Crosstalk between hemostasis and lymphangiogenesis

Abstract

The assembly of lymphatic vessels during development or wound healing is crucial in the formation of a functional lymphatic system. Lymphangiogenesis is a process with similarities to angiogenesis. The relationship between hemostasis and lymphangiogenesis was first uncovered when the role of platelets in blood/lymphatic vessel separation during development was revealed. A fascinating hypothesis about the role of platelets and coagulation/fibrinolysis in lymphangiogenesis has been recently proposed using a new tail‐wounding assay in mice by Lim et al.1.Lim L. Bui H. Farrelly O. et al.Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC.Blood. 2019; 134: 1764-1775Crossref PubMed Scopus (19) Google Scholar During wound healing, vascular endothelial growth factor C (VEGF‐C) is released from activated platelets and undergoes proteolytic activation by thrombin, facilitating lymphatic vessel growth. During the process of wound healing, various hemostatic systems including platelets, coagulation, and fibrinolysis are activated, and in addition to their role in hemostatic plug formation trigger lymphangiogenesis. In this review, the relationship between hemostasis and lymphangiogenesis, not restricted to lymphangiogenesis during wound healing, is broadly discussed. The first report about the relationship between hemostasis and lymphangiogenesis demonstrated the role of platelets in blood/lymphatic vessel separation during embryonic development.2.Suzuki‐Inoue K. Inoue O. Ding G. et al.Essential in vivo roles of the C‐type lectin receptor CLEC‐2: embryonic/neonatal lethality of CLEC‐2‐deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC‐2‐deficient platelets.J Biol Chem. 2010; 285: 24494-24507Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 3.Bertozzi C.C. Schmaier A.A. Mericko P. et al.Platelets regulate lymphatic vascular development through CLEC‐2‐SLP‐76 signaling.Blood. 2010; 116: 661-670Crossref PubMed Scopus (339) Google Scholar, 4.Uhrin P. Zaujec J. Breuss J.M. et al.Novel function for blood platelets and podoplanin in developmental separation of blood and lymphatic circulation.Blood. 2010; 115: 3997-4005Crossref PubMed Scopus (236) Google Scholar During organ development, a cluster of vascular endothelial cells (VECs) in the cardinal vein is committed to become lymphatic endothelial cells (LECs), and these sprout to form primary lymphatic sacs.5.Tammela T. Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise.Cell. 2010; 140: 460-476Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar Thereafter, the peripheral lymphatic vasculature is generated by further centrifugal growth from the primary lymph sacs.5.Tammela T. Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise.Cell. 2010; 140: 460-476Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar Previous studies, including our own, have revealed that mice deficient in the platelet activation receptor C‐type lectin‐like receptor 2 (CLEC‐2) are characterized by blood‐filled lymphatic vessels due to blood/lymphatic vessel misconnection.2.Suzuki‐Inoue K. Inoue O. Ding G. et al.Essential in vivo roles of the C‐type lectin receptor CLEC‐2: embryonic/neonatal lethality of CLEC‐2‐deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC‐2‐deficient platelets.J Biol Chem. 2010; 285: 24494-24507Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 3.Bertozzi C.C. Schmaier A.A. Mericko P. et al.Platelets regulate lymphatic vascular development through CLEC‐2‐SLP‐76 signaling.Blood. 