Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation Shuchi Guptaa,b, Christoph Konradtc, Adam Corkend, Jerry Wared, Bernhard Nieswandte,f, Jorge Di Paolag, Mei Yuh, Demin Wangh, Marvin T. Niemani, Sidney W. Whiteheartj, and Lawrence F. Brassa,b,1 aDepartment of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; bDepartment of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; cDepartment of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, IN 47907; dDepartment of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205; eInstitute of Experimental Biomedicine, University Hospital Wuerzburg, 97080 Wuerzburg, Germany; fRudolf Virchow Center, University of Wuerzburg, 97080 Wuerzburg, Germany; gDepartment of Pediatrics, Washington University School of Medicine in St. Louis, St. Louis, MO 63110; hHematopoiesis and B cell biology lab, Blood Research Institute, Versiti, Milwaukee, WI 53226; iDepartment of Pharmacology, Case Western Reserve University, Cleveland, OH 44106; and jDepartment of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536 Edited by Barry S. Coller, The Rockefeller University, New York, NY, and approved August 6, 2020 (received for review April 20, 2020) Platelets are best known for their vasoprotective responses to and increased microvascular permeability in thrombocytopenic injury and inflammation. Here, we have asked whether they also rabbits and humans (6, 7). Collectively, these older observations support vascular integrity when neither injury nor inflammation is suggest that platelets have a role in maintaining barrier function present. Changes in vascular barrier function in dermal and meningeal but reveal little about the mechanisms involved (2, 3, 8–11). In vessels were measured in real time in mouse models using the hemostasis, platelets are activated by agonists such as collagen, differential extravasation of fluorescent tracers as a biomarker. thrombin, adenosine diphosphate (ADP), and thromboxane A2 Severe thrombocytopenia produced by two distinct methods caused (TxA2), triggering events that include and depend on granule increased extravasation of 40-kDa dextran from capillaries and secretion. In inflammatory conditions, platelets primarily rely on postcapillary venules but had no effect on extravasation of 70-kDa signaling through GPVI and CLEC-2 to prevent bleeding (3), dextran or albumin. This reduction in barrier function required more but granule secretion and platelet activation by thrombin, ADP, CELL BIOLOGY than 4 h to emerge after thrombocytopenia was established, and TxA2 have been shown to be dispensable (8, 12). reverting to normal as the platelet count recovered. Barrier dysfunc- To better understand the role of platelets in maintaining tion was also observed in mice that lacked platelet-dense granules, vascular homeostasis, we began by asking whether severe dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI thrombocytopenia causes vascular leakage in vivo and, if so, signaling effector phospholipase C (PLC) γ2. It did not occur in mice which platelet processes are normally involved in preventing it. lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease acti- Since detection methods like the Miles assay that are based on vated receptor 4 (PAR4). Notably, although both meningeal and der- albumin loss proved to be insufficiently sensitive for measuring mal vessels were affected, intracerebral vessels, which are known for homeostatic vascular barrier function, we have used intravital their tighter junctions between endothelial cells, were not. Collec- microscopy and the differential extravasation of fluorescently tively, these observations 1) highlight a role for platelets in maintain- tagged dextran molecules from capillaries and postcapillary ve- ing vascular homeostasis in the absence of injury or inflammation, 2) nules to detect barrier dysfunction. An initial comparison of provide a sensitive biomarker for detecting changes in platelet- probes confirmed a previous report that there is a slow dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets Significance causes changes in the vessel wall itself, accounting for the time re- quired for dysfunction to emerge. The human circulatory system includes the heart, arteries, veins, and capillaries, all of which are lined by endothelial cells platelets | vascular integrity | homeostasis | GPVI signaling | dense granules to prevent the escape of vascular contents into surrounding tissues. Platelets are blood cells that are best known for their he vascular endothelium forms a continuous barrier whose