Fibrinolytic response in normal subjects to venous occlusion and DDAVP infusion

THROMBOSIS RESEARCH 56; 625-634, 1989 0049-3848/89 $3.00 t .OO Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.

FIBRINOLYTIC RESPONSE IN NORMAL SUBJECTS TO VENOUS OCCLUSION AND DDAVP INFUSION

M. Cugno, L. Uziel, I. Fabrizi, B. Bottasso*. F. Maggiolini. A. Agostoni. Department of Clinical Medicine, University of Milano. “S. Paolo” Hospital, Milano, Italy. * A. Bianchi Bonomi Hemophilia and Thrombosis Centre and Third Institute of Clinical Medicine, University of Milano, Italy (Received 11.3.1989; accepted in revised form 2.10.1989 by Editor P.M. Mannucci)

ABSTRACT

A set of fibrinolytic arameters was measured in 40 healthy subjects before and a?ter a venous occlusion (VO) test lasting 10 min. After VO, plasma levels of tissue-type plasminogen activator (t-PA) antigen increased in all subjects, t-PA activity increased only in 25 subjects who were considered responders and remained unchanged in 15 (non-responders). High levels of plasminogen activator inhibitor (PAI) in the non-responder group explain this discrepancy. The non-responders had basal levels of PAI activity and t-PA anti en higher than responders (
INTRODUCTION The efficacy of the fibrinolytic system depends on the conversion of the plasma protein plasminogen to the protease plasmin by plasminogen activators. Two types of plasminogen activators have been identiiled in plasma: tissue-type plasminogen activator (t-PA), synthesized in the vascular Key Words: Venous occlusion, Desmopressin, activator, plasminogen activator inhibitor. 625

tissue-type

plasminogen

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endothelium (1). and urokinase-type plasminogen activator (u-PA), initially purified from urine. t-PA activity is the resultant of its release from the endothelium (2). he atic clearance (3) and interaction with fibrin (4). The most important inhi %itor of t-PA is plasminogen activator inhibitor 1 (PAI- 1) from the endothelium (5). Under basal conditions, there is little or no plasma activity of t-PA (6) since it circulates mainly complexed to the natural inhibitor PAI- 1 (7). t-PA is released from the endothelium in the bloodstream after various stimuli, such as venous occlusion (VO) (8, 9) or infusion of the vasopressin analogue 1-desamino-8-D-a.rginine vasopressin (DDAVP) (10, 11). VO has been lar ely used as a dia ostic procedure to assess the fibrinolytic otential otgindividuals. Poor fiT rinolytic responses to VO have been descri %ed in several thrombotic diseases, both venous (12, 13. 14) and arterial (15. 16). but the wide overlapping of results for patients and normal controls makes it difficult to interpret the test in the individual patient. During the evaluation of the fibrinolytic responses to VO in normal subjects, to establish reference intervals in our laboratory, we found that an unexpectedly high population of these subjects did not respond to VO. Therefore, we chose to study the causes for this abnormality, and to evaluate whether DDAVP could elicit a response in non-responders to VO. MATEFUALS AND METTHODS Subjects We studied 40 healthy volunteers from our medical staff (27 men and 13 women) grouped for age (16 subjects from 20 to 40 years: 16 subjects from 40 to 60 years and 8 subjects over 60 years). No subject was overwei ht according to the body mass index [(weight in Kg) divided by (height in mf 21, cut off values: 27 for men. 25 for women. Sample collection Blood samples were collected in the morning before 9.00, after an overnight fast and a rest of 15 min. After discarding the first 2 ml of blood, 9 volumes of whole blood were added to 1 volume of 3.8% trisodium citrate. Platelet poor plasma was obtained by immediately centrifuging the blood at 40 C at 2000 g for 30 min. Plasma was then a"ed into plastic tubes, snap frozen in a mixture of solid carbondioxide and methanol and stored at -800 C until tested. Venous occlusion test The VO test consisted of maintaining a pressure midway the diastolic and the systolic arterial ressure for 10 min. in a sph gmomanometer cuff placed around an arm. B Pood samples were collecte cr from an antecubital vein before and at the end of the VO. DDAVP test DDAVP (Minirin, Ferring, Malmo. Sweden) was infused intravenously at a dose of 0.4 pg/Kg. diluted in saline, over 30 min. Blood samples were collected before and immediately after the infusion.

