Proc. Nati. Acad. Sci. USA Vol. 80, pp. 7318-7322, December 1983 Medical Sciences

Fibrinogen Baltimore II: Congenital hypodysfibrinogenemia with delayed release of fibrinopeptide- B and decreased rate of synthesis (/hypofibrinogenemia/catabolism) RAY F. EBERT AND WILLIAM R. BELL Department of Medicine, Division of Hematology, The Johns Hopkins University, Baltimore, MD 21205 Communicated by Curt P. Richter, June 24, 1983 ABSTRACT A congenital hypodysfibrinogenemia, fibrinogen Fibrinogen. Fibrinogen was purified from citrate/phos- Baltimore H, was found in a young asymptomatic Caucasian fe- phate/dextrose plasma by three glycine precipitation steps (9) male. Prothrombin, partial thromboplastin, and euglobulin lysis at room-temperature. Fibrinogen isolated in this way from nor- times were normal, as were platelet function and fac- mal or abnormal plasma was >98% coagulable when assayed by tor assays. Subnormal plasma fibrinogen levels were found using the method of Laki (10) and appeared similarly homogeneous chronometric, rate-independent, and immunologic assay meth- when analyzed by NaDodSO4/polyacrylamide gel electropho- ods. Kinetic analysis of fibrinopeptide release revealed a delay in resis (11) under reducing conditions. Commercial normal hu- the thrombin-catalyzed release of fibrinopeptide B from the ab- man fibrinogen (Kabi, grade L, Stockholm, Sweden) was fur- normal protein. Proteolysis of fibrinopeptide A by thrombin or ther purified as above by glycine precipitation. This preparation -Arvin, monomer polymerization, and fibrin polymer liga- unless otherwise noted. tion occurred at normal rates.-Catabolism of radiolabeled au- was used as "control" fibrinogen tologous and homologous fibrinogen was also normal, but the fi- Thrombin. Thrombin (bovine, topical; Parke-Davis) was brinogen synthetic rate was -less than half the normal value. stored at -200C in a solution [1,000 units (u)/ml] of 25 mM Comparison of the coagulation-characteristics of fibrinogen Bal- barbital buffer (pH 7.5) containing 135 mM NaCl. Human a- timore H with those of other abnormal indicates that thrombin, a generous gift of G. Murano (National Institutes of it represents a unique example of hypodysfibrinogenemia. Health), was prepared by the method of Fenton et al. (12). All references to thrombin activity are in US (formerly NIH) units. Congenital dysfibrinogenemia is an inherited disorder of fi- Coagulation Studies. The following procedures were used brinogen synthesis in which a-presumed structural abnormality for clinical laboratory studies: one-stage prothrombin time (PT) results in altered coagulation characteristics of the protein. The by the method of Quick et al. (13) with rabbit brain throm- clinical manifestations of the disease vary from hemorrhagic boplastin (Pel-Freez; ref. 14); partial thromboplastin time (PTT) diathesis to thrombosis. The characteristic clinical laboratory by the method of Nye et al. (15) with Thrombofax Reagent (Or- finding in dysfibrinogenemia is an apparently low plasma fi- tho Diagnostics); euglobulin lysis time according to von Kaulla brinogen concentration using an assay dependent on the rate and Schultz (16); factors VIII, IX, XI, and XII according to Har- of clot formation. This value usually falls within normal limits disty and MacPherson (17) with the appropriate factor-deficient when a rate-independent method is used. plasma; and Factor XIII according to Tyler (18). Factors VII and In lieu of information concerning the precise location -of a X were measured as follows: citrated patient plasma was diluted molecular defect, dysfibrinogens have been classified on the 1:10 with 0.9% NaCl, and 0.15 ml of this solution was com- basis of their functional characteristics (1-3). The etiology of bined with an equal amount of factor-deficient plasma. A one- dysfibrinogenemia has been