Activated protein C generation is greatly decreased in plasma from newborns compared to adults in the presence or absence of endothelium

Summary

Activated protein C (APC) generation strongly affects sepsis and thrombosis by inhibition of thrombin generation. However, it is unclear if there are age-related differences in effectiveness of protein C (PC). We studied age effects on plasma APC generation ± endothelium. Defibrinated (Ancrod) plasma (from adults or newborns (umbilical cord)) was recalcified with buffer containing tissue factor ± thrombomodulin (TM) on either plastic or endothelium (HUVEC) at 37oC. Timed subsamples of reaction mixture were taken into either heparin-EDTA or FFRCMK-EDTA solutions and analyzed for APC-PC inhibitor (APC-PCI) or APC-α₁antitrypsin (APC-α₁AT) by ELISAs. Since heparin converts free APC to APC-PCI, the difference in APCPCI measured in heparin-EDTA and FFRCMK-EDTA samples was equal to free active APC. APC-α₂macroglobulin (APC-α₂M) was measured as remaining chromogenic activity in heparin-EDTA. Free APC, APC-PCI and APC-α₁AT were decreased in newborn compared to adult plasma on plastic. However, APC-α₂M made up a larger fraction of inhibitor complexes in newborn plasma. On endothelium, significantly more APC, APC-PCI and APC-α₁AT were generated in either plasma compared to that on plastic with excess added TM. APC, APC-PCI and APC-α₁AT were also reduced and total APC-α₂M increased in newborn plasma on HUVEC. Addition of PC to newborn plasma gave APC generation similar to adult plasma. Thus, free APC, APC-PCI and APC-α₁AT generation is reduced in newborn compared to adult plasma with or without endothelium, likely due to reduced plasma PC levels. Endothelium enhances APC generation, regardless of plasma type, possibly because of cell surface factors such as TM, phospholipid and endothelial PC receptor.

Keywords

Activated protein C - newborn - endothelium - α₂macroglobulin - protein C inhibitor

