cyclooxygenase az of

Research Paper

Mediators of Inflammation 2, 385-389 (1993)

FLUID of artificial blisters from erythromelalgic skin areasin primary thrombocythaemia contained a high amount ofprostaglandin-E-like activity. Dazoxiben did not alleviatethe erythromelalgia in patients with primary thrombo-cythaemia despite complete inhibition of platelet mal-ondialdehyde and thromboxane B2 synthesis and no in-hibition of prostaglandin-E-like material. During a 10-daydazoxiben treatment period, persistent erythromelalgiawas associated with a significant shortened mean plateletlife span of 3.2 days. During subsequent treatment withlow dose acetylsalicylic acid daily complete relief of ery-thromelalgia was associated with inhibition of plateletprostaglandin endoperoxide production and correction ofplatelet mean life span to normal, 7.9 days. These observa-tions indicate that prostaglandin E2, or another prostaglan-din endoperoxide metabolite, is involved in the patho-genesisof erythromelalgia. The presented study does not

give one single clue as to the origin (platelet, vessel wall orother) of the prostanoid, but very likely originates fromplatelets because a very low dose of acetylsalicylic acid(250 to 500 mg every other day), which irreversibly inhibitsplatelet cyclooxygenase, is highly effective in the preven-tion of erythromelalgia in thrombocythaemia.

Key words: Aspirin, Cyclooxygenase, Dazoxyben, Erythro-melalgia, Platelets, Prostaglandins, Thrombocythaemia,Thromboxane

Prostaglandin cyclooxygenaseproducts but not thromboxaneAz are involved in thepathogenesis ofewthromelalgia inthrombocythaemia

J. J. Michiels1"cA and F. J. Zijlstraz

1Department of Haematology, University HospitalDijkzigt Molewaterplein 40, 3015 G D, and2Institute of Pharmacology, ErasmusUniversity Medical School, Rotterdam, theNetherlands

ca Corresponding Author

Introduction

Erythromelalgia is characterized by warm, red,congested extremities and painful burning sensa-tions. In what appears to be the antithesis ofRaynaud’s disease, warmth intensifies the dis-comfort and cold provides relief. Acroparesthesiase.g. tingling, pins and needles sensations, andnumbness in the toes and fingers usually precedethe disabling and burning distress. Erythromelalgiamay lead to painful acrocyanosis and peripheralgangrene.

In previous studies we demonstrated thaterythromelalgia is causally related to thrombo-cythaemia and results from platelet mediatedinflammation and microvascular changes (Fig. 1).1-3The histopathological vascular changes are confinedto arterioles and characterized by aspecific inflam-mation, fibromuscular intimal proliferation andocclusive thrombi in the absence of pre-existingvascular disease.4 Both clinical signs and vascularlesions completely disappear by treatment with theplatelet inhibiting drugs acetylsalicylic acid (ASA)and indomethacin, which inhibits platelet ag-gregation by inactivation of platelet cyclooxygenaseactivity.To obtain more information of the pathophysio-

logic mechanism of erythromelalgia, prostaglandin

activity in fluid from artificial blisters fromerythromelalgic areas was investigated. Sub-sequently the authors evaluated the effects ofdazoxiben, a selective inhibitor of thromboxanesynthetase activity, on erythromelalgia, plateletkinetics and prostaglandin synthesis in patients withprimary thrombocythaemia. Evidence is presented

THROMBOCYTHAEMIA VERA

PERIPHERAL GANGRENEACROCYANOSIS

OCCLUSIVEPLATELET

THROMBI INARTERIOLES

PLATELET DERIVEDGROWTH FACTOR

FIBROMUSCuLARINTIMAL PROLIFERATIONOF ARTERIOLES

INFLAMMATORYSYMPTOMS

PROSTAGLANDINSACTIVATION OF

COAGULATION ETC.

FIG. 1. Pathophysiologic pathways in the genesis of platelet-mediatederythromelalgia and acrocyanotic ischaemia in thrombocythaemia.

993 Rapid Communications of Oxford Ltd Mediators of Inflammation. Vol 2. 1993 385

j. j. Michiels and F. J. Zijlstra

that platelet prostaglandin E2 or another prosta-glandin endoperoxide metabolite is involved in thepathogenesis of erythromelalgia in primary throm-bocythaemia.

