Heterogeneous mechanisms responsible for reduced fibrinolytic capacity in patients with a history of venous thrombosis

Fibrinolysis (1994) 8, 87-95 (~)1994 Longman Group UK Ltd

Heterogeneous Mechanisms Responsible for Reduced Fibrinolytic Capacity in Patients with a History of Venous Thrombosis

J. M. Hadjez, S. Combe, M. H. Horellou, J. Conard, G. Nguyen, P. Van Dreden, M. Samama S U M M A R Y . Abnormal response to venous stasis is a frequent finding in patients with previous venous thrombosis. The factors studied to explain the response of the fibrinolytic system to venous occlusion (VO) have included tissue plasminogen activator (t-PA), PAI antigen and activity measurements as additional tests to the conventional euglobulin fibrinolytic activity. However, there is no consensus on the definition of an abnormal response. In order to further investigate the clinical relevance and the definition of good and bad responders (GR, BR), we have used a 10min VO test in 109 patients with well documented venous thromboembolic events. Hematocrit, euglobulin clot lysis time (ECLT), diluted whole blood lysis time (DWBLT), tissue plasminogen activator (t-PA ag), fibrinolytic activity of euglobulins on fibrin plates (EFA), activity of the t-PA inhibitor (PAI act), a complete study of haemostasis, and a platelet count were performed before and after VO. Clinical characteristics did not reveal a significant difference between GR and BR. Among these 109 patients, 46 patients had a GR according to our previous definition (residual PAI-<2 U/mi). In this subgroup, results are homogeneous in the various fibrinolytic tests used. In the 63 remaining patients, results are heterogeneous: in 28 patients a BR was observed whatever the test used and could be attributed to a high basal PAI act in 17 cases, or a poor t-PA release in 11 cases. In the remaining 35 cases, interpretation of results is difficult: in 16 patients the anomaly of the t-PA-PAI balance coexists with a satisfactory response of global fibrinolytic activity judged on the E C L T after VO (<90min) or on the EFA after VO (t-PA>2U/I). This discordance may be linked to a compensating activity of another pathway of the fibrinolytic system. In 19 cases the discordances remain unexplained. In these subgroups of BR, clinical characteristics were not clearly different. However, the subgroup 'concordant BR' seems to be more frequently associated with recurrences or familial thrombotic tendency. This work underlines the difficulties of clinical interpretation of hypofibrinolysis testing. Global tests (ECLT, DWBLT) and EFA are not only dependent on the t-PA-PAI balance but also on the contact phase and u-PA course. Combinations of tests and a better understanding of the multiple pathways involved will be necessary if we wish to prospectively identify patients at significant risk of thrombosis.

INTRODUCTION - GOALS OF T H E STUDY

with hypofibrinolysis do not develop clinical thrombosis. The relationship between hypo or dysplasminogenemia and thrombosis is not well understood;4"5 however, a susceptibility of normal fibrin to fibrinolysis activators seems necessary to prevent venous thrombosis. A normal fibrinolytic system plays a protective role against venous thrombosis. The presence of an abnormal fibrinogen (dysfibrinogenemia), as seen for example in Dusard syndrome in which fibrin clots are resistant to lysis, may be a cause of thrombosis. 6 The diagnostic criteria for hypofibrinolysis have been arbitrary and varied. 7-1z Global tests of the fibrinolytic system such as euglobulin clot lysis time (ECLT) and/or the clot lysis time of diluted whole blood ( D W B L T or Fearnley-

Hypofibrinolysis is defined as reduced or absent fibrinolytic activity in the presence of appropriate stimulus. The frequency of this anomaly is estimated at about 30% in patients with thromboses, a very high percentage compared to deficiencies of antithrombin III, protein C and protein S, which combined account for only 8 to 10% of thromboses.l'2 It is generally accepted that hypofibrinolysis predisposes to venous thrombosis, 3 but many individuals J. M. Hadjez, S. Combe, M. H. Horellou, J. Conard, G. Nguyen, P.

Van Dreden, M. Samama, Laboratoire central d'h6matologie. H6pital H6tel Dieu, 1 place du Parvis N6tre Dame, 75004 Paris, France. 87