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Laboratory tests Fibrinolvtic activitv in the normal euglobulin fraction of plasma was measured by the fibrin plate method (17). with the results expressed as mm of diameter of lysis. Purified Cl-inhibitor (Cl-INH. Immuno. Vienna, Austria) was added to a portion of the euglobulin fraction to inhibit intrinsic fibrinolysis activator and to provide a more specific measurement of tissueFPe plasminogen activator activity (18). Tissue-tvpe nlasminoeen activator A) activity in the normal eu obulin fraction of plasma was measured by the chromogenic method of Verd eijen (19). Tissue-type plasminoeen activator (t-PA) antie measured by a commercial immunosorbe% as::! (American Diagnostica. New York, activator inhibitor (PAI)activitv was measured by the chromogenic substrate, as described by Verheijen (20). Plasminoflen activator inhibitor-l (PA&l) antigen was measured by a commercial enzymelinked immunosorbent assay (American Diagnostica. New York, NY). van Willebrand factor antigen (vwF:Ag) was measured b a commercial enzymelinked immunosorbent assay (Boehringer. MannK eim, West Germany). FibrinoPen was measured by a commercial method (Fibrin0 en reagent, Boehringer, Marmheim, West Germany). mvcerides and choPester01 were measured in serum by commonly used methods (Iatron Laboratories Inc, Tokyo, Japan). C Reactive Protein was measured as a marker of acute phase b a commercial latex method (Behringwerke AG, Marbur , West Germany). Pratelets were counted by a Thrombocounter C (Coulter Ef ectronics Limited, Harpenden, UK). Design of the study All volunteers underwent VO and were tested for fibrinol ‘c activity, t-PA ter the test. To activity, t-PA antigen and PAI activity before and aiyu distinguish responders and non-responders we considered t-PA activity increased when it exceeded 0.05 IU/ml after VO: this cut-off value was derived from the lower detection limit of the assay. In 9 of 15 subjects who had no increase of t-PA activity over baseline after venous occlusion (nonresponders), the DDAVP test was also performed (the remaining 6 nonresponders refused the drug infusion). In these 9 subjects PAI- antigen and vwF:Ag measurements were added to the tests listed previously. Statistical analysis

Differences between roups were evaluated by the non-parametric test of Mann-Whitney: toe Bicients of correlation were determined by linear regression analysis.

RESULT8 Fibrinolytic responses to 10 min VO, for all the 40 healthy subjects are summarized in Table I. Despite a wide overlap of pre- and post-occlusion values, t-PA activity and t-PA antigen were signiiicantly increased (p
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TABLE I Fibrinolytic responses to Venous Occlusion (VO) in 40 healthy subjects. Basal

After VO

Fibrin plates (mm)

13.0 (<5.0-20.0)

19.0* (7.5-26.5)

Fibrin plates+C 1-M-I (mm)

<5.0 (<5.0-13.0)

13.0* (<5.0-21.1)

t-PA activity (IU/ml)

co.05 (cO.O5-0.81)

0.53* (<0.05- 12.66)

t-PA Ag (ng/ml)

7.6 (2.0-26.4)

14.w (5.5-52.0)

PAI activity (IU/mI)

7.0 (0.0-35.0)

5.0 (0.0-33.0)

Values are expressed as median and (range). Significance *pco.o01.

versus basal:

TABLE II Fibrinolytic responses to Venous Occlusion (VO) in 40 healthy subjects divided into Responders and Non-Res onders according to the postocclusion increase o P t-PA activity. RESPONDERS n=25 After VO Basal

NON-RESPONDERS n=15 Basal After VO

Fibrin plates (mm)

14.5 (6.0-20.0)

20.0* (15.5-26.5)

10.0+ 13.0 (<5.0- 13.6) (7.5- 18.0)

Fibrin plates+Cl-INH (mm)

8.0 (<5.0- 13.0)

15.01 (8.0-21.1)

<5.0++ (<5.0-6.0)

<5.0 (<5.0-9.0)

t-PA activity (III/ml)

0.12 2.37* (<0.05-0.81) (0.15-12.66)

<0.05+

co.05

t-PA Ag (ng/ml)

6.0 (2.0-11.7)

13.9* (5.5-50.7)

11.8++ (5.9-26.4)

21.0* (11.5-52.0)

PAl activity (III/ml)

4.35 (O.O-10.5)

o.o* (0.0-7.0)

11.0++ (7.0-35.0)

11.0 (6.4-33.0)

Values are expressed as median and (range). Signiiicance versus responders: +p
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When the subjects were ouped according to their capacity to increase t-PA activity after venous occ Fusion of at least 0.05 IU/mI, we could distin uish 25 responders and 15 non-responders (Table II), In non-responders, %asal levels of PAI activity and t-PA antigen were higher than in responders (p~O.0001) and fibrinolytic and t-PA activities were lower (fibrin lates: p
TABLE III Physical, hematological and biochemical parameters of 40 healthy subjects divided into Responders and Non-Responders to Venous Occlusion. RESPONDERS n=25

NON-RESPONDERS n=15

AGE (years)

43 (24-63)

g-66)

WEIGHT (Kg)

60.5 (50-93)

Z-113)

BODY MASS INDEX (Kg/mz)

2 1.3 (18.8-26.9)

26.1* (23.7-27.0)

HEMATOCRIT (o/o)

42 (36-48)

43.6 (38-50)

PLATELETS (n x 103/mms)

191 (140-312)

196 (138-285)

FIBRINOGEN (mg/dl)

220 (150-315)

258 (180-425)

TRIGLYCERIDES

82 (41-140)

97.5 (47-20 1)

166 (128-243)

(147-251)

CHOLESTEROL

(mg/dl) (mg/dl)

ressed Values are Responders: ?? p< “g .05.

as

median

190

and

(range).

