Plasma Resistance to Activated Protein C Regulates the Activation of Coagulation Induced By

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 determine 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. The five serial measure- (t = 0 h) in citrated plasma without P-PACK ments of TAT and F 1 + 2 were evaluated by and baseline plasma concentrations of protein Friedman's two-way analysis of variance; C, protein S, protein C inhibitor, and plas- when the results were significant, differences minogen activator inhibitor type-1 were deter- within the groups were evaluated by mined in EDTA-plasma without P-PACK. Wilcoxon's signed rank sum test to compare The blood samples were immediately placed appropriate time points. Box and whisker plots on crushed ice until centrifugation (2000 g for were used to present the serial measurements 20 min at 4°C). Plasma specimens of 500 ,l in of TAT and F 1 + 2. The box is defined by the plastic tubes were frozen and kept at - 80°C lower and upper quartiles and the solid square 124 Pedersen, Gram, J7espersen

Baseline characteristics ofthe patients with acute ischaemic heart disease receiving thrombolysis 10° | Median 25-75% Range Characteristic n = 25 9 Median age (y) (range) 62 (52-74)

Male gender (%) 75 Heart: first published as 10.1136/hrt.77.2.122 on 1 February 1997. Downloaded from Median APC-R (1) (range) 3A19(216394) Median duration of symptoms 8 (mm) (range) 160 (50-290) APC-R, activated protein C resistance ratio. 7

in the box indicates the median value. The 6 whiskers attached to the box indicate the mini- 0 mum and maximum values. Otherwise, data E are presented as median values with ranges in V-C 5 brackets. Spearman rank order correlation was U- used to test associations between two vari- 4 ables. To examine the relation between APC-R and the coagulation reaction products, we 3 used two summary measures of the coagula- U ** tion reaction products-that is, the average area under the curve in the 24-hour treatment 2 period and the peak value after initiation of * UI treatment.22 A P value < 0 05 was regarded as statistically significant. 1

II Results 0 2.25 4 12 24 The baseline characteristics of the patients are h presented in the table. Figure 2 Box and whisker plot showing the response of the coagulation reaction product, prothrombin fragment 1 + 2, during thefirst 24 hours after the start ofthrombolysis COAGULATION ACTIVATION INDUCED BY with 100 mg alteplase in 25 patients with ischaemic THROMBOLYSIS heart disease. *F 1 + 20ho, V F 1 + 22,ho.r P < 0 0001; The coagulation activation in the patients was **F 1 + 20o V F 1 + 24-ho, P < 0.0001. F 1 + 2, reflected by significant changes in the plasma prothrombinfragment 1 + 2. concentrations of TAT and F 1 + 2 in the first 24 hours after starting thrombolysis (figs 1 and 1 + 2 in the first four hours after the start of

2). We observed a significant increase in the thrombolysis followed by a decrease and http://heart.bmj.com/ median plasma concentrations of TAT and F return to the pretreatment concentrations.

Figure 1 Box and ASSOCIATIONS BETWEEN APC-R AND THE whisker plot showing the Median CII 25-75% Range COAGULATION REACTION PRODUCTS response ofthe coagulation There was a negative correlation between reaction product, thrombin- 66.6 137.7 218 APC-R and area antithrombin III-complexes under the curve of TAT during thefirst 24 hours 40 r (r, = - 0-60; P < 0.003), between APC-R after the start of AIk and the peak value of TAT (r, = - 055; on September 26, 2021 by guest. Protected copyright. thrombolysis with 100 mg P < 0-005), and a negative correlation between alteplase in 25 patients with ischaemic heart disease. APC-R and area under the curve of F 1 + 2 *TATOhr v TAT2-our, (r, = - 0-37; P = 0 07), between APC-R P < 0-0002; **TATOhr V and the peak value of F 1 + 2 = - 0-42; TAT4,,hu, P < 0 0003. (r, TAT, thrombin- 30H P < 0'04). The associations between APC-R antithrombin III- and the coagulation reaction products are complexes. shown in figs 3 and 4.

