Many of the cellular responses that occur in activated platelets resemble events that take place following activation of cell-death pathways in nucleated cells. We tested the hypothesis that formation of the mitochondrial permeability transition pore (MPTP), a key signaling event during cell death, also plays a critical role in platelet activation. Stimulation of murine platelets with thrombin plus the glycopro-tein VI agonist convulxin resulted in a rapid loss of mitochondrial transmem-brane potential (m) in a subpopulation of activated platelets. In the absence of cyclophilin D (CypD), an essential regulator of MPTP formation, murine platelet activation responses were altered. CypD-deficient platelets exhibited defects in phosphatidylserine externalization, high-level surface fibrinogen retention, membrane vesiculation, and procoagulant activity. Also, in CypD-deficient platelet-rich plasma, clot retraction was altered. Stimulation with thrombin plus H 2 O 2 , a known activator of MPTP formation, also increased high-level surface fibrinogen retention , phosphatidylserine externaliza-tion, and platelet procoagulant activity in a CypD-dependent manner. In a model of carotid artery photochemical injury, thrombosis was markedly accelerated in CypD-deficient mice. These results implicate CypD and the MPTP as critical regulators of platelet activation and suggest a novel CypD-dependent negative-feedback mechanism regulating arterial thrombosis. (Blood. 2008;111:1257-1265)

Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways.Increasing evidence suggests that multiple trans-acting factors and cis-acting elements cooperatively regulate the expression of cardiac muscle-specific genes (reviewed in references 28 and 36), unlike skeletal muscle myogenesis where myogenic basic helix-loop-helix factors can activate the entire myogenic program (reviewed by Olson and Klein [37a]). For example, the cardiac ␣-myosin heavy chain gene (␣-MHC) is synergistically activated by myocyte-specific enhancer factor 2 (MEF2) and thyroid hormone receptor, and this activation depends on the binding of each factor to the DNA target sequences (27). Multiple transcription factors, such as E-box and CArG-box binding factors and Sp1, are required for the muscle-specific expression of the cardiac ␣-actin gene (37b). Cardiac myosin light chain 2v (MLC2v) gene expression appears to depend on several factors, including YB-1 and CARP (44,45).Homeobox genes have been studied extensively in many animal species, where they play fundamental roles in specifying cell fate and positional identity in embryos. The nk-4/msh-2 Drosophila gene, tinman, has been ...

Next-generation RNA sequence analysis of platelets from an individual with autosomal recessive gray platelet syndrome (GPS, MIM139090) detected abnormal transcript reads, including intron retention, mapping to NBEAL2 (encoding neurobeachin-like 2). Genomic DNA sequencing confirmed mutations in NBEAL2 as the genetic cause of GPS. NBEAL2 encodes a protein containing a BEACH domain that is predicted to be involved in vesicular trafficking and may be critical for the development of platelet α-granules.

Background: Inactivation of integrin ␣ IIb ␤ 3 reverses platelet aggregate formation upon coagulation. Results and conclusion: Platelets from patient (Scott) and mouse (Capn1 Ϫ/Ϫ and Ppif Ϫ/Ϫ ) blood reveal a dual mechanism of ␣ IIb ␤ 3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. Significance: These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets.