Fibrinotysis (1990) 4, 41 43 0268 9499/90/0004-0041/$10.00 �9 1990 Lortgman Group UK Ltd

Activation of the Intrinsic Fibrinolytic Pathway with Normal Extrinsic

Responses in a Patient Undergoing Gastric Surgery

P. J. Grant, K. K. Hampton, J. A. Davies, M. Mahmoud-Alexandroni, P. J. Gaffney, C. R. M. Prentice

S U M M A R Y. In a study to investigate the intrinsic and extrinsic fibrin olytic pathways during surgery, 1 patient with elevated activity of the intrinsic fibrinolytic pathway and with increased crosslinked fibrin breakdown products (XL-FDP) was identified. The patient, a 79-year-old man, underwent radical gastrectomy for invasive adenocarci- noma. Plasminogen activator activity measured as euglobulin clot lysis and tissue plasminogen activator responded normally during the operation whilst pro-urokinase and urokinase were elevated throughout at 0.84.0 iu/ml and 0.5-1.9 iu/ml, respectively. Intrinsic activator activity was elevated at 157 to 246% and XL-FDP remained greater than 1000 ng/ml during the operative period. Factor XII activity was 47% preoperatively and fell to 21% by skin clo- sure. Fibrinopeptide A, protein C, az-antiplasmin, plasminogen and fibrinogen reacted as in other patients we have studied. Platelet count, APTT and PT remained normal. The results show a normal extrinsic fibrinolytic response to surgery with elevated levels of IPA activity during the perioperative period. The latter was expressed by the presence of high circulating levels of XL-FDP.

KE YWORDS. Surgery. Intrinsic fibrinolytic pathway. Factor XII. Urokinase. Crosslinked fibrin degradation prod- ucts.

Major abdominal surgery is accompanied by signific- ant intraoperative increases in extrinsic fibrinolytic activity as reflected by a shortened euglobulin clot lysis time 1 and raised tissue plasminogen activator (t-PA) activity. 2 By the first postoperative day, t-PA activity falls to undetectable levels, due to a rise in t-PA inhibitory activity? The effects of major surgery on the intrinsic fibrinolytic system have not been investigated although it is known that acute increases in urokinase (UK) do not generally occur. During a study of the intrinsic and extrinsic fibrinolytic re- sponses during surgery, a patient with gastric adeno- carcinoma was found to have raised UK, pro- urokinase (pro-UK) and high molecular weight fibrin degradation products without accompanying evi- dence of disseminated intravascular coagulation (DIC). We believe that this is the first reported case of sustained disturbance of intrinsic fibrinolytic activity in plasma.

MATERIALS AND METHODS

A 79-year-old man admitted for resection of a gastric

P. J. Grant, K. K. Hampton, J. A. Davies, C. R. M. Prentice, Univer- sity Depar tment of Medicine, Leeds, M. Mahmoud-Alexandroni, P. J. Gaffney, The National Institute of Biological Standards and Con- trol, S. Mimms, UK.

41

adenocarcinoma was 1 of 12 patients recruited to take part in a study investigating the effects of major sur- gery on fibrinolytic activity for which approval had been obtained from the Leeds West Research Ethics Committee. He had a history of transurethral resec- tion for prostatic carcinoma 2 years previously and a 1-year history of glaucoma. His drug therapy on ad- mission was ranitidine, ferrous sulphate and timolol eye drops. At operation a radical total gastrectomy was performed and there was no evidence of meta- stases. Postoperatively the patient suffered a series of severe medical complications including septicaemia, pulmonary embolus and renal failure and he died 7 weeks after operation. By the time the results reported here were available it was too late to obtain tumour and further plasma samples for analysis.

Venous blood samples were taken pre- and post- induction of anaesthesia, immediately after skin in- cision, at bowel manipulation, after bowel clamping, at skin closure and on the first postoperative day. Separate venepuncture sites and multiple syringe tech- nique were used on each occasion and tubes for assays of fibrinolytic activity kept at 4~ Blood was separ- ated by centrifugation at 21509 and 4~ for 15min and plasma snap-frozen in liquid nitrogen and stored at - 70~ Plasma samples were assayed for: euglobu- lin clot lysis time (ECLT) with results expressed in

42 Activation of the Intrinsic Fibrinolytic Pathway with Normal Extrinsic Responses in a Patient Undergoing Gastric Surgery

TaMe 1 Components of the intrinsic and extrinsic fibrinolytic systems during radical gastrectomy in a patient with adenocareinoma of the s tomach

Plasmin- ECLT t-PA t,PAII U K p ro -UK IPA ogen =t2-AP

Sample time (units) (miu/ml) (miu/ml) (iu/ml) (iu/ml) (%) (%) (%)

Pre-anaesthesia 145 100 7.5 0.6 0.8 202 100 92 Post-anaesthesia 96 134 7.5 1.7 4.0 197 97 88 Skin incision 135 998 7.3 1.9 1.25 200 87 91 Bowel manipulat ion 3086 2793 3.9 0.5 1.25 170 85 86 Bowel clamping 1111 2124 6.0 0.5 0.8 157 91 90 Skin closure 17 55 35 0.8 0.8 246 72 84 First postoperative day 10 100 85 0.5 0.8 177 53 85 'Normal 'per ioperat ive range* 6 2 0 6 6 100-6476 (~90 0.07q).32 N D 3 118 ND 78-126

* Range of values obtained over all sampling times in 11 further patients (n = 77). ND: not done.