88 Heterogeneous Mechanisms Responsible for Reduced FibrinolyticCapacity Gallimore test) are relatively insensitive to hypofibrinolysis. They are more useful in the diagnosis of hyperfibrinolysis. 13 Since the 1980s, the E C L T has been performed after various stimulus methods: after 10 or 20min of venous stasis, or after an intravenous injection of D D A V P (Minirin| or after a standardized physical exercise. 14-16 Each method has weaknesses. ~0 9 Venous occlusion (VO) for 10min is the most common method. It is better tolerated than VO for 20min, though the latter may be more sensitive. 9 D D A V P causes side effects such as postinjection flush, a rise in arterial pressure, and fluid retention. 9 The physical exercise method used as a stimulus method requires special equipment. The VO test recognized G R and BR. 14 Shortening of E C L T after the VO to below 90mn in the test tube, 8'~7 or D W B L T to below 120mn were considered by our group 8 and others as a GR. Even better, the demonstration of increased fibrinolytic activity of euglobulin solution on fibrin plates (EFA) (in order to exclude the fibrinogen variable), 18 was also regarded as a GR. Recent advances in the study of fibrinolysis include new more precise methods of investigation. Specifically, 3 pathways of the fibrinolytic system: the t-PA-PAI system, the u-PA course, and the contact phase pathway can be studied separately. In patients with recurrent venous thrombosis, studies of the t-PA-PAI pathway have revealed 2 types of BR: the first has elevated basal PAI levels, and the second an abnormal basal PAI level but reduced fibrinolysis activator release (t-PA in particular). 19'2~ Thus, a biological marker of thrombosis risk may be an imbalance between t-PA-PAI measured by the level of residual PAI activity following the stimulus of VO. 2~ Released t-PA neutralizes the activity of PAI, whose level becomes practically nil following VO in GR. This approach does not require taking into account the t-PA ag level, whose normals vary from one lab to another, and are difficult to determine. 2~'22 To a lesser degree, the same applies for measuring variations in t-PA activity, which requires specific conditions for sampling and measurement. Therefore, it is tempting to suggest as a criterion for good response to the fibrinolytic stimulus of venous stasis, the level of residual PAI. Absent PAI activity indirectly reveals the existence of a persistent free t-PA activity. Before a VO test, the t-PA in the circulating blood is almost entirely complexed with PAI and inactivated, and free PAI activity is present in the blood. In 121 patients with previous DVT, we have compared the new tests (t-PA, PAI) with the old tests ( E C L T , D W B L T , E F A on fibrin plates), to try to explain the causes of hypofibrinolysis in these patients and determine whether such clarification could affect clinical decisions. A clearer understanding of hypofibrinolysis pathways and measurements might lead to improved

methods of prophylaxis and treatment. Lechner et al have proposed long term treatment with oral anticoagulants (vitamin K antagonists) in individuals with venous thrombosis, associated with reduced fibrinolytic capacity measured 6 weeks or more after the thrombotic episode. 23

MATERIALS AND METHODS Study Population 121 consecutive patients with a history of venous thrombosis and/or pulmonary embolism have been studied. Thromboembolism was confirmed by angiography or doppler in 111 patients. Two patients were excluded because of an underlying condition known to predispose to thrombosis (Behcet's disease, collagenosis). The remaining 109 patients (48 men, 61 women) had biological investigations done at least 3 months after the last episode of thrombosis. Each patient was studied before and after a VO of 10min (the pressure applied being midway between the systolic and diastolic pressure). Two groups of patients were then identified: 9 the GR, in whom the PAI activity after VO was below or equal to 2 U/ml. 9 the BR, in whom the PAI activity after V O was above to 2 U/ml. The 2 U/ml limit was chosen because the technique used for the measurement of the PAI activity is not sensitive below this level. The results obtained after VO were not corrected according to the hematocrit as they were in our previous study. 21

Laboratory Methods The blood samples were taken between 09:00-11"00. After a control specimen was drawn from an arm, VO was accomplished by a blood pressure cuff inflation to a pressure midway between systolic and diastolic. Following 10 rain of occlusion, a second sample was drawn from that extremity. Samples were collected in E D T A tubes (fnal concentration 5mmol/L) and in citrated tubes (lvol. 0.11mol/L citrate p H 4.5 and 9 blood vol.). The samples with E D T A were immediately put on ice, a platelet-poor plasma (PPP) was obtained through two consecutive centrifugations at +4~ (10min at 3500g, then 2rain at 10000rpm). Plasma samples were kept in frozen state at -30~ The tests used were: 9 The hematocrit (Hct). A calculation was used to assess the adequacy of the VO: the expected increase in the Hct after VO ranges from 10 to 20%. 9 The euglobulin clot lysis time (ECLT). The plasma was diluted 15-fold, and a p H of 5.9 was obtainedfl 4 9 The diluted whole blood clot lysis time ( D W B L T or Fearnley-Gallimore test).25

Fibrinolysis 89 9 The fibrinolytic (plasminogen activator) activity of the E F A 26 was measured after acidification (pH 5.9) of plasma diluted 10 times. Precipitation of the euglobulins was followed by a redissolving in a buffer solution which was then placed on bovine fibrin plates. The fibrinolytic activity was determined by the diameter of the areas of lysis and expressed in equivalent international units of t-PA after a prior standardization with a titrated preparation of t-PA as recommended by many authors. 9 The tissue plasminogen activator inhibitor activity (PAl act), expressed in units per ml (Verheijen and coll. method). 27 9 The level of t-PA antigen (t-PA ag E L I S A kit Biopool method). 9 All patients had a study of haemostasis: PT, APTF, coagulation factors, fibrinogen, platelets count, level of physiological inhibitors of coagulation pathways (ATIII, ProtC, ProtS), plasminogen. The statistical analysis of the data was performed with software Statview | (calculation of averages, standard deviations, Wilcoxon test, Student t-test, correlation coefficients).

