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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. The studies by Graham et al in this issue of Blood reveal the importance of dense granule secretion for in vivo platelet accumulation following laser injury of cremaster arterioles.8 The most striking observation is that dense granule deficient mice (ruby-eye mice) show almost no platelet accumulation at injury sites, indicating that molecules released by dense granules are necessary for efficient thrombus formation. In VAMP-8⫺/⫺ mice, thrombus formation is delayed and decreased (but not absent) following laser injury, suggesting that the v-SNARE Endobrevin/VAMP-8 is an important component of in vivo platelet granule secretion. The incomplete inhibition of platelet accumulation at sites of injury in VAMP-8⫺/⫺ mice was somewhat surprising because VAMP-8 mediates the release of all platelet granules, hence an additive effect might have been expected. The results can be explained by functional redundancy of SNAREs, whereby VAMP-2 or -3 can mediate granule release in the absence of VAMP-8.7 The authors observe that VAMP-2 and VAMP-8 levels are comparable in mouse platelets, whereas VAMP-3 and VAMP-8 levels are comparable in human platelets. As VAMP-2 levels are greatly diminished in human platelets, this suggests that the major alternative granule v-SNARE in humans is VAMP-3. The dense granule constituents responsible for mediating platelet accumulation in vivo were examined in vitro via aggregation studies of plasma-free washed platelets. Compared with wild-type, platelets from VAMP8⫺/⫺ and ruby-eye mice required 1.5- to 10-fold higher concentrations of thrombin for complete aggregation, while addition of exogenous ADP produced normal aggregation responses at low thrombin concentrations. The function of PKC␣⫺/⫺ mouse platelets (which have impaired dense granule biogenesis and defective secretion of both dense and ␣-granules) can also be restored by ADP in vitro.9 Thus, ADP is a key dense granule component, and its release during activation is likely required for efficient platelet accumulation at vascular injury sites. The thrombus formation defects observed in ruby-eye mice indicate that in vivo thrombin generation following laser injury cannot compensate for the absence of dense granule secretion. Although these studies provide important new insights into the role of platelet granule secretion in vivo, some questions do remain. blood 3 0 J U L Y 2 0 0 9 I V O L U M E 1 1 4 , N U M B E R 5 For example, which SNAREs and regulatory proteins control selective secretion from granules, including the recently discovered distinct populations of ␣-granules?10 What contribution does ␣-granule secretion make to thrombus formation in vivo? The availability of megakaryocyte-restricted gene knockout mice will undoubtedly shed light on some of these questions. Importantly, the current studies suggest that antiplatelet therapies targeting granule secretion could attenuate acute thrombus formation and also reduce the development of chronic atherosclerotic lesions by inhibiting the release of inflammatory and immune-modulating factors from platelets. Conflict-of-interest disclosure: The author declares no competing financial interests. ■ REFERENCES 1. Lo B, Li L, Gissen P, et al. Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet ␣-granule biogenesis. Blood. 2005;106: 4159-4166. 2. Nurden P, Nurden AT. Congenital disorders associated with platelet dysfunctions. Thromb Haemost. 2008;99: 253-263. 3. Flaumenhaft R. Molecular basis of platelet granule secretion. Arterioscler Thromb Vasc Biol. 2003;23:1152-1160. 4. Ren Q, Ye S, Whiteheart SW. The platelet release reaction: just when you thought platelet secretion was simple. Curr Opin Hematol. 2008;15:537-541. 5. Reed GL. Platelet secretory mechanisms. Semin Thromb Hemost. 2004;30:441-450. 6. Südhof TC, Rothman JE. Membrane fusion: grappling with SNARE and SM proteins. Science. 2009;323:474-477. 7. Ren Q, Barber HK, Crawford GL, et al. Endobrevin/ VAMP-8 is the primary v-SNARE for the platelet release reaction. Mol Biol Cell. 2007;18:24-33. 8. Graham GJ, Ren Q, Dilks JR, Blair P, Whiteheart SW, Flaumenhaft R. Endobrevin/VAMP-8 – dependent dense granule release mediates thrombus formation in vivo. Blood. 2009;114:1083-1090. 9. Konopatskaya O, Gilio K, Harper MT, et al. PKC␣ regulates platelet granule secretion and thrombus formation in mice. J Clin Invest. 2009;119:399-407. 10. Italiano JE Jr, Richardson JL, Patel-Hett S, et al. Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released. Blood. 2008; 111:1227-1233. ● ● ● THROMBOSIS & HEMOSTASIS Comment on Sutherland et al, page 1091 When 1 plus 1 equals 3 in VWD ---------------------------------------------------------------------------------------------------------------Anne C. Goodeve UNIVERSITY OF SHEFFIELD In this issue of Blood, Sutherland and colleagues describe an unusual in-frame deletion of exons 4-5 of the VWF gene associated with both dominantly inherited type 1 and with type 3 VWD.1 he mutation was initially identified in homozygous type 3 von Willebrand disease (VWD) patients, subsequently found in further compound heterozygous patients with type 3 VWD, and then sought in a previously studied cohort of type 1 VWD cases.2 It has thus been identified in 8 of 24 (33%) alleles of the type 3 VWD cohort of 12 apparently unrelated index cases (2 homozygotes and 4 compound heterozygotes) and additionally identified in 2 of 34 (6%) type 1 VWD index cases. Most mutations identified in type 3 VWD are “null” mutations, resulting in a lack of expression due to non-sense, frameshift, splice site, and large deletion mutations. Large deletions typically result in a lack of von Willebrand factor (VWF) expression, and penetrance of bleeding symptoms in simple heterozygous individuals with such null mutations is very low. The deletion currently described is unusual in that a truncated VWF T protein is produced, albeit in very low quantity, that has a dominant negative effect on expression of the second allele. The small group of heterozygous type 1 VWD patients with the mutation have mean VWF levels of around 25 IU/dL, and the mutation appears to result in bleeding in the majority of individuals investigated. All but one family where the mutation was identified shared a common haplotype associated with the deletion comprising 25 SNP throughout VWF. A second related haplotype found in heterozygous form in only one type 3 family appeared to have arisen from a subsequent recombination 3⬘ to the deletion. The distribution of the deletion is not yet known; however, its presence in a number of presumed independent type 3 and type 1 VWD families suggests that it may be widespread in families of British origin, possibly including emigrant families. Analysis of the other 2 large 933 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. plicated in the recombination event could also currently likely to be missed, where a hetbe investigated. erozygous deletion is Conflict-of-interest disclosure: A.C.G. remasked by the presence ceived sponsorship from CSL-Behring for mainof an intact second altenance of ISTH-SSC on VWF website. ■ lele. Initial analysis of the partial cloned seREFERENCES quence of VWF indi1. Sutherland MS, Cumming AM, Bowman M, et al. A cated that there were novel deletion mutation is recurrent in von Willebrand dis14 Alu sequences idenease types 1 and 3. Blood. 2009;114:1091-1098. tified,7 but there are 2. Cumming A, Grundy P, Keeney S, et al. An investigation of the von Willebrand factor genotype in UK patients likely to be many more Alu-mediated VWF deletion followed by recombination in one family. The top diagnosed to have type 1 von Willebrand disease. Thromb than this in the entire portion of the figure shows the VWF gene intron-exon structure. Unequal homoloHaemost. 2006;96:630-641. gous recombination between Alu Y repeats in introns 3 and 5 removes 8631bp and gene. Close sequence 3. Goodeve A, Eikenboom J, Castaman G, et al. Phenotype results in an in-frame deletion of exons 4-5. The deletion is located on an ancestral similarity between reand genotype of a cohort of families historically diagnosed haplotype (Hap 1), present in all but one of the alleles investigated by Sutherland et with type 1 von Willebrand disease in the European study, al.1 A subsequent recombination event (panels 2 and 3) results in generation of peat sequences can Molecular and Clinical Markers for the Diagnosis and Manhaplotype 2 (Hap 2). The recombination location is likely to lie between intron 5 and help promote unequal agement of Type 1 von Willebrand Disease (MCMDMexon 8. Professional illustration by Debra T. Dartez. 1VWD). Blood. 2007;109:112-121. homologous recombi4. James PD, Notley C, Hegadorn C, et al. The mutational nation, resulting in deletion of the interventype 1 VWD cohorts collected in Europe and spectrum of type 1 von Willebrand disease: results from a ing sequence. In the current example, Alu Y Canada3,4 will help provide insight into the Canadian cohort study. Blood. 