THE JOURNAL OF B~LO~ICAL CHEMISTRY Vol. 248, No. 4,Issue of February 25, pp.

1395-1407, 1973
Printed in U.S.A.

Human

Factor

XIII
SUBUNIT FIBRIN*

from

Plasma

and
PROTEOLYTIC

Platelets
ACTIVATION, AND CROSS-LINKING

MOLECULAR WEIGHTS, OF FIBRINOGEN AND

STRUCTURES,

(Received for publication,

MARTIN

August 2, 1972)

L. SCHWARTZ,~

SALVATORE

V. PIZZO,$

ROBERT

L. HILL,

AND

PATRICK

A.

&L(EE$

From the Department of Biochemistry, Duke University Medical Center, and Department of Medicine, Veterans Administration Hospital, Durham, North Carolina 27710

SUMMARY The subunit structures of Factor XIII from plasma and platelets have been examined by sedimentation equilibrium in the ultracentrifuge. Plasma Factor XIII has a molecular weight of 320,000 f 20,000 in dilute salt solution at neutral pH. The two different types of subunits of the plasma factor, the a and b chains, have molecular weights of about 75,000 and 88,000, respectively. Platelet Factor XIII has a molecular weight of 146,000 f 10,000, whereas its single type of subunit, the a chain, has a molecular weight of about 75,000. The a chains of the platelet and plasma factors are identical not only in molecular weight but also on gel electrophoresis in The three different systems and in amino acid composition. a chain of platelet Factor XIII combines with the b chain of plasma factor to give a protein which is electrophoretically indistinguishable from native plasma Factor XIII. In addition, the amino acid composition of the plasma factor is essentially identical with twice the average amino acid composition of the (I and b chains. These analyses indicate that the subunit structure of plasma factor is a2bz and that of the platelet factor is u2. The subunits of each factor are combined in the native molecules solely by noncovalent bonds. Thrombin, trypsin, reptilase, papain, and an enzyme contaminating some ancrod preparations activate plasma Factor XIII. In each case, except possibly with papain, Factor XIII activation coincides with a decrease in molecular weight of 4,000 of the a chain. High concentrations of thrombin only slightly inactivate Factor XIII after prolonged incubation, whereas trypsin not only activates but extensively inactivates plasma Factor XIII. Activation of Factor XIII is not autocatalytc. Neither release of the fibrinopeptides nor gelation is required for activated plasma Factor XIII to cross-link fibrinogen. The y chains of fibrin are cross-linked by activated plasma Factor XIII more rapidly than the cz chains, but the rates of cross-linking of the a! (A) and y chains of fibrinogen * These studies were supported by research grants from the National Heart and Lung Institute (HE-06400) and a Clinical Investigatorship from the Veterans Administration. $ Predoctoral Fellow, Medical Scientist Training Program, National Institute of General Medical Sciences (GM-01678). J Clinical Investigator, Durham Veterans Administration Hospital.

are about the same. In addition, the pattern of cross-linking of fibrinogen or fibrin by guinea pig liver transglutaminase is different from that catalyzed by activated Factor XIII. The lysis time of fibrin clots with cross-linked (Y and y chains was considerably greater than that of fibrin clots containing cross-linked y chains but little or no cross-linked CY chains.

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Human blood coagulation Factor XIII, the fibrin-stabilizing factor, is a zymogen which, after activation by thrombin (1, 2), catalyzes the formation of e-(y-glutamyl)lysine cross-links between fibrin monomers so as to produce insoluble fibrin (3-6). Trypsin, papain, and reptilase have also been found to activate Factor XIII (5, 7-12), whereas papain and guinea pig liver transglutaminase have been reported to have clot st,abilizing activity (13-16). Factor XIII has been found not only in plasma (17, 18) and in platelets (19, 20) but also in the placenta (21). Schwartz et al. (22) showed earlier that plasma Factor XIII contained two types of subunits called the a chain and b chain, whereas platelet Factor XIII contained a single type of subunit which appeared to be identical with the a chain. In addition, on activation of Factor XIII with thrombin the a chain decreased in molecular weight by approximately 4000. It was therefore proposed that the subunit structures for plasma and platelet Factor XIII were a2bz and u2, respectively (22). Bohn has reported that Factor XIII from platelets is identical with the Factor XIII from the placenta (21). The pattern of cross-linking of fibrin by either platelet or plasma Factor XIII has been found to be identical (22). Crosslinking of the y chains of fibrin proceeds more rapidly than crosslinking of the a chains (23). It was also proposed that cross-linking of the (Y chains may be the event which makes cross-linked clots more resistant to plasmin than noncross-linked clots (24). We wish to report here further properties of platelet and plasma Factor XIII. The subunit structures of each factor that were proposed earlier have been confirmed by sedimentation equilibrium studies in the ultracentrifuge. The activation of plasma Factor XIII by several proteolytic enzymes has also been evaluated. In addition, the pattern of cross-linking of fibrinogen

