Proteolytic cleavage of carboxypeptidase N markedly increases its antifibrinolytic activity.

BACKGROUND: Carboxypeptidase N (CPN) is a constitutively active basic carboxypeptidase sharing specificity with activated thrombin-activable fibrinolysis inhibitor (TAFIa). Generally, CPN is regarded as being non-antifibrinolytic. However, this assumption has not been thoroughly investigated, particularly with respect to long-term antifibrinolysis. In addition, a recent report has shown that plasmin cleavage increases the catalytic activity of CPN. Therefore, we investigated the antifibrinolytic properties of CPN and plasmin-cleaved CPN (CPNc). METHODS: CPN was incubated with plasmin for various periods of time and the prolongation of clot lysis at various concentrations of CPN/CPNc mixture was investigated in TAFI-depleted plasma. CPN cleavage was analyzed by electrophoresis and catalytic activity was determined by monitoring cleavage of the small substrate, FA-Ala-Lys. RESULTS: CPN exhibited antifibrinolytic properties in plasma clot lysis assays when present at supraphysiological concentrations. Depletion of CPN from plasma decreased the lysis time of clots formed from minimally diluted plasma at low tissue-type plasminogen activator (t-PA) concentrations. Plasmin cleavage of CPN markedly increased the antifibrinolytic properties. CPN and CPNc prolonged lysis in a non-saturable, dose-dependent, and t-PA-dependent manner. At sufficient concentration, CPN and CPNc prolonged lysis at least forty-fivefold. CPNc was 700% more antifibrinolytic than CPN but only 7% more active toward FA-Ala-Lys. The active site inhibitor GEMSA eliminated the antifibrinolytic effects of CPN and CPNc. Antifibrinolytic activity correlated with cleavage of active and/or regulatory subunits, presumably generating heterodimeric CPNc. CONCLUSIONS: Limited proteolysis of CPN by plasmin generates an enzyme with greatly increased antifibrinolytic properties. We speculate that (patho)physiological proteolysis of CPN may generate a long-term antifibrinolytic enzyme.

In vitro and in vivo expression of Galalpha-(1,3)Gal on porcine islet cells is age dependent.

The expression of Galalpha-(1,3)Gal (alphaGal) on porcine islet cells remains controversial. Several groups have reported that porcine islet endocrine cells do not express alphaGal while we have shown in neonatal porcine islets (NPI) that beta cells do express this antigen. We hypothesize that endocrine cells expressing alphaGal on NPI are less mature cells that may have originated from ductal cells and that expression of this antigen disappears as they develop into fully mature beta cells. Thus, we further examined alphaGal expression on various porcine islet cell preparations and correlated this with the proportion of cytokeratin 7 (CK7)-positive ductal cells. In vitro and in vivo expression of alphaGal and CK7 was significantly (P<0.05) higher in less mature NPI cells compared with matured NPI and adult porcine islet cells while the reverse was observed in the proportion of beta cells. Moreover, a significantly higher proportion of CK7-positive cells was detected in the Gal-expressing population compared with non-expressing cells. In contrast, a higher proportion of beta cells was observed in the Gal-negative population compared with the Gal-positive population. These data showed a reduced expression of alphaGal and CK7 as porcine islet cells mature into beta cells suggesting a possible role for alphaGal in the maturation of pancreatic endocrine beta cells.

In vitro maturation of neonatal porcine islets: a novel model for the study of islet development and xenotransplantation.

The mechanisms involved in islet neogenesis have remained largely unexplored due to lack of an appropriate model. Furthermore, with the recent advances in islet transplantation, the need for alternative islet tissue sources is greater than ever. Therefore, the authors have refined a neonatal porcine islet (NPI) maturation model that offers an ideal tool to gain insight into islet growth as well as an alternative source of transplantable tissue. Recent knowledge in islet growth has resulted in endocrine tissue being derived from human pancreatic precursor tissue in vitro. The potential for large scale production of endocrine tissue in vitro has been indicated, however, more investigation must be done on the various signals and pathways involved in pancreatic development to optimize this technique. The authors believe that their NPI in vitro maturation model provides an ideal tool to study islet growth and maturation. Transduction of the NPI to overexpress genes of interest (i.e., PDX-1) or exposure of the NPI to various culture conditions will allow us to determine the effects on islet maturation. An understanding of NPI development gained will not only allow us to mature this unlimited tissue source for optimal xenotransplantation, but also elude to how human pancreatic endocrine precursor cells may be used to solve the current islet tissue supply problem.