,consequently, the mechanism of acute vascular rejection (AVR) and the cellular aspects of xenograft rejection now receive full attention. AVR is characterized by a dense cellular infiltrate, which is dominated not only by natural killer (NK) cells and macrophages, but also by PMN. It is therefore plausible to assume that neutrophils take an active part in the rejection process and this hypothesis is gaining increasing interest. Vercellotti et al. (2) showed that the adhesion of human neutrophils to porcine endothelial cells (PECs) was promoted by humannaturalxenoantibodiesandcomplement,and,particularly, that endothelial deposition of iC3b was critically involvedintheadhesionprocess.Ehrnfeltetal.(3)showedthat humanPMNsadheretoPECs,providedtheseareactivatedby human natural xenoantibodies. These studies indicate that the interaction between neutrophils and xenoendothelium requires preactivation of endothelial cells by complement and/or natural xenoantibodies and, consequently, that the PMN plays a secondary role in HAR. In contrast, others have providedevidencethathumanPMNhavetheinherentcapac

O-LINKED GLYCOSYLATION AND FUNCTIONAL INCOMPATIBILITY OF PORCINE VON WILLEBRAND FACTOR FOR HUMAN PLATELET GPIB RECEPTORS – A PUTATIVE HURDLE TO XENOTRANSPLANTATION

P1052 Aims: 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 α GalNAc2 to cultures. 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 analyses. Native porcine vWF A1-domains had enhanced human platelet activation potential when compared to 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 α1,3 Gal-epitope in mutant swine may have salutatory effects on the primate platelet activation observed in these xenografts.

Cloning and expression of full-length recombinant porcine von Willebrand factor

Cloning and expression of full-length recombinant porcine von Willebrand factor

Von Willebrand factor (VWF)-dependent human platelet activation: porcine VWF utilizes different transmembrane signaling pathways than does thrombin to activate platelets, but both require protein phosphatase function

The interaction between von Willebrand factor (VWF) and glycoprotein (GP) Ib results in platelet agglutination and activation of many signaling intermediates. To determine if VWF-dependent platelet activation requires the participation of pivotal transmembrane signaling pathways, we analyzed VWF-dependent platelet activation profiles following inhibition of several transmembrane signaling intermediates. This was accomplished using porcine VWF, which has been shown to interact with human GPIb independently of shear stress or ristocetin. Platelet alpha (CD62) and lysozomal granule release (CD63), microparticle formation, and platelet agglutination/aggregation were evaluated. The ability of signaling inhibitors to prevent VWF-dependent platelet activation was compared to their ability to inhibit thrombin-dependent activation. The results demonstrate that VWF-dependent platelet activation can occur independently of the activities of protein kinase C (PKC), wortmannin-sensitive phosphatidylinositide 3-kinase, and phospholipase C, as well as independently of elevations in the concentration of intracellular calcium. In sharp contrast, these transmembrane signaling intermediates are required for thrombin-dependent platelet activation. In addition, thrombin-dependent but not VWF-dependent platelet activation was associated with elevations in the concentration of intracellular calcium under the conditions used. The family of signaling intermediates which appeared to be pivotal for both thrombin- and VWF-dependent platelet activation were the protein tyrosine phosphatases and the serine/threonine phosphatases. It is concluded that thrombin-dependent platelet activation relies on the activation of several transmembrane signaling pathways, whereas VWF-dependent platelet activation is dependent upon the activity of protein phosphatases. Inhibition of these phosphatases in vivo may provide a novel therapeutic approach for treating VWF-dependent platelet disorders such as thrombotic thrombocytopenic purpura or arterial thrombosis.

