British Journal of Haematology, 1990, 74, 290-294

.A familial factor XI11 subunit B deficiency

M A S A N O R I S A I T O , HIDESAKU A S A K U R A , TOMOTAKA YOSHIDA,* KEIKO ITO, KAZUHIRO O K A F U J 1 , t

‘I’AKAsHI YOSHIDA A N D TAMOTSU MATSUDA Department of Internal Medicine (111) and *Central Clinical Laboratory, Kanazawa University School of Medicine, Kanazawa, and ?Department of Internal Medicine, Fukui-Saiseikai Hospital. Fukui, Japan

Received 30 August 1989; accepted for publication 26 October 1989

Summary. A 32-year-old woman with a bleeding tendency born of a consanguineous marriage, was found to have factor XI11 subunit B deficiency. An abnormally low level of factor XI11 activity was initially noticed and this finding led to further studies of the proband and her family. The notable features were: undetectable subunit B of factor XI11 in the proband and her brother and reduced levels of subunit B, 34-52%, in her parents and children. The proband’s brother had a markedly decreased level of subunit A protein. The level of factor XI11 subunit A in platelets of the proband was

Factor XI11 is a zymogen which acts in the final stages of coagulation after thrombin converts fibrinogen to fibrin. In its active form, factor XI11 functions as a transglutaminase to stabilize fibrin clots by catalysing the formation of E - ( Y -

j:lutamyl)-lysyl crosslinks (Folk & Finlayson, 1977). Factor XI11 is composed of two non-identical polypeptides, subunits A and B, which associate noncovalently to form a tetrameric complex (AzBz). Recently. Yorifuji et al(1988) suggested that ihe normal concentration of subunit A is 0.13-0.16 pmol/l. all of which is in A2B2 complex form, and total subunit Bconcentration is 0.26 to 0.28 pmol/l. half of which is complexed while the other half is in free form. The A subunit contains the catalytic site(s) of the enzyme. The B subunit appears to stabilize subunit A (Cooke, 1974; Folk & Finlay- son, 1977) or to regulate the rate of Ca2+ dependent activation of subunit A (Lorand et al, 1974). However, the precise function is not known.

Congenital deficiency of factor XI11 is a rare disorder, characterized biochemically by the absence of plasma factor XI11 subunit A and diminished levels of subunit B protein (approximately S O X of normal levels) (Skrzynia et R Z , 1982). The homozygous deficient state results clinically in a mod- erate to severe haemorrhagic diathesis and poor wound healing. Factor XI11 subunit A deficiency is inherited as an

Correspondence: Dr Masanori Saito. Department of Internal Med- icine (111). Kanazawa University School of Medicine, Takaramachi 13-1, Kanazawa 920. Japan.

normal. The half-life of subunit A determined from the disappearance curve of infused factor XI11 subunit A concen- trate was approximately 3 d and this is the shortest estimate of the half-life of factor XI11 to date. From these results, it is suggested that subunit A is unstable in plasma deficient in subunit B and subunit B stabilizes the A protein.

This is the first report of congenital deficiency of factor XI11 subunit B and this disorder is thought to be inherited as an autosomal recessive trait.

autosomal recessive trait. Until now a total deficiency of subunit B protein of factor XI11 has not been reported and the behaviour of plasma A subunit together with clinical findings in the totally deficient state of B protein are not known.

We report here a family with factor XI11 subunit B deficiency who were associated with a bleeding tendency.

CASE REPORT AND FAMILY HISTORY

The proband is a 32-year-old woman. It is uncertain whether bleeding from the umbilical stump was observed after birth. Since her childhood she had complained of occasional subcutaneous haemorrhage, but she had not been given an evaluation of her bleeding tendency. At the age of 26 she became pregnant and a full-term healthy female was deli- vered by caesarean section performed for cephalopelvic disproportion. On this occasion, abnormal bleeding did not occur. At the age of 31 she became pregnant again and showed massive bleeding from the vagina just before term. Emergency caesarean section was performed and a full-term male was delivered. However, laparotomy was carried out again because of abnormal bleeding at the site of suture and she was transfused with S U of concentrated red cells and 5 U of fresh frozen plasma. At the age of 32 she was sent to US

for an evaluation of her bleeding diathesis. The pedigree of the family is shown in Fig 1 and illustrates the fact that the parents of the proband are cousins. The proband’s sister had

290

Factor X I I l Subunit B Dejciency 291 Table I. Coagulation parameters in the proband's plasma

' 'To O T O

I

1 0

k

I I

,q+l ?I" I 1

@* a* Fig 1. Pedigree of the family. The proband is indicated by arrow (t ) and investigated family members by (*). The parents of the proband are cousins. 0. 0 . Moderately reduced levels of factor XJJJ subunit B: B. 0. No detectable factor XI11 subunit B.

two pregnancies and showed postpartum severe haemor- rhage at both times.

