EFFECTS OF HEMENTERIN, A FIBRINO(GENO)LYTIC METALLOPROTEASE FROM LEECH, ON HUMAN PLATELET AGGREGATON. INVOLVEMENT OF NITRIC OXIDE PATHWAY

Chudzinski-Tavassi, A.M.(1), Lazzari, M.A.(2), Rosenstein, R.(3) Faria, F.(1), Keller Sarmiento, M.I.(3), Alberto, F.(2), Bermejo, E.(2)

(1)Laboratório de Bioquímica e Biofísica, Instituto Butantan, SP. Brasil, (2)IIHEMA, Academia  Nacional de Medicina de Buenos Aires, (3)Facultad de Medicina, Dpto. Biología Humana, Universidad de Buenos Aires, Argentina.

Hementerin (HT) is a fibrino(geno)lytic metalloprotease of 80 kDa, purified from salivary complexes of Haementeria depressa leech. In this report was examined the effect of Hementerinon several human platelets´s parameters. Platelet rich plasma (PRP) was preincubated with different concentrations of HT (0 - 10mg/ml), then 1 mg/ml collagen (final concentration) was added and aggregation recorded. HT inhibited platelet aggregation being IC50 of 7.5mg/ml. To perform a time-course PRP was incubated with buffer or HT (7.5 mg/ml) at 37ºC during 0, 2, or 15 min before activation with collagen. The inhibitory effect of HT was already evident at 2 min of preincubation, meanwhile no changes on plasmatic fibrinogen concentration were observed. Hementerin also inhibited secondary platelet aggregation induced by agonists as: 5mM ADP, 10mM adrenaline, 0.5U/ml thrombin, 0.5 mM arachidonic acid and ATP release was completed blocked. Hementerin did not modify membrane glicoproteins as GPIIb-IIIa, Ib, Ia, IIa, IV measured by flow cytometry. This inhibitory pattern prompted us to hypothesize an involvement of the nitridergic pathway. The inhibitory effect of HT on platelet aggregation induced by thrombin was blocked by L-NAME and L-NNA. An increase of Ca²⁺ flux in platelets preincubated with hementerin and stimulated with thrombin was observed, besides a sensitive increase in cGMP concentration, and a conversion of citrulina in L-arginina was evident indicating NO formation in platelets. Taken together these results we postulate the following sequence in the signaling pathway triggered by HT: 1) an increase in intracellular Ca²⁺ flux, 2) an increase in platelet iNOS activity, 3) cGMP levels augmentation, and 4) inhibition of platelet aggregation through a cGMP-dependent mechanism. Therefore, HT, an inhibitor of platelet aggregation, acting presumably through the increase in platelet iNOS activity, could become a useful tool to understand this mechanism and its possible implications on different therapeutics to inhibit platelet aggregation.

CORRESPONDENCE TO:

Fernanda Faria, Rua Ofélia 324 apto 102-A, São Paulo, SP, CEP: 05423-110, Brasil, Email: fe_faria@terra.com.br