Direct flow cytometric measurement of nucleic acid content in individual platelets is possible using the fluorescent dye Thiazole Orange (Becton-Dickinson, San Jose, CA). When applied to studies of thrombocytopenic patients, platelets with elevated nucleic acid content ("reticulated platelets") can be identified and quantitated. Labeling of these platelets is saturable and is abolished by treatment with RNAse. It has been suggested that, similar to the erythrocyte reticulocyte response to anemia, the number of these platelets appearing in the circulation may provide an estimate of the rate of thrombopoiesis. The authors studied 229 thrombocytopenic patients, measuring both reticulated platelets and platelet-associated immunoglobulin. The results show that for the subset of patients with normal levels of platelet-associated immunoglobulin, the average absolute number of reticulated platelets is independent of platelet count and remains in the normal range. For those with elevated levels of platelet-associated immunoglobulin, the absolute number of reticulated platelets increases in patients who are moderately thrombocytopenic (60 to 100 x 10(9)/L) but decreases to normal or subnormal levels as thrombocytopenia worsens. The latter finding has been duplicated in studies of mice made thrombocytopenic by injection of anti-platelet antiserum. These results are consistent with the hypothesis that reticulated platelets are subject to peripheral destruction at the same rate as mature platelets, and that in the severely thrombocytopenic patient their level may decrease despite an appropriate marrow thrombopoietic response.

Transforming growth factor-beta (TGF-beta) signaling in endothelial cells is able to modulate angiogenesis and vascular remodeling, although the underlying molecular mechanisms remain poorly understood. Endoglin and ALK-1 are components of the TGF-beta receptor complex, predominantly expressed in endothelial cells, and mutations in either endoglin or ALK-1 genes are responsible for the vascular dysplasia known as hereditary hemorrhagic telangiectasia. Here we find that the extracellular and cytoplasmic domains of the auxiliary TGF-beta receptor endoglin interact with ALK-1 (a type I TGF-beta receptor). In addition, endoglin potentiates TGF-beta/ALK1 signaling, with the extracellular domain of endoglin contributing to this functional cooperation between endoglin and ALK-1. By contrast, endoglin appears to interfere with TGF-beta/ALK-5 signaling. These results suggest that the functional association of endoglin with ALK-1 is critical for the endothelial responses to TGF-beta.

Using the rat balloon catheter denudation model, we examined the role of transforming growth factor-beta (TGF-beta) isoforms in vascular repair processes. By en face in situ hybridization, proliferating and quiescent smooth muscle cells in denuded vessels expressed high levels of mRNA for TGF-beta1, TGF-beta2, TGF-beta3, and lower levels of TGF-beta receptor II (TGF-betaRII) mRNA. Compared with normal endothelium, TGF-beta1 and TGF-beta2, as well as TGF-betaRII, mRNA were upregulated in endothelium at the wound edge. Injected recombinant soluble TGF-betaRII (TGF-betaR:Fc) localized preferentially to the adventitia and developing neointima in the injured carotid artery, causing a reduction in intimal lesion formation (up to 65%) and an increase in lumen area (up to 88%). The gain in lumen area was largely due to inhibition of negative remodeling, which coincided with reduced adventitial fibrosis and collagen deposition. Four days after injury, TGF-betaR:Fc treatment almost completely inhibited the induction of smooth muscle alpha-actin expression in adventitial cells. In the vessel wall, TGF-betaR:Fc caused a marked reduction in mRNA levels for collagens type I and III. TGF-betaR:Fc had no effect on endothelial proliferation as determined by reendothelialization of the denuded rat aorta. Together, these findings identify the TGF-beta isoforms as major factors mediating adventitial fibrosis and negative remodeling after vascular injury, a major cause of restenosis after angioplasty.