Temporal and Pharmacological Characterization of Angiostatin Release and Generation by Human Platelets: Implications for Endothelial Cell Migration

Abstract

Platelets play an important role in thrombosis and in neo-vascularisation as they release and produce factors that both promote and suppress angiogenesis. Amongst these factors is the angiogenesis inhibitor angiostatin, which is released during thrombus formation. The impact of anti-thrombotic agents and the kinetics of platelet angiostatin release are unknown. Hence, our objectives were to characterize platelet angiostatin release temporally and pharmacologically and to determine how angiostatin release influences endothelial cell migration, an early stage of angiogenesis. We hypothesized anti-platelet agents would suppress angiostatin release but not generation by platelets. Human platelets were aggregated and temporal angiostatin release was compared to vascular endothelial growth factor (VEGF). Immuno-gold electron microscopy and immunofluorescence microscopy identified α-granules as storage organelles of platelet angiostatin. Acetylsalicylic acid, MRS2395, GPIIb/IIIa blocking peptide, and aprotinin were used to characterize platelet angiostatin release and generation. An endothelial cell migration assay was performed under hypoxic conditions to determine the effects of pharmacological platelet and angiostatin inhibition. Compared to VEGF, angiostatin generation and release from α-granules occurred later temporally during platelet aggregation. Consequently, collagen-activated platelet releasates stimulated endothelial cell migration more potently than maximally-aggregated platelets. Platelet inhibitors prostacyclin, S-nitroso-glutathione, acetylsalicylic acid, and GPIIb/IIIa blocking peptide, but not a P2Y12 inhibitor, suppressed angiostatin release but not generation. Suppression of angiostatin generation in the presence of acetylsalicylic acid enhanced platelet-stimulated endothelial migration. Hence, the temporal and pharmacological modulation of platelet angiostatin release may have significant consequences for neo-vascularization following thrombus formation.

Proangiogenic microtemplated fibrin scaffolds containing aprotinin promote improved wound healing responses

“…One proposed mechanism by which it may promote angiogenesis is by the inhibition of angiostatin generation by platelets (38, 39). Angiostatin induces apoptosis in and inhibits migration of endothelial cells (39), and is formed by the auto-proteolysis of plasmin (38).…”

“…One proposed mechanism by which it may promote angiogenesis is by the inhibition of angiostatin generation by platelets (38, 39). Angiostatin induces apoptosis in and inhibits migration of endothelial cells (39), and is formed by the auto-proteolysis of plasmin (38). As an inhibitor of serine proteases (including plasmin), aprotinin has been shown to inhibit angiostatin generation by platelets and subsequently induce an increased angiogenic response (38).…”

Plasminogen and its fragments in rat brain: a plausible role for astrocytes in angiostatin generation

“…It has been well-documented that angiostatins induce anoikis and apoptosis and inhibit proliferation and migration of endotheliocytes, thus restrict vessel tube formation and neovascularization [6,7]. Currently, numerous reports indicate that various cell types are able to gene rate angiostatins in relatively high amounts [8,9]. In the previous papers, angiostatins are described as constantly formed by several types of retina-resident cells [10].…”

Plasminogen modulates formation and release of platelet angiogenic regulators

“…Some authors have found that platelets constitutively generate angiostatins, internalize, store, and release them upon activation [24]. Plateletderived angiostatins are thought to counterbalance angiogene sis stimulatory effects, including VEGF signalling [25].…”