RESULTS

Clinical Characteristics The clinical characteristics did not reveal a significant difference between the G R and the BR, except body mass index (BMI) or 'index of Quetelet' (weight/ height 2 in kg/m 2) which is higher in the B R group compared to the G R (men: 25.5/24.3; women: 24.1/ 22.3kg/m2). However, the difference was not significant. The sex-ratio, average age, age at the occurrence of the first episode, site of the thrombosis, the percen-

tage of recurrences, and the existence of familial antecedents did not differ in the two groups. In this series, we found 3 patients with protein C deficiency, 3 with protein S defciency, and no antithrombin III deficiency. The treatments in use at the time of the study were identical: 47.8% (GR) and 42.8% (BR) of the patients were not on treatment; 39.1% (GR) and 33% (BR) were using oral anticoagulants.

Laboratory Results (Table 1) The adequacy of venous occlusion was similar in the 2 groups. This was demonstrated by the similar increase in the hematocrit. The difference in response between G R and B R groups cannot be attributed to a difference in the quality of the venous occlusion. There was no significant difference in the mean fibrinogen level (3.44g/1 in the two groups). Hyperfibrinogenemia can prolong the ECLT. The PAI activity was significantly higher in the BR group before VO (p=0.0001). It was also higher after VO by definition (Fig.l). The t-PA ag was high before VO in the B R group (p=0.0016), while it is comparable afterwards. Venous stasis for 10min stimulates a release of t-PA and raises the level of t-PA ag: the difference between the level of the t-PA ag after VO minus t-PA ag before VO, which measures the release of t-PA ag, was higher in the G R group than in the B R group (16.9+8.3/13.6_+ 10), but the difference did not reach significance (p=0.077). Similarly, we did not find a significant difference in the ratio of t-PA ag after VO to the preocclusion level of t-PA ag in the two groups. The fibrinolytic activity of the E F A is not significantly different in the two groups before VO, when it is weak or not measurable. However, it rose significantly after VO in the G R (p=0.0001). This variable is fibrinogen independent, and even expressed in

Table 1 Biologicalparameters of fibrinolysisbefore and after VO

Haematocrit increase (%) Fibrinogen (g/I) PAl (U/ml) ECLT (min) DWBLT (min) Fibrinolytic activity: EFA (t-PA U/ml) t-PAag (ng/ml)

Before After Before After Before After Before After Diff aft/be Before After Diff aft/be Ratio aft/be

Good responders n=46

Bad responders n=63

p

15+5 3.4+0.6 5.7+2.3 0.2+0.6 245+ 191 52+25 258+ 163 61+27 0.1 +0.6 7.6+6.6 7.4+6.5 6.0+2.7 22.5+ 10.5 16.9+8.3 3.9+ 1.3

13_+6 3.4+0.7 10.5-+5.0 8.6+5.0 611+379 244+336 468+335 117-+74 0.1 -+0.6 1.2+1.7 1.1_+1.6 9.1 +5.7 22.7+ 14.7 13.6+ 10.0 2.6+0.9

NS NS 0.0001 by definition 0.0001 0.0002 0.0003 0.0001 NS 0.0001 0.001 0.0016 NS 0.077 NS

90 Heterogeneous Mechanisms Responsible for Reduced Fibrinolytic Capacity

PAl before and after in GR g r o u p

40 35 30 25 20 PAl activity after VO

PAl activity before VO

15 10

,

5

U/ml

0. 0 5,

2.5

5

U/ml

O. _~.. 0 2,5

7,5 10 12,5 15 17,5 20 22,5 25

,

,

5

7,5

-

,

-

,

.

.

,

.

,

,

_

.

10 12,5 15 17,5 20 22,5 25

7_ i'1

- j1 n

PAl b e f o r e

PAl

activily

betore

and

after

in BR g r o u p

VO PAl

0 0

2,5

5

7,5 10 12,5 15 17.5 20 22,5 25

0

2,5

5

activity

afterVO

lllm_ll_

7,5 10 12.5 15 17,5 20 22.5 25

Fig. 1 PAI activity in good and bad responders (GR and BR).

arbitrary t-PA units, reflects global fibrinolytic activity of the patients' plasma euglobulins. However, the composition of the euglobulins differs from that of the whole plasma proteins; theoretically, it does not contain the inhibitors of the fibrinolysis. Nonetheless, we found 30 to 50% of the PAI in the euglobulins (results not shown). The global tests (ECLT, DWBLT or Fearnley Gallimore) were significantly prolonged before and after VO in the BR group. These tests reflect global dysfunction in the fibrinolytic system existing in this group (t-PA-PAI balance, and also other pathways of the fibrinolytic system including u-PA pathway). As for the fibrinolytic activity on fibrin plates, a variable quantity of PAl is found in the euglobulins and may affect the results.