2007;109:145-154. sequences surrounding the breakpoint had mutation’s distribution. If it is sufficiently 5. Rudiger NS, Gregersen N, Kielland-Brandt MC. One over 80% sequence similarity. short well conserved region of Alu-sequences is involved in widespread, then the mutation should be human gene rearrangements and has homology with proThe subsequent recombination event idensought first in any mutation screening strategy karyotic chi. Nucleic Acids Res. 1995;23:256-260. tified in one family by Sutherland et al is also for types 1 and 3 VWD, prior to VWF DNA 6. Bernardi F, Patracchini P, Gemmati D, et al. In-frame of interest. SNP analysis suggests that the resequence analysis. A specific long polymerase deletion of von Willebrand factor A domains in a dominant type of von Willebrand disease. Hum Mol Genet. 1993; chain reaction (PCR) described by the authors, combination site lies between intron 5 and 2:545-548. exon 8. There may now be sufficient density of which produces a deletion-specific 1084 bp 7. Mancuso DJ, Tuley EA, Westfield LA, et al. Structure VWF SNP reported that the region involved amplicon along with a wild-type product of of the gene for human von Willebrand factor. J Biol Chem. 1989;264:19514-19527. could be identified and sequence features im1694 bp, will facilitate this analysis. Examination of the sequence surrounding the deletion breakpoint implicated unequal ● ● ● VASCULAR BIOLOGY homologous recombination between 2 Alu Y Comment on Wei et al, page 1123 repeat elements in introns 3 and 5 as the mutation mechanism. There are approximately 100 000 Alu repeats in the human genome, and these have been repeatedly involved in the ---------------------------------------------------------------------------------------------------------------generation of deletion mutations. As the auPeter Oettgen BETH ISRAEL DEACONESS MEDICAL CENTER thors highlight, this is at least the third example of an Alu-mediated deletion in type 3 In this issue of Blood, Wei and colleagues investigate the overlapping roles of Ets1 VWD and in early reports of VWF deletions, and Ets2 in the regulation of endothelial cell function and survival during embrymutation mechanism was not investigated so onic angiogenesis.1 the mechanism is likely underrecognized. Alu ts1 and Ets2 are known to regulate morphic mutant of Ets2, in which the conrepeats may be involved in both homologous endothelial-restricted gene expression served threonine 72 phosphorylation site is and non-homologous recombination, as a core and endothelial function. For example, Ets1 mutated, Ets2T72A/T72A mice, are viable and 26 bp repeat sequence is reported to be recomhas been shown to regulate the expression of appear normal.5 In an elegant series of experibogenic.5 A previously reported type 2 VWD several genes that regulate endothelial funcments, Wei et al uncover the overlapping roles patient (disease subtype not specified) had a tion and angiogenesis including VEGF, of Ets1 and Ets2 during vascular development. large in-frame deletion of 31 kb (exons 26-34) 2 However, homozygous VEGF-R2, and Tie2. First, they cross Ets1⫺/⫺ mice with Ets2T72A/T72A associated with recombination between an Alu inactivation of Ets1 is associated with defects to derive double mutant Ets1⫺/⫺; Ets2T72A/T72A repeat in intron 25 and a short stretch of ho6 in T-cell function, but no defects in vascular mice. The double mutants are embryonal lemologous sequence in intron 34. These ex3 Similarly, development or angiogenesis. thal and begin to develop vascular abnormaliamples suggest that many more large delewhile Ets2 is a critical regulator of trophoblast ties at day E10.5, including a marked reductions, both in-frame and resulting in null alleles, will be identified as techniques for gene function during extraembryonic development, tion in vascular complexity, defective it is dispensable for development of the embranching, with the remaining vessels being dosage (large deletion or duplication) analysis dilated. To further validate that the phenotype bryo proper.4 Mice with a homozygous hypoare developed. Many of these mutations are Functional redundancy of Ets1 and Ets2 E 934 30 JULY 2009 I VOLUME 114, NUMBER 5 blood From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2009 114: 933-934 doi:10.1182/blood-2009-04-218099 When 1 plus 1 equals 3 in VWD Anne C. Goodeve Updated information and services can be found at: http://www.bloodjournal.org/content/114/5/933.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.