Blur, Band 34, Seite 215-222 (1977) Medical Research Unit, 2nd Propaedeutical Medical Department of Athens University, Evangelismos Hospital, Athens.

Platelet Function, Blood Coagulation and Fibrinolysis in Beh.cet's Syndrome Nicolas E. Stathakis, Theofanis C. Economopoulos, Antony G. Papayannis and Demetrios Thomopoulos Summary In 10 patients with Beh~et's syndrome, various parameters ofplatelet function, blood coagulation and fibrinolysis were studied. With varying frequency the following abnormalities were found: increased retention of platelets in glass bead column, reduced platelet aggregation to low concentrations of adenosine diphosphate, elevated plasma levels of fibrinogen concentration and factor VIII activity, increased plasma antiheparin activity and impairment of fibrinolytic activity. The above abnormalities were found long after the last thrombotic episode and were more frequent in patients with a history of thrombophlebitis. It is suggested that certain hemostatic abnormalities accompany or form part of Beh~et's syndrome and that they are related to the thrombotic complications characteristic of this syndrome. Zusammenfassung Bei 10 Patienten mit Behqet-Syndrom wurden verschiedene Parameter der Thrombozyten-Funktion, der Blutgerinnung und der Fibrinolyse untersucht. Bei einem Teil der Patienten fanden sich abnorme Werte: Erh6hte P1/ittchen-Adh~tsion, verminderte P1/ittchen-Aggregation bei niedrigen Adenosin-Diphosphat-Konzentrationen, Erh6hung des Fibrinogens, des Faktor VIII und der Antiheparin-Aktivit~it im Plasma, Verminderung der fibrinolytischen Aktivit/it. Derartige Ver/inderungen wurden 1/ingere Zeit nach dem letzten thrombotischen Ereignis gefunden, waren abet bei Patienten mit friiher durchgemachter Thrombose hfiufiger. Diese Erkenntnisse weisen darauf hin, dal3 StSrungen der Blutgerinnung einen Tell des Behqet-Syndroms bilden oder mit ihm verbunden sind und dab diese Ver~inderungen mit den charakteristischen thrombotischen Komplikationen dieser Erkrankung in Zusammenhang stehen. Key words: Behqet's syndrome, blood coagulation, fibrinolysis, platelet function.

In 1937 Behfet described a syndrome consisting of relapsing iritis with recurrent ulceration of the mouth and genitalia. It has since been recognized that many other systems may be involved. Superficial and deep vein thrombosis is a prominent feature of the disease, with frequency varying from 10 to 46% in various series [9, 11, 12]. Thrombosis of the inferior and superior vena cava is also not rare [15]. The etiology Eingegangen am 21. 6. 1976

