BIOCHIMICA ET BIOPHYSICA ACTA 121

BBA Report

BBA 31116

The non-plasmin, p ro teo ly t ic origin o f h u m a n f ibrinogen he te rogene i ty

DONALD MILLS and SIMON KARPATKIN Department o f Medicine, New York University Medical Center, 550 First A venue, New York, N. Y. 10016 (U.S.A .)

(Received September 9th, 1971)

SUMMARY

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was employed to compare various minor fibrinogen fractions of normal human plasma with fibrinogen products obtained following plasmin or thrombin digestion of purified bulk fibrinogen (Fraction 1-4). I t is concluded that plasmin is not responsible for normal human fibrinogen heterogeneity. It is suggested that a non-plasmin proteolytic agent, possibly thrombin, is responsible.

It has been generally thought that the enzyme plasmin is responsible for the small amounts of fibrinogen or fibrin breakdown products detected immunologically in normal sera 1- 4, as well as for the heterogeneity found in thrombin-clottable material of plasma ("fibrinogen") s- 7. Indeed, some similarities have been found between minor fibrinogen fractions from normal plasma and the products of limited plasmin proteolysis of bulk fibrinogen in vitro 7' 8. We have used the technique of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate 9 to investigate the normal heterogeneity of human fibrinogen and here present evidence that proteolysis by an enzyme(s) other than plasmin is responsible.

Single donor plasmas, or purified fibrinogen fractions from pooled plasma 8, were diluted to fibrinogen concentrations of 0.2-0.5 mg/ml in 9 M urea-0.01 M sodium phosphate buffer, pH 7,0, containing 1% sodium dodecyl sulfate (without reduction of disulfide bonds). After incubation at 37 ° for 1-2 h, 5-15/ag of fibrinogen (or equivalent) were electrophoresed on 5% gels (7 mA/tube for 16 h) a . Molecular weights could be estimated by assuming a linear relationship between log mol. wt. and mobility for protein bands intermediate between the major fibrinogen component (360 000 mol. wt.: sum of the constituent polypeptide chain molecular weights s) and IgG immunoglobulin (150 000 mol. wt.).

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Fibrin clots were obtained by addition of O. 1 ml of bovine thrombin (Parke- Davis, 75 units/ml, purified by the method of Rasmussen a° using buffers containing 0.1 M e-aminocaproic acid) to the following mixture: 0.2 ml of plasma or fibrinogen fraction, 0.05 ml of 10 -2 M p-hydroxymercuriphenylsulfonic acid (Sigma), and 0.15 ml of 0.5 M e-aminocaproic acid, pH 5.5, with HC1, which was then incubated for 1 h at 25 °.

Polyacrylamide gel electrophoresis in sodium dodecyl sulfate without reduction of disulfide bonds

Fig. 1A shows the gel electrophoresis pattern, in the presence of sodium dodecyl sulfate, of a typical normal plasma. Six components are present with molecular weights between 360 000 (the major component, designated "1") and about 300 000. A total of six similar or identical components were also present in purified fibrinogen fractions and their corresponding fibrins (Figs. 1B-1E). Various purified fibrinogen fractions differed in their respective component clot patterns. The clot of Fraction I-4 contained Components 1,2 and 3 (Fig. 1B), while fractions of increasing ethanol solubility were enriched in lower tool. wt. components: primarily Component 3 (Fig. 1C), Components 3, 4, 5 and 6 (Fig. 1D) and primarily Component 6 in the fraction of highest solubility (Fig. 1E). The decrease in component molecular weights as solubility of the fractions increased appears to explain the variety of fractions which were more soluble than I-4 (ref. 5). Small variations in the relative amounts and number of minor components were found with normal plasma. In contrast, plasma from a patient with hepatoma had an altered distribution, Component 3 predominating. The corresponding clot pattern is shown in Fig. IF.

Z

P,. B C D E F

Fig. 1. Polyacrylamide gel electrophoresis (5% gels) in the presence of sodium dodecyl sulfate (non- reduced samples). A. Normal plasma. B. Clot of the bulk fraction of purified fibrinogen (I-4). C-E. Clots of fractions of increasing ethanol solubility comprising about 6%, 0.5% and 1% of the total fibri- nogen, respectively. F. Plasma clot from a patient with hepatoma.

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Digestion of purified Fraction 1-4 with plasmin. To test one possible source of the observed molecular weight heterogeneity, Fraction I-4 was digested with purified human plasmin at 25 ° in 0.05 M citrate buffer, pH 7.4, clotted, and run on sodium dodecyl sulfate polyacrylamide gel electrophoresis. A single major product (Product I) was formed initially followed by a second major product of lower mol. wt., Product II. Both were clottable as shown in Fig. 2A. in mixing experiments with plasma and fibri- nogen fractions, Product I was found to migrate just below Component 3 while Product II migrated below Component 6. Thus, the initial plasmin digest products (I and II) are not identical to lower mol. wt. fibrinogen components. The derivatives of fibrinogen (Fraction I-4) produced by plasmin over the entire course of digestion have been characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (ref. 12 and papers in preparation). Clottable derivatives other than Products I and II have not been observed (except in trace amounts). Neither Product I nor Product II has been found in appreciable amounts in normal plasma.

