Hydrogen sulfide changes adhesive properties of fibrinogen and collagen in vitro

Hydrogen sulfide (H₂S) is a well-known toxic gas that is synthesized from two amino acids: cysteine (Cys) and homocysteine (Hcy). H₂S, like other organic gases–nitric oxide (NO^•) or carbon monoxide (CO) – is a signaling molecule in the cardiovascular system. Some studies have shown that H₂S is a therapeutic agent in cardiovascular diseases [1–3], but the mechanisms involved in the relationship between the action of H₂S and hemostasis process are still unclear. The main aim of this study was to establish the functional changes of two hemostatic proteins (collagen and fibrinogen) induced by H₂S, and also to examine the effects of these changes on the capability of fibrinogen and collagen to interact with human blood platelets (by measuring the platelet adhesion) in vitro.

Thrombin, collagen type I, bovine serum albumin (BSA), and bicinchoninic acid (BCA) solution were purchased from Sigma (St Louis, MO). Sodium hydrosulfide (NaHS), which has been well established as a reliable H₂S donor [4], [5], was from Sigma (St Louis, MO). Fibrinogen isolated from pooled citrated human plasma by the cold ethanol precipitation technique was followed by ammonium sulfate fractionation at 26% saturation at 4°C, according to Doolittle [6]. Its concentration was determined spectrophotometrically at 280 nm using an extinction coefficient 1.55 for 1 mg/ml solution. The concentration of fibrinogen in plasma was 2 ± 0.2 mg/ml. The concentration of purified fibrinogen in the reaction system was also about 2 mg/ml. The concentration of collagen type I was 40 µg/ml collagen (dissolved in 0.05% CH₃COOH). The reaction was initiated by placing a small drop of NaHS, on the side of the tube containing the fibrinogen solution or collagen solution (the incubation time −5, 15, and 30 minutes, 37°C). We have been using NaHS at the final concentrations of 0.00001–10 mM. The physiological concentration of H₂S in plasma and in tissues is about 50 µM; its physiological level in the brain is up to three-fold higher than in plasma.

Human blood was taken from healthy volunteers aged 23–32 (average: 24; SD = 5.5 years) not taking any medications or addictive substances (including tobacco, alcohol, and aspirin or any other anti-platelet drugs) and keeping a balanced diet (meat and vegetables), with similar socio-economic background, using no antioxidant supplementation. Human blood was collected into ACD solution (citric acid/citrate/dextrose; 5:1 v/v) and platelets were isolated by differential centrifugation of blood, as described by Wachowicz and Kustroń [7]. The final platelet concentration was 3 × 10⁸ platelets/ml. The platelets were counted by the photometric method, according to Walkowiak et al. [8]. Adhesion of blood platelets to native fibrinogen or native collagen and fibrinogen or collagen treated with NaHS was determined according to Tuszynski and Murphy [9]. The absorbance of control platelets (with native fibrinogen or native collagen) was expressed as 100%.

All the values in this study were expressed as means ± SD. The statistical analysis (to calculate the differences among the effect of different concentration of NaHS) was performed with an ANOVA test and POST Hoc test (Bonferoni). In order to eliminate uncertain data, the Q-Dixon test was performed.

As shown in Table I

Table I.  Platelet adhesion of resting and thrombin-stimulated platelets to microplate wells coated with native adhesive proteins (fibrinogen and collagen) and NaHS-treated fibrinogen or collagen (5 min, 15 min, and 30 min, 37°C).