216

N. t3. Slathakis, T. C. Economopoulos, A. G. Papayannisand D. Thomopoulos

o f the thrombophlebitis in the disease is not known. Vasculitis, the underlying p a t h o l o g y o f the disease, is p r o b a b l y the initiating event. Hemostatic disturbances may also account for the t h r o m b o p h i l i c tendency [7]. As the disease is rare, little information is available on this point. I n this w o r k we have studied 10 patients with Beh$et's syndrome, with regard to platelet function, b l o o d coagulation and fibrinolysis. Material and methods Ten patients with Behw syndrome, all males, aged from 23 to 45, were studied. The diagnosis was established on clinical criteria. In all patients at least two of the classical triad of symptoms - a) oral or genital ulcers, b) the typical eye lesions, including uveitis and iritis and c) central nervous system involvement - were present. The clinical data of each patient are shown in Tab. 1 a. Five of them had a history of thrombophlebitis: Blood samples were collected early in the morning into plastic syringes using 20 G needles. The blood was anticoagulated with sodium citrate 3.80/0 in a ratio 10 : 1. Platelet rich plasma (PRP) and platelet poor plasma (PPP) were obtained by centrifuging at 240 g for 30 rain and 1.500 g for 30 rain respectively. The PRP was then adjusted to 300,000 platelets per t~l using the patient's own PPP. The plasma for platelet studies was treated at room temperature while the plasma for coagulation and fibrinolysis tests was treated at + 4 ~ C. All glassware was siliconized. All tests were done within two hours after sampling. The platelet count, bleeding time and platelet retention in glass bead column were performed according to Brecher and Cronkite [6], Mieke et al. [18] and Papayannisand Isragls [23] respectively. For the platelet aggregation tests the turbidometric technique of Born [3] and O'Brien [20] was applied using a platelet aggregation meter (ELL, model 169). The following aggregation agents were used: 1) Adenosine diphosphate (ADP) to a final concentration (f. c.) of 1 ptM and 20 ptM. 2) Collagen 0.1 ml of human connective tissue extract prepared according to Hoving [14] was added. 3) Adrenalin to a f. c. of 5 b~M. The degree of aggregation was expressed as a percentage of maximal fall in optical density. In the collagen induced aggregation the "delay period" and the slope of the aggregation curve were also estimated. The adrenalin induced aggregation consisted of two phases, graded 0 to 4 + ; normally the depth of each was 1 to 3 + for the first phase and 2 to 4 + for the second phase. ADP-release from platelets by collagen was estimated by the platelet aggregation method [29]. The platelet factor - - 3 (PLFa) availability was estimated according to Spaet and

Cintrom [26]. The following plasma factors were assayed: fibrinogen according to Ratnoff and Menzie [24], factor V according to Stormorken [27] and factor VIII according to Hardis*y and Macpherson [13]. Antithrombin III activity (at-III) was estimated according to Abildgaard et al. [I]. For the heparin tolerance test the technique of Godal [8] slightly modified was applied. Fibrinolytic activity was estimated by the euglobulin clot lysis time test [28], and the results expressed in units by multiplying the reciprocal of the lysis time in min by 10,000. Plasminogen was measured by the caseinolytic method of Alkjaersig et al. [2] and fibrinogen/ fibrin degradation products (FDP) by the method of Merskey et al. [17]. Inhibitory activity against plasmin and urokinase was screened by the method of Brakman and Astrup [4] and Brakman et al. [5]. Human plasmin (Kabi, Sweden) and urokinase (Koch-Light, England) were used. Plasma levels of the globulins al-antitrypsin and a2-macroglobulin were measured by the single radial immunodiffusion method [16] using specific antisera (Hoechst, Behring Diagnostics). The results were expressed for convenience as the percentage of normal value obtained from 10 normal pooled sera. Each patient was studied on at least two occasions. The mean value of the results of each study is presented here. Matched normal subjects tested simultaneously with the patients served as controls.

43 26 28 31

3 4 5 6

7 8 9 10

Tab. 1 b : Platelet studies (continued).

= = standard deviation,

4.7 • 1.2 3-7

266 • 52 175-350

Controls

= m e a n value,

4.8 • 0.7 3.0 -6.5

279: • 213 220 -450a

Patients

:

Bleeding time (min)

-k q-}q-

~-~ qqq-

-k

genital ulcers

none prednisolone

-q-

-}--

-

-

82 • 4 65-87

26 • 12-38 20.1 • 4.7 17.2-26.6

5.8 • 4.3 5-15 a = range

83 +1 80-87

28 • 24-32

20.4 • 1.0 17.6 -24.5

•

85 8 67-98

88 • 78-95

e

(%)

n

(see)

o

(see)

n

nolle

/'lone

chlorambucil none

Total aggregation

-

-}-

--

-k --

+ --

prednisolone

-}-

+

n o n e

-{-

--

--

n o n e

--

prednisolone

treatment

_Aggregation by collagen Delay Slope period (0 ~

--

--

-~ --

thrombophlebitis

cutaneous lesions

Platelet s availability

-k -~ --. --

+

-

-

-

-

--+

--

gangrene o f the leg

--+

q-

C.N.S. involvement

4.4 +_ 5.2 2.5 -6.5

ADP-release by collagen (tzg/3 • 108 pls)

qqq-k

-~ -b + qq-

qqq+

-k

+

oral ulcers

--qqq-

eye lesions

Platelet counts ( • 10a/~xl)

Tab. I a : Clinical data o f the patients.