MATERIALS AND METHODS

Coagulation assays. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured using an automatic recorder (Auto-Fi, Dade). Plasma fibrinogen con- tent was determined by the method of Clauss (19 5 7). Quantitative determination of activities of factors 11, V, VII and X was completed using factor-deficient plasma, perform- ing the one-stage PT. Activities of factors VTII. IX, XI and XI1 were measured with factor-deficient plasma. performing the one-stage APTT. Levels of von Willebrand factor antigen were determined using an enzyme-linked immunosorbent assay kit supplied by Diagnostica Stago (Cejka. 1984). Clot solubility test in a solution of 1% monocloroacetic acid was performed as previously described (Laki & Lorand. 1948 ). Factor XI11 activity was measured as transglutaminase activity that was shown to catalyse the incorporation of a fluorescent amine. monodansylcadaverine into casein (Lor- and rt al, 1969; Nishida et d, 1984). Concentrations of subunits A and B of factor XI11 were assayed immunologically in agarose gel containing antibodies according to Laurell's technique (Laurell, 1966). Kesults were expressed as a percentage of the values for pooled normal plasma which consisted of donations from 20 healthy individuals.

Measurement offactor XI11 subiinit A in platelets. Platelet-rich plasma was prepared from blood collected into 1/10 volume of 3.8% sodium citrate. Platelets were washed three times in buffered saline containing 80 mM Na2HP04, 3 . 3 mM KCI, 1.1 mM EDTA (Na4) and 1% bovine serum albumin. Washed

Normal Proband range

Clotting times

APTT (s) PT (s)

Coagulation factors Fibrinogen (g/l) Factor I1 activity (%) Factor V activity (%) Factor VII activity (%) Factor VIII activity (%) von Willebrand factor antigen (%) Factor IX activity (%) Factor X activity (%) Factor XI activity (%) Factor XI1 activity (%) Factor XI11 activity (%)

11.9-13.9 12.3 32.9 <45

2.1 100

71 65 84 67 83 76 90 88 24

1'7-4'1 70-130 70-1 30 70-1 30 70-1 30 70-140 70-1 30 70-1 3 0 70-1 30 70-1 30 65-1 30

platelets were resuspended in the same buffer to a final concentration of 4 x lox platelets per ml. Suspended platelets were solubilized in Triton X-100, and after centrifugation the supernatant was used for measurement of factor XI11 subunit A by Laurell's method. Subunit A in pooled platelets from normal subjects was used as a standard.

Infusion study of factor XI11 concentrate in the proband. The proband was given three ampoules (750 U) of factor XIII concentrate (FibrogamminB) which contains only subunit A. Plasma samples were collected before the infusion and at 3.6. 12. 24 h and 2, 3 , 4, 5, 6 . 7 . 8 d after the infusion. Levels of subunits A and B of factor XIII were measured by Laurell's technique.

RESIJLTS

Platelet count and aggregation studies were normal in the proband (data not shown). Coagulation screening tests and levels of coagulation factors in the proband are shown in Table I. Activities of coagulation factors 11, V, VII, VIII, IX, X, XI and XI1 were within normal limits. The plasma clot of the proband did not dissolve in a solution of 1 "/o monochloroacetic acid within 2 4 h. The only abnormality observed in the coagulation study was a low level of activity of factor XI11 (24%). This finding led to further studies of the proband and her family. Activities of factor XI11 and levels of subunits A and Bin the family members are summarized in Table 11. The notable features were undetectable subunit B of factor XIII in the proband and her brother and reduced levels of subunit B at 34-52% of normal level in the plasma of her parents and children. In the proband and her brother and son. levels of factor XI11 subunit A were reduced in parallel with the decrease in activity of factor XIII. Levels of subunit A were normal in the proband's parents.

The level of factor XI11 subunit A in platelets of the proband was also measured. Platelet extract from the proband had a normal level of factor XI11 subunit A (1 3 5%).

292 Masanori Saito et al Table 11. Plasma levels of factor XI11 activity subunits A and B in the family members

Father Mother Sister Proband Brother Daughter Son

Pedigree no. 11-2 II-3 111-1 111-2 111-4 IV-I IV-2 Factor XI11 aciivity (%) 127 75 < 10 24 10 107 30

Factor XI11 subunit B (%) 52 38 n.t. < 2 < 2 4 7 34 Factor XI11 subunit A (%) 130 68 n.t. 14 < 2 96 14

n.t. =Not tested.

- ,. - - 0 Y I I f. -

1 2 3 4 5 6 7 8 I 1 1 I I I I 9

Time (Cays)

Fig 2. Disappearance of subunit A ( 0 ) and subunit B (0 ) of factor XI11 from the plasma of the proband after infusion of factor XI11 subunit A concentrate. The half-life. determined from the less steep portion of the curve, is approximately 3 d.

Fig 2 illustrates changes in factor XI11 subunit concentra- tion after the infusion of factor XI11 subunit A concentrate into the proband. Subunit A showed a prompt increase to a considerably high level, followed by a rapid decrease during the initial 24 h and a slower fall to base line value during the following 6 d. The half-life determined from the later less steep position of the curve, was approximately 3 d.