Digestion of purified Fraction 1-4 with thrornbin. A second possible source of the observed molecular heterogeneity was tested by digestion of Fraction I-4 with purified human thrombin (100 units/ml). After 4 h at 25 ° the gel pattern of clot proteins (not shown) was similar to that found with the hepatoma patient (Fig. IF). After 24 h (Fig. 2B), products of similar or identical mobility to each of the fibrin Components 4, 5 and 6 were present (clotted protein comprised 80% of the digest). Additional products migrating near Component 6 were also present. Only trace amounts of bands with the mobilities of plasmin Products I and II were detectable during thrombin digestion,

!2: • ~ • "

A B

C ~ - - - ~ - - -

7 - - -

Fig. 2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A and B. Non- reduced samples (5% gels). A. Clot of early plasmin digest of Fraction 1-4. B. Clot of 24 h thrombin digest of Fraction I-4. C-F. Mercaptoethanol-reduced samples (9% gels). C. Clot of Fraction 1-4. D. Clot of high solubility fraction corresponding to Fig. 1D.'E. Plasmin digest clot corresponding to Fig. 2A. F. Thrombin digest clot corresponding to Fig. 2B. The arrow indicates the trace of 35 000 tool. wt. polypeptide present in Fraction 1-4 fibrin.

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indicating that a contaminating plasmin was not the major lytic agent. This is in accord with reports that purified bovine thrombin lysed clots even in the presence of plasmin inhibitors 1 s, 14, which we have also confirmed.

Polyacrylamide gel electrophoresis in sodium dodecyl sulfate with reduction o f disulfide bonds

Disulfide bond reduction allowed further comparison of fibrinogen components with plasmin and thrombin digest products. Samples of clotted protein in urea-sodium dodecyl sulfate solution were reduced by addition of 2-mercaptoethanol (to 1% final concentration) and incubation at 37 ° for 1 h. In each case 50-70 pg of the clot proteins were submitted to electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate (3 mA/tube for 16 h). Fig. 2C shows the pattern of Fraction I-4 fibrin with constituent a-,/3- and 3'-chain bands a' 11. A faint band of about 35 000 mol. wt. is indicated by the arrow. The amount of this band was increased in fibrinogen fractions of increased solubility while the amount of a-chains was decreased. For example, the reduced cl0t pattern of the (intermediate) high solubility fraction corresponding to Fig. 1D is shown in Fig. 2D. By comparison (gel patterns not shown), the clot corresponding to Fig. 1C had a greater content of a-chains and less of the 35 000 mol. wt. band while that corresponding to the highest solubility fraction (Fig. IE) showed the greatest content of the 35 000 mol. wt. peptide with a-chains virtually absent. Trace amounts of additional peptides between the 35 000 mol. wt. peptide and 7-chain band were present in high solubility fractions (e.g. Fig. 2D). The clottable peptides were evidently products of a-chain proteolysis in vivo.

Plasmin and thrombin digestion of Fraction I-4. In the course of plasm~n digest- ion of Fraction 1-4 a peptide of about 26 000 mol. wt. was present in clots in increasing amounts as a-chains decreased. The digest chain pattern corresponding to Fig. 2A is shown in Fig. 2E. The (trace of) 35 000 mol. wt. peptide in Fraction I-4 fibrin did not increase during plasmin digestion. However, in contrast to plasmin digestion, during prolonged digestion with thrombin solutions a similar or identical peptide did increase as a-chains decreased. The 4-h thrombin digest clot pattern (not shown) was virtually indistinguishable from that of the high solubility fibrinogen fraction (Fig. 2D). After 24 h (Fig. 2F) additional bands were observed (molecular weights about 53 000, 46 000 and 31 000). Thus, the initial products of thrombin digestion of purified Fraction I-4 closely resembled normal plasma fibrinogen components while those of plasmin digestion did not.

The similarities between plasma fibrinogen components and thrombin digest products indicate that a non-plasmin fibrinolytic activity similar or identical to that present in the purified human thrombin may cause continuous breakdown of fibrinogen or fibrin in vivo and may be of importance for normal fibrinogen turnover. Furthermore, these results suggest that caution be exercised in attributing non-clottable (serum) break- down products of fibrinogen or fibrin to plasmin proteolysis.

We thank Dr. Henriette Lackner for plasma from a patient with hepatoma after partial hepatectomy, Dr. Alan J. Johnson for purified plasmin and Dr. Kent D. Miller for purified thrombin. This work was supported by a grant from the New York Heart Association.

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REFERENCES

1 A.J. Johnson and C. Merskey, Thromb. Diath. Haemorrhag. Suppl., 39 (1970) 229. 2 D.P. Thomas, S. Niewiarowski, A.R. Myers, K.J. Block and R.W. Colman, No Engl. J. Med., 283

(1970) 663. 3 W.R. Pitney, Semin. Hematol., 8 (1971) 65. 4 P.C. Das, A.G.E. Allan, D.G. Woodfield and J.D. Cash, Br. Med. J., 4 (1967) 718. 5 M.W. Mosesson and S. Sherry,Biochemistry, 5 (1966) 2829. 6 M.W. Mosesson, N. Alkjaersig, B. Sweet and S. Sherry, Biochemistry, 6 (1967) 3279~ 7 L.A. Sherman, M.W. Mosesson and S. Sherry, Biochemistry, 8 (1969) 1515. 8 D. Mills and S. Karpatkin,Biochem. Biophys. Res. Commun., 40 (1970) 206. 9 A.L. Shapiro, E. Vifiuella and J.V. Maizel, Jr.,Biochem. Biophys. Res. Commun., 28 (1967) 815.

10 P.S. Rasmussen, Biochim. Biophys. Acta, 16 (1955) 157. 11 P.A. McKee, P. Mattock and R.L. Hill, Proc. Natl. Acad. Sci. U.S., 66 (1970) 738. 12 D. Mills and S. Karpatkin, Fed. Proc., 30 (1971) 1076, Abstr. 141. 13 M.M. Guest and A.G. Ware, Science, 112 (1950) 21. 14 D.C. Triantaphyllopoulos, C.R. Muirhead and E. Triantaphyllopoulos, Thromb. Diath. Haemorragh.

Suppl., 36 (1969) 269. Biochim. Biophys. A cta, 251 (1971) 121-125