related to alterations in the pri- stage PT was then performed as described above. The results mary structure of the protein. Blomback et al. (4) were the first were compared with a standard curve generated in the same to identify such a defect, and more recently four different ab- manner using pooled normal plasma. normal proteins have been similarly characterized (5-8). Platelet aggregometry was performed on a platelet aggre- In this report a congenital hypodysfibrinogenemia, fibrino- gation profiler (BioData, Horsham, PA) at 37C with platelet- gen Baltimore II, is described. To the best of our knowledge, rich plasma (=300,000 platelets per mm3) and the following as this represents the seventh documented case of dysfibrino- aggregating agents in a final volume of 0.5 ml: 2-10 tLM ADP genemia occurring coincidentally with hypodysfibrinogenemia (Sigma), 1.2 mg of ristocetin (Abbott) per ml, 5.5 and 11 ,uM and the only one in which subnormal plasma fibrinogen con- epinephrine (Ellcins-Sinn, Cherry Hill, NJ), 0.26 mg of col- centrations have occurred coincidentally with delayed release lagen (Worthington) per ml, and 0.2-0.4 u of bovine thrombin of B. (Parke-Davis) .per ml. fibrinopeptide Release of Fibrinopeptides. Release of fibrinopeptides A and B was measured by a modification of the -Martinelli and METHODS Scheraga (19) method. The clotting reaction was initiated at 25VC Blood. Blood was obtained by venipuncture and plasma- by adding 10 1ul of human thrombin (1.33 u/ml) or Arvin (A- pheresis performed with citrate/phosphate/dextrose (Fen-wall 38414, Abbott; lot no. 23-126-DH; 3.3-6.7 u/ml) to 50 /.l of Laboratories, Deerfield, IL) as . Patient plasma a fibrinogen solution (4-10 mg/ml) containing 20 mM sodium used for clinical studies was prepared by diluting 9 vol ofwhole phosphate (pH 7.4) with 0.36 M e-aminocaproic acid and 0.15 blood with 1 vol of 3.'8% trisodium citrate dihydrate. M NaCl. At the indicated times the reaction was stopped and the clot was solubilized by adding 15 t.d-of 5% phosphoric acid. The publication costs ofthis article were defrayed in partby page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: u, unit(s); PT, prothrombin time; PTT, partial throm- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. boplastin time; IEF, isoelectric focusing. 7318 Downloaded by guest on October 2, 2021 Medical Sciences: Ebert and Bell Proc. Nati. Acad. Sci. USA 80 (1983) 7319

A 10-I.d aliquot (=60 lug) was then analyzed by reverse-phase Table 1. Coagulation laboratory findings HPLC on a Varian model 5000 liquid chromatograph. Peptides Test Control Baltimore II were detected absorbance at 205 nm and eluted from an oc- by PT 13-17 sec 14.5 sec with an isocratic mobile tadecylsilane column phase containing PIT <100 sec 73 sec 22% acetonitrile in 0.1% trifluoroacetric acid. Additional de- Euglobulin lysis time Normal Normal tails of this method, which permits rapid determination of the Factors VII-Xm Normal Normal kinetics of fibrinopeptide release, will be published elsewhere. Platelet aggregation* Normal Normal Electrophoretic Studies. NaDodSO4/polyacrylamide gel electrophoresis was performed in a Pharmacia horizontal slab * By ADP, ristocetin, epinephrine, collagen, and thrombin. gel apparatus using the Laemmli method (11). Isoelectric focusing (IEF) of reduced fibrinogen was done in riod. The average of all values obtained on days 2-7 is reported 2-mm horizontal slabs of 1% agarose (IEF grade, Pharmacia) in Table 3. Plasma volume (3.04 liter; 37.2 ml/kg) was esti- containing 12% (wt/vol) sorbitol, 6 M urea, 6% (vol/vol) Phar- mated by dividing the total protein-bound radioactivity in- malyte 4-6.5, and 2% (vol/vol) Pharmalyte 3-10. Fibrinogen jected by the radioactivity concentration in the first blood sam- was dissolved in a solution of 6 M urea (freshly deionized by ple. passage