1395

1396
and fibrin by plasma Factor XIII and guinea pig liver transglustated otherwise, Factor XIII was assayed with [14C]putrescine taminase has been compared. Finally, the susceptibility of fibrin and casein as substrates by the method of Chung and Folk (29). to plasmin digestion has been found to vary with the extent of OL Purification of Plasma Factor XIII Subunits-Plasma Factor chain cross-linking. XIII (1.2 mg per ml in 0.05 M Tris-chloride, pII 7.5, containing 0.001 M EDTB) was frozen at -20 and stored for 2 months. EXPERIMENTAL PROCEDURE On thawing, the white flocculent precipitate which formed was separated by centrifugation at 1000 X g for 10 min at 4. The Materials supernatant solution was refrozen and thawed 1 week later. precipitate which formed was Reagents-All protein and enzyme preparations were stored at The small amount of additional removed by centrifugation. The precipitate consisted of pure a -20 unless otherwise noted. Human fibrinogen, 95% clottable, chain, whereas the supernatant solution contained largely b wTas prepared by the method of Blomback and Blomback (25) chain with only a trace of contaminating a chain as judged by from fresh frozen plasma through the I-2 step. Purified human electrophoretic analysis. thrombin (a gift from Dr. D. L. Aronson, National Institutes of Purification of Activated Plasma Factor XIII-Plasma Factor Health) was dissolved in glycerol-O.15 M sodium chloride (60:40, XIII (6.4 ml; 0.45 mg per ml) in 0.05 M Tris-chloride, pH 7.5, v/v) (200 NIH units per ml) to give a solution with an absorbcontaining 0.001 M EDTA and 0.005 M dithiothreitol was mixed ance of 1.0 + 0.2 at 280 nm. Pure guinea pig liver transat 25 with 0.2 ml of thrombin (40 units) for 15 min and then glutaminase (26) was a gift from Dr. J. E. Folk and Dr. S. I. chromatographed on a Sephadex G-200 column (80 x 2.5 cm) Chung (National Institute for Dental Research). Trypsin-n-lat 4. Fractions (4 ml) were collected automatically. Fractions tosylamido-2-phenylethyl chloromethyl ketone (Worthington, 32 to 34 and 35 to 38 were pooled, dialyzed exhaustively against 210 units per mg) and cY-chymotrypsin (Worthington, 48 units water, lyophilized, dissolved in 0.7 ml of 0.1 M sodium phosphate per mg) were dissolved and stored in 0.001 N HCl. Reptilase (4:6, v/v), and stored at -20. Pooled frac(Defibrase) was obtained from Pentapharm and was used as (pH 7.0)-glycerol tions 32 to 34 had an absorbance at 280 nm of 1.4 and were used supplied. Preparations of ancrod of varying purity were supas the source of activated Factor XIII. When 5 ~1 of this plied by Dr. G. H. Barlow of Abbott Laboratories. Urokinase material were incubated with 100 ~1 of normal titrated plasma, (Calbiochem reference standard) was dissolved in 0.15 M NaClno clot formed even after a 24-hour incubation. 0.05 M Tris-chloride, pH 7.4, at a concentration of 460 Ploug Amino Acid Composition-Hydrolyses were performed in units per ml. Plasmin was prepared as described earlier (27) and duplicate with 6 N HCl in evacuated sealed glass tubes at 110 was stored in 0.1 M Tris-chloride, pH 7.4, at a concentration of as described earlier (30). Hydrolysates were analyzed with 123 Committee on Thrombolytic Agents units per ml. Papain Beckman amino acid analyzer (120B or 121) equipped with high was obtained from Worthington (30 mg per ml, 20 units per mg, sensitivity cuvettes. Tryptophan (31) and cysteic acid (32) twice crystallized suspension) and was stored at 4. The Factor were estimated separately as described earlier. S-CarboxyXIII-deficient plasma used in these studies has been previously methyl plasma Factor XIII was prepared as described for (Ydescribed (24). Ultrapure guanidine hydrochloride (Heico) and lactalbumin (33)) and S-carboxymethylcysteine was measured in cr-casein (Worthington) were commercial preparations. Hirudin acid hydrolysates on the amino acid analyzer. (Sigma) was dissolved in 0.05 M Tris-chloride, pH 7.4, containing Partial Specific Volume and Extinction Coeficienl of Plasma 0.1 M sodium chloride at a concentration of 1 unit per ~1. [ClFactor XIII-A solution of plasma Factor XIII was dialyzed Diaminobutane (putrescine) was purchased from New England against 0.05 M sodium citrate, pH 7.1, containing 0.158 M Nuclear (0.05 mCi, 0.4 mg per vial). All other compounds used potassium chloride. After dialysis the absorbance at 280 nm, in this study were reagent grade unless stated otherwise. corrected for scattering, was equal to 3.18. The densities of the Factor XIII-free Fibrinogen-Factor XIII-free fibrinogen was Factor XIII solution and the dialysate were measured at 25.60 f prepared as described earlier (24) except that the 3.3 M urea 0.01 in a DMA 02C digital precision density meter (Anton Paar solutions contained 0.1 M e-amino caproic acid in order to retard K. G., A8054 GRAZ, Austria-Europa). Protein concentration fibrinolysis. The urea was removed by dialysis at 4 rather than was determined after a fixed volume of the Factor XIII solution at room temperature. was dialyzed into doubly distilled water and dried to constant Plasma Factor XIII-Plasma Factor XIII was purified as weight at 107 in a fluorocarbon weighing cup (Cahn Division previously described (22), and the preparation was either Ventron Instruments Corp., Catalog Number 2034). From lyophilized or frozen and stored at -20. these measurements plasma Factor XIII was found to have an Platelet Factor XIII-Platelet Factor XIII was purified as 4% urn = 13.8. The apparent specific volume was calculated previously described (22). This preparation showed a major from the following equation: species which was platelet Factor XIII and several minor com+I=1 _-~ 1 (P - PO) ponents on polyacrylamide gel electrophoresis in sodium dodecyl c PO PO sulfate. For ultracentrifugal studies and amino acid analyses, this preparation was further purified by chromatography on where 4 is the apparent specific volume (cc per g), p is the density DEAE-cellulose as previously described (22). Those fractions of the protein solution (g per cc), po is the density of the dialysate with the highest specific activity were used. (g per cc), and C is the concentration of protein (g per ml). The apparent specific volume was assumed to be equal to the partial Methods specific volume. Factor XIII Aclivity--Factor XIII activity was measured by Partial specific volumes were calculated by the method of either of three methods described earlier. The clot solubility Cohn and Edsall from the apparent specific volume of the assay (22) and the isotope assay of Dvilansky et al. (11, 28) were individual amino acids (34) and carbohydrate residues (35) in used in some experiments as indicated under Results. Unless the molecule. The amino acid compositions reported here and

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1397 the carbohydrate compositions reported by Cohn (36) were used for these calculations. Sedimentation Equilibrium-Weight average molecular weights were measured by sedimentation equilibrium in the ultracentrifuge (Spinco model E) by the high speed meniscus depletion method of Yphantis (37). All analyses were performed essentially as described in earlier studies from this laboratory (38, 39). Native plasma Factor XIII was analyzed at a concentration of -0.22 mg per ml in 0.158 M potassium chloride containing 0.05 M sodium citrate, pH 7.1, at 25 at a rotor (An-D rotor) speed of 12,000 rpm. The molecular weight of plasma factor was also measured in 6.0 M guanidine hydrochloride, pH 7.4, at 25 with a rotor (An-D) speed of 26,000 rpm. Sedimentation equilibrium studies on the plasma Factor XIII a chain were performed in 5.9 M guanidine hydrochloride containing 0.01 M dithiothreitol, pH 8, at 21.7 with a rotor (An-D) speed of 35,600 rpm and the a chain at a final concentration of -0.2 mg per ml. Plasma factor b chain (~0.07 mg per ml) was analyzed in 0.158 M potassium chloride containing 0.05 M sodium citrate, pH 7.1, at 25 with a rotor (An-E) speed of 22,000 rpm. A double sector centerpiece (30 mm) with sapphire windows was used for these analyses. Platelet Factor XIII was analyzed in Tris-HCl containing 0.001 M EDTA, pH 7.5, at 25 with a rotor (An-D) speed of 17,000 rpm and a Factor XIII concentration -0.13 mg per ml. Sedimentation equilibrium analyses were also performed in 6 M guanidine hydrochloride containing 0.01 M dithiothreitol, pH 8, at 24 with a rotor (An-D) speed of 29,550 rpm and a final protein concentration of about 0.25 mg per ml. Polyac~ylamide Gel Elec trophoresis-Analyses in gels at pH 3.2 in urea were performed by the method of Panyim and Samples Chalkley (40) with the following modifications. were lyophilized and dissolved in 9.5 M urea (deionized) con taining 5% acetic acid. The samples were applied directly on the surface of the 7.5% polyacrylamide gels and were overlayed with the tray buffer. The gels were stained with Coomassie blue (Colab) by the technique of Weber and Osborn (41). Analyses on gels at pH 8.9 were performed by the procedure of Davis (42) except that ammonium persulfate rather than riboflavin was used to polymerize the 7.5% resolving gel. Samples in 307, sucrose were layered directly on top of the concentrating gel. The gels were stained with Coomassie blue. Polyacrylamide gel electrophoresis in sodium dodecgl sulfate was performed by the method of Weber and Osborn (41) except that gels were polymerized in 6 M urea as described earlier (24). Such gels were found to give sharper resolution of some proteins although the relative mobility of two proteins was not always the same in the presence and absence of urea. Solid samples such as blotted clots were prepared for electrophoresis by adding 6 M urea containing 0.01 M sodium phosphate, pH 7.1, 0.01 M EDTA, 1% SDS, and 1% mercaptoethanol, so that the final protein concentration was about 1 mg per ml. Liquid samples were prepared for electrophoresis by adding 1 volume of sample to 2 volumes of 9 M urea containing 0.015 M sodium phosphate, pH 7.1, 0.015 M EDTA, 1.5yo SDS, and 1.5% mercaptoethanol. The mercaptoethanol was omitted when nonreduced samples were prepared. 1destained gels were scamled at 562 nm with a Gilford linear transport system connected to a Beckman DU recording spectrophotometer. 1 The abbreviation used is: SUS, sodium dodecyl sulfate.
RESULTS