essential role in preventing blood loss after injury. Here, we Tpermeability helps to maintain normal tissue homeostasis by employed sensitive methods to measure changes in vascular impeding or permitting the passage of plasma molecules and barrier function in vivo and used them to identify a role for cells across the vessel wall (1). Severe disorders of barrier function platelets in maintaining vascular homeostasis. The results can allow erythrocytes as well as solutes to escape and, if the loss highlight the role of platelet collagen receptors and dense exceeds the capacity of the lymphatic system to compensate, will granules, show which vessels are particularly dependent on result in edema and organ dysfunction. Well-described extreme platelets for this purpose, and point to an underappreciated examples include sepsis, systemic inflammatory disorders, and the interchange between platelets and endothelial cells that is lost cytokine release syndrome. Disorders of barrier function can, in severe thrombocytopenia. however, also be relatively subtle, allowing solutes, but not erythrocytes, to escape and, in the process, revealing much about Author contributions: S.G. and L.F.B. designed research; S.G. and C.K. performed research; the ways that basal barrier function is normally maintained. A.C., J.W., B.N., J.D.P., M.Y., D.W., M.T.N., and S.W.W. contributed new reagents/analytic Here, we have focused on understanding the role that platelets tools; S.G. analyzed data; and S.G. and L.F.B. wrote the paper. play when neither injury nor inflammation is present. Platelets The authors declare no competing interest. have a long-established role in the hemostatic response to injury This article is a PNAS Direct Submission. and a more recently established role in preventing the escape of Published under the PNAS license. erythrocytes and plasma proteins in the setting of inflammation 1To whom correspondence may be addressed. Email: [email protected]. (2, 3). The idea that platelets also support vascular homeostasis This article contains supporting information online at https://www.pnas.org/lookup/suppl/ dates back to studies on frogs (4) and isolated organs (5), and to doi:10.1073/pnas.2007642117/-/DCSupplemental. electron micrographs that showed thinning of the endothelium www.pnas.org/cgi/doi/10.1073/pnas.2007642117 PNAS Latest Articles | 1of10 Downloaded by guest on September 29, 2021 Fig. 1. Severe thrombocytopenia produces a reversible defect in homeostatic vascular barrier function. (A) Representative images of the skin microcircu- lation on the dorsal side of the ear captured at 3.5 and 15 min after i.v. infusion of fluorescent 40-kDa or 70-kDa dextran into either control mice that received polyclonal nonimmune IgG (Top), thrombocytopenic mice that were studied 24 h after injection of 0.4 μg/g anti-GPIbα (Middle, platelet count ≤5% of normal), and partially recovered mice that were studied 24 h after injection of 0.3 μg/g anti-GPIbα (Bottom, platelet count at least 30% of normal). (Scale bar: 100 μm.) The intensity bar shown was used to assign a value to each pixel based on the intensity of the fluorophore. Brighter pixels are assigned to higher intensity values and darker areas with lower intenisty values correspond to weaker fluorophore positive pixels. Rainbow intensity scale was used to denote 40 kDa dextran and grey scale was used to for 70 kDa dextran. (B) Increased escape of 40-kDa dextran from thin-walled capillaries and post-capillary venules. On the Left is shown the ratio over time of the MFI in the interstitial tissue to the MFI at 3.5 min. On the Right is shown the leakage rate. (C) Same as B, but with 70-kDa dextran. Data points indicated as mean ± SEM. n = 5 to 7 mice in each group. n.s., nonsignificant. extravasation of 40-kDa, but not 70-kDa, dextran from healthy of barrier function does not require the presence of platelet vessels (13). We now show that the rate of 40-kDa dextran ex- thrombin receptors, CLEC-2, or α-granule secretion. Collectively, travasation is sensitive to the circulating platelet count, increasing these results demonstrate a role for platelets in maintaining vas- in the setting of severe thrombocytopenia and then reverting to cular homeostasis, characterize the limits of the role, and establish normal as the platelet count recovers. This defect in barrier some of the underlying mechanisms that are involved. function is detectable in the dermal and meningeal vascular beds, but not in the intracerebral vasculature, and requires >4hto Results emerge after thrombocytopenia is established.