Catherine Ravanat

A 2-Step Mechanism of Arterial Thrombus Formation Induced by Human Atherosclerotic Plaques

OBJECTIVESnThe aim of this study was to understand the initial mechanism of arterial thrombus formation induced by vulnerable human atherosclerotic plaques to re-assess and improve current antithrombotic strategies.nnnBACKGROUNDnRupture of atherosclerotic plaques causes arterial thrombus formation that might lead to myocardial infarction and ischemic stroke. Atherothrombosis is considered as an inseparable tangle of platelet activation and coagulation processes, involving plaque components such as tissue factor (TF) and collagen as well as blood-borne TF and coagulation factor XIIa (FXIIa). A combination of anticoagulants and antiplatelet agents is the present treatment.nnnMETHODSnHuman atheromatous plaque material was exposed to blood or blood components at physiological calcium/magnesium concentration. Platelet aggregation and coagulation were measured under static and arterial flow conditions by state-of-the-art microscopic and physiological techniques. Plaque TF, plaque collagen, FXIIa, and platelet glycoprotein VI (GPVI) were specifically inhibited.nnnRESULTSnPlaques induced thrombus formation by 2 discrete steps. The rapid first phase of GPVI-mediated platelet adhesion and aggregation onto plaque collagen occurred within 1 min. The second phase of coagulation started after a delay of >3 min with the formation of thrombin and fibrin, and was driven entirely by plaque TF. Coagulation occurred only in flow niches provided by platelet aggregates, with no evidence for a role of blood-borne TF and FXIIa. Inhibition of GPVI but not plaque TF inhibited plaque-induced thrombus formation.nnnCONCLUSIONSnThe major thrombogenic plaque components--collagen and TF--induce platelet activation and coagulation, respectively, in 2 consecutive steps. Targeting specifically the first step is crucial and might be sufficient to inhibit atherothrombus formation.

Preserved functional and biochemical characteristics of platelet components prepared with amotosalen and ultraviolet A for pathogen inactivation

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.

The P2X1 Receptor Is Required for Neutrophil Extravasation during Lipopolysaccharide-Induced Lethal Endotoxemia in Mice

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.

Targeting Platelet GPIbβ Reduces Platelet Adhesion, GPIb Signaling and Thrombin Generation and Prevents Arterial Thrombosis

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 GPIb&bgr;, 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 GPIb&bgr; 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 Ca2+ 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 GPIb&agr; 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.

Use of tandem Biacore-mass spectrometry to identify platelet membrane targets of novel monoclonal antibodies.

The monoclonal antibodies (mAbs) ALMA.17 and ALMA.7 recognize human platelet membrane proteins. ALMA.17 is directed against alpha(IIb)beta(3) integrin, but the target of ALMA.7 was unknown previously. Tandem Biacore micropurification and mass spectrometry (MS) analysis of a platelet membrane lysate was used to identify the target of ALMA.7. Detergent lysates enriched in membrane proteins were perfused over immobilized ALMA.17 or ALMA.7 in a Biacore system. The captured proteins were eluted, concentrated on C3 magnetic beads, and digested with trypsin before nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Critical adjustments needed to be made in (i) the detergent mixture to preserve protein antigenicity and sensor chip integrity and (ii) the method of trypsin digestion to concentrate the proteins and use elution buffers that do not interfere with MS. The target of ALMA.17 was confirmed to be alpha(IIb)beta(3) integrin, whereas that of ALMA.7 was identified as CD226 (PTA-1, DNAM-1, TLiSa-1). This was confirmed by immunoassays comparing ALMA.7 with a commercial anti-CD226 mAb. Thus, a tandem Biacore and nano LC-MS/MS strategy allowed unambiguous identification of an unknown antigen in a complex medium such as a platelet membrane lysate. This strategy may be employed to identify any protein capturable on a sensor chip provided that one uses appropriate experimental conditions.

Amotosalen/UVA pathogen inactivation technology reduces platelet activability, induces apoptosis and accelerates clearance

With interest we read the paper by Stivala et al.,[1][1] recently published in Haematologica, evaluating the structural and functional consequences induced by Amotosalen/UVA treatment using the Intercept Blood System (IBS) on platelets from apheresis platelet concentrates (PCs) during storage. This

Dehydration of blood platelets by zeodration: in vitro characterization and hemostatic properties in vivo

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.