Concentration of NaHS [mM]	Adhesion of resting platelets [%]	Adhesion of thrombin–activated platelets [%]
Incubation time–5 minutes
Native collagen [0 mM NaHS]	100	100
Collagen + 0.00001  mM NaHS	96.2 ± 7.4	95.7 ± 7.8
Collagen + 0.0001 mM NaHS	92.3 ± 7.6	92.1 ± 5.9
Collagen + 0.001 mM NaHS	87.3 ± 8.8	87.4 ± 6.9
Collagen + 0.01 mM NaHS	81.5 ± 7.6	83.3 ± 10.1
Collagen + 0.1 mM NaHS	79.3 ± 8.1	79.4 ± 7.7
Collagen + 1 mM NaHS	75.4 ± 7.0	70.3 ± 6.5
Collagen + 5 mM NaHS	71.4 ± 9.0	65.2 ± 5.9
Collagen + 10  mM NaHS	66.6 ± 4.9	60.4 ± 4.9
Native fibrinogen [0 mM NaHS]	100	100
Fibrinogen + 0.00001 mM NaHS	93 ± 5.4	87.5 ± 10.8
Fibrinogen + 0.0001 mM NaHS	86.9 ± 9.9	82.0 ± 8.6
Fibrinogen + 0.001 mM NaHS	83.7 ± 12.1	79.8 ± 13.7
Fibrinogen + 0.01 mM NaHS	80.7 ± 10.6	74.2 ± 12.7
Fibrinogen + 0.1 mM NaHS	76.3 ± 10.5	69.7 ± 13.6
Fibrinogen + 1 mM NaHS	74.9 ± 8.9	63.2 ± 12.2
Fibrinogen + 5 mM NaHS	70.4 ± 10.2	51.8 ± 8.2
Fibrinogen + 10 mM NaHS	61.5 ± 5.6	49.0 ± 8.9
Incubation time–15 minutes
Native collagen [0 mM NaHS]	100	100
Collagen + 0.01  mM	74.7 ± 8.2	75.9 ± 5.9
Collagen + 10 mM	53.2 ± 5.5	54.3 ± 4.1
Native fibrinogen [0 mM NaHS]	100	100
Fibrinogen + 0.01 mM	70.5 ± 4.9	67.9 ± 7.2
Fibronogen + 10 mM	49.4 ± 7.8	40.4 ± 6.6
Incubation time–30 minutes
Native collagen [0 mM NaHS]	100	100
Collagen + 0.01  mM	70.2 ± 5.9	69.9 ± 7.3
Collagen + 10 mM	44.3 ± 7.1	47.8 ± 4.5
Native fibrinogen [0 mM NaHS]	100	100
Fibrinogen + 0.01 mM	63.7 ± 4.9	60.2 ± 6.6
Fibrinogen + 10 mM	41.3 ± 6.6	38.5 ± 7.4

Notes: Results represent the means of eight values. The effect of nine different concentrations of NaHS (0.00001, 0.0001, 0.001, 0.01 0.1, 1, 5, and 10 mM) vs. control was statistically significant according ANOVA test and Post Hoc test–(for collagen–p < 0.01 (for resting platelets), p < 0.001 (for stimulated platelets); for fibrinogen–p < 0.02 (for resting platelets), p < 0.0001 (for stimulated platelets)).

, both resting as well as thrombin-activated blood platelets demonstrate a reduced ability to adhere to NaHS-treated collagen and NaHS-treated fibrinogen in vitro. The distinct inhibitory effect on the platelet adhesion to modified adhesive proteins (collagen and fibrinogen) was observed when collagen or fibrinogen was treated with NaHS, even at the lowest concentration of NaHS (0.00001 mM) (Table 1). The inhibitory properties of NaHS appears to be concentration-dependent for both adhesive proteins. Moreover, we observed the time-dependent (5, 15, and 30 minutes) inhibition of adhesive properties of proteins either by NaHS (0.01 and 10 mM) (for collagen–p < 0.01 (for resting platelets), p < 0.01 (for stimulated platelets); for fibrinogen–p < 0.02 (for resting platelets), p < 0.001 (for stimulated platelets)) (Table I).

Our initial results have also shown that, the adhesion to collagen and to fibrinogen of platelet preincubated with NaHS was reduced in vitro [10]. Moreover, H₂S caused the reduction of anion radicals generation in these cells [10]. Other latest experiments have demonstrated the inhibitory properties of H₂S on the platelet aggregation in vitro. The total inhibition of platelet aggregation (stimulated by various agonists: collagen, arachidonic acid and ADP) was observed at 10 mM NaHS [11], [12]. The authors of these articles have observed that the H₂S had the effect of inhibiting platelet aggregation without toxic effects (up to 10 mM). Results of Zagli et al. [12] demonstrated that the inhibitory effects of H₂S were not dependent upon the effects of NO^• synthesis, adenylyl or guanylyl cyclase activation, or K⁺ channels. Essex [13] assumed that the thiol-disulphide reactions may play some important role in platelets treated with H₂S. Hu et al. [14] and Morel et al. [10] suggest that phosphoinositides 3-kinase may be involved in the mechanism of H₂S action in these cells. The present experiments for the first time demonstrated that H₂S may change not only the platelet functions, including adhesive properties, but also may modify the adhesive activity of two tested proteins (collagen and fibrinogen). However, significance of these changes caused by H₂S needs more explanation. We assume that the interaction of modified adhesive proteins may cause impaired adhesion. Further studies that may characterize changes in fibrinogen and collagen simulated in H₂S and also their role in hemostasis process are still in progress in our laboratory.

Declaration of interest: This work was supported by grant 506/810 and 545/506 from the University of Lodz. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.