Calreticulin, an antithrombotic agent which binds to vitamin K-dependent coagulation factors, stimulates endothelial nitric oxide production, and limits thrombosis in canine coronary arteries.
Coagulation Factor IX/IXa has been shown to bind to cellular surfaces, and Factor IXa expresses its procoagulant activity by assembling into the intrinsic Factor X activating complex (Factors IXa/VIIIa/X), which also forms on membrane surfaces. This led us to identify cellular proteins which bind Factor IX/IXa; an approximately 55-kDa polypeptide was purified to homogeneity from bovine lung extracts based on its capacity to bind 125I-Factor IX in a dose-dependent and saturable manner. From protein sequence data of the amino terminus and internal peptides, the approximately 55-kDa polypeptide was identified as calreticulin, a previously identified intracellular calcium-binding protein. Recombinant calreticulin bound vitamin K-dependent coagulation factors, 125I-Factor IX, 125I-Factor X, and 125I-prothrombin (Kd values of approximately 2.7, 3.2, and 8.3 nM, respectively), via interaction with its C-domain, although it did not affect the coagulant properties of these proteins. 125I-Calreticulin also bound to endothelial cells in vitro (Kd approximately 7.4 nM), and mouse infusion studies showed an initial rapid phase of clearance in which calreticulin could be localized on the vascular endothelium. Exposure of endothelial cells to calreticulin led to dose-dependent, immediate, and sustained increase in the production of nitric oxide, as measured using a porphyrinic microsensor. In a canine electrically induced thrombosis model, intracoronary infusion of calreticulin (n = 7) prevented occlusion of the left circumflex coronary artery in a dose-dependent manner compared with vehicle-treated controls (n = 5). These results indicate that calreticulin interacts with the endothelium to stimulate release of nitric oxide and inhibit clot formation.
Kuwabara
K
K
Department of Physiology, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA.
Pinsky
D J
DJ
Schmidt
A M
AM
Benedict
C
C
Brett
J
J
Ogawa
S
S
Broekman
M J
MJ
Marcus
A J
AJ
Sciacca
R R
RR
Michalak
M
M
D000818:Animals; D001779:Blood Coagulation Factors / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D037282:Calreticulin; D002417:Cattle; D003328:Coronary Thrombosis / Q000517:prevention & control*; D004285:Dogs; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009569:Nitric Oxide / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012261:Ribonucleoproteins / Q000378:metabolism / Q000494:pharmacology*; D014812:Vitamin K / Q000378:metabolism
D001779:Blood Coagulation Factors; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D014812:Vitamin K; D009569:Nitric Oxide