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Targeting factor VIII expression to platelets for hemophilia A gene therapy does not induce an apparent thrombotic risk in mice

2016; Elsevier BV; Volume: 15; Issue: 1 Linguagem: Inglês

10.1111/jth.13436

1538-7933

Christina K. Baumgartner, Jeremy G. Mattson, Hartmut Weiler, Qizhen Shi, Robert R. Montgomery,

Blood Coagulation and Thrombosis Mechanisms

Essentials•Platelet‐Factor (F) VIII gene therapy is a promising treatment in hemophilia A.•This study aims to evaluate if platelet‐FVIII expression would increase the risk for thrombosis.•Targeting FVIII expression to platelets does not induce or elevate thrombosis risk.•Platelets expressing FVIII are neither hyper‐activated nor hyper‐responsive.AcknowledgementsWe thank R. Bercovitz at our institution for help with establishing whole blood flow cytometry for the detection of platelet activation, and B. Cooley at the University of North Carolina (Chapel Hill) for critical discussion on the ferric chloride‐induced carotid artery injury model. This work was supported by the Novo Nordisk Access‐to‐Insight initiative (C. K. Baumgartner), the BloodCenter Research Foundation (R. R. Montgomery and Q. Shi), the Bayer Hemophilia Award (Q. Shi), National Institutes of Health grants HL‐5P01HL044612 (R. R. Montgomery), HL‐5P01HL081588 (R. R. Montgomery), HL‐7R01HL112641 (R. R. Montgomery), and HL‐R01HL102035 (Q. Shi), the MACC fund (Q. Shi), the Children's Hospital Foundation (Q. Shi), and funding from the Medical College and the Children's Hospital of Wisconsin to the Pediatric Cardiac Hemostasis and Thrombosis (PCH&T) Research Center (R. R. Montgomery).Novo Nordisk Access‐to‐Insight initiativeBloodCenter Research FoundationNational Institutes of HealthHL‐5P01HL044612HL‐5P01HL081588HL‐7R01HL112641HL‐R01HL102035MACC fundChildren's Hospital FoundationMedical College and the Children's Hospital of WisconsinSummaryBackgroundTargeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation.ObjectiveTo evaluate whether platelet‐FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach.MethodsIn this study, platelet‐FVIII‐expressing transgenic mice were examined either in steady‐state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride‐induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D‐dimer, thrombin–antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet–leukocyte aggregates, were assessed.ResultsWe generated a new line of transgenic mice that expressed 30‐fold higher levels of platelet‐expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady‐state conditions and prothrombotic conditions induced by lipopolysaccharide‐mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet‐expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII‐expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation.ConclusionWe conclude that, in mice, more than 30‐fold higher levels of platelet‐expressed FVIII than are required for therapeutic efficacy in hemophilia A are not associated with a thrombotic predilection. Essentials•Platelet‐Factor (F) VIII gene therapy is a promising treatment in hemophilia A.•This study aims to evaluate if platelet‐FVIII expression would increase the risk for thrombosis.•Targeting FVIII expression to platelets does not induce or elevate thrombosis risk.•Platelets expressing FVIII are neither hyper‐activated nor hyper‐responsive.AcknowledgementsWe thank R. Bercovitz at our institution for help with establishing whole blood flow cytometry for the detection of platelet activation, and B. Cooley at the University of North Carolina (Chapel Hill) for critical discussion on the ferric chloride‐induced carotid artery injury model. This work was supported by the Novo Nordisk Access‐to‐Insight initiative (C. K. Baumgartner), the BloodCenter Research Foundation (R. R. Montgomery and Q. Shi), the Bayer Hemophilia Award (Q. Shi), National Institutes of Health grants HL‐5P01HL044612 (R. R. Montgomery), HL‐5P01HL081588 (R. R. Montgomery), HL‐7R01HL112641 (R. R. Montgomery), and HL‐R01HL102035 (Q. Shi), the MACC fund (Q. Shi), the Children's Hospital Foundation (Q. Shi), and funding from the Medical College and the Children's Hospital of Wisconsin to the Pediatric Cardiac Hemostasis and Thrombosis (PCH&T) Research Center (R. R. Montgomery).Novo Nordisk Access‐to‐Insight initiativeBloodCenter Research FoundationNational Institutes of HealthHL‐5P01HL044612HL‐5P01HL081588HL‐7R01HL112641HL‐R01HL102035MACC fundChildren's Hospital FoundationMedical College and the Children's Hospital of Wisconsin •Platelet‐Factor (F) VIII gene therapy is a promising treatment in hemophilia A.•This study aims to evaluate if platelet‐FVIII expression would increase the risk for thrombosis.•Targeting FVIII expression to platelets does not induce or elevate thrombosis risk.•Platelets expressing FVIII are neither hyper‐activated nor hyper‐responsive. We thank R. Bercovitz at our institution for help with establishing whole blood flow cytometry for the detection of platelet activation, and B. Cooley at the University of North Carolina (Chapel Hill) for critical discussion on the ferric chloride‐induced carotid artery injury model. This work was supported by the Novo Nordisk Access‐to‐Insight initiative (C. K. Baumgartner), the BloodCenter Research Foundation (R. R. Montgomery and Q. Shi), the Bayer Hemophilia Award (Q. Shi), National Institutes of Health grants HL‐5P01HL044612 (R. R. Montgomery), HL‐5P01HL081588 (R. R. Montgomery), HL‐7R01HL112641 (R. R. Montgomery), and HL‐R01HL102035 (Q. Shi), the MACC fund (Q. Shi), the Children's Hospital Foundation (Q. Shi), and funding from the Medical College and the Children's Hospital of Wisconsin to the Pediatric Cardiac Hemostasis and Thrombosis (PCH&T) Research Center (R. R. Montgomery).Novo Nordisk Access‐to‐Insight initiativeBloodCenter Research FoundationNational Institutes of HealthHL‐5P01HL044612HL‐5P01HL081588HL‐7R01HL112641HL‐R01HL102035MACC fundChildren's Hospital FoundationMedical College and the Children's Hospital of Wisconsin Background Targeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation. To evaluate whether platelet‐FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. In this study, platelet‐FVIII‐expressing transgenic mice were examined either in steady‐state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride‐induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D‐dimer, thrombin–antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet–leukocyte aggregates, were assessed. We generated a new line of transgenic mice that expressed 30‐fold higher levels of platelet‐expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady‐state conditions and prothrombotic conditions induced by lipopolysaccharide‐mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet‐expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII‐expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. We conclude that, in mice, more than 30‐fold higher levels of platelet‐expressed FVIII than are required for therapeutic efficacy in hemophilia A are not associated with a thrombotic predilection.