8 3 2 3

12 1.5 1 15 1 6

45 26 29 35 23 41

2

1

years f r o m onset

age

patient

g

218

N. E. Staihakis, T. C. Economojooulos~A . G. Papayannis and D. Thomopoulos

Results

Platelet function The results of the platelet studies are shown in Tab. l b and 2. Piatelet retention in glass bead column was increased, the mean value (57.2 • 8.1%) being significantly higher than that of the controls (p < 0.001). The platelet aggregation in response to low doses of A D P (I~,M) was poor in most of the cases. The mean value (18.6 _+ 9.1%) was significantly lower than that of the normal controls (p < 0.01). When a higher concentration of A D P was employed, however, the aggregation was similar in patients (79 _+ 4%) and in controls (77 _+ 9%). Case No.

Platelet retention to glass beads (%)

Aggregation by ADP 1 ptM (%) 20 b~M (%)

1 2 3 4 5 6 7 8 9 10 M _+ S.D.

61 43 50 58 51 60 71 62 64 52 57.2 _+ 8.1

42 12 19 10 9 20 20 16 18 20 18.6 _+ 9.1

Controls iV[ _ S.D. Range P

47.8_+7.0 39-60 < 0.01

28.5_+8.7 19-50 < 0.01

89 73 85 78 79 90 70 80 72 74 79 _+ 4 77_+9 65-90 N.S.

M = Mean; S.D. = standard deviation; N. S. = Nonsignificant Tab. 2: Platelet studies Platelet count, bleeding time, platelet aggregation by adrenalin and collagen, ADP-release from the platelets and platelet factor-3 availability were within normal limits.

Coagulation and fibrino~sis Fibrinogen (350 _+ 48 mg/100 ml) and factor V I I I activity (187 • 92%) were markedly elevated (p < 0.01) (Tab. 3). Factor V and at-III were normal. A significant increase of plasma antiheparin activity was found in 2 out of 6 cases in which the heparin tolerance test was performed (Fig. 1). Fibrinolytie activity, as assessed by the euglobulin clot lysis time, was markedly decreased in 3 cases and around the lower limits of normal in another 3 cases (Tab. 3). Plasminogen and F D P were normal in all cases. In five patients a very significant elevation of the inhibitory activity against plasmin (Fig. 2) and nrokinase was found. The a=-macroglobulin was normal in all, while al-antitrypsin was elevated in some cases. Although 4 out of 5 cases with increased levels of inhibitory activity against

Platelet function, blood coagulation and fibrinolysis in Bebfft's syndrome

l

219

N0r

200-

// 100|

80-

| E

60-

o~

Z

/1/ 40-

/

/ /S//, // ...sYd"

30-

_o U

20-

i

l .6

o~_: =~_/-- - . ~

15-

10

|

I

~/9:~,0 ~/s20

I

1/160

Heparin

I

1/80

dllullon

I

1/40

I

1/20

I

1/10

(I110 ~0.3 ulml )

Fig. 1: Heparin tolerance test performed using plasma-thrombin system and progressively higher dilutions of heparin. Comparison with a normal control. plasmin had p r o l o n g e d euglobulin lysis times, a direct correlation between t h e m was n o t found. N o correlation could be also established between the levels o f fibrinolytic inhibition activity and the levels o f individual globulin - al-antitrypsin and %-macroglobulin - selected for assay because o f their k n o w n antiplasmin effect.

Case No.

Fibrinogen (mg/100 ml)

1 2 3 4 5 6 7 8 9 10 M • S.D.