through a 2.5 X 22 cm column of Rexyn I-300, mixed- RESULTS bed ion exchanger; Fisher) and 5% (vol/vol) 2-mercaptoetha- nol. Complete reduction of disulfide bonds occurred after in- Clinical Laboratory Findings. This 25-year-old Caucasian cubation of this solution for 3-6 hr at 37C. Ten microliters (-50 female has been in excellent health all of her life. At age 18 yr, symptomatic impacted wisdom teeth necessitated ex- ,ug) was applied to the gel near the anode, and focusing was dental carried out at 10 W constant power for 75-120 min at 13'C us- traction, and, because of a history of some post-tonsillectomy bleeding, routine coagulation studies were performed. PT, ac- ing 1 M NaOH as the cathode solution and 10mM glutamic acid tivated PTT, euglobulin lysis time, factor assays, and as the anode solution. Gels were fixed and stained with Coo- platelet massie blue G-250 (Bio-Rad). All protein bands focused be- function were all within normal limits (Table 1). However, as tween pH 5.5 and 7.4. is typical of dysfibrinogenemia, plasma fibrinogen concentra- Studies of Fibrinogen Metabolism. Fibrinogen Baltimore II tions appeared to be low when estimated by two methods de- was labeled with "'lI by the iodine monochloride method of pendent on the rate of coagulation (Table 2). However, when McFarlane (20). Free iodine was removed by gel filtration on a method that did not depend on the rate of fibrinogen coag- ulation was used, the estimate of plasma fibrinogen concen- a 2.4 X 82 cm column of Sephadex G-100 (particle size 40-120 Am) equilibrated with 10 mM sodium citrate buffer (pH 7.0) tration more than doubled but remained subnormal (Fig. 1 and containing 0.25 M NaCl in sterile pyrogen-free water. Three- Table 2). These results were confirmed by serial dilution ex- milliliter fractions were collected into sterile nonpyrogenic glass periments using the Ouchterlony double-immunodiffusion tubes, each containing 0.12 ml of 5% human serum albumin technique (30) (data not shown). (sterile, for injection; NYBCEN, New York) to stabilize the fi- Chronometric fibrinogen determinations (Fig. 1) on the pa- brinogen (21). Protein eluting at the excluded volume (specific tient's 11 relatives were normal except for her father and only son, whose symptoms and clinical laboratory findings closely activity, 3.5 ,Ci/mg; 1 Ci = 3.7 X 1010 Bq) was 98% coagula- ble by thrombin and >99.5% precipitable. Immediately prior resembled those reported here. The pattern of inheritance of to injection the fibrinogen solution was sterilized by passage the disorder (Fig. 1) is consistent with an autosomal dominant through a 0.22-pym filter (Millex-GV, Millipore). mode of transmission. '"I-Labeled normal human was ob- Coagulation Studies. The coagulability of the abnormal plasma fibrinogen (for injection) was studied under a variety For tained from Amersham (IBRIN; lot no. and when ad- of conditions (Fig. 2). each of 0179) these experiments the fibrinogen concentration in the control ministered had a specific radioactivity of 75.3 pCi/mg. This plasma was to material was 92% coagulable by thrombin and 95% of the ra- diluted match that of the proposita (estimated at was 10% trichloroacetic acid. 82 mg/dl), but, because the pH and ionic strength of the re- dioactivity precipitated by action mixtures differ, coagulation times between experiments After providing informed, witnessed written and verbal con- are not comparable. sent, the affected father of the proposita was injected with a Thrombin added in increasing amounts to sterile solution containing 0.6 ml (45.2 uCi, 0.6 mg) of '"I-la- normal and abnormal plasma reduces the coagulation time for beled normal fibrinogen and 2.6 ml (27.4 ,Ci, 7.8 mg) of '31I- both but restores the latter to near normal levels (Fig. 2A), a labeled fibrinogen Baltimore II. Five-milliliter blood samples finding