Platelets and platelet adhesion support angiogenesis while preventing excessive hemorrhage

Abstract

Platelets contain both pro- and antiangiogenic factors, but their regulatory role in angiogenesis is poorly understood. Although previous studies showed that platelets stimulate angiogenesis in vitro, the role of platelets in angiogenesis in vivo is largely uncharacterized. To address this topic, we used two in vivo approaches, the cornea micropocket assay and the Matrigel model, in four animal models: thrombocytopenic, Lyst(bg) (platelet storage pool deficiency), glycoprotein (GP) Ibalpha/IL4R transgenic (lacking extracellular GPIbalpha, the receptor for von Willebrand factor as well as other adhesive and procoagulant proteins), and FcgammaR(-/-) (lacking functional GPVI, the collagen receptor) mice. Adult mice were rendered thrombocytopenic by i.p. administration of an antiplatelet antibody. The number of growing vessels in the thrombocytopenic mice was lower in the cornea assay, and they showed significantly increased appearance of hemorrhage compared with mice treated with control IgG. The thrombocytopenic mice also showed more protein leakage and developed hematomas in the Matrigel model. GPIbalpha/IL4R transgenic mice presented increased hemorrhage in both assays, but it was less severe than in the platelet-depleted mice. FcgammaR(-/-) and Lyst(bg) mice showed no defect in experimental angiogenesis. Intravital microscopy revealed a >3-fold increase in platelet adhesion to angiogenic vessels of Matrigel compared with mature quiescent skin vessels. Our results suggest that the presence of platelets not only stimulates angiogenic vessel growth but also plays a critical role in preventing hemorrhage from the angiogenic vessels. The adhesion function of platelets, as mediated by GPIbalpha, significantly contributes to the process.

Fig. 1.

Effect of platelet depletion on angiogenesis in the cornea micropocket assay. (A) Photographs of corneas of mice injected with IgG (Left) or antiplatelet antibody (Right) 72 and 96 h after implanting hydron pellets containing 80 ng of bFGF. Note undefined borders of the vessels in the platelet-depleted eyes due to hemorrhage (red). (B) Number of angiogenic vessels counted 72, 96, and 120 h after bFGF pellet implantation. (C) Hemorrhage scores 72, 96, and 120 h after bFGF pellet implantation. *, P < 0.05; **, P < 0.001 compared with IgG controls.

Fig. 2.

Effect of platelet depletion on angiogenesis in Matrigel assay. Matrigels containing 80 ng of bFGF were implanted s.c. and recovered on day 7 after implantation. (A) Representative photographs of gross morphology of Matrigel implants from mice injected with IgG (Upper) and mice injected with antiplatelet antibody (Lower). (B) Hematoxylin and eosin staining of Matrigel sections from the mice injected with IgG (Left) and from the platelet-depleted mice (Right). Note the numerous extravascular RBCs in Right.

Fig. 3.

Hb levels in Matrigels of antiplatelet, GPIbα/IL4R, FcγR^–/–, and Lyst^bg mice. Seven days after s.c. implantation, the Matrigels were harvested and homogenized. Hb concentration was measured in the supernatants. *, P < 0.02; **, P < 0.002 compared with the control group (n = 6–8).

Fig. 4.

Mouse cornea assay in GPIbα/IL4R and Lyst^bg mice. (A) Corneas of WT and Lyst^bg mice 96 h after pellet implantation (Upper) and corneas of WT and GPIbα/IL4R mice 72 h after pellet implantation (Lower). Significant hemorrhage is seen only in the GPIbα/IL4R mice. (B) Percentage of eyes showing hemorrhage in the corneas of GPIbα/IL4R mice (gray bar) and WT (black bar) implanted with bFGF hydron pellets. *, P < 0.02 compared with the control group.

Fig. 5.

Visualization of platelets in vivo. Platelet–endothelium interactions were investigated in angiogenic and mature quiescent vessels in a dorsal skin fold chamber by in vivo fluorescence microscopy. (A) Two representative images taken 3 s apart show the same field within the Matrigel. Arrows indicate platelets that remained adherent during this period. Asterisks in Upper indicate examples of nonadherent platelets (moved away in Lower). It is important to note that only ≈5% of the circulating platelets were fluorescent. Thus, the actual number of adherent platelets is possibly much higher than shown. (Scale bars, 50 μm.) (B) Quantitative analysis of platelet adherence. Percentage of adherent platelets from total number of platelets observed in the field was determined as described in Materials and Methods. n = 4 animals in each group.