ABSTRACT
BACKGROUND: Ectopically expressed B-domainless factor VIII in megakaryocytes is stored in α-granules, is effective in a number of murine hemostatic models, and is protected from circulating inhibitors. However, this platelet (p) FVIII has different temporal-spatial availability from plasma FVIII, with limited efficacy in other murine hemostatic models. OBJECTIVES AND METHODS: We sought to improve pFVIII hemostatic efficacy by expressing canine (c) FVIII, which has higher stability and activity than human (h) FVIII in FVIII(null) mice. RESULTS AND CONCLUSIONS: We found that pcFVIII was more effective than phFVIII at restoring hemostasis, but peak pcFVIII antigen levels were lower and were associated with greater megakaryocyte apoptosis than phFVIII. These new insights suggest that pFVIII gene therapy strategies should focus on enhancing activity rather than levels. We previously showed that modification of the PACE/furin cleavage site in hFVIII resulted in secretion of hFVIII primarily as a single-chain molecule with increased biological activity. In megakaryocytes, this variant was expressed at the same level as phFVIII with a lentiviral bone marrow transplant approach to reconstitute FVIII(null) mice, but was more effective, resulting in near-normal hemostasis in the cremaster laser injury model. These studies may have implications for pFVIII gene therapy in hemophilia A.
Subject(s)
Apoptosis , Blood Platelets/cytology , Factor VIII/chemistry , Factor VIII/genetics , Genetic Therapy/methods , Megakaryocytes/cytology , Animals , Carotid Arteries/pathology , Cell Line , Cricetinae , Dogs , Hemophilia A/genetics , Hemostasis , Humans , Lentivirus/genetics , Mice , Mice, TransgenicABSTRACT
BACKGROUND: Stromal interaction molecule 1 (STIM1) was recently identified as a critical component of store-operated calcium entry (SOCE) in platelets. We previously reported the Ca(2+) -sensing guanine nucleotide exchange factor CalDAG-GEFI as a critical molecule in Ca(2+) signaling in platelets. OBJECTIVE: To evaluate the contribution of STIM1/SOCE to Ca(2+) -dependent platelet activation and thrombosis, we here compared the activation responses of platelets lacking STIM1 and platelets lacking CalDAG-GEFI. METHODS: The murine Stim1 gene was conditionally deleted in the megakaryocyte/platelet lineage. CalDAG-GEFI(-/-) and Stim1(fl/fl) PF4-Cre mice, along with littermate control mice, were used for in vitro and in vivo experiments under flow as well as static conditions. RESULTS: Integrin α(IIb) ß(3) -mediated aggregation was markedly impaired in CalDAG-GEFI-deficient but not STIM1-deficient platelets, under both static and flow conditions. In contrast, deficiency in either STIM1 or CalDAG-GEFI significantly impaired the ability of platelets to express phosphatidylserine on the cell surface. When subjected to a laser injury thrombosis model, mice lacking STIM1 in platelets were characterized by the formation of unstable platelet-rich thrombi and delayed and reduced fibrin generation in injured arterioles. In CalDAG-GEFI(-/-) mice, fibrin generation was also delayed and reduced, but platelet accumulation was almost abolished. CONCLUSIONS: Our studies suggest that: (i) STIM1/SOCE is critical for the procoagulant activity but not the proadhesive function of platelets; and (ii) at the site of vascular injury, STIM1 and CalDAG-GEFI are critical for the first wave of thrombin generation mediated by procoagulant platelets.