consistent with a proteolytic phase coagulation defect. were collected at the indicated times over a Addition of calcium to normal or abnormal plasma is also ef- period of 7 days, fective in and 1-2.5 ml of plasma derived therefrom was assayed for ra- reducing the coagulation time (Fig. 2B). However, on a Beckman counter. the net reduction in each case (normal vs. abnormal) is to :60% dioactivity (Gamma 8000) gamma The of the coagulation time in the absence of calcium, thus indi- radioactivity in each of seven 24-hr urine samples collected during cating no the same period was determined. Beginning 3 days prior to the difference in the net effect of calcium on coagulation study and for 17 days thereafter, the patient received daily 200 behavior. The relationship between coagulation time and pH mg of potassium iodide orally. over a range of 5.7-8.5 was similar for the abnormal and control Rates of fibrinogen synthesis and catabolism were calculated plasma (Fig. 2C). The addition of abnormal plasma to normal from plots of plasma radioactivity vs. time, assuming a two- Table 2. Plasma fibrinogen concentration compartment model (22, 23). Kinetic constants were obtained a to the data with the aid Concentration, by fitting biexponential equation (24) Method mg/dl of a computer program by using the Newton-Raphson method for nonlinear parameter estimation (25). A correction for non- Chronometric protein-bound iodide was made (23). The fractional catabolic von Clauss (26) 36 rate was determined from urinary excretion data by dividing Arvin time (27) 44 the total cpm in urine collected during a 24-hr period by the Rate-independent (28) 82 total plasma radioactivity at the midpoint of the collection pe- Immunologic (29) 62-96 Downloaded by guest on October 2, 2021 7320 Medical Sciences: Ebert and Bell Proc. Natl. Acad. Sci. USA 80 (1983)

EID

250 250 36 a (218) (82) 20U1

30 (85) FIG. 1. Family studies of fibrinogen Baltimore II. Numbers indi- cate plasma fibrinogen concentrations (mg/dl) determined by chrono- metric (26) or, if in parenthesis, non-rate-dependent (28) methods. The arrow denotes the proposita. Squares, males; circles, females. Open symbols, normal individuals; cross-hatched symbol, deceased, not stud- ied; solid symbols, affected individuals. o 10 20 30 40 50 60 plasma did not affect the coagulation time of the latter (Fig. Time, min 2D), which indicates that the abnormal plasma does not contain FIG. 3. Time course of thrombin-catalyzed fibrinopeptide release, a circulating anticoagulant. measured by reverse-phase HPLC. Time to form visible clot: 9 min. (A) Determination of the Functional Abnormality. When the Release of fibrinopeptide A; (B) release offibrinopeptide B. *, Control kinetics of individual fibrinopeptide release were examined, fibrinogen; o, fibrinogen Baltimore H. One area count = 1 1WV sec. fibrinopeptide A was released at a rate and to an extent similar to that of the control (Fig. 3A). The release of fibrinopeptide used, the total amount of fibrinopeptide B released was iden- B was delayed, although the total amount of peptide was -80% tical to that of the control. The delay is completely abolished of normal at later time points (Fig. 3B). For other experiments at a final thrombin concentration of >3 u/ml and is substan- (not shown) in which higher concentrations of thrombin were tially prolonged at enzyme concentrations of <0.1 u/ml. The observation of a prolonged Arvin clotting time (Table 2) >2 min A indicates that fibrinopeptide A is released more slowly than B normal in plasma. We attempted to confirm this using HPLC 50 50~ to monitor fibrinopeptide A release from isolated fibrinogen 40 40 but found the rate and extent of peptide generation to be nor- 30 30 mal (data not shown). As expected, no fibrinopeptide B was de- 20- 20 10- 10 . tected under these conditions. Q 0 0 No abnormalities regarding the initial rates of fibrin mono- 0 0 5 10 15 20 0 5 101520 mer polymerization were found (Fig. 4), and the plasma clot e Thrombin, u/ml CaCl2, mM 0.70 0o 0 00 0s0 0 c D O * 00*- asCZ 90 sec 110 0.60 0 0O* 50 t 100 2 0.50 - 40 90o a 30 - 80 0 0.40- 8 20- 70 to 10- 60 0 0 0.30 5.0 6.0 7.0 8.0 0 20 40 6080100 0.20- pH % normal plasma 0. 