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The use of animal organs for transplantation in humans is seen as a potential solution to the short supply of human donor organs available for clinical transplantation. However, while several attempts at clinical xenografting have been made over the last ninety years, xenotransplantation has not matched the success of allotransplantation, because of a vigorous rejection response. Xenograft rejection is mediated by mechanisms that differ from those involved in alloreactivity and which are inadequately controlled by conventional immunosuppressive agents. Xenotransplantation therefore requires the development of specific strategies to overcome rejection, through modification of the host immunity or production of genetically engineered pig organs. This article reviews the cellular and molecular events underlying xenograft rejection and the potential strategies of prevention and gives a brief history of the main attempts at clinical xenotransplantation since the beginning of the century. [ABSTRACT FROM AUTHOR]

Glycoprotein V (GPV) is a highly expressed 82 KDa platelet surface transmembrane protein which is loosely attached to the GPIb-IX complex. Despite remaining questions concerning its function, GPV presents several unique features which have repercussions in hematology, atherothrombosis, immunology and transfusion. GPV is specifically expressed in platelets and megakaryocytes and is an ideal marker and reporter gene for the late stages of megakaryopoiesis. The ectodomain of GPV can be released by a number of proteases, namely thrombin, elastase and ADAM10 and 17. Although it was originally proposed as a thrombin receptor, this hypothesis was abandoned since thrombin activation was preserved after blockade of GPV cleavage and in Gp5 knockout mice. The combined potential of GPV to reflect the direct action of thrombin, platelet exposure to strong agonists and inflammatory conditions has led one to evaluate its utility as a marker in the context of atherothrombosis. Increased plasma levels of soluble GPV have notably been recorded in myocardial infarction, stroke and venous thromboembolism. It is also highly valued in transfusion to monitor platelet storage lesions. GPV presents several polymorphisms, which are a possible source of alloantibodies, while autoantibodies have been frequently detected in immune thrombocytopenia. The real biological function of this glycoprotein nevertheless remains an enigma, despite the respectively decreased and increased responses to low concentrations of collagen and thrombin observed in Gp5 knockout mice. Current studies are exploring its role in modulating general or VWF-induced platelet signaling, which could bear relevance in thrombosis and platelet clearance.

Background: The production of platelet concentrates (PCs) is evolving, and their survival capacity needs in vivo evaluation. This requires that the transfused platelets (PLTs) be distinguished from those of the recipient. Labeling at various biotin (Bio) densities allows one to concurrently trace multiple PLT populations, as reported for red blood cells., Study Design and Methods: A method is described to label human PLTs at two densities of Bio for future clinical trials. Injectable-grade PLTs were prepared in a sterile environment, using injectable-grade buffers and good manufacturing practices (GMP)-grade Sulfo-NHS-Biotin. Sulfo-NHS-Biotin concentrations were chosen to maintain PLT integrity and avoid potential alloimmunization while enabling the detection of circulating BioPLTs. The impact of biotinylation on human PLT recirculation was evaluated in vivo in a severe immunodeficient mouse model using ex vivo flow cytometry., Results: BioPLTs labeled with 1.2 or 10 μg/ml Sulfo-NHS-Biotin displayed normal ultrastructure and retained aggregation and secretion capacity and normal expression of the main surface glycoproteins. The procedure avoided detrimental PLT activation or apoptosis signals. Transfused human BioPLT populations could be distinguished from one another and from unlabeled circulating mouse PLTs, and their survival was comparable to that of unlabeled human PLTs in the mouse model., Conclusions: Provided low Sulfo-NHS-Biotin concentrations (<10 μg/ml) are used, injectable-grade BioPLTs comply with safety regulations, conserve PLT integrity, and permit accurate in vivo detection. This alternative to radioisotopes, which allows one to follow different PLT populations in the same recipient, should be valuable when assessing new PC preparations and monitoring PLT survival in clinical research., (© 2021 AABB.)