2010; 116: 661-670Crossref PubMed Scopus (339) Google Scholar Initially, the mechanism of lymphangiogenesis regulation by CLEC‐2 was proposed as follows: at the separation zone of lymph sacs and cardinal veins during sprouting, platelet CLEC‐2 interacts with podoplanin (PDPN) expressed in lymphatic vessels, leading to platelet activation and the release of TGF‐β family members. These cytokines inhibit migration and proliferation of LECs, facilitating blood‐lymphatic vessel separation.6.Osada M. Inoue O. Ding G. et al.Platelet activation receptor CLEC‐2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells.J Biol Chem. 2012; 287: 22241-22252Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar Mice deficient in PDPN or signaling molecules downstream of CLEC‐2, including Syk, SLP‐76, and phospholipase Cγ2, show abnormalities in lymphatic vessel formation.7.Abtahian F. Guerriero A. Sebzda E. et al.Regulation of blood and lymphatic vascular separation by signaling proteins SLP‐76 and Syk.Science. 2003; 299: 247-251Crossref PubMed Scopus (347) Google Scholar, 8.Schacht V. Ramirez M.I. Hong Y.K. et al.T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.EMBO J. 2003; 22: 3546-3556Crossref PubMed Scopus (554) Google Scholar, 9.Ichise H. Ichise T. Ohtani O. Yoshida N. Phospholipase Cgamma2 is necessary for separation of blood and lymphatic vasculature in mice.Development (Cambridge, England). 2009; 136: 191-195Crossref PubMed Scopus (81) Google Scholar This is consistent with the hypothesis that the interaction between platelet‐expressed CLEC‐2 and LEC PDPN, and subsequent platelet activation/release reaction, play a role in lymphatic vessel development. It is not only granule release that participates in inhibiting LEC migration, but also PDPN‐mediated signaling through ezrin/radixin/moesin family proteins.10.Pollitt A.Y. Poulter N.S. Gitz E. et al.Syk and Src family kinases regulate C‐type lectin receptor 2 (CLEC‐2)‐mediated clustering of podoplanin and platelet adhesion to lymphatic endothelial cells.J Biol Chem. 2014; 289: 35695-35710Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11.Langan S.A. Navarro‐Núñez L. Watson S.P. Nash G.B. Modulation of VEGF‐induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin.Platelets. 2018; 29: 486-495Crossref PubMed Scopus (5) Google Scholar Recently, however, there is widespread acceptance of the concept that backflow of blood to lymphatic vessels is prevented at the lymphovenous junction by thrombi generated by the interaction of PDPN with the platelet CLEC‐2/lymphovenous valve.12.Hess P.R. Rawnsley D.R. Jakus Z. et al.Platelets mediate lymphovenous hemostasis to maintain blood‐lymphatic separation throughout life.J Clin Investig. 2014; 124: 273-284Crossref PubMed Scopus (160) Google Scholar, 13.Bianchi R. Russo E. Bachmann S.B. et al.Postnatal deletion of podoplanin in lymphatic endothelium results in blood filling of the lymphatic system and impairs dendritic cell migration to lymph nodes.Arterioscler Thromb Vasc Biol. 2017; 37: 108-117Crossref PubMed Scopus (26) Google Scholar The importance of fibrin clot formation in this setting has been proven using mice lacking integrin αIIbβ3. These mice are unable to aggregate platelets due to a lack of the fibrinogen receptor αIIbβ3, but their platelets can undergo activation and phosphatidyl serine exposure to their surface, which stimulates the coagulation cascade. Thus, these mice can generate a fibrin clot at the lymphovenous junction and do not develop blood/lymphatic vessel misconnection. This mechanism was not restricted to the embryonic developmental stage but occurs throughout life.12.Hess P.R. Rawnsley D.R. Jakus Z. et al.Platelets mediate lymphovenous hemostasis to maintain blood‐lymphatic separation throughout life.J Clin Investig. 2014; 124: 273-284Crossref PubMed Scopus (160) Google Scholar Both platelets and fibrin generation are capable of participating in lymphangiogenesis during organ development. Recently, it has been reported that the chromatin‐remodeling enzyme CHD4 (chromodomain helicase DNA‐binding 4) inhibits the transcription of the urokinase plasminogen activator receptor (uPAR), which protects thrombi at the lymphovenous junction from plasmin‐mediated fibrinolysis and inhibits backflow of blood to lymphatic vessels.14.Crosswhite P.L. Podsiadlowska J.J. Curtis C.D. et al.CHD4‐regulated plasmin activation impacts lymphovenous hemostasis and hepatic vascular integrity.J Clin Investig. 