0.10 000 FIG. 2. Coagulation behavior of control and abnormal plasma. To determine the effect ofthrombin concentration on coagulation time (A) 0.1 ml of bovine thrombin [in 25 mM sodium diethyl barbiturate (pH 40 80 120 160 200 240 280 7.5) containing 125 mM NaCl] was added to 0.1 ml of citrated plasma. Time, sec The effect ofcalcium on thrombin-catalyzed clotting time (B) was stud- ied by combining 0.2 ml of citrated plasma with 0.1 ml of a 14.5 mM FIG. 4. Polymerization of fibrin monomers. Purified normal and NaCl solution containing 15 u ofbovine thrombin per ml and 5-60 mM abnormal fibrinogen (3.0 ml of a solution at 2.5 mg/ml) was combined CaCl2. The effect ofpH on coagulation time (C) was determined by mix- with human thrombin (5 u in 50 p1L) andthe mixture was allowed to clot ing one part citrated plasma with two parts Michaelis buffer (pH 5.7), for 30-45 min at 250C. The clot was removed with a glass rod, washed after which each solution was diluted to the same final volume with three times by immersion in 7 ml ofdistilled water for 10 min, and dis- water to ensure equivalent fibrinogen concentrations. Coagulation was solved in 1 ml of 20 mM sodium acetate buffer (pH 4.2). The protein initiated by adding 0.1 ml of bovine thrombin (25 u/ml) to 0.2 ml of concentration was determined by absorbance at 280 nm (31) and the buffered plasma solution. e, Control in A-C; o, Baltimore II in A-C. Lowry method (32). Polymerization was initiated by mixing 0.5 ml of The effect of mixing normal and abnormal plasma on thrombin-cata- a fibrin solution at 1 mg/ml (in sodium acetate buffer) with an equal lyzed clotting time (D) was studied by combining 0.2 ml ofnormal plasma volume of 80 mM Na2HPO4, to produce a pH 7.4 solution with an ionic [previously diluted with either patient plasma (0) or normal saline (s)] strength of 0.10. The extent of polymerization was measured by tur- with 0.2 ml of bovine thrombin (10 u/mi). For each of the above re- bidity at 350 nm in a Varian DMS-90 spectrophotometer against a blank actions, clot formation was monitored visually at 370C, and each point containingequal volumes of20 mM sodium acetate (pH4.2) and 80 mM represents the mean of triplicate determinations. Na2HPO4. *, Control fibrinogen; o, fibrinogen Baltimore II. Downloaded by guest on October 2, 2021 Medical Sciences: Ebert and Bell Proc. Nati. Acad. Sci. USA 80 (1983) 7321 0 2.0 AaI 1.8- FIG. 5. IEF in urea/agarose gel. Approximately 50 ug each of re- duced fibrinogen Baltimore II (lane

1) and normal fibrinogen (lane 2) 1.6 - was applied near the anode and subjected to IEF. Subunit identifi- cation was based on results of Na- DodSO4/polyacrylamide gel elec- trophoresis in a second dimension cm 1.4 0~ 2 (not shown).

was stable in 5 M urea or 2% monochloroacetic acid. These re- 1.2 - sults indicate that the polymerization and stabilization phases of coagulation are normal. Electrophoresis of Isolated Fibrinogen. A comparison of the mobility of normal and abnormal fibrinogen using NaDodSO4/ polyacrylamide gel electrophoresis revealed no detectable dif- 0 40 80 120 160 200 ferences in size or relative amounts of the Aa, BP, or y sub- Time, hr units (data not shown). FIG. 6. Plasmaradioactivitydisappearanceprofile.o,Normal(ho- IEF of isolated fibrinogen (Fig. 5) revealed the typical pat- mologous) 12I5-labeledfibrinogen; *, 131I-labeledfibrinogenBaltimore tern of 13-15 protein bands (33). No difference between the II (autologous). normal and abnormal