1)ISCUSSION

Since the initial report by Duckert et a1 (1960), approximately 2100 cases of congenital factor XI11 deficiency have been described; however, most cases were deficiency of subunit A protein. Homozygous patients with deficiency of factor XI11 have no detectable subunit A and 50% of normal levels of subunit B in plasma. In 1977 a tentative classification of factor XI11 deficiency into two groups was proposed by Girolami et a1 (19 77,1978): type I is characterized by the lack of both factor XI11 subunits A and B: type I1 is characterized by

normal or near normal subunit B and no subunit A. Girolami et ul reported two cases of type I deficiency, but they did not demonstrate the behaviour of subunit B in their parents or children and it is uncertain whether the abnormality of subunit B was inherited in these cases. Therefore, it is unknown whether these cases were of familial factor XI11 subunit B deficiency.

Marked decrease in activity of factor XI11 was initially noticed in our proband, but further studies of the proband and her family demonstrated that activities and levels of subunit A of factor XI11 were almost normal in her parents and daughter. In this respect, this case is not compatible with deficiency of factor XI11 subunit A. The notable features in this family were no detectable subunit B in the proband and her brother and reduced levels of subunit B at 34-52% in her parents and children. From this result, a diagnosis was made of congenital deficiency of factor XI11 subunit B. Family pedigree shows the possibility that this disorder is inherited as an autosomal recessive trait.

Factor XI11 Subunit B Deficiencg 293 Factor XI11 has also been found in platelets and placenta, in

addition to its distribution in plasma. Platelet factor XI11 is composed of A protein only (Schwartz et ul, 1973) and approximately 50% of total potential factor XI11 activity in platelet-rich plasma is distributed in the platelet compart- ment. In the few cases tested, patients with factor XI11 deficiency also lack platelet factor XI11 (McDonagh et d, 1969). In the proband of our case, the level of subunit A of factor XI11 in platelets was almost normal. This finding supports the fact that this case is deficiency of subunit B, not subunit A deficiency.

Subunit A of factor XI11 has both the thrombin cleavage site and CaZ+ binding site and it possesses catalytic activity. Subunit B has a molecular weight of 76 000 and consists of 641 amino acid residues, the complete sequence of which was determined by Ichinose et al (1986). Subunit B is synthesized by the liver (Nagy et al. 1986) and it is thought to carry or to protect the A subunit (Cooke, 1974; Folk & Finlayson, 1977) and to regulate the activation of zymogen (Lorand et al, 1974) although the precise function is not clearly defined. Also, the behaviour of the A subunit and clinical findings in the presence of subunit B deficiency are not known. Our family study demonstrated that levels of subunit A were markedly decreased in the proband and her brother who lacked subunit B protein. It is suggested that subunit A is unstable in plasma in the absence of B protein and subunit B stabilizes A in the blood. However. the proband's parents and daughter had normal levels of A. although the levels of B were diminished to approximately 50% of normal. Recently, Yorifuji et aZ(1988) suggested that all A is present in complex form (AzB2) in plasma. In contrast, the total amounts of B are twice as much as A and half B is complexed whilst the other half is free in plasma. On the basis of this finding, it is thought that all subunit B was present in complex form in the family members who had low levels of B (approximately 50% in plasma) and consequently the levels ofA remained within normal limits. However, it is not known why the level of A was only 14% in the proband's son who had B protein at 34% of the normal range. Differentiation between complexed B and free B in this family will be needed to answer this question.

Previously, infusions of factor XI11 have been performed in congenital factor XI11 deficiency and the half-life estimated by several investigators varied from 3 to 12 d (McDonagh et nl, 1969: Miloszewski & Losowsky, 1970: Trobisch & Egbring, 1972). Our study in the proband showed that the half-life of subunit A was biphasic with a rapid fall phase and a slowly decreasing phase. This finding was consistent with previous reports, but the half-life read from the slower portion was approximately 3 d, which is the shortest estimate of the half- life of factor XI11 to date. From this result, it is suggested that infused A is unstable and rapidly disappears from the circulation in the deficient state of subunit B.

Factor XI11 subunit A deficiency results clinically in a moderate to severe haemorrhagic diathesis. In our case, the proband and her sister had apparent bleedings only in relation to obstetric events. The reported level of factor XI11 activity in the proband (24%) is in excess of the minimum required for adequate haemostasis. However, the reported

value of factor XI11 was determined using plasma obtained in a clinically stable state, when no bleeding diathesis was observed. We speculate that the levels of factor XI11 activity and subunit A had been reduced to less than the required levels for haemostasis at the time of an obstetric event. On the other hand, her brother with a low level of factor XI11 activity ( 10%) has no evidence of a bleeding tendency. These findings are different from the pattern of bleeding seen in factor XI11 subunit A deficiency. We think that severe bleeding occurs in this family of factor XI11 subunit B deficiency when an obstetric event or surgery overlaps with a marked decrease in the level of factor XI11 activity or subunit A and it is probable that the levels of factor XI11 activity and subunit A fluctuate with the deficiency of subunit B. In the future, it is necessary to follow up clinical findings and levels of factor XI11 in this family.

The family described here is the first case of congenital deficiency of factor XI11 subunit B and this case gives good information about the function of subunit B protein of factor XIII.

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