350 320 185 758 365 340 305 240 360 280 350 • 48

180 100 75 400 205 218 160 125 250 160 187 • 92

37 45 31 7 51 23 47 27 22 31 32 • 4.2

Controls M + S.D. Range P

230 _+ 44 162-320 200~o Euglobulin clot lysis time < 30 units Antiplasmin increased Antinrokinase increased

4 3 4 4 3 4 4

patients without history of thrombosis (5 cases)

Tab. 4: Correlation of abnormal haemostatic findings with thrombotic phenomena. Discussion The results of the present study revealed that Beh~et's syndrome is accompanied by a number of abnormalities in platelet function, blood coagulation and fibrinolysis. The platelet function abnormalities consisted of increased PAd and reduced responsiveness of platelets to small doses of ADP. Increased retention of platelets in glass bead

Platelet function, blood coagulation and flbri•olyds in Behs , syndrome

221

column has been found in many conditions and diseases predisposing to thrombosis, both venous and arterial. However, the etiological or diagnostic significance of this abnormality, which may reflect platelet or plasma hyperactivity, in relation to thrombosis is not clear. Of interest is the impairment of the platelet responsiveness to low doses of ADP. This may be due to prior exposure to small amounts of ADP, released by the platelets in the circulation, which leads to a refractory state [21,22J. The small vessels affected by vasculitis, which is the underlying pathology of the disease [19] may be the site of this process. Increased plasma antiheparin activity has been reported in patients with thrombophlebitis [ 10]. It has been suggested that the heparin-neutralizing activity of plasma is due to release of platelet factor 4 during intravascular aggregation or damage of the platelets [10,25]. The increased antiheparin activity of our patients may well be attributed to platelet factor 4 which could be released during the circulation of platetets through the inflamed small vessels, i.e. to the same process proposed to explain the refractoriness of platelets to small concentrations of ADP. The blood coagulation and fibrinolysis studies showed elevated levels of fibrinogen and factor VIII as well as hypofibrinolysis due, at least in part, to increased inhibitory activity against plasmin. These findings confirm previous observations of Chajek and Fainnaru [7]. In our patients, however, these defects were less frequent and severe. This may be attributed to differences in the clinicaI status and the treatment of the patients studied. The increased inhibition of urokinase should be considered as uncertain, since that of plasmin could influence the test system used. Normal plasmin inhibition may be a prerequisite for the evaluation of the activator inhibitors. The increased inhibitory activity against plasmin was not correlated with a proportional increase in a2 macroglobulin or al-antitrypsin which are responsible for most of the normal antiplasmin activity of plasma. The data of the present study show that abnormalities of hemostasis are frequently encountered in Beh~et's syndrome. It seems unlikely that acute thrombosis was responsible for their appearance since our patients had not suffered any clinically detectable thrombotic episode for, at least, three months before the study. On the other hand, the hemostatic abnormalities were more frequent in patients with history of thrombophlebitis. This relationship suggests that changes in platelet function, blood coagulation and fibrinolysis may play a role in the pathogenesis of the thrombotic complications of the Behget's syndrome. Acknowledgement We are grateful to Miss Renate Sturm for technical assistance.

Rgfgrgngr163 1. Abildgaard U., Gravem K. & Codal t-l. C. : Assay of progressive antithrombin in plasma. Thromb. Dialh. baemorrh. 24, 224 (1970). 2. Alkjaersig N., Fletcher A. P. & Sherry S. : The mechanism of clot resolution by plasmin.J, din. Invest. 38, 1086 (1959).

3. Born G. V. R.: Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194, 927 (1962). 4. Brakman P. & Astrup T. : Selective inhibition in human pregnancy blood of urokinase induced fibrinolysis. Scand. J. din. lab. Invest. 15, 603 (1963).

