Fibrinogen Baltimore III: Congenital dysfibrinogenemia with a shortened γ-subunit

THROMBOSIS RESEARCH 51; 251-258, 1988 0049-3848/88 $3.00 t .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

FIBRINOGEN

BALTIMORE III: CONGENITAL DYSFIBRINOGENEMIA WITH A SHORTENED T-SUBUNIT

Ray F. Eber@

and William

R. Bellb

*Bionetics

Research Institute, 1330 Piccard Drive Rockville, MD 20850 bThe Johns Hopkins University School of Medicine Baltimore, MD 21205 U.S.A. (Received 23.2.1988; Accepted in revised form 5.5.1988 by Editor B. Kudryk)

ABSTRACT An abnormal fibrinogen has been found in an asymptomatic Negro female. Clinical laboratory findings were normal, except for a prolonged thrombin time which was corrected by addition of calcium. Fibrinopeptide release by thrombin and crosslinking by factor XIII also occurred normally, but fibrin monomer polymerization was delayed. Sodium dodecylsulfate-polyacrylamide gel electrophoresis disclosed that 50% of the T-subunits migrated with an apparent M, of 45,500, approximately 1,500 Da smaller than normal. The evidence suggests that an internal sequence of IO-15 residues is missing from the r-subunit of the abnormal fibrinogen.

INTRODUCTION Congenital dysfibrinogenemia is a disease in which the presence of a structurally altered form of fibrinogen produces abnormalities of fibrinogen function (1). The clinical manifestations of the disease include mild to severe bleeding, thrombosis, and poor wound healing, but in most cases are not severe. Typically, a dysfibrinogen clots slowly due to a decrease in the rates of fibrinopeptide release and/or fibrin monomer polymerization; much less frequently, the functional abnormality is related to defective crosslinking by factor XIII of the fibrin polymer. In all but a few of the approximately 200 reported cases, the affected individuals are heterozygous for the trait, which is transmitted in an autosomal dominant pattern. This is an initial report of a new case of dysfibrinogenemia in which the functional defect involves delayed polymerization of fibrin monomers. Fibrinogen isolated from the proposita exhibits a striking structural abnormality: The presence of a r-subunit that _____-_________ ‘To whom correspondence KEY WORDS:

should

be addressed.

DYSFIBRINOGENEMIA/FIBRIN/FIBRINOGEN 251

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FIBRINOGENBALTIMORE III

is approximately 1,500 Da smaller protein has been published (2).

than

MATERIALS

normal.

A preliminary

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this

AND METHODS

Blood collection, fibrinogen isolation, prothrombin time (PT) partial thromboplastin time (PTT), euglobulin lysis time, coagulation factor assays, and electrophoretic analyses were carried out as described previously (3). Gel scanning densitometry was performed with a Biomed Model SL-504-XL soft laser gel scanner. Control fibrinogen was a commercial preparation of pooled human fibrinogen (Kabi, Grade L, Stockholm, Sweden) further purified to >99% coagulability by glycine precipitation (4). Protein was quantitated by the Lowry method (5).