In vitro quality of amotosalen-UVA pathogen-inactivated mini-pool plasma prepared from whole blood stored overnight

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.

Traitement photochimique d’un mélange de plasmas issus de sang total et congélation dans un délai de 18 à 19 heures après le prélèvement

Le procede de traitement INTERCEPT Blood System (CErus) utilisant l’amotosalen et les rayons UVA a ete mis au point pour l’inactivation d’agents pathogenes dans les plaquettes et le plasma. Le plasma d’apherese traite et congele dans les 18xa0h (PFC-IA) est utilise en France. L’objectif de cette etude etait d’evaluer la qualite de melanges de plasmas issus de sang total traites par amotosalen et congeles dans les 18xa0a 19xa0h suivant la collecte. Methodes Cinq plasmas issus de sang total isogroupe ABO sont melanges, puis divises en 2xa0sous-unites de 650xa0mL pour etre traitees chacune par le procede d’inactivation des pathogenes, generant ainsi 2xa0×xa03xa0unites de 200xa0mL de plasmas frais congeles. Les 2xa0sites ont ainsi prepare 40xa0melanges de 5xa0plasmas (10xa0O, 30xa0non-O). La qualite du plasma est evaluee avant traitement (T1), puis apres 2xa0semaines (T2), 6xa0mois (T3) et 12xa0mois (T4) de conservation a −25xa0°C sur les parametres biologiques definis dans l’«xa0avis aux demandeursxa0» de l’ANSM. Resultats L’evolution des parametres biologiques pendant la conservation est detaillee dans le tableau 1. les taux de recuperation post-traitement varient de 81xa0% a 95xa0% et est egal a 68xa0% pour le facteurxa0VIII. Ces parametres demeurent stables pendant la conservation. Les plasmas sont conformes a 12xa0mois pour le facteurxa0VIII et le fibrinogene avec respectivement 77xa0% des unitesxa0≥xa0a 0,5xa0UI/mL et 90xa0% des unitesxa0>xa02xa0g/L (nxa0=xa030xa0; 33xa0% de Oxa0; ≥xa070xa0% requis). Conclusion Le melange de plasma issu de sang total traite par la methode INTERCEPT presente une qualite biochimique qui repond aux normes de qualite du plasma therapeutique inactive.

Test de génération de thrombine après traitement photochimique et conservation de plasma dérivé de sang total

Le procede INTERCEPT Blood System (Cerus), utilisant de l’amotosalen et des UVA a ete mis au point pour l’inactivation d’agents pathogenes dans les plaquettes et le plasma. La qualite de melanges de plasma issus de sang total traites a ete evaluee avec le test de generation de thrombine (TGT). Il permet de mesurer, en continu, la concentration de thrombine active et non seulement le temps necessaire a la generation des premieres traces comme dans les tests de coagulation classiques. Methodes Cinq plasmas deleucocytes issus de sang total de meme groupe ABO sont melanges puis divises en 2xa0sous-unites de 650xa0mL traitees par le procede INTERCEPT. Les 2xa0sites ont prepare 40xa0melanges donnant chacun apres traitement 2xa0×xa03xa0unitesxa0>xa0200xa0mL congelees dans les 19xa0h. Vingt-huit parametres sont mesures dont le fibrinogene, les facteurs V, VII, VIII, les TQ et TCA et le TGT a 1xa0et 20xa0pM de facteur tissulaire avant traitement (T 1 ), puis apres 2xa0semaines (T 2 ), 6xa0mois (T 3 ) et 12xa0mois (T 4 ) axa0 Resultats Bien que l’on observe ( Tableau 1 ) une baisse des facteurs de coagulation liee au traitement de respectivement 14xa0%, 9xa0%, 24xa0% et 32xa0% pour les facteurs I, V, VII, VIII et un allongement des TQ et TCA de 18xa0% et 10xa0%, aucun des parametres du TGT n’est modifie de maniere marquante avec le traitement pour la concentration de 20xa0pM de FT. La cinetique de la reaction est affectee a la concentration de FT de 1xa0pM sans influence sur la quantite totale de thrombine generee. Les parametres evoluent peu avec la conservation a −25xa0°C. Conclusion Le traitement photochimique n’affecte pas de maniere substantielle le potentiel hemostatique du plasma qui reste intact ou peu modifie egalement apres conservation.