RELATIONS BETWEEN COMPONENTS OF THE PROTEIN C/S SYSTEM, PLASMINOGEN 20 ACTIVATOR INHIBITOR TYPE-I, AND APC-R - In order to elucidate the mechanism regulat- H- ing activated protein C, we also investigated whether the main components of the protein * ** C/S-system or known inhibitors of activated protein C determined at baseline (t = 0 h) 10 had any influence on APC-R. The median * * plasma concentration of protein C determined before the start of thrombolysis was 114% ** (78%-161%). There was no correlation between protein C and APC-R (r, = - 0 1 1; JIL TT P = 0-62). The median plasma concentration 0 - I I I I of protein S determined before initiation of 0 2.25 4 12 24 thrombolysis was 119% (96%-170%) and h there was a trend to a negative correlation Plasma resistance to activated protein C regulates the activation ofcoagulation induced by thrombolysis in patients with ischaemic heart disease 125

Figure 3 Scatter plot of between protein S and APC-R (r, = - 0 39; the associations between P = 0 05). The median plasma concentration activated protein C 0 resistance ratio determined . ofprotein C inhibitor determined before initia- before initiation of 60 H tion of thrombolysis was 99% (36%-163%). thrombolysis with alteplase There was no correlation between protein C Heart: first published as 10.1136/hrt.77.2.122 on 1 February 1997. Downloaded from in 25 patients with acute inhibitor and APC-R = 0-06, P = 075). ischaemic heart disease 45 H (rs and the area under the The median plasma concentration of plas- curve ofthrombin 1--M minogen activator inhibitor type-1 antigen antithrombin-III complexes 0 determined before initiation of thrombolysis in thefirst 24 hours of 0 30 H 0 treatment (r, = - 0-60; 0 was 26-8 ng/ml (7.3 ng/ml-226-0 ng/ml). There P < 0003) (A) and was no correlation between plasminogen acti- between activated protein 0 0 C resistance ratio and the 15 [- 0 vator inhibitor type-i antigen and APC-R peak value ofthrombin- 0 (rs = - O-15; P = 0-51). antithrombin III-complexes *0 .0 * The median value of APC-R determined = - O55; P < 0 005) (r, o L i before initiation of thrombolysis and heparin (B). APC-R, activated 1.5 2.0 2.5 3.0 3.5 4.0 protein C resistance ratio; 4-5 treatment in our 25 patients with ischaemic AUC, area under the APC-R (1) heart disease was 3-19 (2 16-3-94). Our refer- curve; TAT, thrombin- antithrombin III- ence interval determined in 25 healthy volun- complexes. 140 B * ( ,218) teers was 2-99-4-78, with a median value of 4-03. 120

-M 100 -* Discussion Early reocclusion after successful reperfusion H 80 I of the infarct related coronary artery may be a 0 result of the coagulation activation caused by 0 60 I thrombolysis'6 and it has been reported that * *.*plasmin is a key enzyme in this activation X 40 1213 (D I process.7 Thrombolysis also induces gener- a. * * ation of anticoagulant endogenous activated 20 - * protein C, which may put a brake on the * * %4 *degree of activation of the coagulation sys- 0 I I tem.'4 We have now shown that plasma resis- 1.5 2.0 2.5 3.0 3.5 4.0 4.5 tance to activated protein C may be an APC-R (1) important mechanism in the regulation of Figure 4 Scatter plot of 6 A coagulation activation during thrombolysis the associations between with alteplase in patients with acute ischaemic http://heart.bmj.com/ activated protein C resistance ratio determined heart disease. before initiation of * Plasma resistance to activated protein C is thrombolysis with alteplase 0 determined as the anticoagulant response, in 25 patients with acute E 4 measured by a modified APTT test, after a ischaemic heart disease C and the area under the standard amount of activated protein C has curve ofprothrombin been added to plasma. Addition of activated fragment 1 + 2 in thefirst U- *z protein C to plasma enhances degradation of

