Arnaud Dupuis

Immobilized fibrinogen activates human platelets through glycoprotein VI

Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.

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. 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.

A novel platelet-type von Willebrand disease mutation (GP1BA p.Met255Ile) associated with type 2B “Malmö/New York” von Willebrand disease

Interaction between von Willebrand factor (VWF) and platelet GPIbα is required for primary haemostasis. Lack or loss-of-function in the ligand-receptor pair results in bleeding complications. Paradoxically, gain-of-function mutations in VWF or GPIbα also result in bleeding complications as observed in type 2B von Willebrand disease (VWD) and platelet-type- (PT-) VWD, respectively. A similar phenotype is observed with increased ristocetin-induced platelet agglutination and disappearance of the highest molecular weight multimers of VWF. We evaluated a patient with a bleeding disorder and a biological presentation compatible with type 2B VWD. VWF and platelet functional assays, sequencing of the VWF and GP1BA genes, and expression studies in HEK cells were performed. Sequencing of the VWF gene in the propositus revealed a heterozygous p.Pro1266Leu mutation previously found in type 2B VWD Malmö/New York. These variants are characterised by a mild phenotype and a normal VWF multimer composition suggesting the presence of a second mutation in our propositus. Sequencing of the GP1BA gene revealed a heterozygous c.765G>A substitution changing Met at position 255 of GPIbα to Ile. This new mutation is located in the β-switch domain where five other gain-of-function mutations have been reported in PT-VWD. Expression of GPIbα Ile255 in HEK GPIb-IX cells resulted in enhanced VWF binding compared to wild-type, similar to known PT-VWD mutations (p.Val249, p.Ser249 and p.Val255) indicating that it contributes to the propositus defects. This first report associating PT- with type 2B VWD illustrates the importance of combining biological assays with genetic testing to better understand the clinical phenotype.

The contribution of mouse models to the understanding of constitutional thrombocytopenia.

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.

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.

Inherited platelet disorders : Management of the bleeding risk

Inherited platelet disorders are rare bleeding syndromes due to either platelet function abnormalities or thrombocytopenia which may be associated with functional defects. The haemorrhagic symptoms observed in these patients are mostly muco-cutaneous and of highly variable severity. Although 30 to 50% of the platelet disorders are still of unknown origin, the precise diagnosis of these pathologies by specialized laboratories together with haemorrhagic scores enables an assessment of the risk of bleeding in each patient. Depending on the diagnostic elements collected, an appropriate medical procedure can be proposed for each situation: scheduled or emergency surgical interventions and pregnancy follow-up. The pathologies most at risk correspond to Glanzmanns thrombasthenia, Bernard-Soulier syndrome, severe thrombocytopenia (<40,000 platelets/μL) and signalling protein abnormalities affecting the activation of GPIIb-IIIa, a membrane glycoprotein essential for platelet aggregation. For these particular patients, in whom the risk of bleeding can be increased by a factor of 40, management protocols during surgical procedures are generally based on the use of conventional platelet concentrates, for both prophylaxis and the control of active bleeding. The perinatal period in women with platelet disorders and their new-born also require special attention. Indeed, beyond unpredictable delivery haemorrhages, bleeding requiring a blood transfusion is observed after delivery in more than 50% of women with Glanzmanns thrombastenia or Bernard-Soulier syndrome.

In Vitro Binding of [³H]PSB-0413 to P2Y₁₂ Receptors.

The P2Y12 /ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y12 receptor defect and for evaluating P2Y12 receptor agonists and antagonists. In the absence of suitable anti‐P2Y12 antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assessing P2Y12 receptors with [3H]PSB‐0413, a selective antagonist for this site. The saturation and competition assays described herein make possible the determination of P2Y12 receptor density on cells, as well as the potencies and affinities of test agents at this site.

UNIT 1.35 In Vitro Binding of [3H]PSB-0413 to P2Y12 Receptors

The P2Y12 /ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y12 receptor defect and for evaluating P2Y12 receptor agonists and antagonists. In the absence of suitable anti‐P2Y12 antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assessing P2Y12 receptors with [3H]PSB‐0413, a selective antagonist for this site. The saturation and competition assays described herein make possible the determination of P2Y12 receptor density on cells, as well as the potencies and affinities of test agents at this site.

In Vitro Binding of [3H]PSB‐0413 to P2Y12 Receptors

The P2Y12 /ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y12 receptor defect and for evaluating P2Y12 receptor agonists and antagonists. In the absence of suitable anti‐P2Y12 antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assessing P2Y12 receptors with [3H]PSB‐0413, a selective antagonist for this site. The saturation and competition assays described herein make possible the determination of P2Y12 receptor density on cells, as well as the potencies and affinities of test agents at this site.