The role of the porcine von willebrand factor: baboon platelet interactions in pulmonary xenotransplantation

Porcine von Willebrand factor (pvWF) has been shown to bind to human glycoprotein Ib (GPIb) and cause activation of human (or primate) platelets in the absence of shear stress. Pulmonary xenografts develop disseminated intravascular coagulation (DIC) and microvascular thrombosis within hours of reperfusion, and the aberrant interaction between pvWF and human platelets may be a possible cause of xenograft-associated DIC. Experimental baboons (n=3) received mouse anti-human GPIb monoclonal antibody before undergoing orthotopic pulmonary xenotransplantation with porcine lungs expressing human membrane cofactor protein (CD46). Blocking the pvWF-GPIb interaction with a monoclonal antibody to GPIb prevented the agglutination of human and baboon platelets by pvWF in vitro. In vivo, the anti-GPIb antibody prevented platelet deposition and prevented the increases in D-Dimers (P=0.011) seen in control xenograft recipients (n=5). However, there was no difference in elevations of prothrombin times (PT) or improvement in the vasoconstriction associated with the loss of xenograft function. This study indicates that the DIC associated with the hyperacute dysfunction of pulmonary xenografts is a complex phenomenon that is affected by, but not solely dependent on, activation of platelets. Aberrant interactions between pvWF and GPIb play a significant role in DIC associated with pulmonary xenotransplantation.

Platelet activation and hypercoagulability following treatment with porcine factor VIII (HYATE:C).

Activation of platelets and coagulation in vivo was studied in nine patients with hemophilia A and inhibitors to human Factor VIII, prior to and following treatment with porcine Factor VIII (PFVIII; HYATE:C). In addition, six hemophiliac patients were similarly studied after treatment with recombinant Factor VIII (rFVIII). Platelet activation was also examined in vitro using porcine von Willebrand factor (PvWF)-enriched and PvWF-depleted fractions obtained by fractionation of PFVIII. Coagulation was assessed by measuring the concentrations of plasma prothrombin fragment 1+2 concentrations (prothrombinase generation) and Factor Xa-ATIII. Patients treated with PFVIII had significantly increased numbers of circulating platelets expressing CD62 and CD63 (markers of platelet activation) and annexin V (marker of platelet procoagulant activity) compared to patients treated with rFVIII; the former patients also demonstrated an increase in plasma coagulability after therapy. In in vitro experiments it was observed that the platelet-activating and procoagulant capacity of PFVIII resided in the PvWF-enriched fraction, and the same was true for the plasma hypercoagulability following exposure of platelets to PFVIII. These results support the hypothesis that PFVIII-induced platelet activation provides a mechanism for enhancing hemostasis, separate from, and additional to, that due to increased circulating Factor VIII, and it is due to residual PvWF in the PFVIII preparation.

Porcine factor VIII: current status and future developments.

High-purity porcine factor VIII (pFVIII) (Hyate:C, Ipsen, UK), with a specific activity of > 125 U mg(-1) protein, has been shown to be effective in up to 90% of bleeds in patients with FVIII inhibitors. These inhibitors have been shown to have a median 15% cross-reactivity to pFVIII, and even less cross-reactivity in patients with acquired haemophilia. Its use is sometimes associated with a transient fall in platelet count and with transfusion reactions. Furthermore, it is not virally attenuated. Although pFVIII has not been shown to transmit any viral illness to its human recipients, it is extensively screened for porcine viruses using a 4-cell line general screen. The source plasma is also screened for porcine parvovirus. To satisfy the demand for ever-improved side-effect profile and viral safety a third-generation pFVIII is under consideration by Ipsen. This product will be purified from porcine plasma screened for porcine parvovirus, using immuno-purification, ion-exchange chromatography and washing. Spiking experiments using human FVIIIC (hFVIIIC) suggest that these purification steps may be associated with a 6-log viral reduction. The product is also virally attenuated using the solvent-detergent method. This should yield an ultra-pure concentrate, lacking porcine von Willebrand factor, with a specific activity of approximately 5000 U mg(-1) protein which does not require added albumin as stabilizer and which may be stored at 2-4 degrees. One would anticipate that this product should be clinically effective and should be associated with enhanced viral safety and an improved side-effect profile. It should not affect the platelet count and should be associated with a much reduced risk of transfusion reactions. Clinical trials are planned.

