Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation

Abstract

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

Keywords: GPVI signaling; dense granules; homeostasis; platelets; vascular integrity.

Fig. 1.

Severe thrombocytopenia produces a reversible defect in homeostatic vascular barrier function. (A) Representative images of the skin microcirculation 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.

Fig. 2.

Changes in barrier function are independent of the method of induction of thrombocytopenia and emerge gradually. Mice were rendered thrombocytopenic with a single dose of busulfan (20 mg/kg). Vascular permeability studies were performed on 13 d afterwards. The platelet counts in the drug treated mice at the point were 10 to 15% of normal control mice. (A) MFI in the interstitial space area and leakage rate following infusion of 40-kDa dextran. (B) Same as A with 70-kDa dextran. Control mice received vehicle. Data points indicated as mean ± SEM. n = 5 mice each group. n.s., nonsignificant. (C) Vascular barrier function in mice was measured 4 h after induction of thrombocytopenia with 0.4 μg/g anti-GPIbα. Circulating platelet counts fell to ∼5% of control within 30 min of antibody administration. MFI in the interstitial space area following infusion of 40-kDa dextran. (D) Same as C with 70-kDa dextran. Control mice received similar amounts of nonimmune IgG. Data points indicated as mean ± SEM. n = 4 mice in each group. n.s., nonsignificant.

Fig. 3.

Platelet PAR4 signaling is not required for maintaining homeostatic vascular barrier function. Representative images obtained 3.5 and 15 min after infusion of 40-kDa dextran into PAR4^−/− mice and matched controls. Ratio of the MFI normalized to MFI at 3.5 min and leakage rate. Data points indicated as mean ± SEM. n = 6 mice each group. (Scale bar: 100 μm.) n.s., nonsignificant.

Fig. 4.

Loss of GPVI and PLCγ2, but not CLEC-2, increases 40-kDa dextran extravasation. (A, Top) Images of the dermal microcirculation 3.5 and 15 min after infusion of 40-kDa Texas red dextran into mice whose platelets were rendered GPVI-deficient by injecting the anti-GPVI antibody, JAQ1. Controls received equivalent amounts of polyclonal nonimmune IgG. (A, Bottom) Ratio of the MFI normalized to MFI at 3.5 min. n = 5 mice in each group. (B) Same as A for GPVI^−/− mice. n = 6 mice in each group. (C) Same as A but for mice depleted of CLEC-2 by administration of antibody INU1. Matched controls received nonimmune IgG. n = 5 mice in each group. (Scale bars: 100 μm.) Mean ± SEM. (D) Images of the dermal microcirculation 3.5 and 15 min after infusion of 40-kDa Texas red dextran into lethally irradiated WT mice rescued with bone marrow from WT or PLCγ2^−/− mice. Ratio of the MFI normalized to MFI at 3.5 min. Data points indicated as mean ± SEM. n = 6 mice in each group. (Scale bars: 100 μm.)

Fig. 5.

Platelet-dense granules, but not α-granules, are required to maintain vascular homeostasis. (A, Top) Images of the dorsal ear skin microcirculation captured 3.5 and 15 min after infusion of 40-kDa dextran in Nbeal2^−/− mice and matched controls. (A, Bottom) Ratio of the MFI in the interstitial tissue area over time normalized to the MFI at 3.5 min. n = 5 mice in each group. (B and C) Same as A with BLOC-1^−/− (Pallid) mice and Unc13d^Jinx mice. Data points for each group indicated as mean ± SEM. n = 6 mice in each group. (Scale bars: 100 μm.)

Fig. 6.

GPVI depletion also reduces barrier function in meningeal vessels, but not intracerebral vessels. Representative images of (A) meningeal and (B) intracerebral vessels captured 1.5 and 10 min after i.v. infusion of 40-kDa dextran. (Scale bars: 100 μm.) MFI in the interstitial space over time normalized to MFI at 1.5 min (baseline). Meningeal and intracerebral vessels were imaged using multiphoton fluorescence microscopy through a thinned-skull cranial window as one z stack. The top surface of skull comprised of meningeal blood vessels followed by deeper cerebral vessels, separated by avascular subarachnoid space. The meningeal vessels were distinguished from cerebral vessels by their location and structure. Data points indicated as mean ± SEM. n = 4 mice in each group. n.s., nonsignificant.

Fig. 7.

Platelet are essential to maintain vascular homeostasis. Based on the results, we propose a model in which the intermittent appearance of small gaps between endothelial cells is a normal event within a healthy microvasculature. (A) Platelets sense exposed extracellular matrix components including collagen. (B) This leads to transient GPVI-dependent platelet activation and low-level dense granule secretion. Secretion products interact with endothelium and (C) reestablish the normal junctions between endothelial cells. Increased escape of small molecules like 40-kDa dextran from thin-walled capillaries and postcapillary venules is a marker for the persistence of these gaps in the absence of platelets, GPVI, PLCγ2, and dense granules.