Background: Deterioration in quality of platelet concentrates (PCs) during storage results from the appearance of storage lesions affecting the hemostatic functions and posttransfusion survival of platelets. These lesions depend on the preparation and pathogen inactivation methods used, duration of storage, and platelet additive solutions (PASs) present in storage bags., Methods: We investigated the effects of citrate contained in third-generation PAS (PAS-III) on storage lesions in buffy-coat PCs with or without photochemical (amotosalen-ultraviolet A) treatment over 7 days., Results: Platelet counts were conserved in all groups during storage, as was platelet swirling without appearance of macroscopic aggregates. Glycoprotein (GP) IIbIIIa and GPVI expression remained stable, whereas GPIbα declined similarly in all groups during storage. Removal of citrate from PAS-III, resulting in global reduction of citrate from 11 to 5 mM, led to a significant decrease in glucose consumption, which largely countered a modest deleterious effect of photochemical treatment. Citrate reduction also resulted in decreased lactate generation and better maintenance of pH during storage, while photochemical treatment had no impact on these parameters. Moreover, citrate-free storage significantly reduced exposure of P-selectin and the apoptosis signal phosphatidylserine, thereby abolishing the activating effect of photochemical treatment on both parameters. Citrate reduction benefited platelet aggregation to various agonists up to Day 7, whereas PCT had no impact on these responses., Conclusion: Removal of citrate from PAS-III has a beneficial impact on platelet metabolism, spontaneous activation, and apoptosis, and improves platelet aggregation, irrespective of photochemical treatment, which should allow transfusion of platelets with better and longer-lasting functional properties., (© 2021 AABB.)

Background and Objectives: Small batch-pooled (mini-pool) whole blood (WB)-derived plasma could be an alternative cost-effective source of therapeutic plasma (TP), but carries an increased risk of transfusion-transmitted infection due to exposure of the recipient to several donors. This risk can be mitigated by inactivation of pathogens susceptible to the amotosalen-UVA (AUVA)-treatment. We evaluated the conservation of coagulation factors in AUVA-plasma prepared from WB stored overnight under routine operating conditions, to determine its therapeutic efficacy. Thrombin generation (TG) by the AUVA-plasma was used to provide an integrated measure of the hemostatic capacity., Materials and Methods: WB-donations (~450 ml) stored overnight were processed to prepare five leucocyte-depleted plasma mini-pools (1300 ml), which were divided into two parts and treated with AUVA. Each mini-pool yielded six AUVA-plasma units (200 ml) which were frozen (-25°C) within 19 h of WB-collection. Their hemostatic quality was evaluated before and after treatment for up to 12 months of storage., Results: Immediately after AUVA-treatment, the regulatory criteria for FVIII activity and fibrinogen content were met. As compared to untreated plasma there was a reduction in fibrinogen (14%), FV (9%), FVII (25%) and FVIII (32%). However, TG was similar in treated and untreated plasma at all-time-points., Conclusions: Frozen WB-derived AUVA-plasma prepared from mini-pools within 19 h of WB-collection met the quality standards required for TP and retained hemostatic capacity for up to 12 months. This product could provide a cost-effective convenient substitute for apheresis plasma., (© 2018 International Society of Blood Transfusion.)

The combined action of collagen and thrombin induces the formation of COAT platelets, which are characterised by a coat of procoagulant and adhesive molecules on their surface. Although recent work has started to highlight their clinical relevance, the exact mechanisms regulating the formation of procoagulant COAT platelets remain unclear. Therefore, we employed flow cytometry in order to visualise in real time surface and intracellular events following simultaneous platelet activation with convulxin and thrombin. After a rapid initial response pattern characterised by the homogenous activation of the fibrinogen receptor glycoprotein IIb/IIIa in all platelets, starting with a delay of about 2 minutes an increasing fraction transforms to procoagulant COAT platelets. Their surface is characterised by progressive loss of PAC-1 binding, expression of negative phospholipids and retention of α-granule von Willebrand factor. Intracellular events in procoagulant COAT platelets are a marked increase of free calcium into the low micromolar range, concomitantly with early depolarisation of the mitochondrial membrane and activation of caspase-3, while non-COAT platelets keep the intracellular free calcium in the nanomolar range and maintain an intact mitochondrial membrane. We show for the first time that the flow-cytometrically distinct fractions of COAT and non-COAT platelets differentially phosphorylate two signalling proteins, PKCα and p38MAPK, which may be involved in the regulation of the different calcium fluxes observed in COAT versus non-COAT platelets. This study demonstrates the utility of concomitant cellular and signalling evaluation using flow cytometry in order to further dissect the mechanisms underlying the dichotomous platelet response observed after collagen/thrombin stimulation.