24 hours oftreatment on September 26, 2021 by guest. Protected copyright. (r, 0 *z S * activated coagulation factor V and activated = - 037;P = 0-07) 2 (A) and between activated *. S* * coagulation factor VIII and thereby prolongs protein C resistance ratio * * *0*the coagulation time. Plasma resistance to and the peak value of prothrombin fragment 1 + *** activated protein C is expressed as the APC-R, 2 (r, = -0-42; P < in which high values indicate low plasma resis- 004) (B). APC-R, I I I I tance to activated protein C and low values activated protein C 0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 indicate high plasma resistance to activated resistance ratio; A UC, area under the curve; F 1 APC-R (1) protein C.23 + 2, prothrombin fragment 1 +2. 10 B ASSOCIATION BETWEEN APC-R AND COAGULATION ACTIVATION * In accordance with a number of previous stud- 8 -* ies, including our own pilot study,"3 we 0 E observed that thrombolysis with alteplase C

c- caused an activation of coagulation as demon- - + 6 strated by a significant increase in TAT and F cL 1 + 2 (figs l and 2). The results of our present 0 study indicate that it is a genuine plasma fac- 4 4. tor that determines the degree of coagulation * * ** activation during thrombolysis with alteplase. 2 % We observed a negative correlation between 0~ %*- APC-R determined before thrombolysis with alteplase and area under the curve ofTAT, the 1 peak value ofTAT and area under the curve of 1.5 2.0 2.5 3.0 3.5 4.0 4.5 F 1 + 2, and the peak value of F 1 + 2 gener- APC-R (1) ated during the 24 hours treatment (figs 3 and 126 Pedersen, Gram, Jespersen

4). Probably this regulatory mechanism is of cific plasma proteins involved in the regulation general importance, and not specifically of the protein C/S system had any influence on related to treatment with alteplase, because we APC-R. There was no correlation between have observed a similar association between protein C and APC-R, whereas we found a

APC-R and activation of coagulation in six trend to a negative correlation between protein Heart: first published as 10.1136/hrt.77.2.122 on 1 February 1997. Downloaded from patients treated with streptokinase (results not S and APC-R. This association may represent a shown). These negative correlations demon- compensatory increase of protein S as a conse- strate that increased plasma activated protein quence of increased plasma resistance to acti- C resistance (decreased APC-R) is associated vated protein C. Protein C inhibitor and with an increased coagulation activation dur- plasminogen activator inhibitor type-I are ser- ing thrombolytic therapy. It is surprising that pins which have the capacity to inhibit acti- plasma resistance to activated protein C deter- vated protein C,'0 31 but it is not known mines to such a degree the coagulation activa- whether these inhibitors are of clinical impor- tion, but this observation supports the view of tance. We did not observe any association the protein C/S-system as a key anticoagulant between protein C inhibitor and APC-R or system.23 Our observation of increased coagu- between plasminogen activator inhibitor type-i lation reaction products in patients with antigen and APC-R. increased plasma resistance to activated protein C accords with recent reports suggesting LIMITATIONS OF THE STUDY an enhanced activation of coagulation in Unfortunately, few of our patients had low val- homozygous carriers of factor V Leiden point ues of APC-R. These patients in particular mutation.24 respond with marked activation of the coagulation system. Thus inclusion of more patients POSSIBLE CLINICAL IMPLICATIONS could strengthen the analysis of our results. The possibility of predicting the degree of However, our result is strengthened by the fact coagulation activation caused by thrombolysis that the association was found with two vari- from APC-R determined before the start of ables (TAT and F 1 + 2) known to reflect the therapy is interesting. Our results obtained by degree of coagulation activation and evaluated the serial measurements of TAT and F 1 + 2 by two summary measures (area under the demonstrate that patients with the lowest curve and peak value) and not single measure- APC-R respond with the most excessive ments. increase in TAT and F 1 + 2 during throm- In addition, it is important to stress that our bolytic therapy (figs 3 and 4). This observation study was designed to investigate the relation suggests that APC-R determined before the between APC-R and the coagulation activa- start of thrombolysis may be used to tailor tion after thrombolysis and not to evaluate any antithrombotic treatment to the individual and association with clinical endpoints. Therefore,