RESULTS Case report and clinical laboratory findings. This 30 year-old Negro female initially presented with microscopic hematuria during pregnancy, and subsequently experienced moderate post-partum bleeding that did not require transfusion. The proposita has experienced tonsillectomy, adenoidectomy, dental extractions, traumatic skin and deep subcutaneous lacerations (requiring sutures) and three child deliveries (vaginal) without significant bleeding. A subnormal plasma fibrinogen concentration (107 mg/dl) was detected using a method dependent on the rate of clot formation (6). When a rate-independent method was used (7), the plasma fibrinogen concentration was within normal limits (170 mg/dl). An immunologic test for fibrinogen concentration (performed as in ref. 3) disclosed a value of 180 mg/dl. The PT, PTT, euglobulin lysis time, and bleeding time were also within normal limits. Platelet aggregation (by ADP, epinephrine, collagen, and ristocetin) occurred normally, and circulating anticoagulants (8, 9) were not detected. The delay in coagulation was not corrected by adding up to 20 u/ml of thrombin to the abnormal plasma, and mixing of abnormal and normal plasma failed to prolong the thrombin clotting time (c.t.) of the latter. However, calcium concentrations in excess of 7 mM returned the c.t. to normal. Structural analyses. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Fig. 1) disclosed the presence of an undersized T-subunit (M, 45,500), in addition to the normal r-subunit (M, 47,000). Densitometric scans of this gel (Fig. 1, middle lane) indicated that the abnormal r-subunit comprised about 50% of the total. When a preparation of normal fibrinogen was mixed with increasing amounts of fibrinogen Baltimore III, a corresponding increase in the intensity of the smaller r-subunit band was detected by soft laser gel scanning (not shown). The T-subunits were eluted from SDSPAGE gels (8% polyacrylamide) and subjected to gas-phase protein sequencing. Each of the r-subunit bands contained the normal N-terminal sequence through Asp,,. This result confirms the identity of the 45.5 kDa band, and indicates that the N-terminal domains of both normal and abnormal r-subunits were intact. Isoelectric focusing of fibrinogen Baltimore III (Fig. 2) failed to reveal any significant differences from normal. When the focused bands were evaluated by SDSPAGE in a second dimension, each T-subunit lane contained both the native (M, 47,000) and abnormal (M, 45,500) subunits. Reversed-phase HPLC of the reduced, carboxamidomethylated subunits from fibrinogen Baltimore III using a butylsilane column (10) yielded a single symmetrical peak in the same elution position as that for the normal r-subunit (data not shown). When the Y-subunit was digested with CNBr and subjected to comparative peptide mapping (lo), peaks with retention times identical to those of the N-terminal (~r_~s) and C-terminal (~~s~_~ii) fragments were present in apparently normal quantities. Thus, the amino- and carboxyl-terminal domains of rBa,timore rII appear to be intact.

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FIBRINOGEN BALTIMORE III

FIG. SDS-PAGE of fibrinogen [Laemmli fibrinogen; middle lane, fibrinogen Phosphorylase b (92,500), bovine carbonic anhydrase (30,000).

253

1

method (II); 10% resolving gel]. Left lane: control Baltimore III; right lane, molecular weight standards: serum albumin (66,200), ovalbumin (45,000) and

Y

FIG. 2 Isoelectric focusing of fibrinogen in urea/agarose gel (method per ref. 3). Approx. 50 c(g each of reduced fibrinogen Baltimore III (lane 1) or control fibrinogen (lane 2) was applied near the anode. Subunit identification was based on results of SDS-PAGE in a second dimension (not shown).

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0TIME

(min)

FIG. 3 Time course of thrombin-catalyzed fibrinopeptide release measured by reversed-phase HPLC (method per ref. 12). Panel A: release of fibrinopeptide A; Panel B: release of fibrinopeptide B. 0, control fibrinogen; @, fibrinogen Baltimore III.

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0.50-

.

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0

0

0 0

.

.

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40

80

120

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240

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280

FIG. 4 Polymerization of fibrin monomers. Reaction conditions were as described

0, control previously

fibrinogen; (3).

0, fibrinogen

Baltimore

III.

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To determine the effect of this structural Functional defect determination. abnormality on fibrinogen function, experiments designed to assess the three phases of coagulation were undertaken. The initial phase, release of fibrinopeptides A and B by However, fibrin thrombin, occurs at rates indistinguishable from normal (Fig. 3). monomer polymerization was substantially delayed (Fig. 4), with the principal effect on Fibrin stabilization, as evidenced by clot insolubility the “lag” phase of polymerization. As a confirming experiment, in 1% monochloroacetic acid or 5 M urea, was normal. SDS-PAGE was used to determine whether the abnormal T-subunit was crosslinked by The results (Fig. 5) indicate that both ‘I-subunits are converted to the factor XIII. Thus, the functional defect resulting corresponding dimers by plasma transglutaminase. from the shortened T-subunit is limited to delayed polymerization of fibrin monomers. Under the conditions used for SDS-PAGE in this experiment, no heterogeneity was detected in the abnormal T-7 dimer (Fig. 5, lane 6).