Study of the Correlations of Different Biological Parameters in GR and BR (Table 2) There is no correlation between the BMI and the augmentation of hematocrit; the balanced load is therefore not responsible for a poor elevation of the hematocrit. 18 We found a positive correlation between the PAI activity and t-PA ag before VO (BR: r=0.51/GR: r=0.21); the t-PA ag measures the free active t-PA and the t-PA linked to PAI, a large part of the t-PA is

complexed with the PAI in particular before venous stasis. This explains the good correlation between these two parameters, particularly in the BR who show an excess of PAl. We have also found a positive correlation between PAI activity and global lysis tests in the BR group (r=0.64/r=0.68 for the ECLT, before and after VO); (r=0.63/r=0.47 for the DWBLT before and after VO); as already described, 29 there also exists a positive correlation between the PAI activity before VO and the ECLT after VO (r= 0.62), suggesting that increased basal PAl activity is a good predictor of a bad response to VO. However, in the GR group the correlation is weak: (r=0.08/r=0.27 for the ECLT before and after VO); the correlation is also weak for the DWBLT before and after VO. Lastly, there is no correlation between the PAl before and the ECLT after VO in GR group. For the ECLT, about 50% of the PAl persist in the euglobulins, its excess bringing on a lengthening of the ECLT, particularly in the BR group (where there is an excess of residual PAI), as we have already said. We have also found a correlation between PAI activity and DWBLT. In both cases, the suppression of the inhibitors concerns the antiplasmins and only partially the PAl. Blood dilution according to the DWBLT method does not entirely suppress the PAl activity, particularly when it is at a high level in BR group.

Fibrinolysis 91

Table 2 Correlation of biological parameters in BR group BR (n=63) ECLT before after

r= ECLT

Before After

DWBLT

Before After

EFA

Before After

t-PAag

Before After

PAI act

Before After

0.70

DWBLT before after

before

EFA after

t-PAag before after

0.66 0.55

0.46 0.36

-0.16 -0.11

-0.46 -0.37

0.41 0.13

0.40

-0.06 -0.09

-0.26 -0.46

0.54 0.11

0.19

-0.10 -0.09

PAl act before after

0.36 0.11

0.64 0.62

0.65 0.68

0.61 -0.01

0.63 0,32

0.60 0.47

-0.20 -0.23

-0.18 -0.39

0.51 0.57

0.46 0.41

-0.13 0.11 0.89

0.90

Negative correlation means that both parameters varies in opposite ways.

to only minimal increases (ECLT after VO 104/110/ 120min and DWBLT after VO 150min).

Table 3 Comparison of hypofibrinolysis criteria Good responders residual PAI--2 n=46

Bad responders residual PAI>2 n=63

Basal PAI: PAI>10U/ml

2 (4.3%)

29 (46%)

Global lysis times after VO: ECLT>90min DWBLT>120min

3 (6.5%) 1 (2.1%)

29 (46%) 20 (31.7%)

The Bad Responder Group

Comparison of Hypofibrinolysis Criteria in Good and Bad Responders Applying the criteria of hypofibrinolysis and the results of the PAI after VO, two populations emerged: 46 GR (42%) and 63 BR (58%).

The Good Responder Group The results of the different tests were generally consistent with the definition of GR (Table 3). Two patients did show a moderate increase in basal PAl (11.2 and 13 U/ml), but this was well compensated for by other undefined fibrinolytic activity or by an increased release of t-PA (with a difference of 17 and 41ng/ml respectively for t-PA ag). In 4 cases, ECLT and DWBLT did not agree but did correspond in fact

The number of BR was greater than expected (Table 4). This may be explained by the fact that the PAl criterion only takes into account the t-PA-PAI equilibrium, and therefore also includes cases in which the latter is compensated for by another fibrinolytic activity or by an increased release of t-PA, as we will see later. In this BR population, some lack of agreement was observed between various tests. Therefore, subgroups must be examined. The BRs were divided into two subgroups, according to whether the PAl activity before venous occlusion (basal) was increased (>10U/ml, n=29) or normal (-<10U/ml, n=34). In cases where the basal PAl was high, we distinguished between patients whose activity on fibrin plates increased after VO (>2U/ml, n=6) and those whose activity was absent or remained low (-<2U/ml, n=23). These two groups were then compared with their global lysis tests to determine if the latter tests would be different in each groups. The results are as follows:

Subgroup 'PAl before VO> IO U/ml' (n=29): The fibrinolytic activity on fibrin plates is less or equal to 2U t-PA/I and 2 profiles are possible:

Table 4 Heterogeneous mechanisms of hypofibrinolysis

Patients (n) EFA Global lysis times Increased basal PAl (n)