2016; 126: 2254-2266Crossref PubMed Scopus (21) Google Scholar Genetic deletion of CHD4 from LECs results in blood/lymphatic vessel misconnection and blood‐filled lymphatics.14.Crosswhite P.L. Podsiadlowska J.J. Curtis C.D. et al.CHD4‐regulated plasmin activation impacts lymphovenous hemostasis and hepatic vascular integrity.J Clin Investig. 2016; 126: 2254-2266Crossref PubMed Scopus (21) Google Scholar This study again proves the importance of lymphovenous hemostasis to maintain the integrity of lymphatic vessels. Although hemostasis facilitates blood/lymphatic separation, the driving force of lymphatic vessel formation is VEGF‐C.5.Tammela T. Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise.Cell. 2010; 140: 460-476Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar In mice with inactivated VEGF‐C, LECs initially differentiate in the embryonic veins but fail to migrate and form primary lymph sacs.5.Tammela T. Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise.Cell. 2010; 140: 460-476Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 15.Karkkainen M.J. Haiko P. Sainio K. et al.Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins.Nat Immunol. 2004; 5: 74-80Crossref PubMed Scopus (1051) Google Scholar VEGF‐C facilitates LEC migration and proliferation via its receptor VEGF receptor (R)‐3, and its inactivation leads to the complete absence of a lymphatic vascular system in mouse embryos.5.Tammela T. Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise.Cell. 2010; 140: 460-476Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar VEGF‐C is produced as an inactive propeptide, which is cleaved by furin to produce pro‐VEGF‐C.16.Rauniyar K. Jha S.K. Jeltsch M. Biology of vascular endothelial growth factor C in the morphogenesis of lymphatic vessels.Front Bioeng Biotechnol. 2018; 6: 7Crossref PubMed Scopus (53) Google Scholar After pro‐VEGF‐C is secreted, it undergoes a second proteolytic cleavage by a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3), forming mature, active VEGF‐C.16.Rauniyar K. Jha S.K. Jeltsch M. Biology of vascular endothelial growth factor C in the morphogenesis of lymphatic vessels.Front Bioeng Biotechnol. 2018; 6: 7Crossref PubMed Scopus (53) Google Scholar For the proteolytic activation of VEGF‐C, a non‐enzymatic protein, collagen and calcium binding EGF domains 1 (CCBE1) is required.17.Jeltsch M. Jha S.K. Tvorogov D. et al.CCBE1 enhances lymphangiogenesis via A disintegrin and metalloprotease with thrombospondin motifs‐3‐mediated vascular endothelial growth factor‐C activation.Circulation. 2014; 129: 1962-1971Crossref PubMed Scopus (153) Google Scholar Genetic deletion of either ADAMTS3 or CCBE1 in mice results in loss of lymphatic vessels.18.Bos F.L. Caunt M. Peterson‐Maduro J. et al.CCBE1 is essential for mammalian lymphatic vascular development and enhances the lymphangiogenic effect of vascular endothelial growth factor‐C in vivo.Circ Res. 2011; 109: 486-491Crossref PubMed Scopus (153) Google Scholar, 19.Janssen L. Dupont L. Bekhouche M. et al.ADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis.Angiogenesis. 2016; 19: 53-65Crossref PubMed Scopus (65) Google Scholar The major form of mature VEGF‐C is produced by ADAMTS3, whereas the minor form is produced by plasmin cleavage, which is nine amino acids longer compared to the major form.16.Rauniyar K. Jha S.K. Jeltsch M. Biology of vascular endothelial growth factor C in the morphogenesis of lymphatic vessels.Front Bioeng Biotechnol. 2018; 6: 7Crossref PubMed Scopus (53) Google Scholar, 20.McColl B.K. Baldwin M.E. Roufail S. et al.Plasmin activates the lymphangiogenic growth factors VEGF‐C and VEGF‐D.J Exp Med. 2003; 198: 863-868Crossref PubMed Scopus (175) Google Scholar Now, Lim et al have reported that thrombin also supports proteolytic activation of VEGF‐C.1.Lim L. Bui H. Farrelly O. et al.Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC.Blood. 2019; 134: 1764-1775Crossref PubMed Scopus (19) Google Scholar Again, hemostasis regulates VEGF‐C activation in addition to blood/lymphatic vessel separation. Recently, kallikrein‐related peptidase 3 (KLK3) has been reported to be the third proteinase to proteolytically activate VEGF‐C.21.Jha S.K. Rauniyar K. Chronowska E. et al.KLK3/PSA and cathepsin D activate VEGF‐C and VEGF‐D.Elife. 2019; 8Crossref Scopus (20) Google Scholar However, KLK3 is also known as a prostate‐specific antigen (PSA) and is not related to factor XII (FXII) activation. Following tissue injury and subsequent wound healing, prothrombin and plasminogen become activated. Lymphangiogenesis occurs during wound healing with the aim to restore the integrity of the lymphatic system, but the exact mechanisms by which lymphangiogenesis occurs are not fully understood. However, some molecules participating in this process have been reported. Signaling mediated through the prostaglandin E receptors EP3 and EP4 facilitates wound healing and lymphangiogenesis with enhanced recruitment of M2 macrophages in mice.22.Hosono K. Isonaka R. Kawakami T. Narumiya S. Majima M. Signaling of prostaglandin E receptors, EP3 and EP4 facilitates wound healing and lymphangiogenesis with enhanced recruitment of M2 macrophages in mice.PLoS ONE. 2016; 11Crossref Scopus (24) Google Scholar Receptor activity‐modifying protein 1 (RAMP1), a regulatory protein of calcitonin‐receptor like receptor (CRLR), plays a crucial role in wound healing, angiogenesis‐induced wound healing, and lymphangiogenesis.23.Kurashige C. Hosono K. Matsuda H. Tsujikawa K. Okamoto H. Majima M. Roles of receptor activity‐modifying protein 1 in angiogenesis and lymphangiogenesis during skin wound healing in mice.FASEB J. 2014; 28: 1237-1247Crossref PubMed Scopus (39) Google Scholar The relationship between hemostasis and lymphangiogenesis during wound healing has recently been reported by Lim et al.1.Lim L. Bui H. Farrelly O. et al.Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC.Blood. 2019; 134: 1764-1775Crossref PubMed Scopus (19) Google Scholar They utilized a new tail‐wounding assay in mice and found that addition of platelet‐rich plasma (PRP) to the injury site significantly accelerates lymphatic vessel repair when compared with the addition of platelet‐poor plasma (PPP) or PRP from VEGF‐C‐deficient mice.1.Lim L. Bui H. Farrelly O. et al.Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC.Blood. 2019; 134: 1764-1775Crossref PubMed Scopus (19) Google Scholar VEGF‐C is abundant in platelets and released upon activation. Released VEGF‐C is proteolytically activated by thrombin, which is produced at the site of injury.1.Lim L. Bui H. Farrelly O. et al.Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC.Blood. 2019; 134: 1764-1775Crossref PubMed Scopus (19) Google Scholar Using an extensive subcutaneous mouse wound model, Güç et al showed that engineered fibrin‐binding VEGF‐C, but not free VEGF‐C, promotes local lymphangiogenesis. Engineered fibrin‐binding VEGF‐C was released as a function of fibrin degradation and remodeling over a course of 10 to 12 days, which enabled specific targeting of lymphatics without causing the remodeling of downstream vessels.24.Güç E. Briquez P.S. Foretay D. et al.Local induction of lymphangiogenesis with engineered fibrin‐binding VEGF‐C promotes wound healing by increasing immune cell trafficking and matrix remodeling.Biomaterials. 2017; 131: 160-175Crossref PubMed Scopus (67) Google Scholar Although fibrin‐binding VEGF‐C is artificial and it remains unknown whether it binds fibrin in vivo, it is a reasonable assumption that lymphangiogenesis during wound healing is facilitated by hemostatic components, including thrombin, plasmin, and fibrin. Lim et al1.Lim L. Bui H. Farrelly O. et al.Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC.Blood. 