FIG. 5 Crosslinking of fibrinogen assessed by SDS-PAGE. Factor XIII-catalyzed crosslinking was carried out in a 0.26 ml reaction mixture containing: 0.1 M TRIS-HCI, pH 7.4; fibrinogen (2.13 mg/ml); cysteine (12 mM); factor XIII (10 pg/ml); and human thrombin Ethylenediamine-tetraacetic acid (12 mM; EDTA) was added to inhibit (0.5 u/ml). factor XIII activity (lanes 2 and 5). CaCl, (12 mM) was added to promote factor XIII activity (lanes 3 and 6). After 22 h at 37 OC, the reaction was stopped and the clot was solubilized by adding 0.1 ml of a solution containing 6 M urea, 5% 2-mercaptoethanol, III fibrinogen, respectively; and 5% SDS. Lanes 1 and 4 contain control and Baltimore lanes 2 and 5 contain EDTA-treated control and Baltimore III fibrinogen, respectively; lanes 3 and 6 contain calcium-treated control and Baltimore III fibrinogen, respectively. Crosslinked polymers of the o-subunit remained in the stacking gel (not shown).

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DISCUSSION

In accordance with the nomenclature suggested by Beck et al. (13), the dysfibrinogenemia described in the present study has been designated fibrinogen Baltimore III. Family studies to assess the heritability of this disorder have not been possible. Except for the moderate complications of pregnancy noted above, the case h,istory and clinical laboratory findings of the index patient are unremarkable. The most striking feature of fibrinogen Baltimore III is the presence, in approximately equal proportions, of two T-subunits: One with the expected molecular weight of 47,000, and one with a M, of 45,500, approximately 1,500 Da smaller than normal. Following SDS-PAGE each T-subunit was eluted from the gel and sequenced with the result that each amino-terminal domain was intact. That the carboxyl-terminal domain of the abnormal T-subunit is also intact is apparent from two lines of evidence: (i) the presence of at least one crosslink acceptor site (Fig. 5), and (ii) more indirectly, the isolation and complete sequencing of the C-terminal CNBr fragment (Tssr_rll; data not shown). On the basis of these results it is our tentative conclusion that an internal deletion is likely to be responsible for the reduced size of rBa,timore rIr. It remains possible, however, that we failed to detect an abnormal C-terminal CNBr fragment, or that a deletion has occurred distal to rs9,,the most C-terminal crosslink acceptor site. The possibility that the smaller T-subunit results from incomplete or defective glycosylation has also not been excluded. Size heterogeneity in the T-subunit has been reported for at least three dysfibrinogens: Paris I (14), Tochigi (15), and Kyoto (16). As is the case for fibrinogen Baltimore III, these three dysfibrinogens exhibit delayed polymerization of fibrin monomers. Fibrinogen Paris I may be distinguished from fibrinogen Baltimore III in that 7Paris r is 2000 Da larger than normal and is not crosslinked by factor XIII (14). Similarly, 7Tochigi, which contains an Arg --> Cys substitution at Ts,s, is distinguishable from the present dysfibrinogen in that it migrates on SDS-PAGE with an apparent molecular weight approximately 500 Da higher than normal (15). Fibrinogen Kyoto closely resembles fibrinogen Baltimore III: Its T-subunit is approximately 2000 Da smaller than normal and is crosslinked by factor XIII (16). The only apparent difference between these two dysfibrinogens lies in the slopes of their fibrin monomer polymerization curves: That of fibrinogen Baltimore III approaches the normal rate, whereas that of fibrinogen Kyoto is less than one-third of normal. Further differentiation of these two proteins must await identification of their structural defects. There is now convincing evidence that the polymerization site located near the Cterminus of the T-subunit encompasses an extended region including residues 7sos_ros (17, 18). This sequence includes the crosslink acceptor sites for plasma transglutaminase, and a calcium binding site. Furthermore, dysfibrinogens containing the substitution Tsrs Arg --> His also exhibit defective fibrin monomer polymerization and normal crosslinking (19, 20). It is intriguing to note that fibrinogen Baltimore III retains crosslink acceptor site(s), yet exhibits delayed fibrin monomer polymerization which is corrected by calcium. Elucidation of the structural defect in this protein may provide additional insight into the relationships among polymerization, calcium-binding, and crosslinking sites in the carboxyl-terminal domain of the T-subunit of human fibrinogen. ACKNOWLEDGMENTS

This work was supported by 07377, HL 29067, HL 24898) and by Maiyland Affiliate, Inc. Human National Institutes of Health. The and Ms. Judy Phelps is also gratefully

grants from the National Institutes of Health (HL a Grant-in-Aid from the American Heart Association, factor XIII was a generous gift of Dr. S.I. Chung, expert technical assistance of Ms. Karen Burkhardt acknowledged. \

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