BR for all biological tests

Compensation by another pathway

Increased resistance of fibrin to lysis

Discordant results

Group 1

Group 2

Group 3

Group 4

28 ~, ]' 17

16

5

14

N N 4

N ]' 2

N 7

92 Heterogeneous Mechanisms Responsible for Reduced Fibrinolytic Capacity

(1) The global lysis tests are long (n=9) or discordant (n=8): this result may be caused by an excess of basal PAl activity which is not compensated by an elevation in fibrinolytic activity; in this group, there is a concordance between the EFA and the ECLT. As for the discordances between the global lysis tests, in two cases, limited values are involved (DWBLT=120min.) in the other cases, there is a hyerfibrinogenemia (5.1/4.9/4.6/4.2 g/l). (2) The lysis tests are short (n=6): these patients have a basal PAl activity which is only slightly superior to 10U/ml (10.2/10.2/11.6/12.2/12.7/19U/ ml) without a concordance between the EFA and the ECLT. This discordance may be due to the PAl which precipitate in variable quantities in the two tests. This secondary group was previously classed as a GR group according to the old criteria. The fibrinolytic activity on fibrin plates is superior to 2U t-PA/I and we have again anticipated two eventualities: (1) The lysis tests are short (n=4): we deduce from this that the basal excess of PAI activity is compensated for by an augmentation in the fibrinolytic activity independent of the t-PA-PAI balance; in this case, there is a good concordance between the EFA and the ECLT. In this sub-group, the PAl levels are also only slightly superior to 10U/ml (respectivly 10.1/10.5/ll.2U/ml). Because of the global tests these patients were previously classed as good responders. (2) The lysis tests are discordant (n=2): here we are concerned with BR by an excess of basal PAI activity which is not (or badly) compensated for by an augmentation of the fibrinolytic activity. The PAl activity is at a higher level than in the preceding subgroup (15 and 20U/ml) which suggests the inability of the fibrinolytic system to compensate for the excess of PAI activity. But these are two isolated cases. It must be noted that, in a case of disagreement of the global lysis tests, ECLT has a minor deviation from the normal (120 min). The discordance between the lengthened ECLT and the normal EFA could suggest a resistance of patient's fibrin to lysis such as in the Dusard syndrome; the negativity of the familial investigation, and the absence of dysfibrinogenemia caused us to reject this diagnosis. The fibrinogen of these patients is normal and does not therefore interfere with the global tests. This discordance remains unexplained.

Subgroup with 'basal PAI
t-PA could be insufficient. But here we have the problem of the discordance between the ECLT and the EFA, for which there is no satisfactory explanation since in this sub-group there is no excess of basal PAI. The fibrinolytic activity on fibrin plates is above 2 U t-PA/1. (1) The global lysis tests are short (n--12): the imbalance of the t-PA-PAI system seems well compensated for by another fibrinolytic activity, the tests exploring the fibrinolytic activity are in agreement. These patients were previously classed GR. (2) The lysis tests are long (n--l) or discordant (n=2, corresponding to limited values); another pathway of fibrinolysis may be altered (u-PA, contact phase). Here again, we are dealing with 3 isolated cases. A Dusard syndrome is also eliminated here. Finally, if we isolate among the BR patients those with homogenous results of hypofibrinolysis using reduced fibrinolytic activity following VO (EFA), excess of basal PAI, and prolonged clot lysis time, the comparison of the clinical parameters of these patients with those of the GR group does not bring to light any particularity concerning the BMI or the site of the venous thrombosis. Recurrences and the positive family history seem slightly less frequent in the GR group (61%/54% in BR and 53%/47.8% in GR) which suggests the more common existence of familial anomalies in this group as it has been described. 30-31 Laboratory follow up of these groups allows us to assess the persistence of the biological anomaly: (1) In the GR group, 20 of 46 patients were seen again (43%) but only 5 had a second VO. Two patients changed to BR. (2) In the BR group, 32 of the 63 patients were seen again (51%), 21 had a second VO and in 17 patients the anomaly persisted.

DISCUSSION Our study did not find a difference in the clinical characteristics of the two groups of patients; the sex ratio, age at time of the first thromboembolic episode, type and site of thrombosis, number of recurrences, and presence of a positive family history did not differ. Body mass index is higher in the BR group but the difference does not reach statistical significance. The higher weight in the BR group and the good correlation (r=0.51, n--49) between the BMI and the plasma level of PAI activity suggests a relationship between overweight and the increased PAI; perhaps weight reduction could be clinically relevant in these patients. Two subgroups showed particularly interesting results: the first subgroup in which all the biological

