Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans.
Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K(m) (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50-70 nM), whereas the K(m) for the thrombin- or thrombin:thrombomodulin-catalyzed reaction is 10-40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus alpha(2)-antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k(cat)/K(m)). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.
Mao
S S
SS
Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486, USA. shi-shan_mao@merck.com
Cooper
C M
CM
Wood
T
T
Shafer
J A
JA
Gardell
S J
SJ
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000097:blood* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000097:blood* / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism* / Q000494:pharmacology; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000378:metabolism; D006490:Hemostatics / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007700:Kinetics; D011817:Rabbits; D013917:Thrombin / Q000494:pharmacology; D018180:Thrombomodulin / Q000378:metabolism; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D004792:Enzyme Precursors; D005343:Fibrinolytic Agents; D006025:Glycosaminoglycans; D006490:Hemostatics; D007074:Immunoglobulin G; D018180:Thrombomodulin; D006493:Heparin; D001120:Arginine; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin