Standard methods for determining prorenin-renin concentrations in plasma (PRC) and other tissues require the addition of exogenous renin substrate (angiotensinogen) to improve the kinetics of the renin reaction. We studied the effects of substrate prepared from normal human plasma fraction Cohn IV-4, or from nephrectomized (2NX) sheep plasma, on PRC of normal and 2NX human plasmas before and after prorenin activation by acid, cold, and trypsin, and compared the results with plasma renin activities (PRA, no added substrate). Plasmas from 2NX men exhibited negligible basal PRA, indicating that very little, if any, renin had been formed from the extrarenal prorenin they contained, and suggesting the lack of an endogenous prorenin activating mechanism, or "convertase," of probable renal origin. Prorenin was demonstrable by tryptic activation, more than by acid or cold, at up to about 30% of normal. Addition of Cohn IV-4 substrate to 2NX plasma unexpectedly produced (i) a basal PRC value higher than in normal plasma, (ii) total renin values after activation by acid, cold, and trypsin that were much closer to normal values than reflected by PRA methodology, without a commensurate increase (if anything a decrease) in prorenin as a percentage of total renin estimated by all activation methods, and (iii) substantial equalization of activation effects such that trypsin was no longer more effective than acid and cold (and this was also noted with normal plasma). The skewing effect of adding Cohn IV-4 substrate on the PRC of 2NX plasma was much greater than in normal plasma, even though 2NX plasma already had an above normal level of endogenous substrate and should have been influenced less. Enhancement of PRC was very pronounced even when Cohn IV-4 was added to make up only 9% of total (endogenous + exogenous) substrate in the incubation system, suggesting that it was not the added substrate but a renin-generating contaminant that inflated the PRC. Such inflation could be blocked by adding protease inhibitors, suggesting that the responsible protease(s) acted as a prorenin "convertase" that generated new renin from renal and (or) extrarenal prorenin contributed by the added substrate, as well as by the plasma being assayed. One component of convertase could be kallikrein, which was identified by chromogenic assay, the importance of which relative to total convertase activity is unknown. The convertase content of substrate from 2NX sheep plasma seemed to be low (again suggesting loss due to 2NX, i.e., its renal origin), but it contributed extrarenal prorenin from which additional renin was apparently formed by the action of convertase provided by normal human plasma in the PRC assay. Thus, both human Cohn IV-4 and 2NX sheep substrates can apparently contribute renal and (or) extrarenal prorenin and various amounts of convertase which can, along with the actual substrate, greatly influence the PRC assay, and do so to a higher degree in some plasmas than in others. We also found that activation by cold and trypsin did not destroy endogenous plasma substrate (if anything increased it), suggesting activation of a "prosubstrate," while acid activation destroyed only about half of the measurable substrate. The presence of convertase in the Cohn IV-4 fraction suggests it must have come from the original whole plasma. This, along with existing prorenin, has the potential to be a functional renin-producing pathway in normal blood which is lost after nephrectomy owing to the disappearance of a renal "convertase."
Studies on Thrombopoiesis. I. A Factor in Normal Human Plasma Required for Platelet Production; Chronic Thrombocytopenia Due to its Deficiency