Immune Complex Formation after Xenotransplantation: Evidence of Type III as well as Type II Immune Reactions Provide Clues to Pathophysiology

Rejection of renal and cardiac xenografts is initiated when natural antibodies of the recipient bind to donor endothelium, activating complement on the surface of endothelial cells. Pulmonary xenotransplants, however, reveal less evidence of antibody binding and complement activation and, in contrast to other xenografts, fare worse when the complement of the graft recipient is depleted. Accordingly, we asked whether distinct immunochemical reactions might occur after xenotransplantation of the lung and what implications such reactions might have for pulmonary pathophysiology. Analysis of serum from baboons after transplantation with porcine lungs revealed complexes containing baboon IgM and porcine von Willebrand factor. The baboon IgM in these complexes was specific for Galalpha1-3Gal. Immune complexes were also seen, albeit to a lesser extent, in the serum of kidney and heart xenotransplant recipients. Deposits of porcine von Willebrand factor and baboon C3 were detected in livers and spleens of transplanted baboons. These results indicate pulmonary xenotransplantation eventuates in formation of immune complexes and in the deposition of those complexes at distant sites. Immune complex formation could explain the peculiar fate of xenoreactive antibodies after pulmonary xenotransplantation and might contribute to the pathophysiology of the lung and systemic changes not previously considered a complication of xenotransplantation.

Porcine von Willebrand factor and thrombin induce the activation of c-Jun amino-terminal kinase (JNK/SAPK) whereas only thrombin induces activation of extracellular signal-related kinase 2 (ERK2) in human platelets.

The interaction of platelets with subendothelial von Willebrand factor (VWF), especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. The signalling cascade which results from the interaction of VWF and its receptor GPIbIX has only been partially defined. Mitogen-activated protein kinases (MAPKs) are a family of downstream transmembrane signalling serine-threonine kinases and have been demonstrated to be present and functional in platelets; these include the extracellular signal-related kinases (ERKs), c-Jun amino-terminal kinases (JNKs) and p38 MAPK. Previously, we showed that p38 MAPK was not required in VWF-induced human platelet activation. It is not known whether VWF-dependent platelet activation involves the activation of the JNK and ERK family of signalling molecules. This report demonstrates that porcine von Willebrand factor (pVWF) induced a sustained and stable JNK activation measurable by 1 min after activation. Thrombin also induced JNK activation assessed at 1 min after activation. In contrast to thrombin, pVWF did not induce ERK2 activation at any time point tested. To ensure that ERK activation was unnecessary for pVWF-dependent platelet activation, we functionally inhibited ERK-dependent signalling with PD98059, a potent and selective inhibitor of the MAP kinase kinase (MEK-1), which is the upstream kinase of ERK1 and ERK2. Although PD98059 inhibited ERK2 activation in platelets, it had no effect on pVWF- or thrombin-induced platelet alpha or lysozomal granule release, modulation of membrane glycoprotein CD41, microparticle formation, platelet shape change or platelet agglutination. It is concluded that pVWF and thrombin induced JNK activation, but whereas thrombin induced ERK2 activation VWF did not; functional ERK2 activity was also not required for pVWF- or thrombin-dependent platelet activation.

P38 MAPK is activated but not necessary in porcine von Willebrand factor‐dependent platelet activation

We have investigated the role of p38 mitogen-activated protein kinase (MAPK) in von Willebrand factor (VWF)-dependent platelet activation. The interaction of platelets with subendothelial VWF, especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. As a model of VWF-dependent platelet activation, porcine VWF was employed. Porcine VWF induced p38 MAPK activation by 1 min post-addition; assessed by phosphorylation of a recombinant p38 MAPK fusion protein substrate termed glutathione S-transferase-MAPK activated protein kinase-2. To determine if p38 MAPK was necessary for porcine VWF-induced platelet activation, we functionally inhibited p38 MAPK activity with SB203580 before exposure of the platelets to porcine VWF. Inhibition of p38 MAPK had no effect on VWF-induced platelet alpha or lysozomal granule release, expression of activated GPIIb IIIa, modulation of membrane glycoprotein CD41, expression of phosphatidylserine as assessed by annexin V binding, microparticle formation, or platelet agglutination. It was concluded that SB203580-inhibitable p38 MAPK activity induced by porcine VWF is not necessary for platelet activation.

