Fibrinolytic study in a homozygous protein C deficient patient

THROMBOSIS RESEARCH 42; 313-322, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Press Ltd. All rights reserved.

FIBRINOLYTIC

STUDY IN A HOMOZYGOUS

PROTEIN C

DEFICIENT PATIENT 1

J. Aznar,

3 2 A. Dasi, 3F . EspaAa and A. Estelles

D partment of Clinical Pathology Department of Pediatrics and k esearch Centre. Hospital "La Fe". 46009 Valencia, Spain ’ 9

(Received 19.8.1985; Accepted in revised form 23.1.1985 by Editor C.L. Arocha-Pifiango)

ABSTRACT

The fihrinolytic system was evaluated in a patient with hunozygousprotein C deficiencyas well as in severalmembers of his fami3.y with a partial deficiency of this protein. Before anticoagulant therapy the patient showed skin J.esions which ouickly After disappeared after administration of fresh Plasma. anticoagulant treatment, the propositus suffered two clinical episodesof "ecchymotic"lesions, which were controlledwith fresh plasma. The patient has remainedfree of new lesions and other clinicalepisodesup to the present date. The fibrinolyticactivityof both the propositusand his family was normal. The patient'sfather showed adequaterelease of tissue plasminogen activatorafter controlJ.edphysical exercise. According to clinicaland analyticaldata frcm our patient and his family, it is suggestedthat, in spite of the preservationof the fibrinolytic system in this case, a localized deficiency in fibrinolysis could exist in view of the clinicalbehaviourof the skin lesionsdescribed.

Key words : Fibrinolysis,protein C deficiency 'To whom correspondenceshould be addressed.

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INTRODUCTION Protein C is a vitamin K-dependent protein (1) which, when activated by thrcmbin (2), develops anticoagulant (3) and fibrinolytic (4) activities. The activation of protein C needs the presence of an endothelial cofactor, thrombomcdulin (5) and activated protein C activity is regulated by another cofactor, plasma protein S (6,7) and a circulatory inhibitor (8,9). Up to the present date, six cases of hoxxzygous protein C deficiency have been described (lo-15), as well as a further probable case (14) in which protein C was not evaluated. In only three of these cases (10, 11,14) has the homozygocity of the clinical history been established, and only three of the five patients are still living (11,13,15). Protein C deficiency in both homo and heterozygous patients is frequently accompanied by recurrent thrombotic accidents (16-20) which are ccnrmonly attributed to the lack of inhibition of factors Va and VIIIa. Although it appears to have been confirmed experimentally that protein C activates the fibrinolytic system (4,21), it is not clearly known to what degree a deficiency of protein C affects the fibrinolytic activity in these patients, as very few fibrinolytic studies have been made in protein C deficiency. In fact, in only one of the six cases studied of homozygous protein C deficiency the euglobulin lysis time was evaluated (lo), whilst in three cases (11,12,14) a high F'DP/fdp level following a severe episode of purpura fulminans was described. Furthermore, a hanozygous protein C deficiency associated with a displasminogenemia has been reported (15). In heterozygous patients in which the fibrinolytic system has been studied (16,17,22,23), it has been found to be normal in all cases. Even after venous occlusion (22,23) or activation with DDAVP (23), the fibrinolytic response was normal and in only one case of functional protein C deficiency has a reduced fibrinolytic response been detected (24). As previously described in the case history of the propositus (11) it is interesting to note that the "ecchptic" lesions show a rapid improvement after administration of fresh plasma, which could be attributed not only to the anticoagulant effect of protein C but also, mainly, to a local activation of the fibrinolytic system. This clinical fact has induced us to make a s-ore exhaustive study of various clinical and analytical aspects of our patient and in particular of his fibrinolytic system.

MATERIAL ANDMRTHOFX Venous blood was anticoagulated in l/10 volume of 3.8% sodium citrate. Platelet poor plasma was obtained by centrifugation at 2,000 g for 20 minutes at 4nC and stored at -7OQC until used. Protein C was measured by an enzyme-linked inmunosorbent assay mathod (Diagnostic Stago, Prance). Functional protein C was assayed according to the method of Francis and Patch (25). Antithrcmbin III used in this assay was purified by the method A as described by Thaler and Schner (26). 10 mg of kaolin per millilitre of 0.1% soya bean phosphatides (Centrolex-P; the gift of Central Soya Co, Chicago) in 0.15 M NaCl was used for AP'ITdeterminations in functional protein C assay. Protein C inhibitor was determined using a functional assay as described by Francis and Thcxnas(27) in which the ability

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FIBRINOLYSIS 8.PROTEIN C

315

of the plasma test samples to inhibit the anticoagulant activity activated crude protein C in the APTI was measured.