Fibrinolysis 93 parameters are concordant (the 'real BRs'). There seems to be a higher frequency of recurrences and of positive family histories which could be linked to a familial biological abnormality (dys-t-PA, for example as recently described by Petaja et al). 31 In the second subgroup, the anomaly of the t-PA-PAI equilibrium is associated with normal tests of global fibrinolytic activity. This second group requires a biological surveillance in order to verify, either its total normalisation, or a confirmed diagnosis of hypofibrinolysis. Among the BRs with a spontaneous thrombotic accident (29/63 patients=47%), we did not find a greater frequency of hypofibrinolysis than in patients with a thrombotic episode associated with a predisposing risk factor. A different result was found by Harbourne et al. 32 There was no difference in the treatments (anticoagulant, antiplatelet) administered at the time of the biological investigations in BR and GR. Fibrinolytic activity is dependent upon many activating and inhibiting factors, and the mechanism of reduced fibrinolytic capacity is multifactorial. Among the 46 GR (42%), the results of global lysis tests and of other parameters of fibrinolysis (t-PA, PAI, EFA) agree in 87% of the patients. In those cases in which some tests are normal and some abnormal (n--4) it is a question of normal values of basal PAI, or of global lysis tests. This illustrates the difficulty in fixing the normal values for fibrinolytic activity. In contrast the group of BR (58%) defined by the criterion of a residual PAI superior to 2U/ml is a heterogenous group. Among these 63 patients with an anomaly in t-PA-PAI balance, different causes are possible: (Table 4) The fibrinolytic activity on fibrin plates may be abnormally low (n--42). The comparison with the global lysis tests leads to two possibilities: (1) The global lysis times after VO are long or discordant (n--28). Here we are dealing, with bad responder patients according to all the criteria. A poor release of t-PA occurred in 11 cases, and excess of basal PAI was present in 17 cases. (2) The global lysis times after VO are normal (n--14); this finding may be due to a greater sensitivity to lysis of the patients' fibrin as compared to the bovine fibrin used in the plates. However, methodological problems could be responsible for discordant results. The activity on fibrin plates after VO is not diminished (n--21). There is a disagreement between the anomaly of the t-PA-PAI balance and the EFA, and two explanations are possible: (1) A compensating intervention through another pathway of the fibrinolytic system, such as the prekallikrein-kallikrein activity (contact phase), or the u-PA fibrinolytic pathway. We know that the u-PA is liberated after stimulation by DDAVP, while

it is not liberated after a venous stasis of 10 minutes. 33,34 (2) The liberation of t-PA from the t-PA-PAI complex in the presence of fibrin or during the preparation of the euglobulins. Although the t-PAPA1 complex is considered to be irreversible, the dissociation may well be the result of an effective competition for the t-PA by fibrin. In 16 cases the clot lysis times after VO tests are normal, which reflects a compensation for the imbalance by another pathway of fibrinolysis; lastly, the expected relation between the EFA and the ECLT was not found in 5 cases where the lysis tests are long or discordant, which could suggest a Dusard syndrome characterised by a normal fibrinolytic activity on fibrin plates which becomes abnormal in the presence of the patients' own fibrin, because of its resistance to lysis. This diagnosis could not be confirmed in those patients with a negative family history. In summary, in 28 BRs, a bad response was linked to a disturbance of the t-PA-PAI balance, and was associated with an insufficient fibrinolytic response, no matter which test was used. Among these patients we found, in accordance with other authors, 17 patients with an excess of basal PAl, and 11 with a decreased release of the t-PA. In contrast, in the 35 other cases, interpretation of the results is difficult: in 16 of these patients the anomaly of the t-PA-PAI balance coexists with a satisfactory response of fibrinolytic activity, judged on the ECLT (<90min) or on the EFA (tPAact>2 U/I). These discordances could be linked to a compensating activity of another pathway of fibrinolysis, in all likelihood the contact phase, since the pathway of the u-PA doesn't seem to be implicated in the VO. In 5 of these patients the discordances between the EFA and the ECLT required consideration of Dusard syndrome which was not confirmed. In the 14 remaining patients, the discordances are difficult to explain, which illustrates the difficulty in defining and classifying the BRs. In this work, the well established heterogeneity in the BR group contrasts with the homogeneity of the results of the GR group. At least, when basal PAI activity is increased the patient belongs to the group of BR in almost every case, (29/63 BR and 2/49 GR) a finding in good agreement with the results of Grimaudo et al, 3~ who found that basal PAI antigen level is a sensitive (83%) and specific (89%) assay for the detection of patients with hypofibrinolysis. These results suggest that measurement of basal PAI activity and/or antigen could be sufficient as a first step to detect patients with impaired fibrinolytic activity. A large increase of basal PAI activity could make the VO unnecessary. In contrast, normal values for basal PAl activity do not permit exclusion of hypofibrinolysis. The clinical relevance of the different mechanisms responsible for a reduced fibrinolytic capacity requires further investigation.

94

Heterogeneous Mechanisms Responsible for Reduced Fibrinolytic Capacity

CONCLUSION

Our work underlines the difficulties of clinical interpretation of hypofibrinolysis. The clinical characteristics do not differ in the different groups. The body mass index seem higher in BR, and its good correlation with the PAl suggests that weight is a risk factor in hypofibrinolysis. A weight reduction for these patients might lessen the risk of recurrences. Treatment does not differ between the two groups. The percentage of BR is not higher in recurrent versus mono-episodic DVT. However, the 'real BR' condition seems to be more frequently associated with recurrencies or familial thrombotic tendency, although this is not statistically significant. The association of the different criteria for hypofibrinolysis, augmentation of residual PAl activity to above 2U/ml, and/or global lysis time lengthened after VO (ECLT->90min, D W B L T - 1 2 0 m i n ) allowed us to divide 109 patients with a history of venous thrombosis into three groups: (1) GR: 46 patients; (2) BR: 28 patients in whom we found an alteration of t-PA-PAI balance, either to an excess of basal PAl activity, or to a decreased t-PA release and (3) 35 patients in whom either the t-PA-PAI imbalance is compensated, or an anomaly in another pathway of the fibrinolytic system is responsible for the bad response and remains to be characterized. In practice, testing for hypofibrinolysis must be performed some time after the acute episode, because PAl activity may be increased secondary to inflammation. If the PAl is very high, (>15U/ml) stimulation is useless because an abnormal response will be obtained in any case as mentioned before; if the PAl activity rises moderately, there may be either confirmed a hypofibrinolysis or compensation of the excess of PAI activity. At the present time, the evidence that a bad response to venous stasis is a risk factor of venous thromboembolism is weak. A prospective study could determine whether a bad response to VO could be an indication to start or continue anticoagulant treatment. This would be especially difficult in young patients with no risk factors and no obvious other hematologic abnormality that would predispose to thromboembolism. The definition of bad responders on only the t-PAl-PAl pathway ignores the possibility of the presence of an alteration of contact phase which could be of clinical relevance. 35-39 The t-PA-PAI balance is not the only mechanism responsible for hypofibrinolysis. Therefore in many cases an imbalance in the t-PA-PAI system does not agree with global lysis tests of the fibrinolytic activity in blood. The heterogeneity of the causes of hypofibrinolysis does not permit its diagnosis by any single test. Combinations of tests, and a better understanding of the multiple pathways involved, will be necessary if we wish to prospectively identify patients at