Jlol~cular Cleight of Plasma Factor XIII and Its SubunitsThe molecular weight of plasma Factor XIII was measured by sedimentation equilibrium in the ultracentrifuge in dilute salt solution and in 6 M guanidine hydrochloride. The molecular weight for Factor XIII in dilute salt solution in three different analyses was found to be 323,000, 310,000, and 328,000, respectively. The experimentally determined value of 0.73 Z!Z 0.01 ml per g for the partial specific volume was used to calculate the molecular weights. The calculated value for the partial specific volume was 0.72 ml per g. The weight average molecular weight distribution of Factor XIII in guanidine hydrochloride varied from approximately 76,000 to 90,000 assuming a partial specific volume of 0.73 ml per g. Essentially identical results were obtained in the presence and absence of mercaptoethanol. These analyses indicate that the subunits of Factor XIII differ in molecular weight and are combined in the native zymogen by noncovalent bonds. Plasma Factor XIII contains two distinct electrophoretic species as judged by analysis in gels containing SDS (Fig. 1) or in gels at pH 3.2 containing urea (Fig. 2). The two species, which were separated from one another by freezing aud thawing solutions of Factor XIII as described under Methods, corresponded electrophoretically (Figs. 1 and 2) to either the a chain or b chain of Factor XIII (22). From sediment,ation equilibrium of the pure a chain in 5.9 M guanidine hydrochloride containing 0.01 M dithiothreitol at pH 8, and assuming that the partial specific volume for the a chain is 0.73 ml per g, the molecular weight of the chain was found to be 75,100. If correction is made for preferential binding of guanidine hydrochloride (43) by using a value of 0.72 ml per g for the partial specific volume of a chain, then its molecular weight is 70,200. Because the effects of guanidine hydrochloride are unknown and the larger number is in better agreement with the value from SDS electrophoresis it is assumed that the molecular weight of a chain is about 75,000. Ultracentrifugal analyses of the b chain or its S-carboxymethyl derivative in 6 M guanidine hydrochloride were unsatisfactory because of precipitation of protein at the fluorocarbonsample meniscus. Analysis in 0.16 M potassium chloride, however, was successful, and, assuming a partial specific volume of 0.73 ml per g, the molecular weight of the b chain was estimated to be 87,800. These analyses, although subject to uncertainty in the values of the partial specific volume show that the molecular weights of the a and b chains are different, while earlier estimates from gel electrophoresis studies indicated that each chain has a molecular weight of about 81,000 (22). Nevertheless, the molecular weights measured by sedimentation equilibrium and by SDS electrophoresis are in fair agreement. The fact that the chains are not resolved on gels in SDS containing mercaptoethanol may result from the particular gel systems used or the fact that the b chain has an anomalous mobility because of its high carbohydrate content. These studies show that the molecular weight of native plasma Factor XIII can be accounted for if it is assumed that it contains 2 of each of its subunit chains. This is a reasonable assumption because of the observation that about equal amounts of both chains are observed on gel scans as shown in Fig. 1. In addition, the amino acid composition of Factor XIII is about equal to the average These composition of its 2 subunit chains, as shown below. data confirm the earlier conclusion (22) that the subuuit structure of plasma Factor XIII can be designated a2b2.

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1398

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FIG. 1. A, SDS gel electrophoretic patterns of plasma Factor XIII, platelet Factor XIII, and the isolated subunits of plasma Factor XIII on 7.5Gx polyacrylamide gels in the absence of rea, plasma Factor XIII; b, plasma Factor XIII a ducing agent. chain; c, plasma Factor XIII b chain with a small amount of a

chain contamination; d, platelet Factor XIII with a minor contaminant. These gels show the samples which were used for the sedimentation equilibrium studies. R, gel scan of a sample of nonreduced plasma Factor XIII.

FIG. 2. Electrophoretic XIII on polyacrylamide plasma Factor XIII; Factor XIII b chain platelet Factor XIII. aggregated species.

patterns of plasma gels containing 6 M b, plasma Factor ST11 with a trace of a chain The bands at the top

and platelet Factor urea at pH 3.2. a, a chain; C, plasma contamination; d, of Gels b and d are

:IloZecular Weighf oj Platelet Factor XI/I-Ultracentrifugal analyses of platelet Factor XIII were performed in the absence and presence of guanidine hydrochloride. Molecular weights of 143,200 and 148,000 were calculated from two different analyses in dilute salt solution assuming that the partial specific volume is 0.73 ml per g. In contrast, in 6 M guanidine hydrochloride the molecular weight was 69,700 to 74,700, the exact value depending on the value for the partial specific volume used in the calculations. This subunit molecular weight is lower than that of 81,000 estimated from gel electrophoretic analyses (22), but it is in good agreement with the molecular weight obtained for the a chain of plasma Factor

XIII by sedimentation equilibrium. Native platelet Factor XIII must t.herefore contain 2 subunits ccmbined by noncovalent bonds as suggested earlier (22). Since other studies show that platelet subunits are indistinguishable from the a chains of plasma Factor XIII, platelet Factor XIII can be designated as a?. Amino Acid Composition of Plasma and Platelet Factor XIIITable I shows the amino acid composition of platelet Factor XIII, plasma Factor XIII, and the a chain and b chain subunits of plasma Factor XIII based on 24.hour hydrolyses. Also shown is the complete amino acid composition of plasma Factor XIII expressed as residues per molecule based on 24-, 4%, and 120-hour hydrolyses. The data shown for a chain, b chain, and platelet Factor XIII are in fair agreement with previous analyses from slices of polyacrylamide gels (22). The a chain a,nd b chain have distinct compositions with the greatest differences noted for aspartic acid, alanine, half-cystine, valine, and tyrosine. The composition of plasma Factor XIII is in good agreement with the average composition of the a and b chains. In accord with earlier findings (22) the composition of platelet Factor XIII is indistinguishable from that of the a chain. The complete amino acid composit.ion of plasma Factor XIII is in good agreement with that of Bohn (36). A value of 78 residues of half-cystine per molecule of native plasma Factor XIII was measured by converting all of the cysteine and cystine to cysteic acid using dimethylsulfoxide as an oxidant. A value of 8 residues of cysteine per molecule was measured by carboxymethylating plasma Factor XIII in guanidine hydrochloride in the absence of reducing agent. Carboxymethylation of the fully reduced purified b chain yielded a value of 4.8 residues of

1399
TABLE

Amino

acid composition of platelet and plasma Factor XII1 and the isolated subunits of plasma Factor XIII

Amino acid

Plasma Factor XIIP

Platelet actorXII1~
(I -

:F actor XIII* -residues/ mo6ecu6c

Plasma

eyue~~soo

Lysine.. Histidine. . Arginine.. . Aspartic acid. Threonine . . Serine. .. . Glutamic acid. Proline. . . Glycine. Alanine. . . Half-cystine. Valine. . Methionine. Isoleucine Leucine. . . . Tyrosine . . Phenylalanine Tryptophan

. .

. . .

5.8 1.9 6.1 12.8 6.2 7.4 11.4 5.1 7.9 6.3 0.9 7.4 2.6 3.7 7.2 3.6 4.1

5.8 2.2 5.8 12.8 6.0 8.2 12.1 5.1 8.6 6.2 0.8 7.0 1.9 3.5 6.9 3.4 4.0

7.2 2.7 4.4 9.4 7.0 7.8 13.0 6.8 7.2 3.5 5.0 3.7 1.3 3.5 7.7 6.1 3.3

6.5 2.5 5.1 11.1 6.5 8.0 12.6 8.0 7.9 4.8 2.9 5.4 1.6 3.5 7.3 4.8 3.7

6.4 2.3 5.2 10.7 6.7 7.2 12.1 6.0 7.9 4.5 3.1 5.5 1.9 3.5 7.3 5.3 4.0

167.4 60.8 129.1 264.6 177.7 195.8 304.2 150.2 290.2 107.1 77.8 187.8 46.4 118.8 196.0 135.2 105.3 57.ld

FIG. 3. Aggregates of the a, chain of Factor XIII detected by gel electrophoresis at pH 8.9. Native plasma or platelet Factor XIII when thawed after storage at -20 in 0.05 M Tris-HCl0.001 M EDTA, pH 7~5, contains *a flocculent white precipitate which is pure a chain. The precipitated a chain may be dissolved by raising the pH. Gel A, Dative plasma Factor XIII; Gel B, plasma Factor XIII which had been precipitated and redissolved. Note the dissociated b chain and the a chain aggregates (multimew); Gel C, the precipitate from plasma Factor XIII which had been separated by centrifugation and redissolved; Gel D, the redissolved precipitate from platelet Factor XIII.