Methods

Platelet survival studies were performed withsodium 51Cr-chromate labelled autologous plate-lets. 1 The mean survival of platelets was calculatedaccording to the multiple hit model, as re-commended by the International Committee forStandardization in Haematology.2

Clinical and haematological data were obtained Resultsroutinely. Thermography was carried out with aBofors Mark II (Karls Koga, Sweden) camera, thatregisters the skin temperature indirectly. The skinsurface temperature was compared with a referencesource of fixed temperature.

Artificial dermal blisters were produced by asuction blister device according to Kristella,6 whichwas connected to the central suction unit of thehospital. The suction pressure was adjusted to100 mmHg. The suction cups were cleaned with70% alcohol and placed on the skin. Within a fewminutes the pressure was decreased slowly to

--200 mmHg. Blisters of 3 mm developed after 1to 2 h. Blisters were aspirated using a thin needleand syringe. 45 M

Prostaglandin-like material was extracted fromblister fluid according to the method of Unger etal.7 After resuspending the direct extracts in saline 2 73 M(NaC1 0.9%), PGE-like material was assayed againstauthentic PGE2 (Upjohn Co., Kalamazoo, USA) on 3 65 M

the isolated rat stomach strip, using the oil-bathtechnique of Ferreira and de Souza Costa.8 Bothstandard and test prostaglandins were injected in10/1 volumes directly into the Krebs’ solutionsuperfusing the tissue. 9 All values for PGE-likematerial were expressed as ng/ml blister fluid.

Malondialdehyde production by arachidonicstimulated platelets in platelet-rich plasma wasmeasured according to Smith et al. 1 Plasmathromboxane B2 (TxB2) and prostaglandin E.(PGE2) were measured in 10 ml peripheral venousblood samples collected under resting conditions inpolypropylene tubes, containing 20/1 of heparin(500/,/ml thromboliquine, Organon, the Nether-lands) and 50 #1 indomethacin (0.1 mg/ml in 0.1 Mphosphate buffer, pH 8.0). Blood samples werecentrifuged immediately at 1400 x g for 10 minand the plasma stored at -20C until assay. Twoml of plasma was applied to a Sep-Pak C18 cartridge(Water Ass). The prostaglandin-like compoundswere eluted with 2 ml of absolute ethanol, and200/,1 aliquots dried under a stream of nitrogen at40C in a radioimmunoassay tube, then redissolvedin assay buffer. Antibody for TxB2 and PGE2 wereobtained from L’Institute Pasteur (Paris, France),3H-TxB2 and 3H-PGE2 from New England Nuclear(Boston, USA) and standards of TxB2 and PGE2from Sigma. Normal values for TxB2 and PGE2were obtained from blood samples taken fromcontrols (aged 22-78 years) on two occasions.

Clinical and haematological data of three patientswith primary thrombocythaemia at the time ofstudy are summarized in Table 1. Thermographicdocumentation of erythromelalgia in the left upperleg and the sole of the right foot in Case 1 is shownin Fig. 2. The skin surface temperature exceeded31C at places of red painful erythromelalgic hot

Table 1. Pertinent data at time of study

Case Age Sex Plateletsx 109/I)

[Presenting symptoms]

750

1715

930

Disabling burning pain and redswelling of right forefoot sole andburning painful red spots in theskin of the left upper leg as shownin Fig. 2Burning pain and redness in toesand forefoot soleBurning pain in red-bluish big toe,forefoot sole and lateral edge of theright foot

Temperature33

I-- 3230-3128-2926-27

EC3 24-25

B

FIG. 2. Thermographic documentation of erythromelalgia in Case 1. (A)The skin surface temperature of painful red congested areas in the skinof the left upper leg and in the right forefoot sole exceed 31 C comparedwith that of the corresponding contralateral skin. (B) Complete relief oferythromelalgia 2 days after treatment with acetylsalicylic acid (500 mgper day) is associated with disappearance of hot spots on isothermo-grams.

386 Mediators of Inflammation. Vol 2.1993

Prostaglandin cyclooxygenase products in thrombocythaemia

Table 2. Prostaglandin E-like (PGE) activity in fluid of artificialblisters from skin areas with and without erythromelalgia inPatient with primary thrombocythaemia as shown in Fig.

