Effect of hydrogen sulphide (H2S) on human platelet adhesion and clot retraction

BERMEJO, EMILSE; ALBERTO, FABIANA; SAENZ, DANIEL; ROSENSTEIN, RUTH; LAZZARI, MARÍA

Amsterdam, Netherland

Congreso; XXIV Congress of the International Society on Thrombosis and Haemostasis (ISTH); 2013

International Society on Thrombosis and Haemostasis

Background: Hydrogen sulphide (H2S) is increasingly recognized as a member of a growing family of â??gasotransmittersâ??, together with its two counterparts, NO and CO. Actually, H2S is emerging as an important endogenous modulator which exhibits beneficial effects on the cardiovascular system, without producing toxic metabolites. Objective: The present work was designed in vitro to study the effect of H2S on human blood platelets adhesion and clot retraction. Methods: Tests were performed using platelet rich plasma (PRP) or washed platelets (WP) (300 Ã? 109 plat/L). Sodium hydrogen sulfide (NaHS) was used as H2S donor. A platelet suspension was treated with 10mM NaHS or buffer. Adhesion: Microplate wells were coated with 100 μL of fibrinogen solution (20 μg/mL) and blocked with 200 μL of BSA solution. WP (100 μL) preincubated with buffer or 10mM NaHS were added to wells coated with fibrinogen and incubated at room temperature for 1 h. The wells were rapidly filled with 100 μL of0.1 M citrate buffer, pH 5.4, containing5 mM p-nitrophenyl phosphate and 1% Triton X-100 (v/v). After 60 min incubation at room temperature, the reaction was stopped by the addition of 70 μL of 2M NaOH. The p-nitrophenol produced was measured with a microplate reader at 405 nm against a platelet-free blank. The amount of platelets adherent to protein-coated wells was estimated by measurement of the platelet acid phosphatase activity using a calibration curve. Clot retraction: PRP pre incubated with buffer or10 mM NaHS. Plasma was induced to coagulate with 0.4 UI/mL thrombin. The clots were allowed to retract at37°C and were photographed at various times (15, 25, 35 and 45 minutes). Two-dimensional sizes of retracted clots on photographs were quantified using NIH ImageJ software, and retraction was expressed as percentage retraction [1 â?? (final clot size/initial clot size)]. Statistical significance was determined using Mann-Whitney test, control vs. H2S (n=5). Results: H2S significantly inhibited platelet adhesion to fibrinogen coated plates (P< 0.01). In clot retraction, no difference was observed at the end point (45 min). However PRP incubated with NaHS had a delay with statistical significance at 25 and 35 min (p=0.001 and p=0.04 respectively), changing from hyperbola to sigmoid the kinetic curve of clot retraction. Conclusion: It has been demonstrated that ligand binding to integrin aIIbb3 induces outside-in signaling-mediated Rap1b. It is crucial for the activation of a second pool of Rac1, which is important for clot retraction and platelet spreading. Our results suggest that a pathway, such as CalDAG-GEFI/Rap1-Rac1, could be affected by H2S. In conclusion, several evidences are accumulating to demonstrate that H2S donor compounds exert significant effects on antiadhesive activity in prevention of thrombotic states.