Download PDF
Thromb Haemost 1999; 82(05): 1474-1481
DOI: 10.1055/s-0037-1614858
Rapid Communications
Schattauer GmbH

Recombinant Kunitz Protease Inhibitory Domain of the Amyloid β-Protein Precursor as an Anticoagulant in Venovenous Extracorporeal Circulation in Rabbits

Gail Annich
1   From the Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA
5   Surgery, University of Michigan, Ann Arbor, MI, USA
,
Tyler White
2   Scios, Inc. Sunnyvale, CA, USA
,
Deborah Damm
2   Scios, Inc. Sunnyvale, CA, USA
,
Yongqiang Zhao
3   Internal Medicine, University of Michigan, Ann Arbor, MI, USA
,
Fahkri Mahdi
3   Internal Medicine, University of Michigan, Ann Arbor, MI, USA
,
Jürgen Meinhardt
5   Surgery, University of Michigan, Ann Arbor, MI, USA
,
Sam Rebello
4   Pharmacology, University of Michigan, Ann Arbor, MI, USA
,
Benedict Lucchesi
4   Pharmacology, University of Michigan, Ann Arbor, MI, USA
,
Robert H. Bartlett
5   Surgery, University of Michigan, Ann Arbor, MI, USA
,
Alvin H. Schmaier
3   Internal Medicine, University of Michigan, Ann Arbor, MI, USA
› Author Affiliations
Further Information

Publication History

Received 25 March 1999

Accepted after revision 12 July 1999

Publication Date:
09 December 2017 (online)

    Permissions and Reprints

Summary

Investigations were performed to characterize a recombinant Kunitz protease inhibitory domain of the amyloid β-protein precursor (rKPI) as anticoagulants. After a single intravenous infusion of wild type rKPI into dogs, its elimination fit a two compartment model with a t½α and t½β of 5 and 77 min, respectively. Further investigations determined if a variant form of rKPI with 178-fold more potent anti-factor Xa activity (rKPI-DD135, Ki = 0.9 nM) could serve as an anticoagulant in a rabbit model of extracorporeal circulation using a venovenous shunt. A prospective investigation was initiated to compare standard heparin (n = 8) at 400 U/kg with different infusion concentrations of rKPI-DD135. After a single intravenous infusion of 1.89 mg/kg of rKPI-DD135 followed by a constant infusion at 0.003 (n = 3), 0.03 (n = 7), or 0.3 (n = 5) mg/kg/min, the anti-factor Xa activity of the animals’ plasma rapidly reaches a steady state for the two lower infusion concentrations of the agent. All infusions of rKPI-DD135 prolong the activated clotting time with less variation than that seen with heparin administration. rKPI-DD135 anticoagulation does not prevent a drop in the platelet counts. Fibrinogen levels decrease only slightly when the circuit is anticoagulated with rKPI-DD135. rKPI-DD135 markedly prolongs the APTT, has little effect on the PT, and reduces plasma prekallikrein and plasminogen activation. The 0.3 mg/kg/min infusion concentration of rKPI-DD135 results in reduced deposition of 111Indium-labeled platelets on the circuit when compared to heparin. Last, after a steady state level is achieved, 60% of the plasma anti-factor Xa activity of rKPI-DD135 is eliminated within 60 min after stopping the infusion. These data show the rKPI-DD135 can provide single agent anticoagulation in a rabbit extracorporeal circuit. Development of short acting factor Xa inhibitors may be useful anticoagulants for cardiopulmonary bypass.

Abbreviations: CPB: cardiopulmonary bypass; KPI: Kunitz protease inhibitor domain; rKPIWT: recombinant wild type Kunitz protease inhibitory domain of the amyloid β-protein precursor; rKPI-DD135: recombinant Kunitz protease inhibitory domain of the amyloid β-protein which is a better inhibitor of factor Xa; ECC: extracorporeal circulation; CDP: citrate phosphate dextrose anti-coagulant; PRP: platelet-rich plasma; ACT: activated clotting time; PT: prothrombin time; APTT: activated partial thromboplastin time; PPP: platelet-poor plasma.

