In vitro incubation with thrombin. (A) Proliferation, expressed as mean counts per minute (CPM) plus or minus SEM assessed by incorporation of ³H-thymidine by triplicates. CD34⁺ cells were isolated from WT mice 2 days after wire-induced injury and incubated for 5 days either in medium alone or in medium containing active site-inhibited thrombin (FIIa), FIIa, FIIa with PAR-1 antagonist (antAg), FIIa with PAR-4 antagonist, VEGF, or PDGF before a 16-hour incubation with ³H-thymidine. (B) Proportion of cells undergoing apoptosis after incubation in vitro for 1 (□) or 5 days (■). Phenotype was determined by annexin V staining and is expressed as a proportion of the cells remaining. CD34⁺ cells were isolated from WT mice 2 days after wire-induced injury and incubated for 5 days either in medium alone, or in medium containing inactive FIIa, FIIa, VEGF, or PDGF. (C) CD34⁺ cells were isolated from WT mice 2 days after wire-induced injury and incubated for 5 days either in medium alone, medium containing inactive thrombin, thrombin, or thrombin with a PAR-1 or PAR-4 antagonist. Graphs show the proportion of cells expressing CD31 alone (□), both CD31 and α-SMA (▩), or α-SMA alone (■). Phenotype was determined by immunocytochemistry of 3 random fields as in Figure 4 and is expressed as a proportion of the cells remaining. (D) As in panel C, except cells incubated with VEGF, VEGF plus thrombin, PDGF, or PDGF plus FIIa. All in vitro experiments were repeated at least twice. Data are presented as means plus or minus SEM.