significant risk of future thrombosis or who might benefit from long term anticoagulant therapy.

ACKNOWLEDGEMENTS We would like to thank Sydney Hecker, MD, MS (Med), Medical Director Health Plans, Palo Alto Medical Clinic, Palo Alto, CA, USA for his help in the preparation of the manuscript.

REFERENCES 1. Heijboer H, Brandjes D P M, Buller H M. Deficiencies of coagulation-inhibiting and fibrinolytic proteins in outpatients with deep venous thrombosis. N Engl J Med 1990; 323: 1512-1516. 2. Conard J, Horellou M H, Samama M. Incidence of thromboembolism in association with congenital disorders in coagulation and fibrinolysis. Acta Chir Scand 1988; 543 (suppl): 15-25. 3. Lijnen H R, Collen D. Review: Congenital and acquired deficiencies of components of the fibrinolytic system and their relationship to bleeding and thrombosis. Fibrinolysis 1989; 3: 67-78. 4. Dolan G, Preston F E. Familial plasminogen deficiency and thromboembolism. Fibrinolysis 1988; 2 (suppl 2): 26-34. 5. Shigekiyo T, Uno Y, Tomonari A, Satoh K, Hondo H, Ueda S, Saito S. Type I congenital plasminogen deficiency is not a risk factor for thrombosis. Thromb Haemost 1992; 67: 189-192. 6. Lijnen H R, Soria J, Soria C, Collen D, Caen J P. Dysfibrinogenemia (fibrinogen dusard) associated with impaired fibrin enhanced plasminogen activation. Thromb Haemost 1984; 51: 108-109. 7. Wiman B, Hamsten A. The role of fibrinolytic system in deep vein thrombosis. Lab Clin Med 1985; 105: 265-270. 8. Conard J, Veuillet Duval A, Horellou M H, Samama M. Etude de la coagulation et de la fibrinolyse dans t31 cas de thromboses veineuses r6cidivantes. Nouv Rev Fr Hematol 1982; 24: 205-209. 9. Nilsson I M, Ljungner H, Tengborn L. Two different mechanisms in patients with venous thrombosis and defective fibrinolysis: low concentration of plasminogen activator or increased concentration of plasminogen activator inhibitor. BMJ 1985; 290: 1453-1456. 10. Pet~ij~iJ. Fibrinolysis and venous thrombosis. Academic dissertation. Helsinki 1989. 11. Nilsson I M, Tengborn L. Impaired fibrinolysis: new evidence in relation to thrombosis. In: Jespersen J, Kluft C, Korsgardo, eds. Clinical aspects of fibrinolysis and thrombosis. Esbjerg: South Jutland University Press, 1983: 273-285. 12. Juhan-Vague I, Alessi M C, Fossat C, Valadier J, Aillaud M F, Serradimigni A. Clinical relevance of reduced t-PA release and elevated PA inhibitor in patients with spontaneous or recurrent deep venous thrombosis. Thromb Haemost 1987; 57: 67-72. 13. Aoki N. Fibrinolysis. Semin Thromb Haemost 1984; 10: 1-103. 14. Robertson B R, Pandolfi M, Nilsson I M. 'Fibrinolytic capacity' in healthy volunteers as estimated from effect of venous occlusion of arms. Acta Chir Scand 1972; 138: 429436. 15. Prowse C V, Cash J D. Physiologic and pharmacologic enhancement of fibrinolysis. Sem Thromb Haemost 1984; 10:51~:~0. 16. Wiman B, Melibring G, Rhnby M. Plasminogen activator release during venous stasis and exercise as determined by a new specific assay. Clin Chim Acta 1983; 127: 279-288. 17. Hedner U, Nilsson I M. The role of fibrinolysis. Clinics in Haematol 1981; 10: 327-342.