Background: The INTERCEPT Blood System (IBS) using amotosalen-HCl and ultraviolet (UV)A inactivates a large spectrum of microbial pathogens and white blood cells in therapeutic plasma. Our aim was to evaluate to what extent IBS modifies the capacity of plasma to generate thrombin and induces qualitative or quantitative modifications of plasma proteins., Study Design and Methods: Plasma units from four donors were collected by apheresis. Samples were taken before (control [CTRL]) and after IBS treatment and stored at -80°C until use. The activities of plasma coagulation factors and inhibitors and the thrombin generation potential were determined using assays measuring clotting times and the calibrated automated thrombogram (CAT), respectively. The proteomic profile of plasma proteins was examined using a two-dimensional differential in-gel electrophoresis (2D-DIGE) method., Results: Nearly all of the procoagulant and antithrombotic factors tested retained at least 78% of their initial pre-IBS activity. Only FVII and FVIII displayed a lower level of conservation (67%), which nevertheless remained within the reference range for conventional plasma coagulation factors. The thrombin generation profile of plasma was conserved after IBS treatment. Among the 1331 protein spots revealed by 2D-DIGE analysis, only four were differentially expressed in IBS plasma compared to CTRL plasma and two were identified by mass spectrometric analysis as transthyretin and apolipoprotein A1., Conclusion: The IBS technique for plasma moderately decreases the activities of plasma coagulation factors and antithrombotic proteins, with no impact on the thrombin generation potential of plasma and very limited modifications of the proteomic profile., (© 2016 AABB.)

Background: Platelets (PLTs) are currently stored at room temperature (RT) for 5 to 7 days. So far, there exists no validated method for the preparation and long-term storage of dehydrated PLTs suitable for transfusion after rehydration. In this study, a desiccation process, zeodration, was applied to PLTs., Study Design and Methods: A complete procedure of dehydration at RT by zeodration was employed. Zeodrated human and mouse PLTs were characterized in vitro. Zeodrated mouse PLTs were transfused into clopidogrel-treated mice to evaluate their hemostatic properties., Results: The optimal conditions for dehydration of PLTs at RT in a laboratory scale zeodrator were defined as 145 mbar and 20.2 ± 1.5 °C. The recovery rate was 85 ± 2% and the dryness of zeodrated PLTs (Z&#95;PLTs) indicated that they were sufficiently stable for long-term storage. Rehydrated Z&#95;PLTs were round, were not aggregated, and expressed the glycoproteins required for PLT function. Z&#95;PLTs agglutinated in the presence of von Willebrand factor (VWF) and aggregated in response to thrombin or collagen independently of an active metabolism. In a flow system, Z&#95;PLTs could adhere to VWF and form aggregates on a collagen matrix. Thrombin was generated at the surface of Z&#95;PLTs as efficiently as on fresh PLTs. In clopidogrel-treated mice, which exhibited a severely prolonged bleeding time, continuous perfusion of Z&#95;PLTs restored normal hemostasis., Conclusion: Zeodration represents a new strategy to prepare PLTs with partly preserved aggregative properties after storage and rehydration. Z&#95;PLTs have potential hemostatic properties provided it is possible to improve their transfusion efficacy., (© 2015 AABB.)

We studied haemostasis in two mouse models with thrombocytosis caused by different pathogenic mechanisms. In one strain (Yall;Mpl-/-) thrombocytosis is driven by a misbalance between thrombopoietin and its receptor, whereas in the other strain, thrombocytosis is caused by expressing a human JAK2-V617F transgene (FF1) that depends on activation by Cre-recombinase (VavCre;FF1, MxCre;FF1). Thrombotic responses were increased following some, but not all types of challenges. In a vaso-occlusive thrombotic model following collagen-adrenaline injection we found increased mortality in both strains. Arterial thrombosis, examined after ferric chloride-induced carotid injury, was accelerated but with little impact on maximal thrombus size. In a vena cava stasis model, clots were of similar size as in wild-type controls, but exhibited a different composition with a higher platelet to fibrin ratio. Both thrombocytosis strains displayed increased haemorrhagic tendency in a tail bleeding assay. Yall;Mpl and VavCre;FF1 displayed a lower proportion of the more reactive high-molecular-weight forms of von Willebrand factor in their plasma, mimicking essential thrombocythaemia with very high platelet counts. Bleeding could not be explained by clear defects in platelet activation, which were normal or only weakly decreased. In conclusion, these models of thrombocytosis recapitulate several features of the haemorrhagic and thrombotic diatheses in ET and PV demonstrating potentials but also some limitations to study these major complications.