Volume of blister fluid PGE Erythromelalgia(ml) (ng/ml)

Table 3. The effect of dazoxiben (200 mg every 6 hfor 5 days) on platelet malondialdehyde (MDA)synthesis in Patient 2 who suffered from primarythrombocythaemia and erythromelalgia

Day MDA (nmol/109 platelets)

1.19 5.40 Present1.29 6.53 Present1.02 0.80 Absent1.35 0.99 Absent*

*Erythromelalgic skin area during treatment with acetylsalicylicacid for 2 weeks.

Pretreatment value 2 h 4 h

14.3 3.52 1.98 4.813 1.25 5.094 1.98 4.435 1.89 5.51

spots, which completely disappeared by curativetreatment with aspirin.

Prostaglandin measurements in fluid of artificialblisters from erythromelalgic areas in the left upperleg in Patient 1 with primary thrombocythaemiaare shown in Table 2. PGE-like activities in blisterfluid from skin areas with active erythromelalgiameasured on two different occasions are evidently’higher as compared with the value in blister fluidfrom skin areas without erythromelalgia. PGE-likeactivity in blister fluid from erythromelalgic areasduring treatment with ASA declined to the level ofthe symptom-free skin area (Table 2).The effects of platelet inhibiting drugs on

erythromelalgia in typical cases of primarythrombocythaemia are shown in Fig. 3. Relief oferythromelalgic pain from one oral dose of ASA

Erythromelalgia -I’- present

+ + ++

8

c 6

4

2

2 3 4 5

Aspi ri n ndomethacin500 mg 75 mg

absent

+ -++

2 3 4 5 days

Indomethacn25 mg 3td

++++++

10

c 6

4

2 3 4

++ + + + ++ + + +

2 3 4

Dipyridamole Sulfnpyrazone100 mg 4td 200 mg 4td

2 3 4

Sodium salicylate500 mg 3td

days

FIG. 3. The effect of aspirin (acetylsalicylic acid), indomethacin,dipyridamol, sulphinpyrazone and sodium salicylate on erythromelalgiaand on platelet cyclooxygenase activity, as measured by malondialdehyde(MDA) production by arachidonic acid stimulated platelets inplatelet-rich plasma of patients showing primary thrombocythaemia.

*Time lapse after intake of dazoxiben.

(500 mg) lasted 3 to 4 days, but from one oral doseof indomethacin (75mg) less than 24h. Theanalgesic effect of these drugs is in accordance withthe length of inhibition of platelet malondialdehyde(MDA) production by arachidonic acid stimulatedplatelets in platelet-rich plasma (Fig. 3). Continuedindomethacin (25 mg 3 times a day) relieved theerythromelalgia and inhibited MDA, and bothreappeared within 24 h after discontinuation of thedrug (Fig. 3).

Dipyridamole, 400 mg, sulphinpyrazon 800 mg,sodium salicylate 1500 mg (Fig. 3) and ticlopidine,1000mg (data not shown) for 4 days did notalleviate the erythromelalgic symptoms, but alsohad no effect on platelet MDA production (Fig. 3).

Treatment of Patient 2, who suffered fromprimary thrombocythaemia and erythromelalgia,with dazoxiben (200 mg orally every 6 h for 5 days)resulted in incomplete inhibition of platelet MDAsynthesis of 90% at 2 h and of 75% at 4 h after eachingested dose of the drug (Table 3).

Treatment of Patient 3, who suffered fromprimary thrombocythaemia and erythromelalgia,with dazoxiben (400 mg every 6 h for 10 days)resulted in a nearly complete inhibition of MDAsynthesis and complete inhibition of TxB2 forma-tion, but no inhibition of PGE2 synthesis in exposedplatelets (Table 4).

Dazoxiben treatment (400 mg 4 times a day for10 days) did not relieve erythromelalgia and wasassociated with a significant shortened mean plateletsurvival of 3.7 days, indicating platelet consump-tion. Subsequent treatment with one low dose ofASA 500 mg daily, resulted in prompt and completerelief of erythromelalgia, correction of platelet meansurvival to normal (7.9 days) and a change ofplatelet disappearance curves from curvilinear to alinear pattern.