222

~V. 1g.. Stathakis, T. C. Economopoulos, A . G. Papayannis and D. Thornopoulos

17. Merskey C., Kleiner G. J. & Johnson 5. Brakman P., Mohler G. R. Jr. & A. J. : Quantitative estimation of split Astrup T.: A group of patients with products of fibrinogen in human serum: impaired plasma fibrinolytic system and relation to diagnosis and treatment. selective inhibition of tissue activator Blood 28, 1 (1966). induced fibrinolysis. Scand.J. Haemat. 3, 389 (1966). 18. Mielke C. H. Jr., Kaneshiro M. M., 6. Brecher G. & Cronkite E. P.: MorMaher J. A., Weiner J. M. & Rapaport phology and enumeration of human S. L.: The standardized normal Ivy bleeding time and its prolongation by blood platelets. J. appl. PbysioL 3, 365 (1950). aspirin. Blood 34, 204 (1969). 7. Chajek M. & Fainnaru M.: Beh~et's 19. Nazzaro P.: Behget's Disease. (ed. by disease with decreased fibrinolysis and M. Monacelli & P. Nazzaro), p. 15, superior venae cavae occlusion. Brit. Basel (1966). reed. J. I, 782 (1973). 20. O'Brien J. R. : Platelet aggregation. II. 8. Codal H. C. : Heparin tolerance and the Some results from a new method of plasma proteins. Seand. J. din. lab. Invest. study. ]. din. Path. 15, 452 (1962). 13, 314 (1961). 21. O'Brien J. R. : A comparison of platelet 9. Dowling G. B.: Behget's disease. Proc. aggregation produced by seven comroy. Soc. Med. 54, 101 (1961). pounds and a comparison of their in10. Farbiszewski R., Niewiarowski S., Wohibitors, jr. din. Path. 17, 275 (1964.) rowski K. & Lipinski B. : Release of 22. O'Brien J. R., Etherington M. & platelet factor 4 in vivo during intraJamieson S. : Refractory state of platelet vascular coagulation and in thromaggregation with major operations. botic states. Thromb. Diath. haemorrh. 19, Lancet 2, 741 (1971). 575 (1968). 23. Papayannis A. G. & Israels M. C. G. : 11. France R., Buchanan R. N., Wilson The value of the platelet adhesiveness M. W. & Sheldon M. B.: Relapsing test in the assessment of abnormalities iritis with recurrent ulcers of mouth of platelet function. Acta Haemat. 46, 1 and genitalia (Behget's syndrome): Re(1971). view with report to additional case. 24. Ratnoff O. D. & Menzie C.: A new Medicine (Baltimore) 30, 335 (1951). method for the determination of fibri12. Haim S., Barzilai D. & Hazani E.: nogen in small samples of plasma. J. Involvement of veins in Behget's synLab. din. Med. 37, 316 (1951). drome. Brit. J. Derraat. 84, 238 (1971). 25. Sear C. H. J. & Poller L. : Antiheparin 13. Hardisty R. M. and Macpherson J. C. : activity of human serum and platelet A one stage factor VIII (antihaemofactor 4. Thromb. Diath. beamorrh. 30, philic globulin) assay and its use on 93 (1973). venous and capillary plasma. Tbromb. 26. Spaet T. & Cintrom J.: Studies on Diatb. haemorrh. 7, 215 (1962). platelet factor 3 availability. Brit. J. 14. Hovig T. : Aggregation of rabbit blood Haemat. 11, 269 (1965). platelets produced in vitro by saline 27. Stormorken H.: The preparation of "extract" of tendons. Thromb. Diath. proaccelerin (parahaemophilia) plasma haemorrh. 9, 248 (1963). for the assay of proaccelerin. Scand. J. 15. Kansu E., Ozer F. L., Akalin E., Giiller din. lab. Invest. 9, 273 (1957). V., Zileli T., Tanman E., Kaplapan E. 28. Von Kaulla K. N.: Chemistry of & Mtiftiioglu E.: Behget's syndrome thrombolysis: human fibrinolytic enwith obstruction of the venae cavae: a zymes. Springfield, Ill., p. 76, Thomas report of seven cases. Quart. J. Med. 41, (1963). 151 (1972). 29. Weiss H. J. : Platelet aggregation, adhe16. Mancini G., Carbonara A. O. & sion and adenosine diphosphate release Heremans J. F. : Immunochemical quanin thrombopathia (Platelet factor 3 detitation of antigens by single radial ficiency). Amer. J. Med. 43, 570 (1967). immunodiffusion. Int. J. Immunocbem. 2, 235 (1965). Author's address : Ass. Professor Dr. A. G. Papayannis, Evangelismos Hospital, Athens 140, Greece.