that such a strategy may reduce the incidence there is a need for larger studies to investigate http://heart.bmj.com/ of early reocclusion. It should be noted that the relation between APC-R, coagulation acti- plasma resistance to activated protein C is eas- vation, and clinical endpoints after thromboly- ily determined (within 30 min.) by a modified sis. APTT test. There was no association between baseline APTT and the coagulation activation CONCLUSION (results not shown) and to our knowledge our In conclusion, we report a hitherto unde- study is the first study to report that it may be scribed mechanism (plasma resistance to acti- possible to predict the degree of coagulation vated protein C) that is involved in the on September 26, 2021 by guest. Protected copyright. activation by determination of APC-R at base- regulation the coagulation activation triggered line. by thrombolysis. This mechanism is inherent in human plasma and it is associated with CAUSES OF INCREASED PLASMA RESISTANCE TO resistance to activated protein C. ACTIVATED PROTEIN C This study was supported by a grant from Fonden for Mutation in coagulation factor V is the most Lxgevidenskabelig Forskning i Ringkobing, Ribe og definitive cause of increased plasma resistance S0nderjyllands Amter. Dorte Jespersen, Mette Toft, and to activated protein C,25 but otherwise the Kathrine Overgard provided excellent technical assistance. physiological regulation of activated protein C in plasma is poorly understood. The mutation 1 ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomised trial of intravenous is particularly common among patients with streptokinase, oral aspirin, both, or neither among 17187 venous thrombosis,26 whereas the prevalence cases of suspected acute myocardial infarction. ISIS-2. Lancet 1988;2:349-60. of heterozygosity for the mutation of factor V 2 ISIS-3 (Third International Study of Infarct Survival) Arg506 to Gln in patients with ischaemic heart Collaborative Group. ISIS-3: a randomised comparison of streptokinase vs tissue plasminogen activator vs disease is 5-6%.27-29 Because there were no anistreplase and of aspirin plus heparin vs aspirin alone cellular elements in our samples, we were not among 41299 cases of suspected acute myocardial infarction. Lancet 1993;339:753-70. able to determine the prevalence of this muta- 3 The GUSTO Investigators. An international randomized tion in our patients. However, it is unlikely trial comparing four thrombolytic strategies for acute myocardial infarction. N Engl _J Med 1993;329:673-82. that the prevalence is higher than previously 4 Collen D. Towards improved thrombolytic therapy. Lancet reported because our patients had APC-R 1993;342:34-6. 5 Scharfstein JS, George D, Burchenal JEB, Cannon CP, > 2 16, and most of patients with mutation in Becker RC, Eisenberg PR, et al. Haemostatic markers coagulation factor V have APC-R below 2.0.26 predict clinical events in patients treated with rt-PA and adjunctive antithrombotic therapy (abstract). Jf Am Coll The mechanism of an enhanced APC-R as Cardiol 1994;23:56A. observed in the present study remains 6 Gulba DC, Barthels M, Westhoff-Bleck M, Jost S, unknown but we have examined whether spe- Rafflenbeul W, Daniel WG, et al. Increased thrombin Plasma resistance to activated protein C regulates the activation ofcoagulation induced by thrombolysis in patients with ischaemic heart disease 127