Discussion

Erythromelalgia in thrombocythaemia is a

platelet mediated syndrome of arteriolar inflamma-tion and thrombosis of usually acral areas (Fig. 1)

Mediators of Inflammation. Vol 2.1993 ,387

j. j. Michiels and 17. J. Zijlstra

Table 4. The effect of dazoxiben (400 mg every 6 h for 10 days) on platelet malondiadehyde (MDA)synthesis, plasma thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) levels in Patient 3 who sufferedfrom primary thrombocythaemia and erythromelalgia

Day MDA (nmol per TxB2 PGE2109 platelets (pg/ml) (pg/ml)

C* 2 h** 4 h C 2 h 4 h C 2 h 4 h

20.1 2.0 3.06 1667 49 75 95 115 753 1.7 1.9 <15 <15 989 0.2 < 15 < 15 85 68

Pretreatment value.**Time lapse in hours after intake of dazoxiben.

and may occur in the skin of the leg simulatingsuperficial thrombophlebitis.2 Platelet consumptionis evidently increased during active erythro-melalgia. 13’14 The drugs ASA and indomethacin,which inhibit platelet cyclooxygenase activity,15-18induce complete relief of the erythromelalgicsymptoms and recovery of the ischaemic circulationdisturbances. The diagnostic long-lasting effect of asingle low dose of ASA on erythromelalgia canreadily be explained by its irreversible inhibition ofplatelet cyclooxygenase activity. One single dose ofindomethacin improves erythromelalgia for lessthan 24 h, which corresponded with its reversibleinhibition of platelet cyclooxygenase activity.In contrast, sodium salicylate and the plateletinhibiting drugs sulphinpyrazone, dipyridamole,ticlopidine and dazoxiben neither inhibit plateletcyclooxy-genase activity19’2 nor alleviate erythro-melalgia.2 Curative treatment of erythromelalgiawith ASA (500 mg per day) resulted in a significantincrease of shortened platelet survival to nor-mal. 13’14 In contrast, dazoxiben neither affectederythromelalgia nor corrected the shortened plateletsurvival. These data are consistent with the conceptthat prostaglandin cyclooxygenase products but notthromboxane A2 are necessary for the developmentof erythromelalgia.

Evidence of intravascular activation, secretionand aggregation of hypersensitive platelets inthrombocythaemia, taking place at high shear rateconditions in the end-arterial microvasculature,stems from the histopathological demonstration offibromuscular intimal proliferation and thromboticocclusions of arterioles and small arteries in skinareas of active erythromelalgia (Fig. 1). 1-4 Therelease of platelet derived growth factor duringsecretion and aggregation of platelets is supposedto account for the fibromuscular intimal prolifera-tion. The release and activation of vaso-activesubstances, e.g. prostaglandins, during plateletmediated processes in thrombocythaemia may beresponsible for the inflammatory symptoms oferythromelalgia (Fig. 1).2 This assumption issupported by the finding of increased PGE-like

388 Mediators of Inflammation. Vol 2.1993

activity in blister fluid from erythromelalgic areas(Table 2). The presented study does not give onesingle clue as to the origin (platelet, vessel wall orother) of the prostanoid, but very likely originatesfrom platelets, because a very low dose of ASA (250to 500 mg every other day) is highly effective in theprevention of erythromelalgia--a most disablingcondition. It is thought that cyclooxygenase activityin the platelets lacking nuclei is irreversiblyinhibited by ASA, whereas the production ofprostacyclin, which has an antiaggregation effect, ismuch less affected in the endothelial cells containingnuclei for protein synthesis of cyclooxygenase.21’22

Evidence exists that prostaglandin endoperoxidesare important mediators of inflammation.23,24

Therefore, the presented clinical and experimentalobservations indicate that not thromboxane A2, butits direct precursors, the prostaglandin endoper-oxides or another prostanoid, prostaglandin E. inparticular, are responsible for the pain andinflammation associated with erythromelalgia inprimary thrombocythaemia.

References1. Michiels JJ. Erythromelalgia and thrombocythaemia. PhD Thesis, 1981;

Rotterdam.2. Michiels J, Abels J, Steketee J, Vliet HHDM, Vuzevski VD.