2019; 134: 1764-1775Crossref PubMed Scopus (19) Google Scholar reported that platelets facilitate lymphangiogenesis, whereas previous studies showed evidence of anti‐lymphangiogenic effects of platelets.6.Osada M. Inoue O. Ding G. et al.Platelet activation receptor CLEC‐2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells.J Biol Chem. 2012; 287: 22241-22252Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 11.Langan S.A. Navarro‐Núñez L. Watson S.P. Nash G.B. Modulation of VEGF‐induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin.Platelets. 2018; 29: 486-495Crossref PubMed Scopus (5) Google Scholar, 25.Sato H. Higashiyama M. Hozumi H. et al.Platelet interaction with lymphatics aggravates intestinal inflammation by suppressing lymphangiogenesis.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G276-G285Crossref PubMed Scopus (15) Google Scholar, 26.Oka M. Iwata C. Suzuki H.I. et al.Inhibition of endogenous TGF‐beta signaling enhances lymphangiogenesis.Blood. 2008; 111: 4571-4579Crossref PubMed Scopus (177) Google Scholar Platelets contain transforming growth factor (TGF)‐β in abundance, which has anti‐lymphangiogenic effects. TGF‐β is released from platelets by an interaction between platelet CLEC‐2 and PDPN on LECs.2.Suzuki‐Inoue K. Inoue O. Ding G. et al.Essential in vivo roles of the C‐type lectin receptor CLEC‐2: embryonic/neonatal lethality of CLEC‐2‐deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC‐2‐deficient platelets.J Biol Chem. 2010; 285: 24494-24507Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 6.Osada M. Inoue O. Ding G. et al.Platelet activation receptor CLEC‐2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells.J Biol Chem. 2012; 287: 22241-22252Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar Alternatively, PDPN clustering by CLEC‐2 transduces signals to inhibit migration of LECs.10.Pollitt A.Y. Poulter N.S. Gitz E. et al.Syk and Src family kinases regulate C‐type lectin receptor 2 (CLEC‐2)‐mediated clustering of podoplanin and platelet adhesion to lymphatic endothelial cells.J Biol Chem. 2014; 289: 35695-35710Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11.Langan S.A. Navarro‐Núñez L. Watson S.P. Nash G.B. Modulation of VEGF‐induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin.Platelets. 2018; 29: 486-495Crossref PubMed Scopus (5) Google Scholar The antilymphangiogenic effects of platelets have also been proven in vivo. Sato et al25.Sato H. Higashiyama M. Hozumi H. et al.Platelet interaction with lymphatics aggravates intestinal inflammation by suppressing lymphangiogenesis.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G276-G285Crossref PubMed Scopus (15) Google Scholar showed in an inflammatory bowel disease model in mice that platelet interaction with lymphatic cells aggravates intestinal inflammation by suppressing lymphangiogenesis. In contrast, Lim et al1.Lim L. Bui H. Farrelly O. et al.Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC.Blood. 2019; 134: 1764-1775Crossref PubMed Scopus (19) Google Scholar reported the lymphangiogenesis‐facilitating effects of platelets, whereby VEGF‐C is released by activated platelets and proteolytically activated to facilitate lymphangiogenesis during wound healing. Moreover, Hur et al27.Hur J. Jang J.H. Oh I.‐.Y. et al.Human podoplanin‐positive monocytes and platelets enhance lymphangiogenesis through the activation of the podoplanin/CLEC‐2 axis.Mol Ther. 2014; 22: 1518-1529Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar reported that human podoplanin‐positive monocytes and platelets enhance lymphangiogenesis through the activation of the PDPN/CLEC‐2 axis. The supernatant obtained from co‐culture of PDPN‐positive monocytes (PPMs) with platelets was able to enhance the migration, viability, and proliferation of LECs by increasing the levels of LEC markers and lymphangiogenic cytokines. Local injection of PPMs with platelets significantly increased lymphangiogenesis and facilitated wound healing in the full‐thickness skin wounds of nude mice. A