122 Heart 1997;77:122-127 Plasma resistance to activated protein C regulates the activation of coagulation induced by thrombolysis in patients with ischaemic heart Heart: first published as 10.1136/hrt.77.2.122 on 1 February 1997. Downloaded from disease Ole Dyg Pedersen, Jorgen Gram, J0rgen Jespersen Abstract Keywords: thrombolysis; coagulation; activated protein Objective-To determine whether there C resistance; ischaemic heart disease was a relation between plasma resistance to activated protein C and the coagulation Several large scale trials have shown that activation induced during thrombolysis thrombolytic therapy reduces mortality after with 100 mg alteplase in 25 patients with acute myocardial infarction,'-3 and today acute ischaemic heart disease. thrombolytic therapy is a well established Methods-Blood samples were collected treatment for acute myocardial infarction. before (t = 0 h), during (t = 2-25 h), and Failure to reperfuse or reocclusion after suc- after (t = 4 h, t = 12 h, and t = 24 h) cessful reperfusion are still major challenges. thrombolysis to examine the relation Failure to reperfuse was reported in 15-40% between baseline activated protein C of patients and in addition 5-20% of the resistance ratio and markers of coagula- patients in whom thrombolysis was successful tion activation-that is, thrombin- had reocclusion.4 antithrombin III-complexes and pro- Early reocclusion may be a result of activa- thrombin fragment 1 + 2 generated dur- tion of the coagulation system by thromboly- ing thrombolysis. sis.5-7 The activation of coagulation system is Results-There was a negative correlation reflected by the generation of fibrinopeptide A, between activated protein C resistance prothrombin fragment 1 + 2 (F 1 + 2), and ratio and area under the curve of throm- thrombin-antithrombin III-complexes (TAT) bin-antithrombin III-complexes (r. = - during and after the infusion of thrombolytic http://heart.bmj.com/ 0-60; P < 0 003) and there was a trend to a agents.8-"1 The mechanism responsible for this negative correlation between activated apparently paradoxical coagulation activation protein C resistance ratio and area under is incompletely understood, but observations the curve of prothrombin fragment 1 + 2 indicate that plasmin generated during throm- (r. = - 0-37; P = 0.07). This accorded bolysis is the primary trigger.7 1213 In addition with the negative correlation between to activation of coagulation it has recently activated protein C resistance ratio and been reported that thrombolysis generates an the peak value of thrombin-antithrombin endogenous anticoagulant enzyme, activated on September 26, 2021 by guest. Protected copyright. III-complexes (r. = - 055; P < 0.005) and protein C,'4 which may regulate activation of between activated protein C resistance coagulation through the proteolytic degrada- ratio and the peak value of prothrombin tion of central components of the coagulation fragment 1 + 2 (r. = - 0*42; P < 0.04). cascade.'5 Thus there is evidence that the acti- Components of the protein C/S system or vation of coagulation during thrombolysis is known inhibitors of activated protein C determined by a complex balance between Department of Internal Medicine, may influence the activated protein C plasmin mediated coagulant and anticoagulant Ribe County Hospital resistance ratio. There were no associa- mechanisms. in Esbjerg, Esbjerg, tions between the activated protein C To study whether factors in plasma regulate Denmark 0 D Pedersen resistance ratio and protein C, protein C the activation of coagulation during thrombol- or activator resistance to acti- Department of Clinical inhibitor, plasminogen ysis we determined plasma Biochemistry, Ribe inhibitor type-1, whereas there was a vated protein C before thrombolytic therapy County Hospital in trend to a negative correlation between with alteplase in 25 patients with acute Esbjerg, Institute of activated protein C resistance ratio and ischaemic heart disease and correlated the Thrombosis Research, South Jutland protein S. plasma activated protein C resistance values University Centre, Conclusions-The results indicate that with the amount of coagulation reaction prod- Esbjerg, Denmark plasma resistance to activated protein C ucts formed during thrombolysis. J Gram J Jespersen may be one ofthe main mechanisms regu- Correspondence to: lating the activation of coagulation Dr Ole Dyg Pedersen, induced by thrombolysis. This study sug- Patients and methods Department of Clinical Biochemistry, Ribe County gests that it may be possible to single out PATIENTS Hospital in Esbjerg, individuals with a high risk of reocclusion We studied 25 patients admitted to the coro- 0stergade 80, 6700 Esbjerg, Denmark. before the start of thrombolytic therapy. nary care unit with a tentative diagnosis of Accepted for publication acute myocardial infarction and symptoms 2 September 1996 (Heart 1997;77:122-127) lasting less than five hours. Plasma resistance to activatedprotein C regulates the activation ofcoagulation induced by thrombolysis in patients with ischaemic heart disease 123 Originally, the