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a These data are based on duplicate 24-hour hydrolysates. *These data are based on duplicate 24, 48, and 120-hour hydrolysates. A molecular weight of 320,000 minus the 4.9% by weight carbohydrate (36) of the molecule was used in the calculations. Threonine and serine values are based on zero time extrapolations. Valine and isoleucine values are from the 120hour hydrolysates. c Determined as cysteic acid after oxidation with dimethylsulfoxide (32) and directly from the long column analysis of the 24-hour hydrolysate. Carboxymethylation yielded a somewhat lower number of 2.54 residues/RIO residues. d Determined spectrophotometrically by the method of Edelhoch (31).

FIG. 4. Recombination of native platelet Factor XIII with purified b chain from plasma Factor XIII. Electrophoretic patterns on pH 8.9 polyacrylamide gels of: A, native platelet Factor XIII (25 ~1, absorbance at 230 nm of 0.2) ; B, plasma Factor XIII b chain (10 pl, absorbance at 280 nm of 0.57) ; C, native platelet Factor XIII (50 pl) plus plasma Factor XIII 6 chain (10 ~1); D, native plasma Factor XIII (5 ~1, absorbance at 280 nm of 2.1).

half-cystine per 100 residues,while the pure a chain was calculated to have 0.8 residue of half-cystine per 100 residues. From thesedata it was calculated that each b chain contains at least 15 intramolecular disulfide bonds, while each a chain contains at most two intramolecular disulfidebonds. Separationand Recombination of a and b chains-Frozen solutions of platelet and plasmaFactor XIII which had been dissolved in Tris-HCl, pH 7.5, and EDTA contained precipitates when thawed. Electrophoretic analysis indicated that the precipitate was essentially pure a chain, as shown in Fig. lA, Gel 6. The supernatant from thawed solutions of the plasma factor was found to contain essentiallypure b chain (Fig. lA, Gel c). Several important observations can be made because of the easein separating the chains of plasma Factor XIII, although it shouldbe emphasized that frozen and thawed solutions of Factor XIII under these conditions lose considerable activity. Firstly, the a chains prepared in the samemanner from each factor behave identically on electrophoresisat pH 8.9 (Fig. 3) and at pH 3.2 in urea (Fig. 2). The multiple species seenat pH 8.9 result from noncovalent aggregation. Secondly, the gelsat pH 3.2 show that there are two finely resolved species of a chain. Charge heterogeneity for platelet Factor XIII was noted earlier by Bohn (36); however, he found that

the platelet Factor XIII differs in mobility from either of the two chains of the plasmafactor. Finally, as shown in Fig. 4, it was possibleto combine equal amounts of platelet Factor XIII and the purified b chains and generate a specieswhich was identical electrophoretically to plasmaFactor XIII. These results strengthen the conclusionthat platelet factor contains only a chainsand that theseare identical with those of plasma Factor XIII. Stability of Factor XIII-Although the platelet and plasma factors could not be stored frozen in Tris-EDTA buffers because the a chains precipitated, it was found that solutionsof both factors could be stored frozen in 0.05 M sodiumcitrate, pH 7.1, containing 0.158 M potassiumchloride or in distilled water for at least 10 to 12 weekswithout lossof activity. Lyophilized, salt-free preparations lost roughly one-third of their activity when stored at -20 for 3 weeks. Using dithiothreitol in place of cysteine in the clot solubility assay routinely increasedthe Factor XIII titer from 2- to &fold. More striking was the finding that a solution of platelet Factor XIII, the titer of which had dropped from l/20,480 to Jic after freezing in a Tris-EDTA buffer, had its titer increase l/5,120 whencysteine to wasreplacedwith dithiothreitol.

1400 Activation of Factor XIII by Proteolytic Enzymes--It was noted earlier (22) that activation of Factor XIII zymogen by thrombin resulted in a decrease in the molecular weigl$ of the a It was chain although the b chain was apparently unaffected. concluded that the degraded a chain, called the a chain, was the enzymatically active subunit. The activation of plasma Factor XIII by other proteolytic enzymes has been followed by gel electrophoresis in SDS as shown in Fig. 5. The structural changes accompanying thrombin activation are included for comparison. As judged by the [Wlputrescine assay, Factor XIII was fully activated by t.rypsin and by a partially purified preparation of ancrod. Gel analyses of Factor XIII activated by these enzymes showed that the a chain was formed but that the b chain was unaltered as observed on activation with thrombin. In contrast, pure ancrod neither activated Factor XIII nor appeared to alter it structurally. Reptilase only partially activated Factor XIII under the conditions used, and accordingly, smaller amounts of the a chain appeared to have been formed. Although papain activated Factor XIII it appeared to Hecause degrade extensively the b chain as well as the a chain. papain was found to retain some activity in SDS, it was inactivated with iodoacetamide after reaction with Factor XIII and before electrophoretic analysis. Finally, neither a-chymotrypsin nor plasmin activated Factor XIII, in accord with an earlier report (8), and a chains were not observed on electrophoretic analysis. The SDS gel electrophoretic patterns of carboxymethylated plasma Factor XIII zymogen- and thrombin-activated plasma Factor XIII (Fig. 5B) show clearly that only t.hc a chain increases in mobility upon activation. In every case where Factor XIII was activated by a proteolytic enzyme, calcium was not required. Under the conditions of activation described in Fig. 6, thrombin completely activates plasma Factor XIII in 5 min. Activity was subsequently lost very slowly with 85% of the initial activity remaining after 17 hours of incubation. During the incubation the a chain was cleaved in part to fragments of 24,000 and 56,000 molecular weight, but the quantity of these fragments did not increase proportionally with time. Platelet Factor XIII under the experimental conditions in Fig. 6 was largely activated in 5 min, was most active after 120 min, then slowly lost activity with 66% of the maximum activity remaining after 25 hours. The platelet a chain was also cleaved in part to fragments of 24,000 and 56,000 molecular weight. In contrast to thrombin, trypsin extensively digests plasma Factor XIII on prolonged reaction and depending on the experimental conditions selectively degrades either the a chain or the b chain. Fig. 7 shows the effect of trypsin on the activity of the factor as well as the electrophoretic analyses of the reaction mixtures with time. In the presence of Ca*+ and dithiothreitol, trypsin degrades the a chain faster than the b chain, and the extent of inactivation is roughly prol~ortional to the loss of a chain and the appearance of digestion products of lower molecu111 the absence of Ca2+ and dithiothreitol, b chain lar weight. is degraded more rapidly than the a chain and the rate of inactivation is much less. These data provide additional evidence that the a chain has the enzymatic activity of plasma Factor XIII. Finally, plasma Factor XIII zgmogrn was not activated by a&rated Factor XIII. Whnl 30 pccgof plasma Factor XIII

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FIG. 5. Proteolytic activation of plasma Factor XIII. A, SDS electrophoretic patterns on 7.5c/* polyacrylamide gels in the presence of reducing agent of plasma Factor XIII which had been incubated with sodium chloride or a proteolytic enzyme as follows: a, 0.15 M sodium chloride; b, pure human thrombin; c, impure ancrod; d, pure ancrod; e, reptilase; f, trypsin; g, papain. H, SDS electrophoresis of carboxymethylated Fact,or XIII and carboxymethylated, thrombin-activated Factor XIII. Activation mixtures were as follows. Ancrod: To 100 ~1 of plasma Factor XIII (1 mg per ml) in distilled water were added 80 ~1 of 0.01 M cysteine (pH 7.0, adjusted with solid Tris) containing 0.15 M sodium chloride and 200 ~1 (2 units) of ancrod. After 30 min at 25 aliquots were assayed and prepared for electrophoresis. lieptilase: To 150 ~1 of plasma Factor XIII (0.73 mg per ml)

dissolved in 0.05 Y Tris-HCI, pH 7.5, containing 0.01 M EDTA and 0.15 M sodium rhloride were added 30~1 of 0.05 M dithiothreitol in the above buffer and 120 ~1 (0.5 unit) of reptilnse. After 180 min at 25, aliquots were assayed and prepared for electrophoresis. Papain : To 1OOJ of plusma Fact,or XIII (1.5 mg per ml) dissolved in 0.05 M Tris-HCl, pH 7.5, containing O.COl M EDTA, 0.15 M sodium rhloride, and 0.01 M dithiothreitol were added 100 ~1 of papain (20 mg per ml) dissolved in the above buffer containing 0.01 M dithiothreitol. After 60 min at 25, a lo-p1 aliquot was assayed and the remaining solution was added in the dark to 1 ml of 0.1 M Tris-HCl, pH 8.2, containing 0.0021 M iodoacetamide for 15 min and then prepared for electrophoresis. Thrombin and trypsin activstion mixtures are described in the legends to Figs. 9 and 10.