of

an

Plasma euglobulin lysis time was determined according to Buckell's method (28). Plasminogen levels were measured by imnunologic (29,30) and amydolytic (31) methods. Fast-acting antiplasmin activity was measured by a chromogenic substrate method (32). Fibrinogen degradations products (fdp/FDP) were determined using a hemagglutination inhibition method (33). Tissue-type plasminogen activator (t-PA) activity was determined by a functional method (34) as previously described (35). t-PA antigen was measured by an enzyme-linked imnunosorbent assay method (36) (Biopcol, Sweden). t-PA inhibitor activity was assayed as previously described (37). The pedigree of the propositus, as reported (ll), comprises seven members with heterozygous protein C deficiency (mother,father, brother, two sisters, maternal and paternal grandfathers) and six members who do not present protein C deficiency (maternal and paternal grandmothers, aunt, male paternal cousin and female paternal cousin). The propositus's father underwent a syml?tcnn limited bicycle ergometer test in a sitting position starting with 30 W. The sutxnaximalwork load was performed, reaching 85% of the individually expected maximm working capacity (125 W). RESULTS The previously reported (11) inmunological protein C evaluation of the members of the family is now ccxnplementedby a functional determination of both protein C and its plasma inhibitor (Table I). TABLE I Protein C and protein C inhibitor in several n-embersof the family Protein C Protein C (%) Antigen Functional inhibitor(%) Mother Father Brother Sister (III-4)* Sister (III-5)* Maternal grandfather Paternal V Matemalgrantither Paternal II Maternal aunt (11-5) Paternal aunt (11-2) Cousin (111-2) Cousin (III-l) Normal values (mean f 1SD) (n) * Reference

(11)

52 53 45 45 52 53 43 120 130 88 96 96 82

51 50 47 49 60 57 56 100 75 101 92

98 f 15 (29)

97 f 10 (22)

135 103 91 88 103 88 116

98 96

86

94 * 15 (18)

Activity %I1 %X 120 92 85 93 92 87 85 99 92 83 78 91 97

95 f 11 (22)

99 103 86 86 74 113 88 125 96 105 88 89 130

97+11 (23)

(%)

F.X

65

84

21 13

80 88

98 74

79

0.1 <5 -

10

105

200 100

0.4 <5

3.4

5.6

100

170 80

* Hours or ** Days post-administration of fresh frozen plasma

(Fun&)

(%)

F.11 (Fun&)

0.4 -

23

23

Protein C,antigen (%) Protein C,functional (%) Protein C,inhibitor (%)

82

85

Fast-acting antiplasmin (%)

150 101

150 98

Plasminogen - Antigen (p/ml) - Functional (%)

0

0.6

-

t-PA inhibitor (U/ml)

0.5

1.6

-

5.5

108

12d**

t-PA in acidified plasma (ng/ml)

8.2

32 h

2.0

6.7

10 h

0.1

125

130

0

0.75

1.6

0.2 <1

130

100 82

0

1.9

4.5

-

162d

ingestion

8.8

80

47d

With cowin

t-PA in acidified blood (ng/ml)

t-PA antigen (ng/ml)

Euglobulin lysis time (min)

36 h*

Without coumarin ingestion

FIBRINOLYTIC PARAMETERS IN HCMOZYGOUS PROTEIN C PATIENT

TABLE II

(22) (23)

211 +11 97

(29) (22) (18)

(29)

(59) (37)

(44)

(34)

(26)

(31)

(50)

(n)

95

98.2 +15 97.8 k10.2 +_15 94

10

98.2 +15

182 k30 109 k13.9

0.57kO.68

0.31kO.12

0.58AO.26

9.8i3.9

> 180

Normal Values (mean + 1SD)

”

w

z?

N

P

z -

0

PO

G

-n

118 107 104 108 99 80

fc?w2 Maternal Matemalgrandwkher Paternal aunt grandfather (II-5)* 200

rl2 Paternal aunt (II-2)* 200 !!u cousin (111-2)" 250 o"o cousin (III-l)* 250 z

* Reference (11)

93 89 126 112

185 135 212 200

Sister (III-4)* Sister Maternal (III-5)* Paternal grandfather

u)8.3 ? 2.3 '=I&% asa dEM

105.7 97

201 135 191

G&Sir

Plasminoqen Eknctional Antigen (%) oKJ/d)

94 85 100

100 91 90

105 99 91 68

103 95 99

1.8 2.8 2.8

1.8

3.7 5.6 3.7 1.8

3.7 2 2.4

Fast-acting antj.plasmin (%I FDP/fdP

FIBRINOLYTIC PCTIVITY IN SEVERAL MEMBERS

TABLE III

8.16 7.32 10.64

10.79 8.96 5.84

4.72 4.17 6.08 10.22

5.9 9.4 7.2

-

-

-.