Table 2 Plasma concentrations of factor XII, protein C, fibrinogen, fibrinopeptide A and erosslinked fibrin degradation products during radical gastrectomy in a patient with adenocarcinoma of the s tomach

FXII PC Fbg FPA XL-FDP Sample time (%) (%) (g/l) (pmol/ml) (ng/ml)

Pre-anaesthesia 47 108 2,2 35 > 1000 Post-anaesthesia 32 114 2.2 8.1 > 1000 Skin incision 28 116 2.3 4.2 > 1000 Bowel manipulat ion 31 118 2.0 4.4 > 1000 Bowel clamping 28 114 2.0 5.9 > 1000 Skin closure 21 76 1.7 22 > 1000 First postoperative day 30 58 2.2 16 > 1000 'Normal ' perioperative range N D 52-136 ND 0 .3 -> 60 35-845

* Range of values obtained over all sampling times in I 1 further patients (n =77). ND: not done.

units (106/ECLT 2) of plasminogen activator activity; 4 t-PA activity was measured by a spectrophotometric assay 5 and t-PA, UK and pro-UK using ELISA tech- niques;7,8 factor XII by a one-stage assay based on the partial thromboplastin time 9 with results expressed as a percentage of a normal pool; fibrinopeptide A (FPA) by radioimmunoassay 1~ using the antibody supplied by IMCO (Stockholm, Sweden) and crosslinked fibrin degradation products (XL-FDP) using an enzyme- linked immunosorbent assay; 11 protein C by Laurell 'rocket' electrophoresis; t2 ez-antiplasmin using the methods and reagents of the Coatest kit (KabiVi- trum); and fibrinogen by the method of Claus. 13 Intrinsic plasminogen activator (IPA) was measured by the method of Kluft. 14 Briefly this consists of aug- mented precipitation of intrinsic and extrinsic activ- ator activity from plasma and stimulation of activator activity by addition of dextran sulphate during euglo- bulin precipitation. Flufenamate was added to sup- press inhibitory activity. This step measures total activator activity (factor XII-dependent, UK related and t-PA). Following addition of C1 inhibitor and in the absence of dextran sulphate, residual t-PA related activity is measured and IPA determined by subtrac- tion from total activity. ~s These assays were carried out using fibrin plates that were read after 18 h incuba- tion at 37~ The surface area of the lysis zone was cal- culated and plotted against standard values.

RESULTS

The results of assays of the coagulation and fibrinoly- tic factors are shown in Tables 1 and 2. Plasminogen activator activity measured as ECLT rose from 145 and 96 units before operation to 3086 units at bowel clamping and fell to 10 units on the first postoperative day. Similarly, t-PA rose from 100 to 2793 miu/ml at bowel clamping and t-PAI fell from 7.5 to 3.9 miu/ml over the same time.

pro-UK and UK were both high preoperatively and remained elevated during the operation. IPA concen- trations were high preoperatively at 202 and 197% and remained high during the operation whilst factor XII activity gradually fell from 47% preoperatively to 21% after skin closure. Plasma fibrinogen and plas- minogen both fell during the operation to levels of 1.7g/l and 72% respectively. Protein C and ~2-anti- plasmin remained unchanged whilst FPA rose to 22 pmol/ml at skin closure. XL-FDP was elevated pre- operatively at 1000 ng/ml and remained high through- out the operation. The APTT, and platelet count remained normal pre- and postoperatively.

DISCUSSION

The results from this patient demonstrate a sustained

Fibrinolysis 43

increase in activity of the intr insic f ibrinolytic pa thway with raised p ro -UK, UK, IPA and X L - F D P over the operative period. The method used to assay IPA measures bo th factor XII-dependent , and UK-l ike act ivator activities, and it seems probable that the high IPA values reflected main ly UK- l ike activity. It is likely that these values represent p r imary fibrinolysis derived from the intr insic pathway, and were no t secondary to t h rombin generat ion or DIC, as F P A and f ibr inogen responded normal ly dur ing surgery, whilst the A P T T and platelet count were unaffected. In contras t the extrinsic pa thway responded in a sim- ilar m a n n e r to that described previously 1 with intra- operative increases in p lasminogen act ivator activity and tPA, followed by fibrinolytic 'shut down ' on the first postoperat ive day.

Several types of t u m o u r have been reported to con- tain urokinase in increased amoun t s 16'17 and it has been suggested that this might cont r ibu te to t u m o u r spread. 18 A recent s tudy has reported high levels of U K in the p lasma and ur ine of pat ients with gastro- intest inal mal ignancy which tend to fall postoperat- ively. 19 Levels remained high in those pat ients with disseminated mal ignancy. Al though there was no clin- ical evidence of metastases in our patient , the cont inu- ing high p lasma levels of p r o - U K and U K immediately after resection suggest that the t u m o u r had already disseminated. Unfor tunate ly , firm evi- dence on this point was unavailable. The presence of high levels of X L - F D P with no rma l f ibr inogen con- centra t ions indicates that degradat ion of crosslinked fibrin was taking place, and this suggests that the U K was funct ional ly active in vivo.

A C K N O W L E D G E M E N T S

We are grateful to Mrs M. E. Smith for secretarial assistance. Dr Hampton was supported by a Research Training Fellowship from the Wellcome Trust.

R E F E R E N C E S

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Received: 30 November 1988 Accepted after revision: 15 September 1989 Offprint orders to: Dr P. J. Grant, University Department of Medi- cine, General Infirmary, Leeds LS1 3EX, UK.