Pharmacologic Protein Kinase CαInhibition Uncouples Human Platelet-Stimulated Angiogenesis from Collagen-Induced Aggregation

Abstract

Platelets promote angiogenesis by releasing angiogenesisregulating factors from their a-granules upon aggregation. This effect has both physiologic and pathologic significance as it may contribute to carcinogenesis. Platelet a-granule release and aggregation are regulated, in part, via protein kinase C (PKC) a and b signaling. Our study investigated the effects of PKC inhibition on aggregation, angiogenesis-regulator secretion from a-granules, and platelet-stimulated angiogenesis. We hypothesized that selective PKCa inhibition may preferentially suppress angiogenesis-regulator secretion from a-granules but not aggregation, limiting platelet-stimulated angiogenesis. Human platelets were aggregated in the presence of conventional PKC inhibitors myr-FARKGALRQ and Ro 32-0432 (2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide). Immunofluorescence microscopy of PKC translocation was used to determine the specificity of PKC-inhibitor targeting. Enzyme-linked immunosorbent assay was used to measure vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) release from platelets. Platelet effects on angiogenesis were tested using a capillary-formation assay. Ro 32-0432, but not the peptide inhibitor myr-FARKGALRQ (myristoylated-pseudosubstrate peptide inhibitor), inhibited aggregation in a concentration-dependent manner, while both Ro 32-0432 and myr-FARKGALRQ preferentially suppressed VEGF over TSP-1 secretion. Suppression of angiogenesis-regulator release occurred at inhibitor concentrations that did not significantly affect aggregation. Immunofluorescence microscopy revealed that PKCa targeting to a-granules is inhibited when angiogenesis-regulator secretion is uncoupled from aggregation. At concentrations that uncoupled a-granule release from aggregation, Ro 32-0432 and myr-FARKGALRQ inhibited platelet-stimulated angiogenesis. Hence, selective PKCa inhibition suppresses angiogenesisregulator release from platelet a-granules with minimal effects on aggregation. Thus, selective PKCa inhibitors may have pharmacologic significance to regulate platelet-promoted angiogenesis.

The Wnt Antagonist Dickkopf-1 Promotes Pathological Type 2 Cell-Mediated Inflammation

“…Activation of human platelets with sLMAG induced Dkk-1 within 1 hour in a dose-dependent manner ( Figure 6K ). Inhibition of PKCα ( Figure 6L ) blocked secretion of Dkk-1 upon sLMAG stimulation of human platelets, confirming that activated platelets secrete Dkk-1 from α-granules (Moncada de la Rosa et al, 2013). In contrast, stimulation of human peripheral blood mononuclear cells (PBMCs) or vascular endothelial cells with sLMAG or HDM extract failed to induce secretion of Dkk-1 ( Supplementary Figure 4 ).…”

Effect of Platelet Lysate on Human Cells Involved in Different Phases of Wound Healing

“…The differential release of angiogenesis stimulators and inhibitors from platelets was also studied, showing that activation with adenosine diphosphate stimulated the release of VEGF but not endostatin, promoting capillary tube formation [26]. A very recent study evaluated platelet-stimulated angiogenesis focusing mainly on the release of angiogenesis-regulating factors from their α-granules upon aggregation [27]. …”

Inhibition of protein kinase C enhances angiogenesis induced by platelet-derived growth factor C in hyperglycemic endothelial cells

“…However, the role of PKC for angiogenesis still remains elusive because the effect of PKC on angiogenesis seems to be different or even opposite depending on PKC isoforms [ 39 ]. Moreover, there are some conflicting reports indicating that PKC-α promotes [ 40 , 41 ] or inhibits [ 42 ] angiogenic activity of ECs, and it has been known that inhibition of PKC leads to suppression of VEGF release from platelets [ 43 ]. We show here that inhibition of PKC by Bis I leads to increased PDGFR-α expression, resulting in potentiation of intracellular signaling, and augmentation of angiogenesis induced by PDGF-C, but not by VEGF-A (Figures 3 , 4 , 5 ).…”