clinical study on 132 patients with esophageal cancer suggested that platelets have prolymphangiogenic effects.28.Schoppmann S.F. Alidzanovic L. Schultheis A. Perkmann T. Brostjan C. Birner P. Thrombocytes correlate with lymphangiogenesis in human esophageal cancer and mediate growth of lymphatic endothelial cells in vitro.PLoS ONE. 2013; 8Crossref PubMed Scopus (14) Google Scholar Stromal thrombocyte clusters (STC) were evident in 82 samples (25.6%), and vascular thrombotic clusters (VTC) in 56 (17.5%), both of which were associated with a significantly higher peripheral blood platelet count (PBPC). The presence of STC was associated with higher lymphatic microvessel density (P < .001). LEC proliferation was enhanced by co‐culture with human platelets in a dose‐ and time‐dependent manner and mediated by the release of PDGF‐BB and VEGF‐C, contradictorily to findings of the previous studies.6.Osada M. Inoue O. Ding G. et al.Platelet activation receptor CLEC‐2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells.J Biol Chem. 2012; 287: 22241-22252Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 25.Sato H. Higashiyama M. Hozumi H. et al.Platelet interaction with lymphatics aggravates intestinal inflammation by suppressing lymphangiogenesis.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G276-G285Crossref PubMed Scopus (15) Google Scholar Platelets contain both pro‐ and antilymphangiogenic cytokines, including TGF‐β and VEGF‐C/platelet‐derived growth factor (PDGF)‐BB in their α‐granules. It has been reported that distinct granule contents can be released differentially in response to individual platelet agonists, although this concept remains controversial. Activation signals from proteinase‐activated receptor (PAR)1 (thrombin receptor), adenosine diphosphate receptors, and glycoprotein VI favor the release of proangiogenic factors (eg, VEGF), whereas PAR4 and TxA2 promote anti‐angiogenic factor (eg, endostatin) release.29.Chatterjee M. Huang Z. Zhang W. et al.Distinct platelet packaging, release, and surface expression of proangiogenic and antiangiogenic factors on different platelet stimuli.Blood. 2011; 117: 3907-3911Crossref PubMed Scopus (148) Google Scholar, 30.Battinelli E.M. Markens B.A. Italiano Jr, J.E. Release of angiogenesis regulatory proteins from platelet alpha granules: modulation of physiologic and pathologic angiogenesis.Blood. 2011; 118: 1359-1369Crossref PubMed Scopus (263) Google Scholar Differential packaging of pro‐ and anti‐angiogenic proteins in distinct α‐granules in platelets has also been reported in addition to differential release upon platelet activation.31.Battinelli E.M. Thon J.N. Okazaki R. et al.Megakaryocytes package contents into separate alpha‐granules that are differentially distributed in platelets.Blood Adv. 2019; 3: 3092-3098Crossref PubMed Scopus (27) Google Scholar Similar mechanisms may be applicable to lymphangiogenesis, in that pro‐ and antilymphangiogenic proteins are differently packed in distinct α‐granules and are differentially released upon platelet activation. Alternatively, the presence of a third set of cells (such as PPMs) other than platelets and LECs may facilitate lymphangiogenesis. Platelets may play pro‐ or antilymphangiogenic roles depending on the stage of the life cycle and the place—that is, lymphangiogenesis during embryonic development, and tumor lymphangiogenesis during wound healing. Platelets prevent blood/lymphatic vessel mixing by preventing backflow at the lymphovenous junction. Is this, however, the only mechanism involved? It has been reported that the first lymphovenous valves are formed in juxtaposed sites of the cardinal vein, and from the primordial thoracic duct at embryonic day (E) 12.32.Marchiò S. Astanina E. Bussolino F. Emerging lymphae for the fountain of life.EMBO J. 2013; 32: 609-611Crossref PubMed Scopus (4) Google Scholar However, blood‐filled lymph sacs are developed in E11.5 in PDPN−/− embryos3.Bertozzi C.C. Schmaier A.A. Mericko P. et al.Platelets regulate lymphatic vascular development through CLEC‐2‐SLP‐76 signaling.Blood. 