Glycoprotein V : the unsolved GPV puzzle.

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.

Human platelets labeled at two discrete biotin densities are functional in vitro and are detected in vivo in the murine circulation: A promising approach to monitor platelet survival in vivo in clinical research.

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.

Removal of citrate from PAS-III additive solution improves functional and biochemical characteristics of buffy-coat platelet concentrates stored for 7 days, with or without INTERCEPT pathogen reduction.

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.

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

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.

Delayed-onset of procoagulant signalling revealed by kinetic analysis of COAT platelet formation.

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.

Hemostatic properties and protein expression profile of therapeutic apheresis plasma treated with amotosalen and ultraviolet A for pathogen inactivation.

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.

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

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.

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.

Haemorrhagic and thrombotic diatheses in mouse models with thrombocytosis.

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.

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 GPIbβ, diminished adhesion of platelets and chinese hamster ovary cells transfected with the human GPIb-IX complex to von Willebrand factor under flow conditions.

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

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.

A central role of GPIb-IX in the procoagulant function of platelets that is independent of the 45-kDa GPIbalpha N-terminal extracellular domain.

Activated platelets become procoagulant and efficiently promote the conversion of prothrombin to thrombin. A role of the GPIb-V-IX complex has long been postulated in view of the decreased prothrombin consumption in Bernard-Soulier patients. We evaluated the impact of GPIb-V-IX deficiency and the requirement for the GPIbalpha extracellular domain.

A 2-step mechanism of arterial thrombus formation induced by human atherosclerotic plaques.

Objectives: The 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.

Background: Rupture 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).

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.

Reduced atherosclerotic lesions in P2Y1/apolipoprotein E double-knockout mice: the contribution of non-hematopoietic-derived P2Y1 receptors.

Background: The P2Y(1) receptor plays a key role in arterial thrombosis and is widely expressed in many cell types involved in atherosclerosis. The aim of this study was to evaluate its potential involvement in the development of atherosclerotic lesions.

Methods And Results: Apolipoprotein E-deficient (ApoE(-/-)) and P2Y(1)(-/-)/ApoE(-/-) mice were maintained on regular chow for 17 or 30 weeks before analysis of atherosclerotic lesions.

Ex vivo inhibition of thrombus formation by an anti-glycoprotein VI Fab fragment in non-human primates without modification of glycoprotein VI expression.

Objectives: Glycoprotein (GP)VI is an attractive target for the development of new antithrombotic drugs. Its deficiency protects animals in several models of thrombosis, arterial stenosis and ischemia--reperfusion while inducing no major bleeding tendency. The Fab fragment of one anti-GPVI monoclonal antibody (9O12.

Megakaryocyte-restricted MYH9 inactivation dramatically affects hemostasis while preserving platelet aggregation and secretion.

Mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain IIA result in bleeding disorders characterized by a macrothrombocytopenia. To understand the role of myosin in normal platelet functions and in pathology, we generated mice with disruption of MYH9 in megakaryocytes. MYH9Delta mice displayed macrothrombocytopenia with a strong increase in bleeding time and absence of clot retraction.

Synthesis of GPIb beta with novel transmembrane and cytoplasmic sequences in a Bernard-Soulier patient resulting in GPIb-defective signaling in CHO cells.

The molecular defect of a new Bernard-Soulier patient, originating from Morocco and presenting thrombocytopenia with large platelets and an absence of ristocetin-induced platelet agglutination, has been identified and reproduced in transfected heterologous cells. Gene sequencing revealed insertion of a guanine in the domain coding for the transmembrane region of the glycoprotein (GP) Ib beta subunit. This mutation causes a translational frame shift, which creates putative novel transmembrane and cytoplasmic 37 and 125 amino acids domains, respectively.

Flow cytometric analysis of intraplatelet VASP phosphorylation for the detection of clopidogrel resistance in patients with ischemic cardiovascular diseases.

Interindividual variability of the inhibitory effect of clopidogrel on platelet functions leading to clopidogrel resistance has been described in some patients with ischemic cardiovascular disease. A reliable laboratory test is therefore needed to identify patients insufficiently protected by this antiplatelet treatment. The phosphorylation of vasodilator-stimulated phosphoprotein (VASP), an intraplatelet actin regulatory protein, is dependent on the level of activation of the platelet P2Y12 receptor, which is targeted by clopidogrel.

The plaque lipid lysophosphatidic acid stimulates platelet activation and platelet-monocyte aggregate formation in whole blood: involvement of P2Y1 and P2Y12 receptors.

Despite the fact that lysophosphatidic acid (LPA) has been identified as a main platelet-activating lipid of mildly oxidized low-density lipoprotein (LDL) and human atherosclerotic lesions, it remains unknown whether it is capable of activating platelets in blood. We found that LPA at concentrations slightly above plasma levels induces platelet shape change, aggregation, and platelet-monocyte aggregate formation in blood. 1-alkyl-LPA (16:0 fatty acid) was almost 20-fold more potent than 1-acyl-LPA (16:0).

Differential involvement of the P2Y1 and P2Y12 receptors in platelet procoagulant activity.

Objective: In vivo, activated platelets contribute to the initiation of thrombin generation through the exposure of phosphatidylserine to form a procoagulant catalytic surface and through platelet-leukocyte interactions, which lead to the exposure of leukocyte tissue factor (TF). On the basis of observations that the platelet P2Y1 and P2Y12 receptors both contribute to thrombosis and thrombin formation in an in vivo model of TF-induced thromboembolism, we further characterized the role of these receptors in thrombin generation.

Methods And Results: By using the selective P2 antagonists MRS2179 and AR-C69931MX, the P2Y12 receptor was found to be involved in thrombin-induced exposure of PS on isolated platelets and consequently in TF-induced thrombin formation in platelet-rich plasma.

Endotoxin-free heat-shock protein 70 fails to induce APC activation.

Previous work has suggested that the peptide-carrier, heat-shock protein (hsp)70, could directly activate APC. Here we show that this ability is related to endotoxin contamination of the human rhsp70 produced in Escherichia coli. Hence, the ability of 1-3 microg/ml of rhsp70 to induce the maturation of human monocyte-derived DC is abrogated in the presence of the LPS-antagonist polymyxin B or when the rhsp70 contains less than 60 IU/mg endotoxin.

Hemostasis imbalance in experimental hypertension.

Background: The rat model of chronic intoxication by N(G) -nitro-L-arginine methyl ester (L-NAME) induces severe systemic arterial hypertension and progressive ischemic lesions in the central nervous system and kidneys. We investigated the possible molecular basis of these thrombotic events.

Methods And Results: Administration of L-NAME increased plasma markers of thrombin generation, thrombin-antithrombin complexes, and soluble glycoprotein V, measured by specific ELISA.