Erythromelalgia caused by platelet-mediated arteriolar inflammation andthrombosis in thrombocythemia. Ann Int Med 1985; 102: 466-471.

3. Michiels J, van Joost T. Erythromelalgia and thrombocythemia: causalrelation. J Amer Acad Dermatol 1990; 22: 107-111.

4. Michiels j, Ten Kate FWJ, Vuzevski VD, Abels J. Histopathology oferythromelalgia in thrombocythaemia. Histopatholog 1984; 8: 669-678.

5. Dale J, Thaulow E, Myhre E, Parry J. The effect of thromboxanesynthethase inhibitor, Dazoxygen, and acetyl-salicylic acid plateletfunction and prostaglandin metabolism. Thromb Haemostas 1983; 50: 703-705.

6. Kristella U. Suction blister device for separation of viable epidermis fromdermis. J Invest Dermatol 1968; 50: 129-132.

7. Unger WG, Stamford IF, Bennett A. Extraction of prostaglandins fromhuman blood. Nature 1971; 233: 336-337.

8. Ferreira H, DeSouza Costa E. A laminar superfusion technique with muchincreased sensitivity for the detection of smooth muscle stimulatingsubstances. Eur J Pbarmaco11976; 39: 377-381.

9. Bult H, Bonta IL. Prostaglandin endoperoxides, serotonin and the superfusedrabbit aorta. Agents andActions 1976; 6: 712-720.

10. Smith JB, Ingermann CM, Silver MJ. Malondialdehyde formationindicator of prostaglandin production by human platelets. J Lab Clin Med1976; 88: 167-172.

11. International Committee for Standardization in Hematology. Recommendedmethods for radioisotope survival studies. Blood 1977; 50:1137-1144.

12. International Committee for Standardization in Hematology. Recommendedmethod for platelet survival studies. J Nucl Med 1988; 29: 564-566.

Prostaglandin cyclooxygenase products in thrombocythaemia

13. Michiels j, Lindemans J, Vliet HHDM, Abels J. Survival kinetics ofplatelets in thrombocythaemia related to erythromelalgia. Brit J Haematol1982; 50: 691.

14. Michiels JJ. Platelet mediated inflammation and thrombosis in thrombo-cythemia. Blood 1990; 76 (Suppl 1): 515.

15. Roth GJ, Majerus PhW. The mechanism of the effect of aspirin on humanplatelets. J Clin Invest 1972; 9: 97-103.

16. Burch JW, Brenziger ML, Stanford M, Majerus PhW. Sensitivity of fattyacid cyclooxygenase from human aorta to acetylation by aspirin. Proc NatlAcad Sci USA 1978; 75: 5181-5184.

17. Crook D, Collins AJ. Comparison of effects of aspirin and indomethacinhuman platelet prostaglandin synthethase. Ann Rheum Dis 1977; 36: 459-463.

18. Fitzgerald GA. Dipyridamole. N EnglJ Med 1987; 316: 1247-1257.19. Moncada S, Korbut J. Dipyridamole and other phosphodiesterase inhibitors

act antithrombotic agents by potentiating endogenous prostaglandinmetabolism. Lancet 1978; i: 1286-1289.

20. Lagarde M, Ghazi I, Dochavanne, M. Effects of ticlopedine plateletprostaglandin metabolism. Prostaglandins Med 1979; 2: 433-440.

21. Moncada S, Vane JR. Arachidonic acid metabolites and the interactionsbetween platelets and blood-vessel walls. N EnglJMed 1979; 300:1142-1147.

22. Kallmann R, Nieuwenhuis HK, de Groot PG, Gijn J, Sixma JJ. Effectsof low dose aspirin, 10 mg and 30 mg daily, bleeding time, thromboxaneproduction and 6-keto-PGFl= excretion in healthy subjects. Thromb Res 1987;45: 355--361.

23. Kuehl FA, Humes JL, Egan EA. Role of prostaglandin endoperoxide in

inflammatory processes. Nature 1977; 265: 170-173.24. Vane JR. Prostaglandins mediators of inflammation. Adv Prost Thromb

Res 1976; 2: 791-801.

Received 23 June 1993;accepted 12 August 1993

Mediators of Inflammation. Vol 2.1993 389

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