A novel role of h2-calponin in regulating whole blood thrombosis and platelet adhesion during physiologic flow.

Calponin is an actin filament-associated protein reported in platelets, although the specific isoform expressed and functional role were not identified. The h2-calponin isoform is expressed in myeloid-derived peripheral blood monocytes, where it regulates adhesion. Our objective was to characterize the presence and function of the h2 isoform of calponin in platelets. H2-calponin was detected in human and mouse platelets via Western blotting. Immunofluorescent staining demonstrated h2-calponin and actin colocalized in both human and wild-type mouse platelets at rest and following collagen activation. The kinetics of platelet adhesion and whole blood thrombosis during physiologic flow was evaluated in a microfluidic flow-based thrombosis assay. The time to initiation of rapid platelet/thrombus accumulation (lag time) was significantly longer in h2-calponin knockout versus wild-type mouse blood (130.02 ± 3.74 sec and 72.95 ± 16.23 sec, respectively, P < 0.05). There was no significant difference in the rate of platelet/thrombus accumulation during the rapid phase or the maximum platelet/thrombus accumulation. H2-calponin knockout mice also had prolonged bleeding time and blood loss. H2-calponin in platelets facilitates early interactions between platelets and collagen during physiologic flow, but does not significantly affect the rate or magnitude of platelet/thrombus accumulation. H2-calponin knockout mice take 2.3 times longer to achieve hemostasis compared to wild-type controls in a tail bleeding model. The ability to delay platelet accumulation without inhibiting downstream thrombotic potential would be of significant therapeutic value, thus h2-calponin may be a novel target for therapeutic platelet inhibition.

Macrophages induce the adhesion phenotype in normal peritoneal fibroblasts.

OBJECTIVE: To determine whether macrophages, exposed to hypoxia, stimulate primary cultures of fibroblasts to acquire the adhesion phenotype. The adhesion phenotype has been previously characterized, in part, by increased fibroblast expression of transforming growth factor (TGF) ß1, vascular endothelial growth factor (VEGF), and type I collagen. DESIGN: Media collected from human macrophages cultured under hypoxic conditions (2% O(2)) were used to treat human peritoneal fibroblasts. Additionally, human peritoneal fibroblasts were treated with varying concentrations of TGF-ß1. Real-time reverse-transcription polymerase chain reaction and Western blot analysis were used to measure mRNA and protein levels, respectively, for select adhesion markers: TGF-ß1, VEGF, and, type I collagen. We hypothesized that macrophage secretion, under hypoxic conditions, is responsible for inducing the adhesion phenotype in human peritoneal fibroblasts. SETTING: University research laboratory. PATIENT(S): Human macrophages and peritoneal fibroblasts. INTERVENTION(S): Macrophage-fibroblast interaction. MAIN OUTCOME MEASURE(S): Ability of macrophages to induce the adhesion phenotype in human peritoneal fibroblasts. RESULT(S): Hypoxia treatment resulted in a significant increase in TGF-ß1 expression in human macrophages. Additionally, hypoxia treatment resulted in a significant increase in TGF-ß1, VEGF, and type I collagen mRNA and protein levels in normal peritoneal fibroblasts compared with normoxic conditions. Similarly, normal peritoneal fibroblasts treated with media collected from macrophages cultured under hypoxic conditions resulted in a significant increase in TGF-ß1, VEGF, and type I collagen mRNA and protein levels compared with normal peritoneal fibroblasts treated with media collected from macrophages cultured under normoxic conditions. Additionally, human peritoneal fibroblasts exposed to varying concentrations of TGF-ß1 exhibited a dose-dependent response in the expression of TGF-ß1, VEGF, and type I collagen. At a low TGF-ß1 concentration (12.5 ng TGF-ß1/mL medium), TGF-ß1, VEGF, and type I collagen were significantly increased. In contrast, at higher TGF-ß1 concentrations (25 and 50 ng TGF-ß1/mL media), TGF-ß1, VEGF, and type I collagen mRNA levels were significantly reduced compared with 12.5 ng TGF-ß1/mL medium. CONCLUSION(S): Human macrophages, cultured under hypoxic conditions, release factors that induce the expression of the adhesion phenotype in normal peritoneal fibroblasts. Particularly, TGF-ß1 reproduces this response by regulating the expression of TGF-ß1, VEGF, and type I collagen in a dose-dependent manner. Therefore, these findings highlight an important role for human macrophages in peritoneal wound healing.