cohort consisted of 34 until assay. Determination of plasma resis- patients, recruited from one arm of a ran- tance to activated protein C is liable to errors domised placebo controlled trial of thromboly- if the plasma samples are contaminated with SiS.13 Nine patients were excluded from the platelets.'8 Thus a centrifugation load of at present study: two patients died within a few least 1000 g and a centrifugation time of 20 Heart: first published as 10.1136/hrt.77.2.122 on 1 February 1997. Downloaded from hours after inclusion; in five patients no min or above this should be used, and plasma plasma samples were available for determina- should be carefully sampled to avoid platelet tion of plasma resistance to activated protein contamination.'9 C, in one patient it was not possible to deter- mine plasma resistance to activated protein C ASSAYS because there was marked prolongation of Plasma concentration of TAT and F 1 + 2 clotting time, and one patient who responded were determined by enzyme-linked immuno- with abnormal high coagulation reaction prod- sorbent assays (ELISA; Behringwerke, ucts died of cardiac rupture 31 hours after Marburg, Germany) and expressed in ug/l and inclusion. None of the patients received treat- nmol/l, respectively. Plasma resistance to acti- ment with heparin or vitamin K antagonist vated protein C was determined by a modified before admission or at the time of admission APIT test (Coatest, activated protein C resis- to hospital. The activated partial-thrombo- tance, Chromogenix, Molndal, Sweden). plastin times (APTT) in the 25 patients Briefly, 50 4l plasma and 50 jul APTT-reagent detemined at baseline (t = 0 h) were 29-3 s- (phospholipids and activator) were incubated 44-8 s and the median value was 37-6 s. These at 37°C for 5 min. Fifty ,ul of CaCl2 (APTT) values were similar to the values obtained in or activated protein C/CaCl2 (APTT+acti- 25 healthy volunteers (range = 34.5 s- vated protein C) was added and the coagula- 46-2 s; median value = 39.4 s). tion time was determined by the use of a Lode The patients were treated with a 5000 IU coagulometer (Groningen, The Netherlands). bolus injection of intravenous heparin (porcine Activated protein C resistance ratio (APC-R) mucosa, Leo Pharmaceuticals, Copenhagen, was calculated as the ratio of (APTT+acti- Denmark) followed by a 3 h treatment with a vated protein C)/APTT. The analytic impreci- alteplase, administered as an initial bolus sion of the assay was examined at two levels by injection of 10 mg, followed by a continuous control plasma delivered by the manufacturer. infusion of 50 mg and 20 mg in each of the The interassay coefficient of variance was subsequent hours (total amount 100 mg). 3-1% (mean APC-R = 3-32; SD = 0-10; After the alteplase was given all the patients N = 7) and 4 0 % (mean APC-R = 1 90; SD were treated with 1000 IU of heparin per hour = 0-08; N = 7). for the next 21 hours. Plasma concentration of protein C was The study was approved by the local ethics determined by an enzyme-linked immunosor- committee and performed according to the bent assay (ELISA) according to a previously http://heart.bmj.com/ principles of the Declaration of Helsinki. described procedure and expressed relative Informed consent was obtained from all par- (%) to pooled normal plasma.20 Plasma con- ticipants before they entered the study. centration of protein S was determined by electroimmunodiffusion using 1 2% poly- BLOOD SAMPLING clonal antibody against human protein S Blood samples were collected from a venous (Dako, Glostrup, Denmark) in a 1% agarose cannula inserted into the forearm contralateral gel and expressed relative (%) to pooled nor- to the infusion arm before (t = 0 h), during mal plasma.2' Plasma concentration of protein on September 26, 2021 by guest. Protected copyright. (t = 2-25 h), and after (t = 4 h, 12 h, and C inhibitor was determined by electroimmun- 24 h) treatment with alteplase. Blood samples odiffision using 1-5% antiserum against at 0 h were collected before heparin treatment. human protein C inhibitor (Nordic, Tilburg, The first 10 ml of blood was discarded and The Netherlands) in a 1% agarose gel and blood samples for determination of TAT and expressed relative (%) to pooled normal F 1 + 2 were collected into 5 ml siliconised plasma. Plasma concentration of plasminogen evacuated glass tubes containing sodium cit- activator inhibitor type-i antigen was deter- rate (0-5 ml of 0-129 mol/l sodium citrate; mined by using an ELISA (Tinteelize, Vacutainer A 3206 SBW-E, Becton Biopool, Umea, Sweden) and expressed in Dickinson, Heidelberg, Germany) by a stan- ng/ml. All assays were done in dublicate. dard technique. 16 Immediately after blood collection 10 41l of a 5 0 mmol/l solution of D- STATISTICAL ANALYSIS Phe-Pro-Arg-CH,Cl (P-PACK, Calbiochem, We used non-parametric statistics to evaluate San Diego, CA, USA) was added to prevent in the results, because most of our data showed vitro activation reaction.17 Plasma resistance to non-Gaussian distribution and our group of activated protein C was determined at baseline patients was small.