Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39.
Excessive platelet accumulation and recruitment, leading to vessel occlusion at sites of vascular injury, present major therapeutic challenges in cardiovascular medicine. Endothelial cell CD39, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ATP and ADP released from activated platelets, thereby abolishing recruitment. Therefore, a soluble form of CD39, retaining nucleotidase activities, would constitute a novel antithrombotic agent. We designed a recombinant, soluble form of human CD39, and isolated it from conditioned media from transiently transfected COS-1 cells and from stably transfected Chinese hamster ovary (CHO) cells. Conditioned medium from CHO cells grown under serum-free conditions was subjected to anti-CD39 immunoaffinity column chromatography, yielding a single approximately 66-kD protein with ATPase and ADPase activities. Purified soluble CD39 blocked ADP-induced platelet aggregation in vitro, and inhibited collagen-induced platelet reactivity. Kinetic analyses indicated that, while soluble CD39 had a Km for ADP of 5.9 microM and for ATP of 2.1 microM, the specificity constant kcat/Km was the same for both substrates. Intravenously administered soluble CD39 remained active in mice for an extended period of time, with an elimination phase half-life of almost 2 d. The data indicate that soluble CD39 is a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses.
Gayle
R B
RB
Immunex Corporation, Seattle, Washington 98101, USA. gayler@immunex.com
Maliszewski
C R
CR
Gimpel
S D
SD
Schoenborn
M A
MA
Caspary
R G
RG
Richards
C
C
Brasel
K
K
Price
V
V
Drosopoulos
J H
JH
Islam
N
N
Alyonycheva
T N
TN
Broekman
M J
MJ
Marcus
A J
AJ
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000494:pharmacology*; D001081:Apyrase / Q000235:genetics / Q000493:pharmacokinetics*; D001792:Blood Platelets / Q000187:drug effects*; D016466:CHO Cells; D019556:COS Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D005343:Fibrinolytic Agents / Q000493:pharmacokinetics*; D006207:Half-Life; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000493:pharmacokinetics; D012995:Solubility; D013923:Thromboembolism / Q000517:prevention & control
D015703:Antigens, CD; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D001081:Apyrase; C067869:CD39 antigen