Key words

Amyloid β-protein precursor - factor Xa inhibitor - anticoagulant - ECC
 

  • References

  • 1 Trossaert M, Gaillard A, Commin PL, Amiral J, Vissac AM, Fressinaud E. High incidence of anti-heparin/platelet factor 4 antibodies after cardiopulmonary bypass surgery. Brit J of Haemat 1998; 101: 653-5.
    PubMedGoogle Scholar
  • 2 Royston D, Bidstrup BP, Taylor KM, Sapsford RN. Effect of aprotinin on need for blood transfusion after repeat open-heart surgery. Lancet 1987; 2: 1289-91.
    PubMedGoogle Scholar
  • 3 Van Nostrand WE, Wagner SL, Farrow JS, Cunningham DD. Immunopurification and protease inhibitory properties of protease nexin-2/amyloid beta-protein precursor. J Biol Chem 1990; 265: 9591-4.
    PubMedGoogle Scholar
  • 4 Smith RP, Higuchi DA, Broze Jr GJ. Platelet coagulation factor XIa-inhibitor, a form of Alzheimer amyloid precursor protein. Science 1990; 248: 1126-8.
    CrossrefPubMedGoogle Scholar
  • 5 Schmaier AH, Dahl LD, Rozemuller AJM, Roos RAC, Wagner SL, Chung R, Van Nostrand WE. Protease nexin-2/amyloid β protein precursor. A tight-binding inhibitor of coagulation factor IXa. J Clin Invest 1993; 92: 2540-5.
    CrossrefPubMedGoogle Scholar
  • 6 Schmaier AH, Dahl LD, Hasan AAK, Cines DB, Bauer KA, Van Nostrand WE. Factor IXa inhibition by protease nexin-2/amyloid β-protein precursor on phospholipid vesicles and cell membranes. Biochemistry 1995; 34: 1171-8.
    CrossrefPubMedGoogle Scholar
  • 7 Mahdi F, Van Nostrand WE, Schmaier AH. Protease nexin-2/amyloid β-protein precursor inhibits factor Xa in the prothrombinase complex. J Biol Chem 1995; 270: 23468-74.
    CrossrefPubMedGoogle Scholar
  • 8 Van Nostrand WE, Schmaier AH, Farrow JS, Cunningham DD. Protease nexin-II (Amyloid β-protein precursor): A platelet α-granule protein. Science 1990; 248: 745-8.
    CrossrefPubMedGoogle Scholar
  • 9 Wagner SL, Siegel RS, Vedvick TS, Raschke WC, Van Nostrand WE. High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid beta-protein precursor. Biochem Biophys Res Comm 1992; 186: 1138-45.
    CrossrefPubMedGoogle Scholar
  • 10 Chase Jr T, Shaw E. p-Nitrophenyl-p’-guanidinobenzoate HCl: A new active site titrant for trypsin. Biochem Biophys Res Commun 1967; 29: 508-14.
    CrossrefPubMedGoogle Scholar
  • 11 Bieth JG. Theoretical and practical aspects of proteinase inhibition. Methods Enzymol 1995; 248: 59-84.
    PubMedGoogle Scholar
  • 12 Bieth JG. In vivo significance of kinetic constants of protein proteinase inhibitors. Biochem Med 1984; 32: 387-97.
    CrossrefPubMedGoogle Scholar
  • 13 Rand ML, Packham MA, Mustard JF. Survival of density subpopulations of rabbit platelets: use of 51Cr- or 111In-labeled platelets to measure survival of least dense and most dense platelets concurrently. Blood 1983; 61: 362-7.
    PubMedGoogle Scholar
  • 14 Timmons S, Hawiger J. Separation of human platelets from plasma proteins including Factor VIII by a combined albumin gradient-gel filtration method using Hepes buffer. Thromb Res 1978; 12: 297-306.
    CrossrefPubMedGoogle Scholar
  • 15 Schmaier AH, Claypool W, Colman RW. Crotalocytin: recognition and purification of a timber rattlesnake platelet aggregating protein. Blood 1980; 56: 1013-9.
    PubMedGoogle Scholar
  • 16 Rowland M, Tozer T. Clinical pharmacokinetics: concepts & applications. 2nd Edition. Lea & Febiger; Philadelphia, PA: 1989
    Google Scholar
  • 17 DeLa Cadena R, Scott CF, Colman RW. Evaluation of a functional microassay for determination of plasma prekallikrein. J Lab Clin Med 1987; 109: 601-7.
    PubMedGoogle Scholar
  • 18 Stassen JM, Lambeir AM, Matthyssens G, Ripka WC, Nystrom A, Sixma JJ, Vermylen J. Characterization of a novel series of aprotinin-derived anticoagulants. I. In vitro and pharmacological properties. Thromb Haemost 1995; 74: 646-54.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Harker LA, Hanson SR, Kelly AB. Antithrombotic benefits and hemorrhagic risks of direct thrombin antagonists. Thromb Haemost 1995; 74: 464-72.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 20 Gitlin SD, Deeb GM, Yann C, Schmaier AH. Intraoperative monitoring of danaparoid sodium anticoagulation during cardiovascular operations. J Vasc Surg 1998; 27: 568-75.
    CrossrefPubMedGoogle Scholar
  • 21 Van Nostrand WE, Schmaier AH, Siegel RS, Wagner SL, Raschke WC. Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitz-type protease inhibitor domain of the amyloid b-protein precursor. J Biol Chem 1995; 270: 22827-30.
    CrossrefPubMedGoogle Scholar