No abstract

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four-step procedure: stimulation of its release from platelets by thrombin, cation-exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin-Sepharose CL-6B chromatography, and reverse-phase HPLC on a C 4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a heat-and acid-labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS-PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS-PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet-derived growth factor (PDGF).

Whether hematopoietic stem cells can proliferate without limit, or whether their regenerative capacity declines with repeated division, has been debated for decades. Prevailing opinion favours an intrinsic 'decline', a view based on the finite degree to which murine bone marrow can be serially transplanted, the diminished self-renewal of spleen colony-forming cells (CFU-s) subjected to repeated passage, and the failure of stem cells to regenerate to normal levels after even a single transplantation. However, serial transfer experiments did not specifically monitor input and output of long-lived stem cells (long-term reconstituting cells, LTRCs), leaving competing interpretations unresolved. We have re-examined the issue by quantitating 7-12 month LTRCs during sequential transplantations. Although these cells recovered to only 4% of normal levels after primary bone marrow transplantation, at each passage they increased around 10-fold relative to the amount transplanted, attaining an estimated cumulative expansion of 8400-fold over the original input after four transfers. Expansion was limited by transfer of increasing numbers of marrow cells and specifically of LRTCs, suggesting an extrinsically determined ceiling to stem cell growth. Conversely, expansion was enhanced in vivo by administration of stem cell factor (SCF, c-kit ligand) and interleukin-11. The results challenge the view that expansion of passaged stem cells is limited by exhaustion, and indicate that augmentation after transplant is limited by extrinsic mechanisms whose effects are reversible either by further transfer of the stem cells into irradiated hosts or by administration of exogenous cytokines.

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SummaryPrevious studies on recombinant human soluble thrombomodulin (rsTM) from Chinese hamster ovary cells revealed that rsTM was expressed as two proteins that differed functionally in vitro due to the presence (rsTMp) or absence (rsTMa) of chondroitin-4-sulfate. The current study evaluates the in vivo behavior of rsTM in rats and in a rat model of tissue factor-induced disseminated intravascular coagulation (DIC). rsTMp was more potent than rsTMa for prolongation of the activated partial thromboplastin time (APTT) and their in vivo half-lives determined by ELISA were 20 min for rsTMp and 5.0 h for rsTMa. Injection of a tissue factor suspension (5 mg/kg) resulted in DIC as judged by decreased platelet counts and fibrinogen concentrations, prolonged APTT, and increased fibrin and fibrinogen degradation products (FDP) levels. A bolus injection of either rsTM (0.2 mg/kg) 1 min before induction of DIC essentially neutralized effects on platelets, fibrinogen, and FDP levels, and had only a moderate effect on APTT prolongation. The dose of anticoagulant to inhibit the drop in platelet counts by 50% (ED50) was 0.2 mg/kg rsTMa, 0.07 mg/kg rsTMp, and 7 U/ kg heparin. The effect of increasing concentrations of rsTM and heparin on bleeding times were compared in experiments involving incision of the rat tail. Doubling of the bleeding times occurred at 5 mg/kg rsTMa, 3 mg/kg rsTMp or 90 U/kg heparin. These values represent a 25-fold increase over the ED50 for rsTMa, 43-fold for rsTMp and 13-fold for heparin. These results suggest that rsTMp is a potent anticoagulant to inhibit the platelet reduction when injected prior to the induction of DIC in rats.