Direct measurement of a second fibrinogen alpha chain in lamprey blood plasma

THROMBOSIS RESEARCH 68; 489-493, 1992 00493848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

BRIEF

COMMUNICATION

DIRECT MEASUREMENT OF A SECOND FIBRINOGEN CHAIN IN LAMPREY BLOOD PLASMA

ALPHA

Russell F. Doolittle, Marcia Riley and Yang Pans Center for Molecular Genetics University of California, San Diego La Jolla, California 92093-0634 U.S.A.

(Received 10.9.1992; accepted in revised form 15.10.1992 by Editor M.W. Mosesson)

Recently we reported the cDNA sequence of a second lamprey fibrinogen a chain; it was unique in having a carboxyl-terminal region that is homologous to those of 8 and ychains (1). As such, the putative chain had characteristics of an archetypal fibrinogen. Its low message abundance in lamprey liver indicated that the encoded protein must occur at significantly lower concentrations than ordinary a chains, however. We now report direct measurements of this novel a chain in lamprey plasma, where it accounts for about 10% of the fibrinogen a chains. We were able to quantitate both the well characterized major type of a chain and the newly discovered minor form by taking advantage of two fortuitous features inherent in the system. First, it has long been known that lamprey thrombin releases both fibrinopeptides A and B from lamprey fibrinogen, whereas mammalian thrombins release only the lamprey fibrinopeptide B (2,3). Second, the cDNA sequence of the second lamprey a chain (1) indicated that its fibrinopeptide A was two amino acids longer than that of the major kind of a chain (3,4), suggesting that the two might be distinguishable when isolated from clot liquors. The expected sequences were DDSSIDIR (fibtinopeptide A2) and DDISLR (fibrinopeptide A). EXPERIMENTAL We began by examining the peptides released from lamprey fibrinogen that had been prepared by a single cold-ethanol precipitation step. Aliquots were clotted with either lamprey or bovine thrombin, a strategy that allowed us to pinpoint quickly the fibrinopeptide A peaks. Clot liquors were acidified to pH 2 and aliquots applied to a Vydac Cl8 reversed-phase HPLC column equilibrated with 0.1% TFA. Elutions were conducted with acetonitrile gradients. The appropriate peaks were taken to dryness in a Speed-Vat and subjected to total acid hydrolysis for 24 hrs. Amino acid analyses were conducted on suitable aliquots on a Beckman Model 6300 amino acid analyzer capable of reproducible analyses at subnanomolar levels. These initial experiments showed that bulk fibrinogen preparations contain both of the expected fibrinopeptides A, the minor (A2) occurring as a small peak emerging just in advance of the major (A) on the Cl8 reversed-phase HPLC column (Fig. 1). Key words: lamprey fibrinogen a chains, archetypal protein $ Present address: Howard Hughes Medical Institute (U426), Univ. Calif., San Francisco, San Francisco, CA 94143 USA 489

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These experiments also revealed that the fibrinopeptide A2 is actually 10 residues in length (Table I), not eight as had been supposed. The discrepancy has to do with the cleavage site for removing the signal peptide. We had presumed, reasonably, that the fibrinopeptide A2 began at the same position as the fibrinopeptide A. In actuality, it begins two residues sooner. Both the presumed and the actual sites conform to reported consensus sequences for signal cleavage sites (5). As a result, the sequence of the fibrinopeptide A2 turns out to be SGDDSSIDIR. Given the possibility that not all the fibrinogen containing the novel a chains precipitated under the same conditions as the major form of fibrinogen, we set about devising a procedure that would allow us to measure the A2 released from plasma itself. To this end we used a scaled down version of an ion exchange procedure originally described by Blombtik and Vestermark (6). Thus, peptides were first concentrated by passing clot liquors over small (25 ml bed volume) Dowex 50X2 columns that had been equilibrated with 0.1 M ammonium formate, pH 3.0. Adsorbed peptide material was eluted with 0.2 M ammonium acetate, pH 6.9. Eluates were freeze-dried repeatedly to remove volatile salts, after which aliquots were applied to the HELC column. In the case of the bulk-purified fibrinogen, the yields of fibrinopeptide A2 were essentially the same by this procedure as when aliquots were taken directly from the clot liquor. The yields of fibrinopeptide A were greatly reduced when clots were generated directly from plasma, however. Suspecting that the fibrinopeptide A might be more amenable to degradation by plasma carboxypeptidases, we compared yields for plasma that had been dialyzed against 0.2M NaCl with and without 10-3 EDTA, inasmuch as carboxypeptidases are invariably metal-dependent. As a subsidiary experiment, then, a comparison of fibrinopeptide recoveries was made after clotting (a) bulk fibrinogen, (b) plasma, and (c) plasma in the presence of 2 x 10m3M EDTA.

...

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FIG. 1 HPLC tracing of clot liquor from bulk fibrinogen preparation showing peaks corresponding to lamprey fibrinopeptides A2, A and B, the amino acid compositions of which are listed in Table I.

