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Annals ofthe Rheumatic Diseases 1991; 50: 567-571

In vitro platelet aggregability studies: lack of evidence for platelet hyperactivity in systemic sclerosis J

E Price, P S Klimiuk, M I V

Jayson

Abstract Systemic sclerosis is characterised by vascular endothelial damage. Platelets adhering to the exposed subendothelium may contribute to the inflammatory changes found in the vessel wail. Increased in vitro platelet aggregability in systemic sclerosis has been reported. In vitro platelet aggregation of platelet rich plasma obtained from patients with systemic sclerosis (CREST (calcinosis, Raynaud's phenomenon oesophageal dysmotility, sclerodactyly, telangiectasia) variant) and from controls matched for age and sex was compared. Collagen, ADP, and platelet activating factor were used as aggregating agents. The actions of a platelet activating factor antagonist, BN52063, were also examined. Each agonist caused dose dependent platelet aggregation; there was no difference in either rate of primary aggregation or maximum percentage aggregation between platelets derived from patients with systemic sclerosis and from the control group (analysis of variance). BN52063 was shown to be a dose dependent, competitive antagonist of platelet aggregation induced by platelet activating factor; there was no difference in its action on platelets derived from patients with systemic sclerosis or controls. These results do not support the hypothesis that platelets from patients with systemic sclerosis are hyperactive and may explain the disappointing results obtained with antiplatelet drugs in systemic sclerosis.

Rheumatic Diseases Centre, University of Manchester, Hope Hospital, Salford M6 8HD J

E Price

P S Klimiuk M I V Jayson Correspondence to: Professor Jayson. Accepted for publication 4 July 1990

Systemic sclerosis is characterised by vascular endothelial damage.' Endothelial damage in smaller arteries and arterioles leads to exposure of the subendothelium. Platelets adhering to the subendothelium may contribute to the inflammatory changes seen in the vessel wall by releasing inflammatory mediators.2 In vitro studies have suggested that platelets from patients with systemic sclerosis are hyperactive.3 6If this is the case, thrombus formation at injured sites on vessel walls would be more extensive and platelets would be more likely to aggregate in the circulation on exposure to aggregating agents. Antiplatelet drugs may be clinically useful. Collagen is the basement membrane component responsible for platelet adherence, release, and aggregation. After adhesion to collagen the platelets release ADP. Platelet activating factor is a phospholipid considered to be an important inflammatory mediator, participating in many complex inflammatory and

allergic disorders. It is a potent platelet aggregatory agent. We compared in vitro platelet aggregation in response to collagen, ADP, and platelet activating factor of platelet rich plasma obtained from patients with systemic sclerosis (CREST variant) and from controls matched for age and sex. The effects of BN50263, a specific platelet activating factor antagonist, on aggregation induced by platelet activating factor were also investigated. Patients and methods PATIENTS

Patients with systemic sclerosis (CREST variant) and normal controls matched for age and sex were selected for the study. Subjects more than 300/o overweight as defined by Health Education Council guidelines7 were excluded. All subjects were asked to avoid a fatty breakfast and to refrain from smoking on the day of the study. Blood sampling took place at 9 00 am using a 19 gauge needle and with minimal tourniquet and trauma. PREPARATION OF PLATELET RICH AND PLATELET POOR PLASMA

To prepare platelet rich plasma 20 ml blood was added to 3 ml acid-citrate-dextrose (34-7 mM citric acid, 74-8 mM trisodium citrate, 123-6 mM D-glucose-monohydrate, pH 4 8) and centrifuged at 110 g for 20 minutes at room temperature. The top 75-85% platelet rich plasma was removed with a plastic pipette, avoiding contamination by red blood cells. Platelet count, mean platelet volume, and platelet distribution width were measured in whole blood and platelet rich plasma with a Coulter counter model S-plus-IV. Platelet poor plasma was prepared by centrifuging whole blood anticoagulated with acidcitrate-dextrose at 2000 g for 10 minutes at room

temiperature. PLATELET ACTIVATING FACTOR, ADP, AND COLLAGEN INDUCED PLATELET AGGREGATION

Platelet aggregation studies using a Bio/Data Corporation aggregometer, model PAP4, were carried out within three hours of blood sampling. The platelet count in platelet rich plasma was adjusted to 250x 109/l with autologous platelet poor plasma. Platelet activating factor (Sigma) 0 1-500 Ftmol/l was prepared in Tyrodes buffer containing 0-38% bovine serum albumin. ADP (Sigma) 0-31-50 imol/l was prepared in TRIS-saline. Lyophilised soluble calf skin

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collagen (Bio/Data Corporation) was reconsti- Table 3 Platelet distribu n width in samples of whole blood and platelet rich plasma prepared from 12 patients tuted with distilled water to a working concen- with systenic sclerosis and from 12 control subjects. Results tration of 19 mg/ml. Aliquots (450 1) of are expressed (in arbitrary units) as mean (SD) platelet rich plasma were incubated in the Platelet distribution width (arb units) aggregometer at 37°C for three minutes before Whole Platelet rich the addition of 50 [d platelet activating factor, plasma blood was The or aggregation profile collagen. ADP, 17-1 (0-5) NS 17-1 (0-5) followed for at least five minutes. Lag phase, Systemic sclerosis 16-9 (0-4)* 17-4 (06) rate of primary aggregation (measured by slope Normal controls at the steepest part of the curve), and maximum *p

In vitro platelet aggregability studies: lack of evidence for platelet hyperactivity in systemic sclerosis.

Systemic sclerosis is characterised by vascular endothelial damage. Platelets adhering to the exposed subendothelium may contribute to the inflammator...
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