Extracellular ATP is becoming increasingly recognized as an important regulator of inflammation. However, the known repertoire of P2 receptor subtypes responsible for the proinflammatory effects of ATP is sparse. We looked at whether the P2X1 receptor, an ATP-gated cation channel present on platelets, neutrophils, and macrophages, participates in the acute systemic inflammation provoked by LPS. Compared with wild-type (WT) mice, P2X1(-/-) mice displayed strongly diminished pathological responses, with dampened neutrophil accumulation in the lungs, less tissue damage, reduced activation of coagulation, and resistance to LPS-induced death. P2X1 receptor deficiency also was associated with a marked reduction in plasma levels of the main proinflammatory cytokines and chemokines induced by LPS. Interestingly, macrophages and neutrophils isolated from WT and P2X1(-/-) mice produced similar levels of proinflammatory cytokines when stimulated with LPS in vitro. Intravital microscopy revealed a defect in LPS-induced neutrophil emigration from cremaster venules into the tissues of P2X1(-/-) mice. Using adoptive transfer of immunofluorescently labeled neutrophils from WT and P2X1(-/-) mice into WT mice, we demonstrate that the absence of the P2X1 receptor on neutrophils was responsible for this defect. This study reveals a major role for the P2X1 receptor in LPS-induced lethal endotoxemia through its critical involvement in neutrophil emigration from venules., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Objective: The glycoprotein (GP) Ib-V-IX complex regulates the adhesion, activation, and procoagulant activity of platelets. We previously reported that RAM.1, a rat monoclonal antibody directed against the extracellular domain of mouse GPIbβ, diminished adhesion of platelets and chinese hamster ovary cells transfected with the human GPIb-IX complex to von Willebrand factor under flow conditions. Here, we further evaluated the functional importance of GPIbβ by studying the impact of RAM.1 on GPIb-mediated platelet responses and in vitro and in vivo thrombus formation., Approach and Results: We show that RAM.1 dramatically reduced GPIb-mediated filopodia extension of chinese hamster ovary GPIb-IX cells after adhesion to von Willebrand factor. RAM.1 also reduced filopodia extension and GPIb-mediated Ca(2+) signaling after adhesion of mouse platelets to von Willebrand factor. RAM.1 inhibited thrombin generation in platelet-rich plasma without impairing phosphatidylserine exposure. In addition, RAM.1 reduced thrombus formation after perfusion of mouse whole blood over collagen in a shear-dependent manner. This effect was confirmed in vivo, because injection of F(ab)'2 fragments of RAM.1 diminished thrombus formation induced by laser beam injury of mesenteric arterioles and forceps injury of the abdominal aorta. In contrast, RAM.1 F(ab)'2 did not prolong the tail-bleeding time or increase the volume of blood lost., Conclusions: These findings are the first evidence that targeting a subunit other than GPIbα can lead to an antithrombotic effect via the GPIb-V-IX complex. This could represent an alternative way to reduce thrombus formation with a minor impact on hemostasis.

Background: Platelet concentrate (PC) functionality decreases during storage. This is referred to as the storage lesion. Pathogen inactivation may accelerate or induce lesions, potentially accounting for reduced viability. Our aim was to characterize functional and biochemical properties of platelets (PLTs) from photochemically treated buffy-coat PCs (PCT-PCs) compared to those from conventional PCs., Study Design and Methods: Four PCT-PCs and four conventional PCs were stored for 6.5 days and PLT function and proteomic profiles were examined at various time points during storage. To evaluate their intrinsic properties, samples of stored PLTs were taken, washed, and suspended in Tyrode's buffer before testing., Results: PLT counts and morphology were conserved although a slight increase in the PLT volume was observed after PCT. Glycoprotein (GP) IIbIIIa, IaIIa, and VI expression remained stable while GPIbα declined similarly in both types of PCs. A steep decrease (50%) in GPV occurred on Day 1.5 in PCT-PCs and Day 2.5 in control PCs. For both PCT- and control PCs, P-selectin expression and activated GPIIbIIIa remained low during storage. PCT- and control PCs were fully responsive to aggregation agonists up to Day 4.5 and exhibited similar perfusion functionality. Mitochondrial membrane potential and annexin A5 binding of PCT-PCs and control PCs were comparable. Two-dimensional differential in-gel electrophoresis and mass spectrometry profiles for 1882 protein spots revealed only three proteins selectively changed in PCT-PCs compared to control-PCs., Conclusion: Washed treated and untreated PCs have similar functional, morphologic, and proteomic characteristics provided that PLTs are suspended in an appropriate medium during testing., (© 2012 American Association of Blood Banks.)