• References

• 1 Harris KW, Esmon CT. Protein S is required for bovine platelets to support activated protein C binding and activity. J Biol Chem 1985; 260: 2007-10.
• 2 Krishnaswamy S, Williams EB, Mann KG. The binding of activated protein C to factors V and Va. J Biol Chem 1986; 261: 9684-93.
• 3 Pescatore SL. Clinical management of protein C deficiency. Expert Opin Pharmacother 2001; 02: 431-9.
• 4 Aiach M, Borgel D, Gaussem P. et al. Protein C and protein S deficiencies. Semin Hematol 1997; 34: 205-16.
• 5 Dahlback B, Villoutreix BO. Molecular recognition in the protein C anticoagulant pathway. J Thromb Haemost 2003; 01: 1525-34.
• 6 Federman DG, Kirsner RS. An update on hypercoagulable disorders. Arch Intern Med 2001; 161: 1051-6.
• 7 Sheppard D. Activated protein C resistance: the most common risk factor for venous thromboembolism. J Am Board Fam Pract 2000; 13: 111-5.
• 8 Salem HH, Maricyama I, Ishii H. et al. Isolation and characterization of thrombomodulin from human placenta. J Biol Chem 1984; 259: 12246-51.
• 9 Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem 1982; 257: 7944-51.
• 10 Esmon NL. Thrombomodulin. Semin Thromb Hemostas 1987; 13: 454-63.
• 11 Esmon BL. Thrombomodulin. Semin Thromb Haemost 1987; 13: 454-63.
• 12 Segel GB, Francis CA. Anticoagulant proteins in childhood venous and arterial thrombosis: a review. Blood Cells Mol Dis 2000; 26: 540-60.
• 13 Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA 1981; 78: 2249-54.
• 14 Esmon CT. Protein C: Biochemistry, physiology, and clinical implications. Blood 1983; 62: 1155-8.
• 15 Delvos U, Meusel P, Preissner KT. et al. Formation of activated protein C and inactivation of cell-bound thrombin by antithrombin III at the surface of cultured vascular endothelial cells a comparitive study of two anticoagulant mechanisms. Thromb Haemost 1987; 57: 87-91.
• 16 Taylor Jr FB, Peer GT, Lockhart MS. et al. Endothelial protein C receptor plays and important role in protein C activation in vivo. Blood 2001; 97: 1685-8.
• 17 Liaw PC, Mather T, Oganesyan N. et al. Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor. J Biol Chem 2001; 276: 8364-70.
• 18 Pipe SW, Kaufman RJ. Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa. Proc Natl Acad of Sci USA 1997; 94: 11851-6.
• 19 Suzuki K, Stenflo J, Dahlback B. et al. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914-20.
• 20 Regan LM, Stearns-Kurosawa DJ, Kurosawa S. et al. The endothelial cell protein C receptor. Inhibition of activated protein C anticoagulant function without modulation of reaction with proteinase inhibitors. J Biol Chem 1996; 271: 17499-503.
• 21 Heeb MJ, Gruber A, Griffin JH. Identification of divalent metal ion-dependent inhibition of activated protein C by alpha-2-macroglobulin and alpha-2-antiplasmin in blood and comparisons to inhibition of factor Xa, thrombin, and plasmin. J Biol Chem 1991; 266: 17606-12.
• 22 Hoogendoorn H, Toh CH, Nesheim ME. et al. alpha-2-macroglobulin binds and inhibits activated protein C. Blood 1991; 78: 2283-90.
• 23 Scully MF, Toh CH, Hoogendoorn H. et al. Activation of protein C and its distribution between its inhibitors, protein C inhibitor, alpha 2-antitrypsin and alpha 2-macroglobulin, in patients with disseminated intravascular coagulation. Thromb Haemost 1993; 69: 448-53.
• 24 Andrew M, David M, Adams M. et al. Venous thromboembolic complications (VTE) in children: first analyses of the Canadian Registry of VTE. Blood 1994; 83: 1251-7.
• 25 Schmidt B, Andrew M. Neonatal thrombosis: Report of a prospective Canadian and International registry. Pediatrics 1995; 96: 939-43.
• 26 Nowak-gottl U, Von Kries R, Gobel U. Neonatal symptomatic thromboembolism in Germany: two year survey. Arch Dis Child 1997; 76: F163-7.
• 27 van Ommen CH, Heijboer H, Buller HR. et al. Venous thromboembolism in childhood: A prospective two-year registry in the Netherlands. J Pediatr 2001; 139: 676-81.
• 28 Andrew M, Schmidt B, Mitchell L. et al. Thrombin generation in newborn plasma is critically dependent on the concentration of prothrombin. Thromb Haemost 1990; 63: 27-30.
• 29 Andrew M, Paes B, Milner R. et al. Development of the human coagulation system in the full-term infant. Blood 1987; 70: 165-72.
• 30 Schmidt B, Mitchell L, Ofosu F. et al. Alpha-2-macroglobulin is an important progressive inhibitor of thrombin in neonatal and infant plasma. Thromb Haemost 1989; 62: 1074-7.
• 31 Ling X, Delorme M, Berry L. et al. Alpha-2-macroglobulin remains as important as antithrombin III for thrombin regulation in cord plasma in the presence of endothelial cell surfaces. Pediatr Res 1995; 37: 373-8.
• 32 Andrew M, Vegh P, Johnston M. et al. Maturation of the hemostatic system during childhood. Blood 1992; 80: 1998-2005.
• 33 Andrew M, Paes B, Milner R. et al. Development of the human coagulation system in the healthy premature infant. Blood 1988; 72: 1651-7.
• 34 Petaja J, Fernandez JA, Fellman V. et al. Upregulation of the antithrombotic protein C pathway at birth. Pediatr Hematol Oncol 1998; 15: 489-99.
• 35 Menashi S, Aurousseau MH, Gozin D. et al. High levels of circulating thrombomodulin in human foetuses and children. Thromb Haemost 1999; 81: 906-9.
• 36 Towbin H, Gordon J. Immunoblotting and dot immunobinding- Current status and outlook. J Immunol Methods 1984; 72: 313-40.
• 37 Hoogendoorn H, Nesheim ME, Giles AR. A qualitative and quantitative analysis of the activation and inactivation of protein C in vivo in a primate model. Blood 1990; 75: 2164-71.
• 38 Kurosawa S, Stearns-Kurosawa DJ, Hidari N. et al. Identification of functional endothelial protein C receptor in human plasma. J Clin Invest 1997; 100: 411-8.