fibrinogens was found. Immunoelectro- phoresis (3% agar, pH 8.6) of fibrinogen Baltimore II also failed it is apparent in the present case that no serious bleeding, to reveal any differences from normal (data not shown). thrombotic episodes, or abnormal wound healing could be at- Metabolic Studies. The plasma clearance curves for radio- tributed to the abnormal fibrinogen. This is consistent with, if labeled autologous and homologous fibrinogen were virtually not predicted from, the functional defect, for it is generally ac- identical (Fig. 6). The corresponding half-lives, intravascular cepted that cleavage of fibrinopeptide B is not essential for fi- distributions, and fractional catabolic rates were similar and brinogen coagulation (3, 31, 36). within normal limits (Table 3). However, the rate of fibrinogen The results in Fig. 2A suggest that the delay in plasma co- synthesis was well below normal for the autologous fibrinogen agulation can be abolished at increased thrombin concentra- Cable 3). Thus, the subnormal fibrinogen concentration results tions. Consistent with these data is the observation that the ki- from a decreased rate of fibrinogen synthesis or secretion. netics of fibrinopeptide B release are indistinguishable from normal at thrombin concentrations >3 u/ml. Thus, the func- DISCUSSION tional defect is corrected in vitro by relatively high thrombin In accordance with the nomenclature suggested by Beck et al. concentrations, and this characteristic may contribute to the (35), the hypodysfibrinogenemia described in the present study unremarkable case history of the index patient. has been designated fibrinogen Baltimore II. Clinical labora- Experiments to assess the initial rates of fibrin monomer tory studies of the proposita were unremarkable, except for the polymerization revealed no difference between the abnormal presence of subnormal amounts of fibrinogen with a prolonged and control fibrinogen. However, the structure of the abnor- clotting time. Examination of all 11 immediate blood relatives mal fibrin polymer may well be different from normal, as evi- revealed that the father and only son of the proposita were sim- denced by the divergence in solution turbidity at later time points ilarly affected, thereby confirming the heritability of the dis- (Fig. 4), despite the fact that the initial protein concentration order and suggesting that the dual traits of hypo- and dysfi- (measured by two methods) was identical to that of the control. brinogenemia are not transmitted independenty. Only 5 of the =100 congenital dysfibrinogenemias have been Assessment of fibrinopeptide release kinetics reveals that fi- found to exhibit delayed release of fibrinopeptide B. Fibrin- brinogen Baltimore II is functionally abnormal in the proteo- ogens Bethesda I (37), New York (38), and New Orleans (39) lytic phase of coagulation, the specific defect being a delay in were reported to have slow release of both fibrinopeptides A release of fibrinopeptide B. Although dysfibrinogenemia may and B and are thus different from the present dysfibrinogen. sometimes be accompanied by severe bleeding or thrombosis, A review of the procedure used to measure fibrinopeptide re- Table 3. Metabolic studies of normal and abnormal fibrinogen Intravascular Fractional Half-life, Mean residence distribution, catabolic rate* Synthesis rate, Fibrinogen days* time, dayst % of total pool Plasma Urine mg/kg per day Autologous 3.48 3.45 70 29 31.8 9.2 Homologous 3.83 2.86 74 35 25.8 - Normal mean (34) 4.14 4.17 71 24 23.0 28.0 Normal range (34) 3.1-5.6 2.9-5.9 45-87 17-35 NR 16-64 NR, not reported. * Calculated from slope of line determined by least-squares regression on last five points in Fig. 6. tThe reciprocal of the plasma-derived fractional catabolic rate. *Data were derived from plasma and urine and are expressed as % of