Significance

versus

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Nine non-responders to VO underwent DDAVP infusion, and in seven subjects this induced good res onses with significant increases in both functional and antigenic t-PA an f a fall in functional PAl (Table IV: figure 1).

TABLE IV Fibrinolytic and vwF:Ag responses to Venous Occlusion and DDAVP infusion in 9 Non-Responders to Venous Occlusion. VENOUS OCCLUSION n=9 Basal After

DDAVP INFUSION n=9 Basal After

(<5*0-13.0)

10.0

13.0 (7.5-15.5)

9.4 (<5.0-12.5)

~5.0

c5.0

<5.0

t-PA activity (IU/ml)

<0.05

co.05

t-PA Ag (rig/ml)

12.0 (5.9-26.4)

21.0* (11.6-34.4)

9.8 (5.6-16.3)

21.5** (10.4-42.6)

PAI activity (W/ml)

10.0 (6.4-33.0)

14.4 (7.5-25.0)

16.0 (4.6-26.0)

o.o** (O.O-16.0)

PA&l Ag (rig/ml)

20.1 (11.5-30.4)

24.1 (13.3-38.8)

22.9 (5.6-35.4)

12.1** (2.0-25.4)

vwF:Ag (%)

131 (83- 170)

191* (129-271)

110 (60- 174)

267** (152-377)

Fibrin plates (mm) Fibrin plates+Cl-INH (mm)

(<5.0-8.0)

(<5.0-8.1)

18.8** (5.4-21.3) l&4** (c5.0-19.0)

co.05 2.66** (<0.05-0.07)(<0.05- 12.94)

Values are expressed as median and (range). Significance versus basal: *p
There was a good correlation between PAI activity and PAI- antigen levels (x-=0.89. pcO.001). Although only DDAVP increased median t-PA activity (Table IV). both VO and DDAVP increased t-PA antigen and another marker of endothelial release, i.e., vwF:Ag (Table IV). Moreover there was a correlation between t-PA antigen and vWFAg (r=O.70; p
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W/ml

631

W/m 31

ia

3a

mm E!s

15

10

S

0

oJ

Fibrin plates

Fibrin plates + Cl-INH

t-PA activity

0

t-PA antigen

PAI activity

FIG. 1 Fibrinolytic parameters in 9 Non-Responders to Venous Occlusion before and after DDAVP infusion.

DISCUSSION We demonstrate that the lack of fibrinolytic response to venous occlusion in our health subjects is associated with high plasma levels of PAI and not with a de Ktit in release of t-PA. The majority of non-responders to VO became responders when DDAVP infusion was used as the stimulus (7 of 9 tested). The deiinition of non-responders is not standardized so far and this makes it difficult to corn are data of different studies. A poor fibrinolytic response to VO was descrig ed in 33-35% of patients with recurrent deep vein thrombosis (12- 14) and in 37% of patients who had survived a myocardial infarction (15) with wide overla between normals and patients (12.15). (In one paper , however, normal v apues are not indicated (14)). Recently Sultan et al. re orted a hi percentage of non-responders to VO (44%) in a small group o P normal su%h jects (21). In our grou of 40 normal subjects, 37% did not increase t-PA activity after VO, althoug R for the whole group there was a significant increase. Analyzing separately the results for the two groups, responders and non-responders, a release of t-PA antigen was common to

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both groups, but this increase was not associated with an increase in t-PA activity in non-responders. Thus the lack of response is not due to defective synthesis or release of the t-PA molecule, but to the higher values of PAI activity present in basal condition in non responders than in responders. Moreover in non-responders PAI activity remained stable after VO, while it fell to 0 in the majority of responders (Table II). As a consequence of the t-PA release, one would expect some decrease in PAI activity levels also in nonresponders. A lack of PAl activity decrease after venous occlusion has already been* reported (2 1.22) and possible lanations involve endothelial (22) and latelet PAI release or a delay in“fs1 e interaction between t-PA, released rf?om endothelium, and circulating PAI (21). This last hypothesis does not fit with our data because in non-responders, after VO. t-PA activity remained unchanged despite an increase in t-PA antigen. Measurement of PAIantigen by an immunoenzymatic method did not add further information, because this method mainly measures free PAL Among the h sical parameters selected, only age and weight were significantly lx g er in non-responders. Correlation between age and PAI levels has been well demonstrated (23). while, for weight, a reduction of flbrinol ‘c response to VO has been reported in obese patients (24). Despite the sli 2 tl higher weights of non-responders, none of our subjects was truly obese an cy all had triglycerides within the normal range. The fact that VO succeeded in inducing endothelial release of vWFAg in non-responders rules out the possibili that the lack in fibrinolytic response to VO may be due to a generalized en 8 othelial d sfunction. Our findings extend the Pmding of Sultan et al. (21) that VO indicates too many non-responders among normal subjects, makin its interpretation difficult when used clinical1 . DDAVP overcomes this lac% of sensitivity and seems to be preferable, al x ough its diagnostic use may be limited by the need of intravenous infusion and by the risk of side effects (25).

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