1401 result of contaminating traces of thrombin converting the fibrinogen to fibrin, control studieswere performed in the presence of hirudin, a potent antithrombin. This agent effectively inhibited thrombin conversionof fibrinogen to fibrin and did not affect the courseof cross-linkingof fibrinogen as judged by gel
electrophoretic analyses as shown in Fig. 8. Finally, it is note-

FIG. 6. Action of thrombin on Factor XIII ss seen by SDS electrophoresis of reduced samples on 7.5y0 polyacrylamide gels. A, plasma Factor XIII incubated with thrombin. A mixture of 0.9 ml of plasma Factor XIII (0.5 mg per ml) in 0.05 M Tris-HCl containing 0.001 M EDTA, pH 7.5, and 0.05 ml 110 units) of pure human thrombin was incubated at 25, and at intervals aliquots and preparedfor SDS electrophoresis. R, platelet were assayed Factor XIII incubated with thrombin. A mixture of 0.3 ml of platelet Factor XIII (0.8 mg per ml) in 0.015 M sodium chloride, 0.03 ml of 0.3 M Tris-HCl containing 0.1 M dithiothreitol, pH 7.5, 0.03 ml (6 units) of pure human thrombin, and 0.24 ml of 0.1 M Tris-HCI containing 0.002 M EDTA, pH 7.5, was incubated at 25; at intervals aliquots were either assayed or prepared for SDS electrophoresis. The arrows indicate impurities in the prepsretion examined.

symogenwere incubated with 3 pg of activated plasmaFactor XIII in a pH 7.5 Tris buffer for 1 hour in the presence and absenceof 0.01 M calcium chloride, there was no increasein activity nor did additional a chain appearwith time asjudged by electrophoreticanalysis. Cross-linkingof IGrimgen and Fib&i by Factor XIII and Guinea Pig Liver Transglutaminase-Fibrin is rapidly a.nd extensively cross-linkedby Factor XIII (23) and is its normal substrate, but it has been found that fibrinogen can also be cross-linkedby Factor XIII. Fig. 8 showsa time courseof the cross-linkingof fibrinogen under conditions which would result in the complete cross-linkingof the y chains of fibrin within 1 min if thrombin were present. The pattern of cross-linkingis somewhatdifferent from that of fibrin. The y chainsare crosslinked very slowly and coincidently with the cross-linkingof the a(A) chains. Under these conditions all of the y chains and ar(A) chainshave become cross-linked 24 hours. The amount by of y dimers was much lessthan the amount of /3(U) chains. Thus, somey chains may be in the extensively cross-linked species the top of the gel. The /3(U) chainsdid not appear at to be cross-linkedas is the casewith fibrin. To exclude the possibility that the apparent cross-linkingof fibrinogen was the

worthy that during cross-linkingof fibrinogen no gel was formed and only a finely dispersedprecipitate was observed after 24 hours reaction. These results indicate that neither releaseof fibrinopeptidesnot gelation is essential cross-linkingalthough for they markedly influencethe rate and the pattern of cross-linking. Fibrinogen was also cross-linkedby guinea pig liver transglutaminase,as shownin Fig. 8B, Gel c. Not only the y chains and a(A) chains but also the P(R) chains becamecross-linked after incubation with a high concentration of guinea pig liver transglutaminase. Fig. 9 compares cross-linkingof human fibrin by activated the plasma Factor XIII and by guinea pig liver transglutaminase. Pure human thrombin, Factor XIII-free fibrinogen, and either pure plasma Factor XIII or pure guinea pig liver transglutaminase were usedin thesestudies. Cross-linkingof the y chains is catalyzed by trace amounts of Factor XIII, while a much higher concentration is required to cross-link the LYchains. y dimerization is complete before cross-linking of the a! chain commences. With guinea pig liver transglutaminase the a! chainsare cross-linked somewhatfaster than the y chains. If y dimers are formed at all, they are only a transient species. Eventually all of the (Ychainsand y chainsare cross-linked into a very high molecular weight complex which barely enters the gel. Activated plasma Factor XIII did not appear to cross-link any human serumproteins in contrast to guinea pig liver transglutaminase which cross-linked several of the human serum proteins (Fig. 10). Lysis Time Versus Degree of Cross-1inkiwFig. llii shows that the lysis time is prolongedby a factor of two for fibrin which is formed in the presence high concentrationsof Factor XIII of when comparedto fibrin formed in the presence low concenof trations of Factor XIII. Furthermore, the latter have lysis times which are indistinguishable from those of completely noncross-linked clots. Fig. 11B showsthe SDS gel patterns of fibrin clots corresponding the points on the graph in Fig. 11A. to It is evident that the prolongation of lysis times coincideswith the appearanceof extensively cross-linked(Y chains and that thoseclots with the longestlysis times have virtually all of their (Y chains cross-linked. The cross-linkingof y chains doesnot appear to increasethe lysis time sinceno differencein lysis time was noted for completely uncross-linkedfibrin and fibrin with completely cross-linkedy chainsbut essentiallyno cross-linked g chains.
DISCUSSION

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of Plasmaand Platelet Factor XIZZ-Several observations reported here provide additional support for the earlier suggestion(22) that plasmaFactor XIII is composed, of, 2 nonidentical subunits, which are designatedas the a and b chains, and that 2 of each of the subunits are combinednoncovalently in the native moleculeso asto give the subunit structure eb2. (a) The molecularweights of the a and 6 chainsare approximately 75,000 and 88,000, respectively, as judged by sedimentationequilibrium analyses,and twice the sum of their weights accounts well for the molecular weight of the native molecule. (b\ The weight average molecular weight distribuSubunit Structure

1402 --A.

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PRESENT 0 I ABSENY

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F r\

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CALCIUM-DifHtOTHRElTOL PRESENT (l I

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01

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FIG. 7. The action of trypsin on human plasma Factor XIII. A, SDS electrophoretic patterns on 7.5% polyacrylamide gels in the presence of reducing agent. The first gel shows plasma Factor XIII zymogen. The next three gels show the activation and digestion of plasma Factor XIII by trypsin in the presence of calcium and dithiothreitol. The final three gels show the activation and digestion of plasma Factor XIII by trypsin in the absence of calcium and dithiothreitol. R, Factor XIII activity as a function of time of reaction with trypsin. The Factor XIII activity at each time has been divided by the activity of an equivalent tion of the Factor XIII varies from about 76,000 to 90,000 in guanidine hydrochloride in the presence or absence of mercaptoethanol, indicating that the subunits are not combined through disulfide bonds. (c) The a and b chains are the fundamental subunits of plasma Factor XIII, because they are not reduced

sample that had been fully activated by thrombin. The following incubation mixtures were used: (a) 0.62 ml of plasma Factor XIII (1 mg per ml) in 0.05 M Tris-HCl containing 0.001 M EDTA, pH 7.5, plus 0.01 ml of trypsin (1 mg per ml) at 25; (5) 0.57 ml of plasma Factor XIII (0.87 mg per ml) in 0.05 M Tris-HCl containing 0.001 M EDTA and 0.15 M sodium chloride, pH 7.5, plus 0.02 ml of trypsin (1 mg per ml) and 0.06 ml of 0.03 M dithiothreitol containing 0.15 M sodium chloride and 0.1 M calcium chloride.