2010; 116: 661-670Crossref PubMed Scopus (339) Google Scholar before lymphovenous junction is established. Moreover, blood‐filled lymphatics has not been reported in mice deficient in factor VIII, factor IX, or fibrinogen, in which fibrin clot formation is impaired.33.Janardhan H.P. Trivedi C.M. Establishment and maintenance of blood‐lymph separation.Cell Mol Life Sci. 2019; 76: 1865-1876Crossref PubMed Scopus (8) Google Scholar It is difficult for thrombi in the lymphovenous junction to prevent backflow of blood into the thoracic duct without preventing lymphatic flow into the vein. These findings contradict the “prevention of backflow at the lymphovenous junction” theory. The other theory is that the TGF‐β family proteins released from platelets activated by platelet CLEC‐2/LEC PDPN interaction inhibit the migration and proliferation of LECs, facilitating blood‐lymphatic vessel separation. This theory also has its limitations. Blood/lymphatic vessel misconnection has not been reported in patients with Gray platelet syndrome, whose platelets lack α‐granule contents.33.Janardhan H.P. Trivedi C.M. Establishment and maintenance of blood‐lymph separation.Cell Mol Life Sci. 2019; 76: 1865-1876Crossref PubMed Scopus (8) Google Scholar It has been reported that the peripheral lymphatic vasculature is generated by further centrifugal growth from the primary lymph sacs.5.Tammela T. Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise.Cell. 2010; 140: 460-476Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar It is tempting to speculate that during lymphatic elongation, lymphatic vessels encounter vascular vessels and their mixture is avoided by association between LEC PDPN and platelet CLEC‐2. To support this idea, multiple anastomotic sites between blood vessels and lymphatic vessels were observed in the peripheral skin of CLEC‐2−/− fetuses.2.Suzuki‐Inoue K. Inoue O. Ding G. et al.Essential in vivo roles of the C‐type lectin receptor CLEC‐2: embryonic/neonatal lethality of CLEC‐2‐deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC‐2‐deficient platelets.J Biol Chem. 2010; 285: 24494-24507Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar A recent publication reported another mechanism of blood/lymphatic vessel separation. Lymphatic development in the mesentery and the intestine is relatively late; immature lymphatics become detectable in the mesentery but not in the intestinal tube, around E13.5‐E14.5, while organized lymphatic vessel plexuses and capillaries are observed in the intestinal tube and villi around E17.5.34.Kim K.E. Sung H.K. Koh G.Y. Lymphatic development in mouse small intestine.Dev Dyn. 2007; 236: 2020-2025Crossref PubMed Scopus (48) Google Scholar During mesenteric vascular development, the integrity of these vessels is transiently lost and red blood cells (RBCs) are extravasated. These RBCs are trapped by the developing lymphatic vessels and cleared by lymph flow. Platelet adhesion to the endothelium and activation at the endothelial cell gaps are mediated by platelet‐expressed CLEC‐2 and prevent excessive RBC extravasation. In the absence of platelet‐expressed CLEC‐2, extravasated RBCs and platelets are trapped in the developing lymphatic vessels but cannot be removed due to a lack of lymph flow, resulting in blood‐filled lymphatics.35.Zhang Y. Daubel N. Stritt S. Makinen T. Transient loss of venous integrity during developmental vascular remodeling leads to red blood cell extravasation and clearance by lymphatic vessels.Development (Cambridge, England). 2018; 145Google Scholar Thus, platelets regulate blood/lymphatic vessel separation via various mechanisms, which are not mutually exclusive and may be spatiotemporally regulated. None of the authors has any conflict of interest. Katsue Suzuki‐Inoue, Nagaharu Tsukiji, and Shimon Otake prepared the manuscript.