title: Carbon monoxide released by CORM-3 inhibits human platelets by a mechanism independent of soluble guanylate cyclase.
abstract: OBJECTIVE
  Carbon monoxide (CO) modulates several physiological functions through activation of a cGMP-dependent pathway similar to that of nitric oxide (NO). Here we investigated the possible involvement of soluble guanylate cyclase in the anti-aggregatory effect of micromolar concentrations of CO released by a novel, water-soluble, CO releasing molecule (CORM) in human platelets.
  
  
  METHODS
  Human platelet aggregation was induced by collagen or thrombin, and the effects of CO releasing molecule (CORM-3) and an NO donor on platelet aggregation were compared.
  
  
  RESULTS
  CORM-3 liberated CO in a time- and concentration-dependent manner as evidenced by the formation of carbon monoxy myoglobin (MbCO) using a spectrophotometric assay. When added to washed platelets, CORM-3 (10-300 microM) inhibited collagen- and thrombin-induced aggregation in a concentration-dependent manner. The anti-aggregatory effect of CORM-3 was reversed by deoxy-Mb (50 microM). Interestingly, in the presence of an inhibitor of guanylate cyclase (ODQ, 5 microM), inhibition of collagen-induced aggregation by CORM-3 was not blocked but potentiated. Under the same experimental conditions, inhibition of platelet aggregation by an NO donor (SNAP, 1 microM) was prevented by ODQ. In collagen-induced or thrombin-induced platelet aggregation, a stimulator of guanylate cyclase (YC-1, 0.3 microM) did not alter the effect of CORM-3, whereas it markedly potentiated the inhibition of platelet aggregation mediated by SNAP. Notably, CORM-3-induced inhibition of platelet aggregation was of similar degree when platelets were activated by a low (20 mU/ml) or by high concentration of thrombin (100-200 mU/ml), whereas NO donors (SNP and SNAP)- or carbaprostacylin (cPGI(2))-induced effects were considerably attenuated when platelets were activated by high concentrations of thrombin.
  
  
  CONCLUSIONS
  Inhibition of platelet aggregation by CO released by a novel, water-soluble CORM is not mediated by activation of soluble guanylate cyclase. In contrast to NO and PGI(2), CO effectively inhibits platelets even when cells are activated excessively. We suggest that despite the fact that CO is not a potent inhibitor of platelet activation, it may gain importance when NO and PGI(2) alone are insufficient to overcome excessive platelet activation.
authors.affiliation: Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University Medical College, 31-531 Krakow 16 Grzegorzecka, Poland. s.chlopicki@cyfronet.krakow.pl
authors.forename: Stefan
authors.initials: S
authors.lastname: Chlopicki
authors.forename: Rafal
authors.initials: R
authors.lastname: Olszanecki
authors.forename: Ewa
authors.initials: E
authors.lastname: Marcinkiewicz
authors.forename: Magdalena
authors.initials: M
authors.lastname: Lomnicka
authors.forename: Roberto
authors.initials: R
authors.lastname: Motterlini
mesh_terms: D000704:Analysis of Variance; D001792:Blood Platelets / Q000378:metabolism*; D002248:Carbon Monoxide / Q000494:pharmacology*; D003094:Collagen / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009942:Organometallic Compounds / Q000494:pharmacology*; D010069:Oxadiazoles / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D015539:Platelet Activation; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011450:Prostaglandin Endoperoxides, Synthetic; D011810:Quinoxalines / Q000494:pharmacology; D013053:Spectrophotometry; D018709:Statistics, Nonparametric; D013917:Thrombin / Q000502:physiology; D013997:Time Factors
chemical_list: C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D007191:Indazoles; D020030:Nitric Oxide Donors; D009942:Organometallic Compounds; D010069:Oxadiazoles; D010975:Platelet Aggregation Inhibitors; D011450:Prostaglandin Endoperoxides, Synthetic; D011810:Quinoxalines; C110881:S-nitro-N-acetylpenicillamine; C476530:tricarbonylchloro(glycinato)ruthenium(II); C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D002248:Carbon Monoxide; D003094:Collagen; D013917:Thrombin; D006162:Guanylate Cyclase; D010396:Penicillamine