• 39 Laurell M, Christensson A, Abrahamsson PA. et al. Protein C inhibitor in human body fluids. Seminal plasma is rich in inhibitor antigen deriving from cells throughout the male reproductive system. J Clin Invest 1992; 89: 1094-101.
• 40 Zelvyte I, Sjogren HO, Janciauskiene S. Effects of native and cleaved forms of alpha1-antitrypsin on ME 1477 tumor cell functional activity. Cancer Detect Prev 2002; 26: 256-65.
• 41 O’Brodovich H, Berry L, D’Costa M. et al. The influence of fetal pulmonary epithelium on thrombin activity. Am J Physiol (Lung Cell Mol Physiol) 1991; 05: L262-70.
• 42 Friedrich U, Blom AM, Dahlback B. et al. Structural and energetic characteristics of the heparin-binding site in antithrombotic protein C. J Biol Chem 2001; 276: 24122-8.
• 43 Espana F, Zuazu I, Vicente V. et al. Quantification of circulating activated protein C in human plasma by immunoassays-enzyme levels are proportional to protein C levels. Thromb Haemost 1996; 75: 56-61.
• 44 Franzoi M, Simioni P, Luni S. et al. Effect of delivery modalities on the physiologic inhibition system of coagulation of the neonate. Thromb Res 2002; 105: 15-8.
• 45 Kueppers F, Offord KP. Alpha-1 antitrypsin elevation in heathly neonates. J Lab Clin Med 1979; 94: 475-80.
• 46 Ninomiya M, Makuuchi H, Ohtsuka T. et al. Ischemic heart disease associated with protein C deficiency. Eur J Cardiothorac Surg 2001; 20: 883-5.
• 47 Dellinger RP. Inflammation and coagulation: implications for the septic patient. Clin Infect Dis 2003; 36: 1259-65.
• 48 Wiedemann HP. Activated protein C was costeffective for prolonging survival in a subgroup of patients with severe sepsis. ACP J Club 2003; 138: 81.
• 49 Matthay MA, Zimmerman GA, Esmon C. et al. Future research directions in acute lung injury: summary of a national heart, lung, and blood institute working group. Am J Crit Care Med 2003; 167: 1027-35.
• 50 Laterre PF, Wittebole X, Dhainaut JF. Anticoagulant therapy in acute lung injury. Crit Care Med 2003; 31: S329-36.
• 51 Riewald M, Ruf W. Science review: Role of coagulation protease cascades in sepsis. Crit Care 2003; 07: 123-9.
• 52 Esmon CT. Protein C pathway in sepsis. Ann Med 2002; 34: 598-605.
• 53 Nagaraja D, Christopher R. Pro-thrombotic states in stroke. Neurol India 2002; 50: S21-9.
• 54 Oh D, Kim SH, Kang MS. et al. Acquired activated protein C resistance, high tissue factor expression, and hyper-homocysteinemia in systemic lupus erythematosis. Am J Hematol 2003; 72: 103-8.
• 55 Curvers J, Thomassen MC, Rimmer J. et al. Effects of hereditary and acquired risk factors of venous thrombosis on a thrombin generation-based APC resistance test. Thromb Haemost 2002; 88: 5-11.
• 56 Belicova M, Lukac B, Dvorsky J. et al. Thromboembolic disease and present oral contraception. Clin Appl Thromb Hemost 2003; 09: 45-51.
• 57 Zangari M, Saghafifar F, Anaissie E. et al. Activated protein C resistance in the absence of factor V Leiden mutation is a common finding in multiple myeloma and is associated with an increased risk of thrombotic complications. Blood Coag Fibrinol 2002; 13: 187-92.
• 58 Schneider DM, von Tempelhoff GF, Herrle B. et al. Maternal and cord blood hemostasis at delivery. J Perinat Med 1997; 25: 55-61.
• 59 Marlar RA, Kressin DC, Madden RM. Contribution of plasma proteinase inhibitors to the regulation of activated protein C in plasma. Thromb Haemost 1993; 69: 16-20.
• 60 Standberg K, Kjellberg M, Knebel R. et al. A sensitive immunochemical assay for measuring the concentration of the activated protein C-protein C inhibitor complex in plasma: use of a catcher antibody specific for the complexed/cleaved form of the inhibitor. Thromb Haemost 2001; 86: 604-10.
• 61 Watanabe R, Wada H, Sakakura M. et al. Plasma levels of activated protein C-protein C inhibitor complex in patients with hypercoagulable states. Am J Hematol 2000; 65: 35-40.
• 62 Shen L, Villoutreix BO, Dahlback B. Involvement of Lys 62(217) and Lys 63(218) of human anticoagulant protein C in heparin stimulation of inhibition by the protein C inhibitor. Thromb Haemost 1999; 82: 72-9.
• 63 Shen L, Dahlback B, Villoutreix BO. Tracking structural features leading to resistance of activated protein C to alpha 1-antitrypsin. Biochemistry 2000; 39: 2853-60.
• 64 Aznar J, Espana F, Estelles A. et al. Heparin stimulation of the inhibition of activated protein C and other enzymes by human protein C inhibitor—influence of the molecular weight of heparin and ionic strength. Thromb Haemost 1996; 76: 983-8.
• 65 Andrew M, Mitchell L, Berry L. et al. An anticoagulant dermatan sulfate proteoglycan circulates in the pregnant woman and her fetus. J Clin Invest 1992; 89: 321-6.
• 66 Esmon CT, Gu JM, Xu J. et al. Regulation and functions of the protein C anticoagulant pathway. Haematologica 1999; 84: 363-8.
• 67 Fukudome K, Esmon CT. Identification, cloning, and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994; 269: 26486-91.
• 68 Crawley JT, Gu JM, Ferrell G, Esmon CT. Disruption of endothelial cell protein C/activated protein C receptor (EPCR) during mouse embryo development. Thromb Haemost 2002; 88: 259-66.
• 69 Gu JM, Crawley JT, Ferrell G. et al. Disruption of the endothelial cell protein C receptor gene in mice causes placental thrombosis and early embryonic lethality. J Biol Chem 2002; 277: 43335-43.
• 70 Cvirn G, Gallistl S, Koestenberger M. et al. Alpha-2-macroglobulin enhances prothrombin activation and thrombin potential by inhibiting the anticoagulant protein C/protein S system in cord and adult plasma. Thromb Res 2002; 105: 433-9.
• 71 Cvirn G, Gallistl S, Muntean W. Alpha-2-macroglobulin inhibits the anticoagulant action of activated protein C in cord and adult plasma. Haemostasis 2001; 31: 1-11.
• 72 Cvirn G, Gallistl S, Muntean W. Effects of antithrombin and protein C on thrombin generation in newborn and adult plasma. Thromb Res 1999; 93: 183-90.