Factors in xenograft rejection.

Important mechanisms underlying immediate xenograft loss by hyperacute rejection (HAR), in the pig-to-primate combination, have been recently delineated. There are now several proposed therapies that deal with the problem of complement activation and xenoreactive natural antibody (XNA) binding to the vasculature that have been shown to prevent HAR. However, vascularized xenografts are still lost, typically within days, by delayed xenograft rejection (DXR), alternatively known as acute vascular rejection (AVR). This process is characterized by endothelial cell (EC) perturbation, localization of XNA within the graft vasculature, host NK cell and monocyte activation with platelet sequestration and vascular thrombosis. Alternative immunosuppressive strategies, additive anti-complement therapies with the control of any resulting EC activation processes and induction of protective responses have been proposed to ameliorate this pathological process. In addition, several potentially important molecular incompatibilities between activated human coagulation factors and the natural anticoagulants expressed on porcine EC have been noted. Such incompatibilities may be analogous to cross-species alterations in the function of complement regulatory proteins important in HAR. Disordered thromboregulation is potentially relevant to the progression of inflammatory events in DXR and the disseminated intravascular coagulation seen in primate recipients of porcine renal xenografts. We have recently demonstrated the inability of porcine tissue factor pathway inhibitor (TFPI) to adequately neutralize human factor Xa (FXa), the aberrant activation of both human prothrombin and FXa by porcine EC and the failure of the porcine natural anticoagulant, thrombomodulin to bind human thrombin and hence activate human protein C. The enhanced potential of porcine von Willebrand factor to associate with human platelet GPIb has been demonstrated to be dependent upon the isolated A1 domain of von Willebrand factor. In addition, the loss of TFPI and vascular ATPDase/CD39 activity following EC activation responses would potentiate any procoagulant changes within the xenograft. These developments could exacerbate vascular damage from whatever cause and enhance the activation of platelets and coagulation pathways within xenografts resulting in graft infarction and loss. Analysis of these and the other putative factors underlying DXR should lead to the development and testing of genetic approaches that, in conjunction with selected pharmacological means, may further prolong xenograft survival to a clinically relevant extent.

Effects of human recombinant, plasma-derived and porcine von Willebrand factor in pigs with severe von Willebrand disease

The effects of the infusion of a human recombinant von Willebrand factor (vWF) preparation in pigs homozygous for von Willebrand disease (vWD) were evaluated on serial measurements of von Willebrand factor antigen and activity, FVIII activity, vWF multimer analysis, in-vivo bleeding time and platelet adhesion and thrombus formation on collagen at high shear rates in an ex-vivo model of experimental thrombosis. Plasma-derived human and porcine vWF were used for comparison. Before infusion, the pigs were characterized by undetectable plasma vWF levels, a low level of FVIII, prolonged bleeding time, severely impaired platelet adhesion and thrombus formation. After infusion of the human recombinant vWF, in-vivo recovery of vWF activity ranged from 58% to 82%, depending on the dose infused, and its half-life was longer than for the plasma-derived concentrates. The highest-molecular-weight forms of human recombinant vWF were removed from the circulation gradually. Infusion of the three vWF concentrates produced inconsistent effects on bleeding time and moderate improvement of platelet adhesion and thrombus formation. After infusion, a prolonged increase of FVIII (> 48 h) was observed, suggesting that human recombinant vWF is able to bind and to stabilize porcine factor VIII and that porcine vWD is a good model for studying such interactions.