plasma pool per day. Downloaded by guest on October 2, 2021 7322 Medical Sciences: Ebert and Bell Proc. Natl. Acad. Sci. USA 80 (1983) lease from fibrinogen Detroit (40) indicates that under similar 8. Morris, S., Denninger, M. H., Finlayson, J. S. & Menache, D. conditions of low thrombin concentration (0.05 NIH unit/ml), (1981) Thromb. Haemostasis 46, 104 (abstr.). fibrinogen Baltimore II would also have not released fibrino- 9. Kazal, L. A., Grannis, G. F. & Tocantins, L. M. (1964) in Blood Coagulation, Hemorrhage and Thrombois, eds. Tocantins, L. & peptide B to any appreciable extent. However, a comparison Kazal, L. (Grune & Stratton, New York), pp. 232-239. of other factors, such as the plasma fibrinogen concentration, 10. Laid, K. (1951) Arch. Biophys. 32, 317-324. PT, PIT, and the patient's clinical presentation (41), serves to 11. Laemmli, U. K. (1970) Nature (London) 227, 680-685. distinguish these two abnormal proteins. Unlike the present 12. Fenton, J. W., Fasco, M. J., Stackrow, A. B., Aronson, D. L., dysfibrinogen, fibrinogen Seattle (42, 43) was present in plasma Young, A. M. & Finlayson, J. S. (1977)1. Biol. Chem. 252, 3587- re- 3598. at normal concentrations, repolymerized abnormally, and 13. Quick, A. J., Hussey, C. V. & Geppert, M. (1963) Am.J. Med. Sci. leased only 54% of fibrinopeptide B. Thus, we are able to con- 246, 517-526. clude that fibrinogen Baltimore II is functionally unique among 14. Brambel, C. E. (1945) Arch. Surg. 50, 137-147. those dysfibrinogens with delayed release of fibrinopeptide B. 15. Nye, S. W., Graham, J. B. & Brinkous, K. M. (1962) Am. J. Med. Congenital hypodysfibrinogenemia has been described in six Sci. 243, 279-287. families in addition to that reported here (44-49). Fibrinogen 16. von Kaulla, K. N. & Schultz, R. L. (1958) Am. J. Clin. Pathol. 29, II a slight delay in total fibrino- 104-112. Bethesda (44), which manifests 17. Hardisty, R. M. & MacPherson, J. C. (1962) Thromb. Diath. Hae- peptide release, also exhibits a major delay in fibrin monomer morrh. 7, 215-229. polymerization and is therefore dissimilar to fibrinogen Balti- 18. Tyler, H. M. (1966) Thromb. Diath. Haemorrh. 16, 61-68. more II. The remaining proteins (45-49) are delayed only in 19. Martinelli, R. A. & Scheraga, H. A. (1979) Anal. Biochem. 96, 246- the polymerization phase of coagulation. Thus, fibrinogen Bal- 249. timore II is readily distinguished from the other hypodysfi- 20. McFarlane, A. S. (1963)J. Clin. Invest. 42, 346-361. 21. Krohn, K., Sherman, L. & Welch, M. (1972) Biochim. Biophys. brinogens on the basis of its functional defect, a delay in fi- Acta 285, 404-413. brinopeptide B release. 22. Matthews, C. M. E. (1957) Phys. Med. Biol. 2, 36-53. The steady-state plasma fibrinogen concentration is essen- 23. Reeve, J., Russell, B., Gibbs, G. P. & Peters, D. K. (1982) Clin. tially a function of its synthetic rate and rate of plasma clear- Sci. 63, 175-185. ance. We found the catabolic rate for fibrinogen Baltimore II 24. Gibaldi, M. & Perrier, D. (1975) Pharmacokinetics (Dekker, New to be within normal limits and conclude that the hypofibri- York), pp. 48-86. 25. Powell, J. B. (1967) in Numerical Analysis: An Introduction, ed. nogenemia results from a synthetic rate about one-third of nor- Walsh, J. (Thompson, Washington, DC), pp. 143-157. mal (Table 3). 26. von Clauss, A. (1957) Acta Haernatol. 