the compositionof the native factor is essentiallyidentical with twice the average compositionof the two chains. (e) The two chains appear to be present in about equal amounts as judged by gel electrophoresis (Fig. 1B). Platelet Factor XIII clearly contains only a chains and has in size by reduction and carboxymethylation. (d) The amino the subunit structure oz. Earlier studiesby Schwartz et al. (22) acid compositions of the a and b chains differ significantly, while showedthat plasmaFactor XIII a chain and the singletype of

1403

Fro. 8. The cross-linking of fibrinogen by Factor XIIIa and guinea pig liver transglutaminase. A, SDS electrophoretic patterns on 7.59& gels of fibrinogen cross-linked by Factor XIIIa. To 250 ~1 of urea-treated fibrinogen (2 mg per ml) in 0.3 M sodium chloride at 25 were added 250 pl of 0.1 M Tris-HCl containing 0.01 M dithiothreitol and 0.04 M calcium chloride, pH 7.5, and 25 pl of Factor XIIIa. At the intervals shown, aliquots were subsubunit

mitted to electrophoresis. B, SDS electrophoretic patterns of fibrin formed from a mixture of urea-treated fibrinogen, Factor XIIIa, and thrombin (a) ; urea-treated fibrinogen cross-linked by Factor XIIIa in the nresence of 10 units of hirudin and 0.4 unit of thrombin (5); and-urea-treated fibrinogen cross-linked by the liver enzyme (fibrinogen-enzyme, 8:1, w/w).

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workers have of platelet Factor XIII had similar amino acid composi- ing in a Tris-EDTA buffer, may explain why some tions and were not stained by the periodic acid-Schiff baserea- found much lower values for the molecular weight. There hasbeengeneralagreement that the molecularweight of gent. They alsohad the samemobility on SDS polyacrylamide gel electrophoresisand had the same decreasein molecular platelet Factor XIII is lessthan that of plasmaFactor XIII, and from its sedimentationcoefficient and carbohydrate content weight upon thrombin activation (22). In addition, the patto tern of fibrin cross-linking by plasmaFactor XIII could not be I3ohnpredicted a value of 150,000 200,000(36). Cther values distinguishedfrom that by the platelet factor. The data given between 110,000 and 150,000 have been estimated from gel in here show that platelet and plasma Factor XIII a chain have filtration (50, 51). The value of 146,000f 10,000measured additional properties in common. They have the samemolecu- this study by sedimentation equilibrium is based on a more lar weightsas measured sedimentationequilibrium, the same reliable technique than used in earlier work. Nevertheless, by mobility on pH 3.2 urea gels, form noncovalently linked ag- Bohn (36) suggestedthat platelet Factor XIII has subunits gregateswhen frozen in a Tris-EDTA buffer, and are converted since its sedimentation coefficient decreasedfrom 7.4 under by thrombin to an active species with a molecular weight of native conditions to 3.25 in 1 K propionic acid. This is in accord 77,OOO, which is subsequently degradedto species with molecular with the studiesreported here which show a subunit molecular weight of 81,000 by SDS polyacrylamide gel electrophoresis weightsof 56,000and 24,000. In addition, plasmaFactor XIII b chain combineswith native platelet Factor XIII to give a and 69,700to 74,700by sedimentationequilibrium in guanidine species indistinguishablefrom plasma Factor XIII on disc gel hydrochloride. Loewy et al. (45) found that on dilution or after storagean electrophoresis pH 8.9. These observations taken together at of provide compelling evidence that plasma Factor XIII a chain inactive 110,000molecular weight subunit with an ezo,W 4.6 from plasmaFactor XIII, and concludedthat plasma is identical with the single type of subunit of platelet Factor dissociated Factor XIII is composedof 3 identical subunits. Bohn (49) XIII unless subtle differences in amino acid sequence are shown later showedthat plasma Factor XIII sedimented 5 M urea in by further analysis. The identity of the a chain of plasma Factor as two different species with sedimentationcoefficientsof 3.45 XIII with platelet Factor XIII also provides a simple explanation for the observation that patients with a congenital Factor and 4.95 (36). In addition, he noted that plasmaFactor XIII XIII deficiency lack both the plasma and the platelet factor dissociatedreleasinga subunit which he called split product with an ~20,~ f 4.7 and a molecular weight of approximately o (44). 100,000, It would appear that the dissociating subunit of The studiespresentedhere may clarify earlier conflicting reLoewy and the split product of Bohn are the same the plasma as ports about the molecular weights and subunit structures of Factor XIII b chain. Furthermore, the subunit of Bohn (36) plasmaand platelet Factor XIII. Loewy et al. (45) reported a which has a sedimentationcoefficient of 3.45 in urea appearsto molecular weight of 35O,OOO Bohn (36) a value of 290,000 and be the sameas the a chain of plasmaFactor XIII. Although by the methodsof sedimentation-diffusion. From gel filtration Bohn (36, 52) concluded earlier that the platelet and plasma studiesothers (46,47) have estimatedthat the molecularweight factors differ, his immunological data support the structures wasbetweenlOO,OOO 32O,OOO.Lorand hasestimatedvalues proposedhere. Rabbit anti-plasmaFactor XIII immunoserum and ranging from 100,OOO to 156,000to 195,000(5). The value formed two precipitin lines againstplasmaFactor XIII but only (48) of 320,000 f 20,000obtained by the sedimentationequilibrium one against,platelet Factor XIII, which also formed a line of measurements reported here representsthe most careful deter- identity with one of the precipitin lines against plasmaFactor mination to date and is based on a measuredpartial specific XIII. The secondprecipitin line against plasmaFactor XIII volume. The fact that plasmaFactor XIII can be dissociated formed a line of identity with that formed by anti-split product at low pH, by heating in the presence EDTA (49) or by freez- (b chain) and plasmaFactor XIII. of It was also shownthat an

1404

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FIG. 9. The cross-linking of fibrin by purified plasma Factor XIII and guinea pig liver transglutaminase. A, SDS electrophoretic patterns on 7.5% gels of human fibrin cross-linked by The ratio of Factor XIII to fibrin human plasma Factor XIII. by weight in the samples from left to right in the figure was 1:430,000; 1:27,300; 1:3,400; 1:430; and 1:107. The fibrin was formed for 2 hours by adding 10 ~1 of human thrombin (7 units per ml) in 0.8 M calcium chloride to 300 rl of solution containing a variable concentration of Factor XIII and the following invariant components: urea-treated fibrinogen (1 mg per ml), 0.15 M sodium chloride, 0.0005% (v/v) mercaptoethanol, 0.05 M Tris-HCl, pH 7.5. B, SDS electrophoretic patterns on 7.5% gel of human fibrin cross-linked by the liver enzyme. The ratio of liver enzyme to fibrin by weight in the samples from left to right is 1:240, 1:60, 1:30, and 1:15. The fibrin was formed for 2 hours by simultaneously adding 10 ~1 of thrombin (50 units per ml) in 0.5 M calcium chloride and 5 ~1 of varying concentrations of the liver enzyme in 0.01 M EDTA, pH 7.1, to 200 pl of urea-treated fibrinogen in 0.15 M Tris-HCl, pH 7.5.

