EVIDENCE THAT PLATELETS PROMOTE TUBE FORMATION BY ENDOTHELIAL-CELLS ON MATRIGEL

1 The involvement of platelets in neovascularization was investigated in the matrigel tube formation assay, an in vitro model of angiogenesi s. 2 Platelets promoted the formation of capillary-like structures (ex pressed as relative tube area) number- and time-dependently. Relative tube area increased from 0.98+/-0.02 (n = 8) in the presence of 6.25 x 10(4), to 3.21 +/- 0.12 (n = 8) in the presence of 10(6) platelets/we ll compared to 0.54+/-0.04 (n = 8) in their absence. This increase was unaffected by acetyl salicylic acid (ASA), apyrase, and hirudin. Phot ographs from representative experiments, showed that platelets adhered along the differentiating endothelium. 3 Addition of alpha-thrombin ( 0.1-1 i.u. ml(-1)), the nitric oxide (NO) donor sodium nitroprusside ( SNP; 1-100 mu M) or the NO synthase inhibitor, L-NG-arginine-methylest er (L-NAME, 30-300 mu M) to the assay, had no effect on tube formation compared to that seen with platelets alone. 4 Neuraminidase (0.01 i.u ./10(7) platelets), which strips sialic acid residues from membrane gl ycoproteins, abolished the promoting effect of platelets on tube forma tion. The relative tube area in the presence of neuraminidase-treated platelets was 0.81+/-0.03 (n = 8), in the presence of untreated platel ets 1.69+/-0.09, P<0.001 (n=8) and in the absence of platelets, 0.80+/ -0.04 (n=8). The tetrapeptide Arg-Gly-Asp-Ser (RGDS; 20-200 mu M) whic h inhibits von Willebrand factor, fibrinogen and fibronectin-mediated adhesion, had no effect on the promoting effect of platelets on tube f ormation. 5 These results indicate that platelets promote angiogenesis in vitro. This effect is largely independent from activation by alpha -thrombin, is not modified by manipulating NO and prostaglandin metabo lism and proceeds possibly through adhesion of the platelets to the di fferentiating endothelium.