effect of methylmercuric chloride (mmc) on fibrin polymerization

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Effect of Methylmercuric Chloride (MMC) on Fibrin Polymerization

MARTA MICHALSKA AND RYSZARD WIERZBICKI*

34edical University, Institute of Environmental Research and Bioanalysis, Department of Biochemistry, Aluszyffskiego 1,

90-151 L6di, Poland

ABSTRACT

Methylmercuric chloride (MMC) in concentrations 0.1-10}xM re- duces the amount of fibrinopeptides released from thrombin- activated human fibrinogen. However, the fibrin clot formation is not discriminated and the turbidity of the fibrin gel is even augmented. MMC does not cause such changes in the process of repolymerization of fibrin monomers. The addition of fibrinopeptides to the fibrin monomers results in a similar increase of turbidity of the repolymerizing sample in the presence of MMC as in the case of fibrinogen clotting. These experiments indicate that MMC modifies the structure of fibrin in the presence of fibrinopeptides.

Index Entries: Methylmercuric chloride, fibrin, fibrinopeptides, fibrin polymerization.

INTRODUCTION

Mercuric compounds cause disorders in hemostasis that lead to hypercoagulability. In animals exposed to such compounds , the follow- ing phenomena can be observed: an increase of fibrinogen content in the plasma (1,2), thromboelastograms typical for hypercoagulation (3,4), and an increase in the activity of fibrin-stabilizing factor (5). The mercurials, however , do not cause increased thrombin generation in the plasma (6). The mechanism of the abovemenfioned disorders still remains unclear.

*Author to whom all correspondence and reprint requests should be addressed.

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158 Michalska and Wierzbicki

Mercuric compounds can affect plasma coagulation factors and can change biological functions of the blood platelets (7-9). Thrombin conversion of fibrinogen and the fibrin clot formation constitute the coagulation stages, which can be modified by mercuric compounds. Mercuric chloride modifies the structure of fibrin in the presence of fibrinopeptides and may cause "binding" of the fibrinopeptides to fibrin (10).

In this study we examined the influence of methylmercuric chloride on the thrombin-induced release of fibrinopeptides and the process of fibrin monomers polymerization.

MATERIALS AND METHODS

Bovine thrombin from the Serum and Vaccine Factory (Lublin, Pol- and) was purified according to Brosstad (11). Human fibrinogen was obtained from Immuno AG (Vienna, Austria). Human fibrin monomers were prepared by the method of Gonias et al. (12). The concentration of fibrinogen as well as fibrin monomers themselves were calculated from the absorption coefficient, respectively A I'y' - 15.0 and A I~y" 28o - 280 = 15.5 (12). Fibrinopeptides (FPA + FPB) from human fibrinogen were isolated chromatographically as described by Yuan et al. (13). Fibrinopeptides were obtained as a result of thrombin-induced fibrinogen activation (0.75 NIH U/rag fibrinogen), whereas fibrinopeptide A (FPA) was obtained from fibrinogen activated by Atroxin (Sigma; 0.63 IJ, g/mg) (14). The sample containing 80 mg fibrinogen was incubated for 1.5 h at 35~ After discarding the clot, the pH of the hydrolizate was adjusted to 3.5 and the hydrolizate was applied onto Dowex W8 (200-400 mesh, Serva). Fi- brinopeptides identification was performed in polyacrylamide gel (15) with gel staining according to Steck et al. (16).

Quantitative analysis of the fibrinopeptides was carried out according to Laugen and Bithell (17). The process of fibrin polymerization was spectrophotometrically controlled by measuring the turbidity of the gel- ating sample. In the case of thrombin-induced activation of fibrinogen 0.9 mL PBS (0.01M phosphate buffer, 0.14M NaCL, pH 7.0 at 37~ 0.1 mL methylmercuric chloride, 1 mL fibrinogen (0.6 mg/mL), and 1 mL thrombin (1 NIH U/mL) were mixed and the turbidity (optical density) was measured immediately at 600 nm with a Spekol 11 spectrometer (Carl Zeiss) at 37~ (d = 3 cm).