FIG. 10. The cross-linking of human serum proteins. SDS electrophoretic patterns on 7.5y0 gels in the presence of mercaptoethanol. A, serum; B, serum plus Factor XIIIa; and C, serum plus guinea pig liver transglutaminase. Human serum was prepared by adding 0.1 ml of 0.2 M calcium chloride containing 0.15 M sodium chloride to 0.9 ml of fresh frozen plasma. The clot which formed was removed at the end of 30 min, and the serum was diluted 1: 1 with 0.1 M Tris-HCl containing 0.2 M sodium chloride, 0.01 M dithiothreitol, 0.04 M calcium chloride, and 0.002 M EDTA, pH 7.5. To 100 ~1 of the diluted serum were added either 20 ~1 of plasma Factor XIIIa or 5 ~1 of the liver enzyme (2.66 mg per ml) and the mixtures were incubated at 25 for 120 min.

antiserumto plasmaFactor XIII inhibited platelet Factor XIII activity and vice versa. It was concluded that the active site regionsof platelet and plasma Factor XIII are identical, but that the two factors not only differ in their physicochemical propertiesbut alsohave nonidenticalsubunits(36,52). Ganguly (51) also has not recognized the relationship between platelet and plasmaFactor XIII. Several lines of evidence indicate that only the a chain of plasmaFactor XIII is catalytically active. Bohn (36) showed that a rabbit immunoserumprepared against the split product (b chain) did not inhibit plasmaFactor XIII activity and that purified split product had very low enzymatic activity. Loewy et al. (45) alsofound that their 4.6 820,~ dissociated subunit had no enzymatic activity. Chung and Folk (53) have found that the plasmaand platelet factor have similar Michaelis constants, but that the maximum velocity per mg of the platelet factor is roughly twice that of the plasmafactor. This is in accord with the observationsreported here that digestion of plasmaFactor XIII with trypsin under conditions in which the b chain was selectively degraded resulted in only slight loss of enzymatic activity, but that under conditions in which the a chain was

selectively degraded there was a marked lossof enzymatic activity. It was also found that the specific activity of platelet Factor XIII was almost twice that of plasmaFactor XIII. It is of interest that the mobility of the b chain on SDS polyacrylamide gel electrophoresis decreases markedly in the presenceof mercaptoethanoland even further after carboxymethylation (Fig. 12). In the absenceof reducing agent two clearly resolvedpolypeptide chainsare found in plasmaFactor XIII on SDS gel electrophoresis. The polypeptide chain with greater mobility is the b chain. In the presence reducingagent there of is only a negligiblechangein mobility of the a chain, while the mobility of the b chain is decreased such an extent that it is to not resolved from the a chain, This changein mobility is that expected since, as noted by Griffith (54), reduction usually decreases mobility of a cystine-containingpolypeptide during the electrophoresis SDS polyacrylamide gels and the magnitude in of the decrease mobility increases in with the number of cystine residuesthat was originally present. The nonreducedb chain contains at least 15 intramolecular disulfide bondsper molecule whereasthe a chain contains at most two such bonds. After carboxymethylation the mobility of the a chain is slightly decreased while the mobility of the b chainis significantly decreased. Maleylation has been reported to decrease mobility of prothe teins in SDS (55), while succinylation and glyoxalation have

k
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FIG. 12. SDS electrophoresis of nonreduced, reduced, and carboxymethylated plasma Factor XIII on 7.5% polyacrylamide gels. A, nonreduced sample; R, reduced sample; and C, reduced carboxymethylated sample. The number to the right of each band is the relative mobility which was measured according to the method of Weber and Osborn (41).

Y-dtmer u-crlolns @-chain Y-cha.n

-~Mmlb--rr*r----

such that not all of the (Y chains were cross-linkedin 2 hours after the addition of thrombin, the addition of dithiothreitol to the clotting mixture resulted in completecross-linkingof the a! chainsin 2 hours. Apparent proteolytic digestion of plasma or platelet Factor XIII was not found with purified preparationsstored at -20
either lyophilized, in glycerol, or frozen. Intact a chain was

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FIG. 11. The effect of cross-linking on the lysis time of a fibrin clot. A 14 tube serial dilution of plasma Factor XIII in Factor XIII-deficient plasma was set up as follows. To the first tube were added 380 ~1 of Factor XIII-deficient plasma plus 20 ~1 of plasma Factor XIII (2.25 mg per ml) in 0.158 M potassium chloride-0.05 M sodium citrate (pH 7.1, adjusted with HCl). To each subsequent tube were added 200 ~1 of Factor XIII-deficient plasma. From the first test tube, 200 ~1 were transferred to the second. The contents of the second tube were mixed, and 200 ~1 were transferred to the third tube, and so on. The contents of each tube were divided into two 100-J aliquots yielding two identical serial dilutions. The contents of each tube were clotted by the addition of 10 1.r1of a mixture of 50 ~1 of human thrombin M (200 units per ml) with 450 pl of 0.2 M calcium chloride-O.15 sodium chloride. After a a-hour incubation at 25 one set of clots To each of the second set of was prepared for electrophoresis. M sodium chloride, clots were added 1 ml of 0.05 M Tris-HCl-0.15 pH 7.4, plus 20 pl of stock urokinese. The clots were dislodged, the tubes were agitated, the tubes were placed in ~37 water bath, Tube number 15 and the lysis time for each clot was recorded. A, clot lysis time as a function contained no added FactorXIII. of degree of cross-linking. Factor XIII activity and the extent of cross-linking decrease with increasing tube number. The data shown are the averages from two separate experiments. B, SDS electrophoretic patterns on 7.5% gels of fibrin from tubes corresponding to those in the graph of lysis time versus tube number. been reported to decrease the SDS binding of a protein (56). Since carboxymethylation introduced approximately 33 carboxyl groups into each b chain, the decrease in mobility of the carboxymethylated b chain could be attributed to a conformational change or altered SDS binding. Lability of Factor SZIZ-Many workers have noted a loss of

Factor XIII activity upon storage and the regenerationof activity by the addition of cysteine. It wasfound in the present is much more effective than cysteine study that dithiothreitol in regenerating Factor XIII activity. Even freshly prepared Factor XIII showsa 2- to &fold increasein activity when assayed with dithiothreitol. In addition, when Factor XIII was added to Factor XIII-deficient fibrinogen in a concentration

found even in preparationsstored for over 1 year. The alterations on storageof platelet Factor XIII describedby Ganguly (51) were alsonot observed. Aggregation of Factor XIII preparations could be a serious problem for future studiesif suitable precautionsare not taken. When either platelet or plasmaFactor XIII is frozen overnight in Tris-EDTA at pH 7.5, a precipitate forms, although no such problem is encountered on storage at 4. Since this type of buffer is recommendedfor chromatography of plasma Factor XIII (57) care must be taken not to freeze the solutions. As shown here, the factor remainssoluble in distilled water or in sodium citrate-potassium chloride buffers even after freezing and thawing. Precipitation is not the result of Factor XIII cross-linkingitself or of disulfideinterchange, sinceonly a single band is observedwhen the precipitate is examinedby SDS polyacrylamide gel electrophoresis the absence reducing agent. in of Activation of Factor XIII-Platelet Factor XIII was shown by Buluk (1) to be a zymogen which is activated by thrombin, and later a similarobservationwasnoted for plasmaFactor XIII (2). The earlier suggestionthat calcium is required for the activation step (1, 2) hasbeenshownto be incorrect (12). Only indirect evidence has been provided heretofore that the activation by thrombin is accompaniedby proteolytic cleavage of both factors. The earlier evidence was basedon charge electrophoresis (5, 49) and immunological (51, 58) differencesbetween the factors before and after treatment with thrombin, while the studies reported here using electrophoresis SDS, in clearly showthat activation involves only a limited proteolysis and that when both factors werefully activated the only change found was a decrease molecular weight of the a chain from in 81,000 to about 77,000. The exact nature of the activation process,including the number of bonds split and the size of the peptides released, requires further study. On prolonged incubation of both factors with a high concentration of thrombin only a smalldecrease activity was noted after several hours, in and the a chain was partially degraded into fragments with molecularweightsof 24,000and 56,000during this time. These studies suggestthat thrombin, alone, may not be responsible