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18. Robertson B R, Pandolfi M, Niisson I M. Response of local fibrinolytic activity to venous occlusion of arms and legs in healthy volunteers. Acta Chir Scand 1972; 138: 437--440. 19. Juhan-Vague I, Alessi M C, Fossat C, Valadier J, Aillaud M F, Serradimigni A. Clinical relevance of reduced t-PA release upon venous occlusion in patients with idiopathic/ recurrent deep venous thrombosis. Fibrinolysis 1988; 2 (suppl 2): 112-113. 20. Nilsson I M, Ljungner H, Tengborn L. Two different mechanisms in patients with venous thrombosis and defective fibrinolysis: low concentrations of plasminogen activator or increased concentration of plasminogen activator inhibitor. BMJ 1985; 290: 1453-1456. 21. Nguyen G, Horellou M H, Kruithof H K O, Conard J, Samama M. Residual plasminogen activator inhibitor activity after venous stasis as a criterion for hypofibrinolysis: A study in 83 patients with confirmed deep venous thrombosis. Blood 1988; 72: 601-605. 22. Samama M. Hypofibrinolysis and venous thrombophilia. Fibrinolysis 1980; 4 (suppl 3). 23. Korninger C, Lechner K, Niessner H, G6ssinger H, Kundi M. Impaired fibrinolytic capacity predisposes for recurrences of venous thrombosis. Thromb Haemost 1984; 52: 127-130. 24. Kluft C, Brakman P. Effect of Flufenamate on euglobulin fibrinolysis, involvement of Cl-inactivator. In: Davidson J F, Samama M, Denoyers P C, eds. Progress in chemical fibrinolysis and thrombosis, vol 1. New York: Raven Press, 1976, 375. 25. Fearnley G R, Balmforth G, Fearnley E. Evidence of a diurnal rhythm with a simple method of measuring fibrinolysis. Clin Sci 1957; 16: 645-657. 26. Kluft C, Brakman P. Screening of fibrinolytic activity in plasma euglobulin fractions on the fibrin plates. In: Davidson J F, Samama M, Denoyers P C, eds. Progress in chemical fibrinolysis and thrombosis, vol 2. New York: Raven Press, 1976, 57. 27. Verheijen J H, Chang G T G, Kluft C. Evidence for the occurrence of a fast acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemost 1984; 51: 392-395. 28. Juhan-Vague (personal communication). 29. Urano T, Sakakibara K, Rydzewski A, Urano S, Takada Y,

Received: 26 March 1993 Accepted after revision: 13 July 1993 Offprint orders to: Dr M. Samama, Laboratoire Central d'h6matologie, H6pital H6tel Dieu, 1 place du Parvis N6tre Dame, 75004 Paris, France.

Takada A. Relationships between euglobulin clot lysis time and the plasma levels of tissue plasminogen activator and plasminogen activator inhibitor 1. Thromb Haemost 1990; 63: 82-88. 30. Grimaudo V, Bachmann F, Hauert J, Christie M A, Kruithof E. Hypofibrinolysis in patients with a history of idiopathic deep venous thrombosis and/or pulmonary embolism. Thromb Haemost 1992; 67: 397-401. 31. Pet~ij~iJ, Rasi V, Vahtera E, Myllyla G. Familial clustering of defective release of t-PA. Br J Haematol 1991; 79: 291-295. 32. Harbourne T, O'Brien D, Nicolaides A N. Fibrinolytic activity in patients with idiopathic and secondary deep venous thrombosis. Thromb Res 1991; 64: 543-550. 33. Levi M, Ten Cate J W, Dooijewaard G, Sturk A, Brommer E J P, Agnelli G. D.D.A.V.P induces systemic release of u-PA. Thromb Haemost 1989; 62: 686-689. 34. Van Dreden P, Conard J, Sasportes T, Horellou M H, Samama M. Changes in t-PA activity and in free t-PA antigen after venous occlusion in patients with venous thromboembolic disease. Fibrinolysis 1992; 6 (suppl 2): 48. 35. Jennings I, Luddington J, Harper P L. Changes in endothelial-related coagulation proteins in response to venous occlusion. Thromb Haemost 1991; 65: 374--376. 36. Levi M, Hack E C, de Boer J P, Brandies D P M, Biiller H R, ten Cates J W. Reduction of contact activation related fibrinolytic activity in factor XII deficient patients. Further evidence for the role of the contact system in fibrinolysis in vivo. J Clin Invest 1991; 88: 1155-1160. 37. von K~inel R, Wuillemin W A, Furlan M, L~immle B. Factor XII clotting activity and antigen levels in patients with thromboembolic disease. Blood Coagul Fibrinolysis 1992; 3: 555-561. 38. Halbmayer W-M, Mannhalter C, Feichtinger C, Rubi K, Fischer M. The prevalence of factor XII deficiency in 103 orally anticoagulated outpatients suffering from recurrent venous and/or arterial thromboembolism. Thromb Haemost 1992; 68: 285-290. 39. Binnema D J, Dooijewaard G, van Lersel J J L, Turion P N C, Kluft C. The contact system dependent plasminogen activator from human plasma: identification and characterization. Thromb Haemost 1990; 64: 390-397.