Fibrin monomers dissolved in NaBr (1.0M) and acetate buffer (0.05M), pH 5.3, were repolymerized by 20-fold dilution in PBS: 2.65 mL PBS, 0.1 mL methylmercuric chloride, and 0.1 mL fibrinopeptides, and 0.15 mL concentrated fibrin monomers were mixed and the turbidity estimated as described above. Methylmercuric chloride was a Merck product, other chemicals were analytical grade.

Statistical analysis of data in Table 1 was performed by means of Student's t-test. Differences between the curves in Figs. 1-4 were evalu- ated with use of Cochran-Cox's test.

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Table 1 Amount of Fibrinopeptides (FPA + FPB) Released

from Thrombin-Activated Human Fibrinogen in the Presence of Methylmercuric Chloride

HMC ( p , M ) Fibrinopeptides n

0 (control) 3.03 _+ 0.04 11 0.1 3.07 + 0.10 10 1.0 2.54 + 0.04* 10 10.0 2.42 _+ 0.02* 10

Mean values _+ SD are expressed as p.moles of fibrinopeptides per lamol of fibrinogen.

*Differences statistically significant at p < 0.01 related to the control value.

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Fig. 1. Effect of MMC on fibrin polymerization in 0.01M phosphate buffer, 0.14M NaC1, pH 7.0, at 37~ Fibrinogen (final concentration 0.2 mg/mL) was activated by thrombin (0.33 NIH U/mL) in the presence of methylmercuric chloride. 1. Control (without MMC); 2. 0.1; 3. 1.0; 4. 101xM MMC; 5. Fibrinogen preincubated with 10 I~M MMC; 6. Without thrombin (fibrinogen + 10tzM MMC). Curves 4 and 5 are statistically different from curve 1; n = 6-10. The calculations have been performed on the significance level 0.05.


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160 Nichalska and Wierzbicki

I I 10 20

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Fig. 2. Repolymerization of fibrin monomers in the presence of MMC at 37~ Concentrated fibrin monomers were 20-fold diluted with 0.01M phosphate buffer, 0.14M NaC1, pH 7.0 (final concentration 0.43 mg/mL). 1. Control (without MMC); 2. 0.1; 3. 1.0; 4. 10~M MMC; n = 6.

RES(ILTS

The measurement of the amount of fibrinopeptides released from human fibrinogen under the influence of thrombin in the presence of methylmercuric chloride (Table 1) was carried out. The amount of fibrinopeptides released from control fibrinogen (without mercurials in the reaction medium) is 3.03 + 0.04 ~mol/~mol fibrinogen. Mercurial in the concentration 0.1-10~M causes a pronounced and statistically significant decrease in the amount of released fibrinopeptides (2.54 + 0.4 and 2.42 +- 0.02 ~mol/p, mol fibrinogen, respectively) for higher concentrations (1.0-10.0 ~M).

The investigation of fibrin polymerization and fibrin monomers repolymerization in the presence of MMC was also performed. The great- est turbidity increase in fibrinogen clotting in the presence of thrombin was observed for the highest concentration of methylmercuric chloride (IO~M). The increase in absorbance was lower for the concentration of 1.0~M, and no differences that could be seen in this method as compared with the control were observed for the concentration 0.1M (Fig. 1). No turbidity differences were observed in the case of fibrin monomer re-

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Effect of/W~IC on Fibrin 161

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1

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Fig. 3. Repolymerization of fibrin monomers (0.43 mg/mL) in the presence of fibrinopeptides (A + B, 13 I~g/mL) and MMC at 37~ 1. Control (without fibrinopeptides and MMC); 2. Control (without MMC); 3. 0.1; 4. 1.0; 5. 101~M MMC. Curves 3-5 are statistically different from curves 1 and 2; n = 6. The calculations have been performed on significance level 0.05.

polymerization under the influence of the investigated compound (Fig. 2).

At the subsequent stage of the study attempts were undertaken to show whether changes in fibrin monomers repolymerization in the presence of fibrinopeptides and MMC could be observed. Monomers at concentration 0.43 mg/mL and fibrinopeptides at 13 ~g/mL or fibrinopeptide A at 3.7 I~g/mL were used for the assay. A marked increase of turbidity in fibrin monomers repolymerization was observed for the mercurial at concentrations 0.1-10.01zM both for the mixture of FPA + FPB (Fig. 3) and for FPA (Fig. 4).