1406 for the rapid inactivation of Factor XIII in vivo. Finally, the studies reported here not only confirm earlier observations that plasma Factor XIII is activated by trypsin (5, 7, 8, 5Q), reptilase (s-12), and papain (9) but, in addition, show that activation by these different enzymes is accompanied by formation of the a chain and also that plasma Factor XIII is activated in the same manner by an enzyme in the venom of Agkistrodon rhodostoma which is a contaminant in some ancrod preparations. Pure ancrod does not activate Factor XIII (60). In contrast to earlier reports, however, activated Factor XIII does not activate Factor XIII zymogen (7) nor is papain able to replace Factor XIII as a clot-stabilizing enzyme (13, 14). Cross-linlcing of Fibrinogen and Fibrin-McKee et al. (23)
showed earlier that when human fibrinogen cont.aminated with

Factor XIII is clotted with bovine thrombin, cross-linking of the y chains of fibrin is complete in about 1 min but cross-linking of the a chains occurs much more slowly. Studies reported here show essentially the same results in a system containing pure human thrombin, pure human Factor XIII-free fibrinogen, and pure human plasma Factor XIII. Clots incubated for 2 hours had all of the 01 chains cross-linked when the ratio of fibrinogen to plasma Factor XIII (mg per mg) was 107 : 1. When the ratio was 6800: 1 all of the y chains were converted to y dimer, and even when the ratio was 110,000: 1 a distinct y dimer band could still be seen. It is of interest that fibrinogen as well as fibrin can be crosslinked by activated plasma Factor XIII although the rate of cross-linking is extremely slow. Lorand et al. (61) have previously demonstrated that activated plasma Factor XIII can incorporate [14C]glycine ethyl ester into fibrinogen but at a much slower rate than into fibrin. In addition, Sasaki eCal. (62) have demonstrated that fibrin-fibrinogen complexes can be crosslinked by activated Factor XIII. Lorand et al. (61) have em-

lysis times which were no longer than completely soluble clots It appeared that this might be explained on the basis that insoluble clots can have markedly varying degrees of (Y chain crosslinking as shown in earlier studies (24), and that resistance. to plasmin might be correlated with the degree of (Y chain crosslinking but not y chain cross-linking. The study reported here demonstrates that increased clot lysis times coincide with crosslinking of cy chains and that, c1ot.s with fully cross-linked LYchains but only a trace of cross-linked a! chains have lysis times only slightly different from those of noncross-linked clots. These studies were performed with human Factor XIII-deficient plasma, pure human Factor XIII, and pure human thrombin, with the only variable being Factor XIII concentration. Urokinase was added at the end of 2 hours rather than before the addition of thrombin because the my chain of fibrinogen is the subunit which is most rapidly degraded by plasmin (72, 73) ; hence, it would have been impossible to study the effect of cross-linking of LY chain on lysis time if the a(a) chain had been degraded before cross-linking had commenced.
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REFERENCES 1. BULUI~ K., JANUSZICO, T., .IND OLBHOMSI<I, J. (1961) :Yatzdre 191, 1093-1094 2. LOR~ND, L., AND KONISHI, K. (1964) Arch. Biochem. Biophys. 106, 58-67 3. PIS~NO, J. J., FINL~YSON, J. S., AND PEYTON, M. P. (1968) Science 160, 892-893 4. MATAEIF, S., AND LOEWY, A. G. (1968) Biochem. Biophys. Res. Commun. 30, 356-362 5. LOIXAND, L., DOWNICY, J., GOTOH, T., JXOBSEN, A., .~ND To~un.4, S. (1968) Biochem. Biophys. Res. Commun. 31, 222-230 6. CHEN, I~., AND DOOLITTLE, IL. F. (1970) Proc. Nat. Acad. Sci. U. S. A. 66, 472-479 7. KONISHI, K., AND TIICIGI, T. (1969) J. Riochem. 66, 281-284 8. KOPI+ M., La~atto, Z. S., ST.IHL, M., .IND WI~:GRZYSO~ICZ, A. (1969) Biochim. Biophys. Acta 181, 437-445 9. BULUK, K., AND ZUCH, A. (1967) Biochim. Biophys. Acta 147, 593-594 10. Josso, Z. (1963) in Fibri,loge,L arid Fibrin Turnover of Clotting Factors (KOLLE:R, A., ed) p. 138, Schattauer Verlag, Stuttgart 11. DVIL~NSKY, A., BRITTEN, A. F. H., .\ND LOEWY, A. G. (1970) . Brit. J. kaerkalol. 18, 399-410 12. TYLICR, H. M. (1970) Biochim. Biophys. Acta 222, 396-404 13. LORAND, L., >~ND KONISHI, K. (1962) Biochem. Biophys. Res. Commun. 7, 457-460 14. LORAND, L., AND KONISHI, K. (1964) Biochemistry 3, 915-919 15. TYLER, H. M., AND L.4~1, K. (1966) Biochem. Biophys. Res. Commun. 24, 506-512 16. LORAND, J. B., UR;!Y.\M.Y, T., SND LOI~~ND, L. (1966) Biochem. Biophys. Res. Commun. 23, 828-834 17. ROHBINS, K. C. (1944) Amer. J. Physiol. 142, 581-588 18. LAKI, K., AND LOI~~ND, L. (1948) Science 108, 280 19. BULUIC, K. (1955) Pal. Tyg. Lek. 10, 191 20. L~SCHER, E. F. (1957) Schweiz. Med. Wochenschr. 67, 1220-1221 21. BOHN, H., AND SCHWICK, H. G. (1971) Arzneimittel-Forschung 21, 1432-1439 22. SCHWARTZ, M. L., Przzo, S. V., HILL, R. L., AND MCKEE, P. A. (1971) J. Biol. Chem. 246, 5851-5854 23. MCKEE, P. A., MATTOCK, P., AND HILL, R. L. (1970) Proc. Nat. Acad. Sci. U. S. A. 66, 738-744 24. SCHWARTZ, M. L., PIZZO, S. V., HILL, R. L., AND MCKEE, P. A. (1971) J. Clin. Invest. 60, 1506-1513 25. BLOMBBCK, B., AND BLOMBSCK, M. (1956) Ark. Kemi lC, 415443 26. FOLK, J. E. (1970) Methods Enzymol. 17A, 889-894 27. PIZZO. S. V.. SCHWARTZ. M. L.. HILL. 1%. L.. AND MCKEE. P. A. (197i) J. Biol. Chem. 24i, 636-645 28. DVILANSKY, A., BRITTEN, A. F. H., AND LOF>WY, A. G. (1970) Thromb. Diath. Haemorrh. 24. 256-264

phasized the importance of the release of the fibrinopeptides in order to expose the cross-linking sites of the fibrinogen molecule; however, it has not been proven that it is the release of the fibrinopeptides rather than the specific orientation of the fibrin monomers in the fibrin gel that accounts for the difference in the rat.e of cross-linking observed for fibrin and fibrinogen. Konishi and Takagi (7) noted an increase in turbidity of a fibrinogen solution when activated Factor XIII was added and concluded that the Factor XIII had a proteolytic effect on fibrinogen. It is more likely that this increase in turbidity was a result of the formation of colloidal size cross-linked networks of fibrinogen. No proteolytic effect of activated Factor XIII
011 fibrinogen

was

found

in this

study;

furthermore,

it has now

been shown rather convincingly that activated Factor XIII is a transglutaminase (3, 4, 53) and not a transpeptidase. Guinea pig liver transglutaminase cross-links both human fibrinogen and fibrin as noted earlier (15, 16, 63, 64) and in this study. In accord with Chung (64) it was found that this enzyme cross-links the cu(A) chains, /3(U) chains, and y chains of human fibrinogen into a very high molecular weight network. Although it was shown previously that the transglutaminase stabilizes fibrin clots (15, 16)) the studies here and those of Chung et al. (65) show that the pattern of cross-linking of fibrin is different from that catalyzed by Factor XIII, the most striking difference being the absence of cross-linking between two y chains (y dimers) . Lysis Time Versus Degree of Cross-Linking-There have been several reports that cross-linked clots are more resistant to fibrinolysis than noncross-linked clots (65-71). It was noted by Gormsen et al. (69), however, that some insoluble clots had

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