levels during thrombolytic therapy in acute myocardial 18 Sidelmann J, Gram J, Pedersen OD, Jespersen J. Influence of infarction. Circulation 1991;83:937-44. plasma platelets on activated protein C resistance assay. 7 Gram J, Munkvad S, Leebeek FWG, Kluft C, Jespersen J. Thromb Haemost 1995;74:993-4. Reactive coagulation induced by plasmin in patients 19 Sidelmann J. Influence of centrifugation load on platelet treated with recombinant tissue-type plasminogen activa- number and PAI-1 antigen concentration in human tor. Coron Artery Dis 1993;4:371-7. plasma. Fibrinolysis 1994;8(suppl 2):148-9 8 Galvani M, Abendschein DR, Ferrini D, Ottani F, 20 Boyer C, Othschild C, Wolf M, Amiral J, Meyer D, Larrien Heart: first published as 10.1136/hrt.77.2.122 on 1 February 1997. Downloaded from Rusticali F, Eisenberg PR. Failure of fixed dose intra- MJ. A new method for the estimation of protein C by venous heparin to suppress increases in thrombin activity ELISA. Thromb Res 1984;36:579-89. after coronary thrombolysis with streptokinase. JAm Coil 21 Jespersen J, Nielsen MT. Levels of protein S during the Cardiol 1994;24:1445-52. normal menstrual cycle and in women on oral contracep- 9 Weaver WD, Hartmann JR, Anderson JL, Reddy PS, tives low in estrogen. Gynecol Obstet Invest 1989;28:82-6. Sobolsli JC, Sasahara AA. New recombinant glycosy- 22 Matthews JNS, Altman DG, Campbell MJ, Royston P. lated prourokinase for treatment of patients with acute Analysis of serial measurements in medical research. myocardial infarction. JAm Coi Cardiol 1994;24: 1242-8. BMJ 1990;300:230-5. 10 Andreotti F, Kluft C, Hackett DR, Davies GJ, Maseri A. 23 Svensson PJ, Dahlback B. Resistance to activated protein C Thrombin generation after fast or prolonged regimens of as a basis for venous thrombosis. N Engl J Med 1994; tissue-type plasminogen activator. Lancet 1993;342: 330:517-22. 937-8. 24 Greengard JS, Eichinger S, Griffin JN, Bauer KA. 11 Rapold HJ, Grimaudo V, Declerck PJ, Kruithof EKO, Variability of thrombosis among homozygous siblings Bachmann F. Plasma levels of plasminogen activator with resistance to activated protein C due to an Arg-Gln inhibitor type 1, ,B-thromboglobulin, and fibrinopeptide mutation in the gene for factor V. N Engl Jf Med 1994; A before, during, and after treatment of acute myocardial 331:1559-62. infarction with alteplase. Blood 1991;78:1490-5. 25 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, 12 Eisenberg PR, Miletich JP, Sobel BE. Factors responsible Dirven RJ, de Ronde H, et al. Mutation in blood coagula- for the differential procoagulant effects of diverse plas- tion factor V associated with resistance to activated pro- minogen activators in plasma. Fibrinolysis 1991;5: tein C. Nature 1994;369:64-7. 217-24. 26 Zoller B, Svensson PS, He X, Dahlback B. Identification of 13 Munkvad S, Gram J, Jespersen J. Possible role of vascular the same factor V gene mutation in 47 out of 50 throm- intima for generation of coagulant activity in patients bosis prone families with inherited resistance to activated undergoing coronary thrombolysis with recombinant tis- protein C. Jf Clin Invest 1994;94:2521-4. sue-type plasminogen activator. A randomized, placebo- 27 Emmerich J, Poirier 0, Evans A, Marques-Vidal P, Arveiler controlled study. Scand J Clin Lab Invest 1991;51: D, Luc Gerald, et al. Myocardial infarction, Arg 506 to 581-90. Gln factor V mutation, and activated protein C resis- 14 Gruber A, Pal A, Kiss RG, Sas G, Griffin JH. Generation tance. Lancet 1995;345:321. of activated protein C during thrombolysis. Lancet 1993; 28 Ridker PM, Hennekens CH, Lindpainmer K, Stampfer 342:1275-6. MJ, Eisenberg PR, Miletich JP. Mutation in the gene 15 Esmon CT. Protein-C: biochemistry, physiology, and clini- coding for coagulation factor V and the risk ofmyocardial cal implications. Blood 1983;62:1155-8. infarction, stroke and venous thrombosis in apparently 16 Kluft C, Verheijen JH. Leiden fibrinolysis working party. healthy men. N EnglJ Med 1995;332:912-7. Blood collection and handling procedures for assessment 29 Editorial. Special report on mutation in the gene coding for of tissue-type plasminogen activator (t-PA) and plas- coagulation factor V. Circulation 1995;91:2693. minogen activator inhibitor-I (PAI-1). Fibrinolysis 30 Suzuki K, Deyashiki Y, Nishioka J, Toma K. Protein C 1990;4(suppl 2):155-61. inhibitor. Structure and fumction. Thromb Haemost 1989; 17 Seifried E, Tanswell P. Comparison of specific antibody, 61:337-42. D-Phe-Pro-Arg-CH2CI and aprotinin for prevention of in 31 Fay WP, Owen WG. Platelet plasminogen activator vitro effects of recombinant tissue-type plasminogen acti- inhibitor. Purification and characterization of interaction vator on haemostasis parameters. Thromb Haemost with plasminogen activators and activated protein C. 1987;58:921-6. Biochemistry 1989;28:5773-8. http://heart.bmj.com/ on September 26, 2021 by guest. Protected copyright.