DISCUSSION

Despite intensive investigation of the toxic effects on organisms of methylmercuric compounds, their influence on the hemostasis is still


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162 ~ichalska and Wierzbicki

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Fig. 4. Repolymerization of fibrin monomers (0.43 mg/mL) in the presence of fibrinopeptide A (3.7 p,g/mL) and MMC, at 37~ 1. Control (without fibrinopeptide and MMC); 2. Control (without MMC); 3. 0.1; 4. 1.0; 5, I0~M MMC. Curves ,3-5 are statistically different from curves 1 and 2; n = 6. The calculations have been performed on the significance level 0.05.

unknown. Methylmercuric chloride generates disorders in the cardio- vascular system (18) and higher mortality of spontaneously hypertensive rats (19). This compound influences biological functions of blood platelets: It induces platelet shape changes, release reaction, and aggregation (7,9,20). There are some data that prove that this cumulative environmental poison, similar to other mercuric compounds (3,6,21), causes changes in the blood clotting system (4). Methylmercuric chloride can activate blood clotting in relatively small concentration in the plasma, below 1raM (4). Such concentration approximates the content of mercury observed in groups of people exposed to it in occupational and environmental conditions (22). It suggests, therefore, that methylmercury similar to other mercurials can be regarded as a risk factor in development of hemostatic disorders.

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Effect of/r on Fibrin 163

Fibrin clot formation is one of the coagulation stages that can be modified by mercurials. They increase turbidity of the fibrin polymerizing sample, which is a symptom of changes in the structure of fibrin gel (10). During fibrin formation in the presence of mercuric chloride the amount of fibrinopeptides released by thrombin into medium decreases, which may point to their "binding" into fibrin (10). A similar effect has been obtained for methylmercuric chloride. At concentration 0.1-10.0 txM this compound causes lower as compared with mercuric chloride but also statistically significant differences both in the amount of released fibrinopeptides and the turbidity of fibrin polymerizing sample. There are no changes in fibrin monomers repolymerization. However, the increase in the turbidity of the repolymerizing monomers was stated when fibrinopeptides were added to the repolymerizing mixture in amounts approximating their content in the fibrinogen molecule (molar ratio of FPA + FPB to monomers = 6.2 and FPA -- 3.8). It is possible, therefore, that procoagulant activity of methylmercuric ions appears only in the presence of the fibrinopeptides, similar to the case of mercuric chloride (10). Simultaneously a smaller amount of the fibrinopeptides released during thrombin-activation of fibrinogen in the presence of MMC suggests retention of the fibrinopeptides in the fibrin clot. How- ever, existing models of fibrin polymerization do not take into account the participation of the fibrinopeptides in the fibrin structure (23-25).

Results presented in this paper do not explain the mechanism by which methylmercuric chloride modifies fibrin polymerization. The increased turbidity during fibrin formation may exemplify the alteration of the fibrin fibers' thickness (26,27). Divalent cations exert a considerable influence on the clotting time and the turbidity of fibrin gels (26,28,29). In the presence of calcium and zinc, soluble fibrin oligomers undergo a phase change (27). Zinc, which is more effective than calcium in increas- ing the clot turbidity, also decreases the rate of fibrinopeptide A release (29). According to recent studies (26-29) Zn 2 + and Ca z~ ions accelerate the polymerization step in fibrin assembly. The linear assembly of oligomers is cation-insensitive, whereas the lateral aggregation can be augmented by these ions. Thus, it is probable that procoagulant properties of CH3Hg' ions result from the acceleration of the lateral aggregation caused by these ions. Keeping in mind the occupational and environmental pollution of mercurials, as well as the frequency of circulation diseases, methylmercury-dependent disorders in fibrinogen conversion to fibrin seem to be worth emphasis.

ACKNOWLEDGMENTS

This study was supported by Grant CPBR 11.11.79 "Occupational Medicine." The authors thank Anna Szadowska and Radost'aw Piestrzyfiski for their assistance in preparing the manuscript.

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164 ~ichalska and Wierzbicki

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effect of methylmercuric chloride (mmc) on fibrin polymerization

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