17, 237-246. Plasma fibrinogen survival has been studied in four of the six 27. Allison, J. V. (1972) Proc. Univ. Otago Med. Sch. 50, 41-42. previously described hypodysfibrinogenemias. Two of these, 28. Ratnoff, 0. D. & Menzie, C. (1951) J. Lab. Clin. Med. 37, 316- fibrinogens Bethesda III (45) and Philadelphia (46), were mark- 320. edly hypercatabolized. A fibrinogen Bethesda 11(44), was 29. Merskey, C., Lalezari, P. & Johnson, A. J. (1969) Proc. Soc. Exp. third, Biol. Med. 131, 871-875. reported to have a slightly increased fractional catabolic rate, 30. Ouchterlony, 0. (1962) Prog. Allergy 6, 30-154. a somewhat shortened plasma half-life (70-72 hr), and a normal 31. Doolittle, R. F. (1975) in The Plasma Proteins: Structure, Func- synthetic rate. Thus, the etiology of the subnormal fibrinogen tion, and Genetic Control, ed. Putnam, F. W. (Academic, New levels in these three cases differs from that of fibrinogen Bal- York), Vol. 2, pp. 110-161. timore II. In the fourth instance (49), subnormal plasma fi- 32. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. brinogen concentrations were not due to hypercatabolism and (1951)J. Biol. Chem. 193, 265-275. 33. Kuyas, C., Haeberli, A. & Straub, P. W. (1982)J. Biol. Chem. 257, thus resulted from a decreased synthetic rate. However, the 1107-1109. functional and solubility characteristics of this protein readily 34. Collen, D., Tytgat, G. N., Claeys, H. & Piessens, R. (1972) Br.J. distinguish it from the present one. Haematol. 22, 681-700. On the basis of the foregoing we conclude that fibrinogen 35. Beck, E. A., Charache, P. & Jackson, D. P. (1965) Nature (Lon- Baltimore II is unique among the dysfibrinogens and suggest don) 203, 143-145. that a similarly unique structural defect is present in this pro- 36. Shainoff, J. R. & Dardik, B. N. (1979) Science 204,200-202. 37. Gralnick, H. R., Givelber, H. M., Shainoff, J. R. & Finlayson, J. tein. S. (1971)J. Clin. Invest. 50, 1819-1830. 38. Al-Mondhiry, H. A. B., Bilezikian, S. B. & Nossel, H. L. (1975) We gratefully acknowledge the expert technical assistance of Ms. Blood 45, 607-619. Daniele Cornell for routine coagulation studies and Ms. Mary Jo Fack- 39. Chavin, S. I., Andes, W. A., Beltran, W G. & Stuckey, W J. (1979) ler for routine coagulation studies as well as those presented in Fig. 2. Thromb. Haemostasis 42, 15 (abstr.). We also thank Drs. Deborah L. Higgins and Jules A. Shafer for helpful 40. Blomback, B. & Blomback, M. (1970) Nouv. Rev. Fr. Hematol. 10, discussions regarding HPLC offibrinopeptides and Dr. Dennis Noe for 671-678. assistance in numerical analysis of the fibrinogen clearance results. This 41. Mammen, E. F., Prasad, A. S., Bamhart, M. I. & Au, C. C. (1969) work was supported in part by Research Grants HLO1601-33 and J. Clin. Invest. 48, 235-249. 42. Branson, H. E., Schmer, G. & Dillard, D. H. (1977) Am.J. Clin. HL24898-02 and Training Grant HL07377 from the National Heart, Pathol. 67, 236-240. Lung, and Blood Institute and by the Whitehall Foundation. 43. Branson, H. E., Theodor, I., Baumgartner, R., Schmer, G. & Pirkle, H. (1979) Thromb. Haemostasis 42, 138 (abstr.). 1. Flute, P. T. (1977) Br. Med. Bull. 33, 253-259. 44. Gralnick, H. R., Givelber, H. M. & Finlayson, J. S. (1973) Thromb. 2. Marder, V. J. (1976) Thromb. Haemostasis 361, 1-8. Diath. Haemorrh. 29, 562-571. 3. Ratnoff, 0. D. & Forman, W. B. (1976) Semmn. Hematol. 13, 141- 45. Gralnick, H. R., Coller, B. S., Fratantoni, J. C. & Martinez, J. 157. (1979) Blood 53, 28-46. 4. Blomback, M., Blomback, B., Mammen, E. F. & Prasad, A. S. 46. Martinez, J., Holburn, R. R., Shapiro, S. S. & Erslev, A. J. (1974) (1968) Nature (London) 218, 134-137. J. Clin. Invest. 53, 600-611. 5. Henschen, A. C., Southan, C., Kehl, M. & Lottspeich, R. (1981) 47. Rupp, C., Kuyas, C., Haberli, A., Furlan, M. & Beck, E. R. (1981) Thromb. Haemoasis 46, 181 (abstr.). Thromb. Haemostasis 46, 104 (abstr.). 6. Henschen, A., Southan, C., Soria, J., Soria, C. & Samama, M. 48. Verhaeghe, R., Verstraete, M., Vermylen, J. & Vermylen, C. (1974) (1981) Thromb. Haemostasis 46, 103 (abstr.). Br.J. Haematol. 26,421-433. 7. Higgins, D. L. & Shafer, J. A. (1981)J. Biol. Chem. 256, 12013- 49. McDonagh, R. P., Carrell, N. A., Roberts, H. R., Blatt, P. M. & 12017. McDonagh, J. (1980) Am. J. Hematol. 9,23-38. Downloaded by guest on October 2, 2021