0.45 0.8

t-PA (ng/ml) acidif. Antigen pkl.SIWi

OF THE FAMILY

-

0.60 0.75

acidif. blood

1.5 1.5 1.5

0.0 2.0 1.0

1.0 0.0 2.3 0.8

0.5 0.7 1.5

IIlh. t-PA U/ml

z 0

:

A"

2 z w

z E

-l-i

w

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It should be noted that none of the other members of the family had ever received oral anticoagulant treatment. In view of the data obtained, a functional protein C anomaly can be excluded. The protein C inhibitor was also found to be normal in all cases. The study of the propositus's fibrinolytic system (Table II) shows the moderate fibrinolytic hyperfunction during the time the existence of a patient is receiving oral anticoagulants. This is shown by a slight decrease in the euglobulin lysis time as well as a moderate increase in the t-PA activity. In relation to this moderate fibrinolytic hyperfunction, it should be noted that the patient was always agitated and sometimes crying during the extraction of the samples. The fibrinolytic study of the family (Table III) shows that, in physiological conditions, both the t-PA and its inhibitor are normal in all members of the family studied. After controlled physical exercise (ergometry), the release of t-PA measured immunologically was normal in the patient's father (before = 9.4 ng/ml; after= 17.5 ng/ml), when comparedtothat of a control group of six normal subjects of the same age and sex (before= lO.Lt 3.2 ng/ml; after= 15.9 + 5.1 ng/ml).

DISCUSSION According to the clinical data of our patients described in the literature, we wish to remark that:

and

those

previously

In accordance with other authors (16-20), in the members of our family with heterozvgous protein C deficiency, recurrent thrombotic phenomena did not systemic fibrinolytic activity was normal in these appear. Besides, the members, in agreement with other authors (16,17,22). In relation to the case history of the propositus and in addition to the findings already mentioned (ll), it is interesting to note the fact that since June 1984, when oral anticoagulant treatment started and plasma was administration discontinued as previously described (38), the patient has suffered two clinical episodes which included tk appearance of "ecchymosis" as previously described (11). These were controlled by administration of fresh plasma and the patient has remained free of new episodes up to the present date. The appearance of new lesions was halted a few minutes after initiating fresh plasma administration (10 ml/Kg weight) and the existing lesions were rapidly reduced, disappearing within a few hours or even, on occasions, while the plasma was being administered. Also, as previously described (11) and in accordance with other authors severe disseminated intravascular coagulation accidents were (12.14), the followed by a favourable reaction of the systemic fibrinolysis, shown by an increase in the. FDP/fdp. This would appear to indicate that the role of activated protein C is not essential in the systemic fibrinolysis. However, once a thranbotic accident has been produced in the microvasculature, the nonprotein C dependent fibrinolytic response is insufficient to dissolve the microthrcmbus formed. This suggests that it is here, at tk microcirculation, where activated protein C-dependent fibrinolytic activity is necessary to maintain an adequate hemostatic balance.

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319

On the other hand, the fact that the skin lesionsand DIC do not appear in heterozygouspatientsnor in hcmozygousones with more than 1% of proteinC (15,39), could be because in these patients, although "ecchymotic" lesions were initiateddue to their anticoagulantdeficiencycaused by a partial lack of protein C, the local fibrinolyticsystem would respond adeouately by dissolving the microthrcmbusformed. However, in homozygous patients with protein C levels of less than 1%. the additionalfailure of the local fibrinolytic system would favour the thrunbosisin the microvasculatureand the developmentof DIC. This supportsthe hypothesisthat, for protein C to develop its fibrinolytic activity in the microvasculature, only small quantities of protein C are reguired, in contrast to the concentrations requiredto develop its anticoagulantfunction.Hence, patientswith levels of protein C above l%, aproximately,tend to show deep venous thrombosiswithout developingskin lesions.

With regard to the mechanismof the fibrinolyticaction of proteinC, it has been suggested that the activatedproteinC may either increase the release of t-PA (4,6,40),enhance the specificreleaseof t-PA inhibitorfrom endothelium(41) or inhibitthe t-PA inhibitoractivity (41-44), althoughthe last of these mechanismsappears to be the most probable (41,44). However the role of proteinC in relationwith "in viva" fibrinolysishas not yet been defined. ACKNCWLEXEMENTS We thank J.Solano,A.Serralbo,P.Escamillafor their technicalassistance.

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C

in