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TABLE I. Amino Acid Compositions of Fibrinopeptides A, A2 and B Isolated Directly from Clot Liquors by HPLC * Fp A2

FP A

Asparaginelaspartate Threonine Serine Glutarnine/glutamate Proline Glycine Alanine Cysteine Wine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total

2.15 [2.0] (0.03) 1.07 [l.O] (0.05)

0.50 [3.0] (0.03) 0.49 [2.9] (0.05) (0.22) [ 1.31

(0.12) (0.04)

(0.04) 1.06 [l.O] 1.09 [l.O]

0.29 [1.7] (0.04)

IAIDPIEY

Fibrin

FP B 4.83 1.21 0.85 1.34 0.81 0.87 2.00

[11.8] [2.9] 12.11 [3.2j 12.01 [2.1] [4.8]

0.90 [2.2] 1.22 [2.9] 0.50 [1.2] (0.01)

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(0.01) (0.02) 1.10 [l]

0.17 [0.9]

0:39 [0.9]

6.47 [6]

1.45 [lo]

14.92 [36]

*Values are given in nanomoles observed without correction; the numbers in brackets represent the calculated molar content. The aliquot applied in the case of fibrinopeptide A2 was 2x the amount that applied for the fibrinopeptide A, which in turn was 3x the amount of fibrinopeptide B applied. The fibrinopeptides A and A2 compositions were verified by repeated analyses of samples that were isolated by the Dowex 50 procedure before HPLC (Fig. 1).

To this end, a 33 ml-sample of titrated plasma from a single lamprey was divided into three portions, two of which were dialyzed against 0.2M NaCl and the third against 0.2M NaCl containing 2 x 10 -3 M. EDTA. One of the former preparations was bulk-precipitated by the addition of 2.8 mls of cold 50% ethanol (final COW. = 10%). The precipitate was removed by centrifugation and redissolved in a starting volume of 0.2M NaCl. Each of the three preparations was then clotted with lamprey thrombin (final cont. = 0.2 mg/ml). After 15 min., the clot liquors were acidified with formic acid and passed over Dowex 50 columns as described above. The HPLC profiles of equivalent aliquots from the three preparations are shown in three panels of Fig. 2. The recovery of the fibrinopeptide A2 was about the same in all three cases amounting to about 0.8 nmoles per ml of starting plasma. In contrast, the yields of fibrinopeptide A were greatly reduced in the preparations made from plasma. In line with our supposition, the loss was significantly less when EDTA was present, however (Table II). DISCUSSION The discovery (1) of a fibrinogen a chain with a carboxyl-terminal segment similar to those found in l3and y chains was the culmination of a long search for a prototypic molecule composed of equivalent and wholly homologous chains. Nonetheless, a number of questions about its occurrence and role were raised by that finding, including its function and whereabouts (1). Now

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FIG. 2 HPLC tracings of material purified by ion exchange on Dowex 50. In all runs a Vydac Cl8 reversed phase column was equilibrated with 0.1% TFA and eluted with a linear acetonitrile gradient. The top panel is from purified fibrinogen; the middle from plasma, and the bottom from EDTA-treated plasma. we know that the chain occurs in the plasma of adult creatures and is precipitated along with ordinary fibrinogen. In our earlier report (l), we presented a phylogenetic tree constructed with sequences from various fibrinogens and fibrinogen-related proteins showing that the lamprey minor a chain carboxyl-terminal region clusters with a putative sequence found as a second open reading frame in a bipartite message for the chicken fibrinogen a chain (7). The fact that corresponding forms TABLE II. Relative Recoveries of Fibrinopeptides A and A2 After Clotting Lamprey Fibrinogen and Plasma*

Fibrinopeptide A2 Fibrinopeptide A Fibrinopeptide A

fibrinogen

plasma

132:;

4.1 3.6 1.2

EDTA-plasma :*: 2:5

*Fibrinopeptide A’lacks arginine. Recoveries were determined by photocopying the HPLC traces shown in Fig. 1 and weighing the excised peaks. In each case the fibrinopeptide A2 peak amounts to about 1.5 nanomoles as shown by amino acid analysis.

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occur in creatures as diverse as lampreys and chickens suggested that equivalent a chains would be found throughout the vertebrates. In this regard, we learned recently that Fu et al (8) have identified a similar fibrinogen a chain in humans. In their case the product is the result of an alternative splice, rather than a separate gene. Acknowledgements We thank Gerd Grieninger for informing us of his results in advance of their publication. Our work was supported by N.I.H. grant HL26873. REFERENCES 1. PAN, Y. and DOOLI’ITLE, R.F. cDNA sequence of a second fibrinogen a chain in lamprey: an archetypal version alignable with full-length p and y chains. Proc Nat1 Acad Sci, USA 89,2066-2070, 1992. 2. DGOLI’ITLE, R. F. Differences in the clotting of lamprey fibrinogen by lamprey and bovine thrombins. B&hem. 94,735741, 1965. 3. COTTRELL, B. A. and DOOLITTLE, R. F. Amino acid sequences of lamprey fibrinopeptides A and B and characterization of the junctions split by lamprey and mammalian thrombins. B&hem. Biophys. Acta 453,426-438, 1976. 4. WANG, Y-Z., PATTERSON, J., GRAY, J.E., YU, C., COT-IRELL, B.A., SHIMIZU, A., GRAHAM, D., RILEY, M. and DOOLI’ITLE, R.F. Complete sequence of the lamprey fibrinogen a chain. Biochemistry 28,9801-9806, 1989. 5. VON HEINJE, G. A new method for predicting signal cleavage sites. Nucleic Acids Res., 14, 4683-4690, 1986. 6. BLOMBACK, B. and VESTERMARK, A. Isolation of tibrino-peptides by chromatography. Arkiv Kemi, 12, 173-182, 1957. 7. WEISSBACH, H. and GRIENINGER, G. Bipartite mRNA for chicken a fibrinogen encodes an amino acid sequence homologous to p and y chains. Proc. Natl. Acad. Sci. USA 87, 5198-5202, 1990. 8. FU, Y., WEISSBACH, S.N., REDMAN, CM. human fibrinogen a subunits. Biochemistry,

L., PLANT, P.W., ODDOUX, C., CAO, Y., LIANG, T.J., ROY, and GRIENINGER, G. Carboxy terminus extended variant of the subunit: a novel exon conferring marked homology to p and y 1992, In Press,